American Pharmaceutical

The Review of American Pharmaceutical Business & Technology

Volume 22 Issue 1 | January/February 2019

SHOW ISSUE:

PDA Annual Meeting

Pittcon 2019
DCAT Week Õ19

Transmission
Raman Spectroscopy for
Pharmaceutical Analysis
Excluding
Burkholderia cepacia
Complex from Aqueous,
Non-Sterile Drug Products

Facility Design for

Continuous Bioprocessing
and Smart Manufacturing
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 January/February 2019 | Volume 22, Issue 1

COVER FEATURES
18 SPECTROSCOPY
Transmission Raman Spectroscopy for Pharmaceutical Analysis
Johannes Kiefer
Technische Thermodynamik, Universität Bremen, Germany

36 MICROBIOLOGY
Excluding Burkholderia cepacia complex from Aqueous, Non-Sterile Drug Products
Tony Cundell, PhD, Principal Consultant,
Microbiological Consulting, LLC

50 BIOPHARM PROCESSING
Facility Design for Continuous Bioprocessing and Smart Manufacturing: Attributes for Success
Jeff Odum, CPIP
NCBiosource

www.americanpharmaceuticalreview.com | | 3
IN THIS ISSUE »
12 MANUFACTURING 60 FORMULATION AND DEVELOPMENT

Does the International Council for Harmonization Offer a Solution Liquid-Fill Capsules – Benefits for Highly Potent API Formulation
via ICH Q12? and Scale-Up
Robert Dream, Managing Director Alyn McNaughton
HDR Company LLC Lonza Pharma & Biotech

22 DRUG DELIVERY 66 BIOPHARM DEVELOPMENT

Kolliphor® HS 15 - An Enabler for Parenteral and Oral Formulations
Shaukat Ali and Karl Kolter
BASF Corporation and BASF SE
45 SEPARATION PURIFICATION
Application of a Small EF Hand Affinity Tag for Expression, Purification
and Biophysical Studies of G Protein-Coupled Membrane Receptors
Alexei A. Yeliseev1 and David J. O’Connell2
1
National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health
2
School of Biomolecular and Biomedical Science, Conway Institute,
University College Dublin
54 FORMULATION AND DEVELOPMENT
Modeling in Drug Metabolism for Drug Discovery and Development
Kristen Cardinal, Project Coordinator, Drug Metabolism, Covance
Hao Sun, PhD, Principal Pharmacokineticist, Translational Sciences, Seattle Genetics
Self-Cleaving Tags Based on Split Inteins: Increased Reliability Enabling
Higher Throughput Applications
Izabela Gierach, Co-founder, Protein Capture Science
David W. Wood, The Ohio State University, Department of Chemical & Biomolecular
Engineering, Columbus, OH and Co-founder, Protein Capture Science
70 SPECTROSCOPY
Assessing Aluminum Vaccine Adjuvant Filling, Sedimentation, and
Resuspension in Sealed Vials using Water Proton NMR
Marc B. Taraban1, Christopher B. Fox2,3 and Yihua Bruce Yu1
1
Department of Pharmaceutical Sciences, and Bio- and Nano-Technology Center,
University of Maryland School of Pharmacy
2
Infectious Disease Research Institute
3
Department of Global Health, University of Washington

SPECIAL FEATURES REGULAR FEATURES

8 Social Media Connections 6 A Message from the Editor
9 CNPerspectives 7 Editorial Advisory Board
42 Eurofins Lancaster Laboratories Facility Tour 82 P.I.N. Points
58 An Interview with Quality Executive Partners, Inc. 84 Advertiser's Index
64 An Interview with Evonik
74 IFPAC-2019 Program Preview
78 DCAT Week '19 Preview
80 INTERPHEX 2019 Preview

4 | | January/February 2019
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 » A Message from the Editor »
It’s the Little Things

There are many big issues facing the world, the United States, and the pharmaceutical industry. Wars, climate change, and government
shutdowns all take up the majority of the daily headlines and news stories we read about.
For the pharmaceutical industry, topics such as drug pricing, government oversight, and costs associated with bringing a product to market,
are always bubbling at the surface.
And while there are many pundits who can (and do) talk about these topics frequently, and probably with more insight than I can muster, right
now, in this little bit of editorial real estate that’s all mine, I’m going to talk about a topic that I’m positive won’t be discussed anywhere else in
the media.
But first a little about me.
I like things I can count on. For example: when putting on or taking off a lid, nut, or anything else – it’s always “righty tighty – lefty loosey”. It’s
always been that way and it always should be that way. Starting your car with a key – turn to the right. Light switches: up for on, down for off.
These actions are embedded in our minds, in our muscle memory, probably in our DNA.
Screw tops for ointment tubes adhere to this law – turn right to tighten, left to loosen.
But…
Why are they different shapes? (see photo for example)
Both incorporate the little foil lining puncturing device in the top
(a great little feature) but the different shapes lead to frustration.
Putting a top back on a tube should require very little mental effort.
I shouldn’t have to stop and think which end goes down.
Numerous times I have attempted to put the top back on one only
to feel the top spinning uselessly around and around because I got
it backwards.
I’ve looked closely at both tops and tried to figure out if one design is
better – but to no avail. One might be better for gripping with fingertips,
and the other might be a bit more streamlined and therefore easier to
slide into a box. Is it a marketing device, designed to give the product an edge, something unique? Is it tube size specific? Smaller tubes only
get one of the designs, larger tubes get the other?
In any case, solving this riddle won’t make any of the bigger issues we are facing any easier.
But a logical explanation would certainly allow me to check it off my list.
So, if you know, drop me an email.

Sincerely,

Mike Auerbach
Editor-In-Chief
mauerbach@comparenetworks.com

6 | | January/February 2019
» Editorial Advisory Board »
Shaukat Ali, PhD John Finkbohner, PhD Daniel L. Norwood, MSPH, PhD
Technical Support Manager Director, Regulatory Affairs Distinguished Research Fellow
BASF Corporation MedImmune Boehringer Ingelheim Pharmaceuticals, Inc.

Ghulam Shabir Arain, PhD, CSci, CChem, Adam S. Goldstein Mehul Patel
FRSC, FCQI Senior Manager, Clinical Purification, Operations/Development Global Marketing Director
Managing Director Genentech Endotoxin and Microbial Detection
DGS Pharma Consulting Ltd., UK Charles River
Davy Guillarme, PhD
Katherine Bakeev, PhD Senior Lecturer, School of Pharmaceutical Sciences David Radspinner
Director of Analytical Services and Support University of Geneva, University of Lausanne Director of Marketing and Applications Support for BioProcess
B&W Tek, Inc. Production
Chris Halling
Douglas J. Ball, MS Thermo Fisher Scientific
Senior Manager, Global Communications
Diplomate, American Board of Toxicology; Research Fellow, and European Marketing
Drug Safety Research & Development Aniruddha M. Railkar, PhD
Catalent Pharma Solutions
Pfizer Global Research & Development Director of CMC
Tarsa Therapeutics
Brian Lingfeng He
Suraj Baloda, PhD
Research Investigator Gary E. Ritchie
Founder and President
Bristol-Myers Squibb President
SARMICON, LLC.
Council For Near Infrared Spectroscopy
Ronald Iacocca, PhD
Rory Budihandojo
Senior Research Advisor, Product and Process Performance Rodolfo J. Romañach, PhD
Director, Quality Systems Audit
Eli Lilly & Co. Professor of Chemistry
Boehringer Ingelheim Shanghai Pharmaceuticals
Co., Ltd. University of Puerto Rico, Mayagüez Campus
Maik W. Jornitz
Vice President of Business Development Shouvik Roy, PhD
Harsh Chauhan, PhD
G-Con Manufacturing, LLC Principal Scientist, Organizational Unit Leader in
Assistant Professor
School of Pharmacy & Health Professions Hemant N. Joshi, PhD, MBA Drug Product Engineering
Creighton University Principal Amgen
Tara Innovations LLC Jim Rydzak
Robert V. Chimenti
Sr. Strategic Applications Engineer Ian Lewis, PhD Investigator, Strategic Technology Division
Innovative Photonic Solutions Director of Marketing GlaxoSmithKline
Adjunct Professor Kaiser Optical Systems, Inc. Ronak Savla
Rowan University
Ralph Lipp, PhD Fellow
Emil W. Ciurczak, PhD President and CEO Catalent Applied Drug Delivery Institute
Doramaxx Consulting Lipp Life Sciences LLC
Ken Seufert
Rick E. Cooley Jack Lysfjord Managing Director, North America
Retired Principal Consultant MEGGLE USA Inc.
Eli Lilly & Co., Inc. Lysfjord Consulting LLC
Jaleel Shujath
Weiguo Dai, PhD Steven R. Maple, PhD Industry Strategist, Life Sciences
Scientific Director, Janssen Fellow, Drug Product Development Head of Pharmaceutical Technology Development Dept. OpenText
Johnson and Johnson Lilly Research Laboratories, Eli Lilly & Co., Inc.
Donald C. Singer
Nila Das, PhD Jerold M. Martin GSK Senior Fellow, R&D
Senior Research Investigator Senior Vice President, Scientific Affairs GlaxoSmithKline
Bristol-Myers Squibb Pall Life Sciences
Onkar N. Singh, PhD, MBA
Vivek Dave, PhD John P. Mayer Director, Pharmaceutical Development at CONRAD
Assistant Professor, Pharmaceutical Sciences Senior Research Scientist Eastern Virginia Medical School
St. John Fisher College, Wegmans School of Pharmacy Indiana University
Allen Templeton, PhD
Michael Dong, PhD Michael J. Miller, PhD Associate Vice President
Consultant President Formulation Sciences Merck Research Laboratories
MWD Consulting Microbiology Consultants, LLC
Zhenyu Wang, PhD
Dr. Thomas Dürig Ronald W. Miller, PhD, MBA Associate Principle Scientist and Group Leader
Sr. R&D Director, Pharmaceutical and Food Ingredients President , Technology Consultant Respiratory Product Development
Ashland Inc. Miller Pharmaceutical Merck & Co.

Walter Dziki, PhD Ganapathy Mohan, PhD Wayne K. Way, PhD

Associate Research Fellow Head of Global CMC Regulatory Affairs Analytical Business Marketing Manager
Abbott Laboratories Merck, Sharp and Dohme Corp. MilliporeSigma

Stuart Farquharson, PhD Shane R. Needham, PhD Larry Wigman, PhD

President & CEO Laboratory Director Principal Scientific Manager
Real-Time Analyzers, Inc. Alturas Analytics, Inc. Genentech

www.americanpharmaceuticalreview.com | | 7
 AMERICAN PHARMACEUTICAL REVIEW

Social Media Connections

FDA Drug Information Harvard Medical School

@FDA_Drug_Info @harvardmed
#FDA releases draft Guidance for Industry on Market-
Using bioinformatics analysis and a novel zebrafish model
ing Status Notifications Under Section 506I of the
of multiple sclerosis, researchers identified environmental
Federal Food, Drug, and Cosmetic Act; Content and
factors that boost neuro-inflammation
Format: https://go.usa.gov/xE8db

Alzheimer's Assoc.
Stand Up To Cancer @alzassociation
@SU2C
"SPRINT MIND 2.0 provides genuine, concrete hope that
we will have answers for future generations on how to
Promising news for the future of personalized im-
prevent dementia." @DrMariaALZ on why we're investing
mune therapies from #StandUpToCancer Convergence
more than $800K to extend the SPRINT MIND study.
Research Team Leader Harlan Robins’ and @Genentech.
http://bit.ly/2B7Z4TO #ENDALZ
Learn more here: https://bloom.bg/2S1ihQy

NYU Langone Health CDC_eHealth

@CDC_eHealth
@nyulangone
Clinicians and pharmacists: Looking for a simple way to
A new study from @nyuschoolofmed shows that inject-
determine which #pneumococcal vaccines your patients
ing wounded skin with stem cells can speed up healing
need? Download new PneumoRecs VaxAdvisor app and
by more than 50 percent in mice with diabetes. Learn
get guidance tailored for each patient. Available for iOS
more about this research: https://bit.ly/2GRFTmw
and Android devices.

Memorial Sloan Kettering

Cancer Center HHS.gov
@sloan_kettering @HHSGov

ICYMI - Cancer DNA taken from spinal fluid could serve You can help reduce the #OpioidCrisis by safely disposing
as a liquid biopsy that provides information on brain of unused medications. Learn how with step-by-step guide
tumor mutations. @nature from @NIDAnews: http://bit.ly/2RcFURT #NDAFW

Connect with American Pharmaceutical Review at:

facebook.com/AmericanPharmaceuticalReview
twitter.com/ampharmrev
linkedin.com/groups/American-Pharmaceutical-
Review-3889659

8 | | January/February 2019
CNPerspectives
American Pharmaceutical Review is one of several outstanding publications available from CompareNetworks, Inc.
Here is a look at the insightful content our readers may enjoy from four of our sister publications: Pharmaceutical Outsourcing,
Labcompare, American Laboratory and Biocompare.

Selecting and Qualifying a Regenerative Medicine CMO

Among the most remarkable technical advances seen within the healthcare sector over the last decade is the fast growing sector of regenerative
medicine. Breakthrough technologies are empowering regenerative medicine through the introduction of new, more accurate, easy-to-use tools
and new drug therapies with the potential to be dis- ease modifying, capable of reversing the damage of certain diseases. It follows that adapting
these novel processes to an outsourcing strategy requires careful consideration. Evaluating and selecting a contract manufacturing organization
(CMO) is one of the seminal decisions that can influence the overall success of a development program.
https://bit.ly/2FUD6bK

Treat With Heat

One of the oldest tricks in the book in science is to “heat up a sample.” It’s an old trick, but a good one, and one that is getting better. That’s
especially true for differential scanning calorimetry (DSC), which analyzes how heat impacts a sample. With DSC analysis, a sample can be cooled
to –180° Celsius and heated up to 2400° Celsius. That provides a wide range for analyzing changes.
https://bit.ly/2FML2uF

Statistics in the Laboratory: Pooling

In statistics, “pooling” describes the practice of gathering together small sets of data that are assumed to have the same value of a characteristic
(e.g., a mean) and using the combined larger set (the “pool”) to obtain a more precise estimate of that characteristic. In medicine, for example, a
statistical “meta analysis” pools the results of many clinical studies to try to find a deeper truth that might not be revealed consistently in each of
the individual studies.
In this column, we’ll see how pooling variances can work to the advantage of analytical chemists and others who carry out routine measurements.
When properly applied, it can save a lot of money.
https://bit.ly/2P2535p

Why Automate Sample Management?

Bottlenecks in drug discovery drive the need for automated sample management. In small molecule discovery, hardware and software determine
how efficiently samples can be stored and used. The options for automation cover a wide range of platform sizes and throughput capabilities,
but today’s key element could be flexibility.
https://bit.ly/2FMGtR3

www.americanpharmaceuticalreview.com | | 9
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» MANUFACTURING »

Does the International

The world economies are increasingly threatened by barriers to trade,
while the pharmaceutical sector through the International Council
for Harmonization (ICH) has been stepping up the effort to dismantle

Council for Harmonization obstacles to global trade in medicines.

Offer a Solution via Regulatory Point of View

From 2005 through 2011 ICH published Q8-Q11 guidelines that intro-

ICH Q12? duced Quality by Design (QbD) concepts to product development and
manufacturing. Industry expected regulatory flexibility and harmoni-
zation from these ICH guidelines, but the guidelines did not convey
methods for optimal processes and planning. Regulatory filings still
contain more information and raise more questions than ever before.
Globally, applicable changes are a logistical challenge due to different
timelines and submission requirements for post-approval changes.
Hence, the risk of drug shortages, supply deviations and noncompli-
Robert Dream ance situations are increased. In summary:

Managing Director 1. Lack of a harmonized approach on technical and regulatory

HDR Company LLC considerations for lifecycle management

2. Several gaps exist that limits full realization of intended

benefits of ICH Q8-Q11

3. Post-approval “operational flexibility” has not been

achieved

4. Main emphasis at ICH to date has focused on early stages

of the product lifecycle

Hence, to alleviate the gaps, ICH Q12 introduced and issued for
comments in December 2017; The draft guidance ICH Q12 entitled
“Technical and Regulatory Considerations for Pharmaceutical Product
Lifecycle Management,”

https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/
Guidelines/Quality/Q12/Q12_Draft_Guideline_Step2_2017_1116.pdf

12 | | January/February 2019
 « MANUFACTURING »

has been published for comment. It applies for products, including
development and manufacture of drug substances (chemical entities
and biotechnological/biological entities) - ICH Q11 and drug-device
combinations – ICH M3 (R2), Step 4, June 11, 2009.
ICH Q12 explicitly addresses post-approval changes for products
already on the market. Following, ICH Q12 most manufacturing and
analytical changes (Figure 1) can be managed efficiently under the
pharmaceutical quality system (PQS) of a company without regulatory
approval prior to implementation. Therefore, cooperation of regulators
(assessors and inspectors) is required by ICH Q12.
Incompatibility with Established EU
Regulatory Framework
During the final framing of drafting ICH Q12 guideline, the European
Commission discovered that after a review of the EU rules, parts of
the guideline could not be implemented in the EU without changes
in legislation. These differences mainly centered on the guideline
concept of established conditions (ECs) for manufacturing and control
for categorizing quality elements requiring regulatory submission if
changed, and product lifecycle management (PLCM) which establishes
a repository on details of established conditions.
marketing authorization holders (MAH) to commit, with agreement of
their regulatory agency to their own lifecycle management protocols
for manufacturing and issues related to lifecycle management,
particularly to CMC.
Key Aspects of ICH Q12
• ECs - Established Conditions
The concept of ECs, provides a clear understanding between
the Marketing Authorization Holder (MAH) and regulatory
authorities regarding the necessary elements to assure
product quality and identify the elements that require a
regulatory submission.
• PACMP - Post-Approval Change Management Protocol
For planned changes, the PACMP is a tool that provides
predictability regarding the information to support a CMC
change and the type (category, which is usually one step
lower than without PACMP) of regulatory submission based
on prior agreement between the MAH and the responsible
competent authority.
• PLCM - Product Life Cycle Management

Both the ECs and PLCM concepts are at the core of Q12 guideline. ECs The PLCM document is for proactive management of the
provide a basis for key objectives behind the guideline of enabling product lifecycle.

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» MANUFACTURING »
Figure 1. Decision Tree for Identification of Established
Conditions (ECs) and Associated Reporting Categories for
Manufacturing Process parameters
Regulatory Tools and Enablers -
Chapter 1.3
ICH Q12 has the potential to reduce costs and time burdens for
regulators and the industry. One of the real benefits could be the
potential regulatory commitment among ICH regions related to
what is “supportive information” within regulatory submissions. This
should also lead to greater application of innovative technologies in
manufacturing and control (i.e. analytical methods) in a timely manner.
Categorization of Post Approval CMC
Changes - Chapter 2
Categorization of Post-Approval CMC Changes is a framework
that encompasses a risk-based categorization for the type of
communication expected of the Marketing Authorization Holder
(MAH) with the regulatory authority regarding CMC changes.
Harmonizing change management in a more transparent and efficient
manner facilitates risk-based regulatory oversight. Harmonized
expectations across the ICH regions emphasize control strategy as a
key component of the enhanced use of regulatory tools for prospective
change management and enabling strategic management of post-
approval changes.
There are some examples of the CTD Sections that contain ECs
(Established Conditions) listed in Appendix I of ICH Q12. The table
does not contain a complete list of ECs for a product. The intention
of the table is to provide general guidance about the elements of
manufacture and control that constitute ECs and their location within
the CTD structure.
14 | | January/February 2019
Established Conditions (ECs) -
Chapter 3
• ECs are legally binding information considered necessary to
assure product quality.
• As a consequence, any change to ECs necessitates a
submission to the regulatory authority.
• All regulatory submissions contain a combination of
ECs and supportive information.
i. Supportive information is not considered to be
ECs, but is provided to share with regulators the
development and manufacturing information at an
appropriate level of detail, and to justify the initial
selection of ECs and their reporting category.
• ECs in a submission are either implicit or explicit:
• Implicit ECs are elements that are not specifically
proposed by the applicant but are derived from and
revised according to regional regulation or guidance
related to post-approval changes.
• Explicit ECs are specifically identified and proposed by
the applicant together with their proposed reporting
category as part of a regulatory submission.
Post Approval Change Management
Protocol (PACMP) - Chapter 4
• A PACMP provides predictability and transparency in terms of
the requirements and studies needed to implement a change.
• An approved PACMP needs to be maintained and assessed
routinely:
• ensure that the outcomes of the initial risk assessment
are still valid.
• confirm that the control strategy continues to ensure
that the product will be produced consistently
following implementation of the change(s).
• The use of a PACMP is enabled through an effective PQS
that incorporates quality risk management principles and an
effective change management system.
• Whenever a CMC change is to be introduced under a PACMP
regulatory requirements with respect to GMP compliance,
an inspection or licensing status should be considered.
Product Lifecycle Management
(PLCM) - Chapter 5
• The PLCM document outlines the specific plan for product
lifecycle management, and includes key elements:
 « MANUFACTURING »

• Summary of Product Control Strategy

• Proposed ECs for the product

• Reporting category for making changes to approved ECs

• PACMPs to prospectively manage and implement one

or more post approval changes

• Post-approval CMC commitments

• Use of a PLCM encourages prospective lifecycle

management planning and facilitate regulatory assessment
and inspection.

• The PLCM document should be updated throughout the

product lifecycle, as needed.

Pharmaceutical Quality System (PQS) Figure 2. Connection Between Knowledge Management and
Change Management Process
and Change Management - Chapter 6
An effective PQS as established in ICH Q10 and in compliance
with regional cGMPs is the responsibility of the firm. Q12 does not
require a specific inspection assessing the state of the PQS before Relationship Between Regulatory
the principles can be used. In the event that the PQS is found not to Assessment and Inspection - Chapter 7
be compliant, it may result in restrictions on the ability to utilize the
flexibility in this guideline. Regulatory assessment and inspection are complementary activities
and communication between assessors and inspectors can facilitate
Consistent with the basic requirements of ICH Q10, an effective change regulatory review of a specific product submission.
management system is necessary for implementation of this guideline
(ICH Q12).

Use of knowledge is the responsibility of the firm and should be
described in the PQS (for more detailed information reference is
made to ICH Q8, Q9, Q10, Q11, Q/IWG Q&A). As described in ICH Q10,
there is no added regulatory requirement for a formal knowledge
management system.
Post Approval Changes for Marketed
Products - Chapter 8
A structured approach to analytical procedure changes, incentiv-
izes structured implementation of equivalent analytical procedures

www.americanpharmaceuticalreview.com | | 15
» MANUFACTURING »
that are fit for purpose (some complex prod-
ucts and methods would be out of scope).
Specific criteria are defined for changes to
analytical procedures used to test marketed
products is described, if followed, the ana-
lytical procedure change can be made with
immediate or another post-implementation
notification, as appropriate.
The data needed for submission to the regu-
latory authority in support of a post-approval
change is established by regional regulations
and guidance. This guideline provides sci-
ence- and risk-based approaches that can be
used to develop strategies for confirmatory
stability studies supporting post-approval
changes to enable more timely filing, approv-
al, and implementation of the changes.
As a note comparing Figure 1 and Figure 3;
we note that there is similarity between se-
lecting ‘Parameter Criticality’ and ‘Established
Conditions’. Changes in ‘CPP’ illustrate a re-
quirement for ‘Prior Approval’; changes in
‘KPP’ requires ‘Notification’; and changes in
‘Non-KPP’ is ‘Not-Reported’.
Figure 2. Decision Tree for Determining Parameter Criticality
16 | | January/February 2019
Q12 Technical and
Regulatory Considerations
for Pharmaceutical
Product Lifecycle
Management; Annex
The Technical and Regulatory Considerations
for Pharmaceutical Product Lifecycle Manage-
ment guideline is issued under a separate
document to covey ICH Q12 guidance.
https://www.fda.gov/downloads/Drugs/
GuidanceCompliance RegulatoryInformation/
Guidances/UCM609366.pdf, Endorsed
16 November 2017, Currently under public
consultation.
ICH Q12, Annex Illustrative Examples;
Annex I: ECs –Illustrative Examples
• Annex I A: Chemical Product
• Annex I B: Biological Product
Annex II: PACMP –Illustrative Examples
• Annex II A: PACMP Example 1
• Annex II B: PACMP Example 2
Annex III: Product Lifecycle Management
Document
• Illustrative Example
Conclusion
As described by ICH Q12, a harmonized ap-
proach regarding technical and regulatory
considerations for lifecycle management will
benefit patients, industry, and regulatory au-
thorities by promoting innovation and con-
on
tinual improvement in the biopharmaceutical
sector, strengthening quality assurance and
improving supply of medicinal products. This
guideline provides a framework to facilitate the
management of post-approval CMC changes
in a more predictable and efficient manner. It
is also intended to demonstrate how increased
product and process knowledge can contrib-
ute to a reduction in the number of regulatory
submissions. Effective implementation of the
tools and enablers described in this guideline
should enhance industry’s ability to manage
many CMC changes effectively under the firm’s
Pharmaceutical Quality System (PQS) with less
need for extensive regulatory oversight prior
to implementation.
References
1. ICH Q12, Step 2, Draft, “TECHNICAL AND REGULATORY
CONSIDERATIONS FOR PHARMACEUTICAL PRODUCT
LIFECYCLE MANAGEMENT,” https://www.ich.
org/fileadmin/Public_Web_Site/ICH_Products/
Guidelines/Quality/Q12/Q12_Draft_Guideline_
Step2_2017_1116.pdf
2. ICH Q12 Annex, Draft, “Technical and Regulatory
Considerations for Pharmaceutical Product Lifecycle
Management,” https://www.fda.gov/downloads/
Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM609366.pdf, Endorsed on 16 November
2017
3. ICH Q8-Q11, 2005-2011
4. ICH M3(R2), Step 4, June 11, 2009; “GUIDANCE ON
NONCLINICAL SAFETY STUDIES FOR THE CONDUCT
OF HUMAN CLINICAL TRIALS AND MARKETING
AUTHORIZATION FOR PHARMACEUTICALS,” https://
www.ich.org/fileadmin/Public_Web_Site/ICH_
Products/Guidelines/Multidisciplinary/M3_R2/Step4/
M3_R2__Guideline.pdf
» SPECTROSCOPY »
Transmission Raman Spectroscopy for
Pharmaceutical Analysis
Johannes Kiefer
Technische Thermodynamik, Universität Bremen, Germany
Abstract
Raman spectroscopy is an established analytical method in the
pharmaceutical industry. Micro-spectroscopic setups utilizing the
backscattered signal are common in the analysis of solid samples
such as tablets. However, backscattering arrangements typically
share the disadvantage of collecting signal from a very small spot
and thus potentially missing important information. Transmission
Raman spectroscopy has been proposed as a solution to this problem.
The present article introduces the concept of transmission Raman
spectroscopy and discusses its applications in pharmaceutical analysis.
Background
Many pharmaceutical products are comprised of a rather small
amount of the active ingredient and a number of excipients, which
may act as stabilizers/protectors, fillers, or therapeutic enhancers.
Due to the potentially small amount of the pharmaceutically active
compound (PhAC), its distribution in the overall formulation may not
be homogeneous. For example, a tablet could contain micrometer-
sized aggregates of the PhAC embedded in a filler matrix. This poses a
challenge to the chemical analysis of the product.
In many analytical applications in engineering and science, high
spatial resolution is an important requirement. Therefore, methods
18 | | January/February 2019
are developed to obtain information from a very small area or
volume. For example, spectroscopic techniques such as infrared,
near-infrared, fluorescence, and Raman spectroscopy are capable
of providing detailed chemical information. They are pushed
towards high resolution so that objects and structures at the
micrometer and sub-micron level can be resolved. Larger areas
and volumes are scanned stepwise in order to characterize entire
objects. This scanning, however, takes time and thus is not suitable
for rapid screening of products. In order to overcome this problem,
spectroscopic approaches based on transmission are very promising
and have been developed for pharmaceutical analysis. Amongst the
four techniques mentioned above, near-infrared (NIR) absorption and
Raman scattering spectroscopy are typically the methods of choice
for the application in transmission mode. Transmission (mid-)infrared
spectroscopy is difficult to apply to products like tablets as the strong
absorption in the mid-IR would call for additional sample preparation,
e.g. to prepare thin pellets with a thickness significantly smaller
than a millimeter. Fluorescence spectroscopy on the other hand
usually uses ultraviolet light, which may not be able to penetrate the
sample due to either strong absorption or scattering. NIR and Raman
spectroscopy are better suited. In the last couple of years, however,
there was a strong push for the development of transmission Raman
methods as the Raman signal is more specific than the NIR spectrum.
Moreover, Raman is advantageous in aqueous environments such as
solutions, emulsions, and suspensions, which are common types of
pharmaceutical products as well. In summary, transmission Raman

spectroscopy (TRS) is a favorable method for pharmaceutical analysis.
In the following, the concept of TRS for pharmaceuticals, which was
pioneered by Matousek and Parker about a decade ago,1 will be
introduced and discussed.
Method
Raman spectroscopy is a means of vibrational spectroscopy, in which
monochromatic light, typically from a laser source, is scattered by the
molecules of a sample. The majority of the scattered photons exhibit
the same wavelength as the incident light, but a small fraction is
scattered inelastically, i.e. the Raman scattering. This means that an
energy transfer takes place during the process and, consequently,
the Raman signal is frequency shifted. Usually, the molecules are
initially in the vibrational ground state and are transferred into a
vibrationally excited state via a virtual intermediate state. The energy
of the scattered photon is therefore reduced by the energy difference
of the two vibrational states involved. As these energy differences are
highly specific for any molecule, the Raman spectrum represents a
molecular fingerprint. It can be employed to determine the chemical
composition and quantify the individual components.
The common Raman micro-spectroscopy approach that utilizes the
backscattered signal is illustrated in Figure 1a. A laser beam is focused
+1-302-368-7824 marketing@bwtek.com
« SPECTROSCOPY »
onto the sample surface by a lens, e.g. a microscope objective, and
the scattered signal is collected and collimated by the same lens.
This arrangement is usually referred to as confocal microscopy. It
enables recording spectra from a tiny measurement spot of about 1
µm diameter when appropriate spatial filtering is applied. In order to
separate the laser and signal paths, a dichroic mirror reflecting the
laser wavelength and transmitting the signal can be used. However,
as mentioned above, a high spatial resolution or a pure surface
measurement is not desirable for a rapid quality control during
pharmaceutical production as it may miss important information.
The transmission Raman approach illustrated in Figure 1 is a suitable
alternative. In TRS, the laser travels through the entire sample, giving
rise to Raman scattering in an extended volume. The signal emitted
in direction of the laser beam is collected and analyzed. Sufficiently
blocking the laser radiation is of special interest here in order to avoid
severe interference. This can be achieved by using suitable dichroic
mirrors and Notch filters. The wavelength of the laser needs to be
selected carefully, because the sample must be transmissive for both
the laser and the signal. Near-infrared radiation has been used in most
TRS applications to date as it also reduces the likelihood of photo-
damage and fluorescence interference.
An important advantage of this approach is the large probe volume.
Not only does this ensure sufficient sampling, which is helpful in
All New QTRam®
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www.americanpharmaceuticalreview.com | | 19
» SPECTROSCOPY »
analyzing heterogeneous samples, but also enhances the sensitivity
of the measurement resulting in an improved limit of detection. The
large probe volume contains a higher number of target molecules,
which is proportional to the signal intensity. If sensitivity is not
an issue, the measurement time can be reduced instead, which is
beneficial for rapid screening of products. A detailed comparison
between the backscattering and transmission approaches can be
found in reference 1.
TRS spectra can be evaluated with the traditional univariate and
multivariate tools. In simple cases, it may be sufficient to calibrate
the intensity of a given spectral signature against the concentration
Figure 1. Schematic illustration of Raman spectroscopy in
backscattering microscopy and transmission mode.
20 | | January/February 2019
of a target compound. Complex matrices may be dealt with using
chemometrics in terms of regression approaches such as partial
linear least squares regression (PLSR), support vector machines (SVM),
and artificial neural networks (ANN). The method of choice can be
selected based on the complexity of the samples to be analyzed and
the availability of reliable data for training the algorithm: the more
complex the sample the larger the required training data set.
Applications
Transmission Raman spectroscopy has already found many applica-
tions in the pharmaceutical sector during the past couple of years.
The majority of them has been summarized in several recent review
articles.2-5 To give an idea, the list of applications includes:
• Monitoring of the settling dynamics of
pharmaceutical suspensions.
• Quantification of the pharmaceutically active compound in
model and commercial tablet and capsule formulations.
• Determination of the state in terms of polymorphs,
crystalline and amorphous content, and co-crystals inside
tablets and capsules.
• At-line quality control of content uniformity of
pharmaceutical tablets.
• Process monitoring, e.g. during the growth of PhAC crystals.
The data evaluation in virtually all practical applications has been
achieved using chemometrics because of the complexity of the data.
 « SPECTROSCOPY »

Scotland, and he holds a guest professor-

Conclusion Author Biography ship of the Erlangen Graduate School in
Advanced Optical Technologies (SAOT)
In conclusion, this paper described Prof. Dr. Johannes Kiefer is Chair Professor at the University Erlangen-Nuremberg,
transmission Raman spectroscopy, which is and Head of the division Technische Germany. His research interests are the areas
an interesting approach for at-line and in- Thermodynamik at the University of Bremen, of developing and applying spectroscopic
line applications in the pharmaceutical and Germany. In addition, he is an Honorary techniques for the characterization of ad-
chemical industries. A key advantage over Professor at the University of Aberdeen, vanced materials and processes.
traditional Raman techniques is the large
probe volume, which allows compensation
for inhomogeneous distributions of the target
compound as well as low concentrations.
It also minimizes contributions from thin
surface layers and capsule shells. Moreover,
it is worth mentioning that Raman speed
sen
spectroscopy is a non-destructive method siti
vit
y
and, hence, tested specimen do not need to
be disposed. With the first TRS instruments
already on the market, the number of real-
world applications will rapidly grow in the

re
s
olu
foreseeable future.

tio
n
Acknowledgment
The author gratefully acknowledg-
es financial support from Deutsche
Forschungsgemeinschaft (DFG) through
grant KI1396/4-1.

References 3D Raman image of a pharmaceutical ointment.

1. P. Matousek, A.P. Parker, Bulk Raman analysis of

pharmaceutical tablets, Applied Spectroscopy 60
(2006) 1353-1357.
2. J.A. Griffen, A.W. Owen, D. Andrews, P. Matousek,
Recent advances in pharmaceutical analysis using
transmission Raman spectroscopy, Spectroscopy 32
3D Raman Imaging
(2017) 37-43. Turn ideas into discoveries
3. K.A. Esmonde-White, M. Cuellar, C. Uerpmann,
B. Lenain, I.R. Lewis, Raman spectroscopy as a Let your discoveries lead the scientific future.
process analytical technology for pharmaceutical
Like no other system, WITec’s confocal 3D Raman
manufacturing and bioprocessing, Analytical and
Bioanalytical Chemistry 409 (2017) 637-649. microscopes allow for cutting-edge chemical
4. K. Buckley, P. Matousek, Recent advances in the imaging and correlative microscopy with AFM,
application of transmission Raman spectroscopy to SNOM, SEM or Profilometry. Discuss your ideas
pharmaceutical analysis, Journal of Pharmaceutical with us at info@witec.de.
and Biomedical Analysis, Journal of Pharmaceutical
and Biomedical Analysis 55 (2011) 645-652.
5. C.C. Corredor, C. Vikstrom, A. Persson, X. Bu, D. Both,
Development and robustness verification of an at-
Raman • AFM • SNOM • RISE www.witec.de
line transmission Raman method for pharmaceutical
tablets using quality by design (QbD) principles, MADE IN GERMANY
Journal of Pharmaceutical Innovation 13 (2018)
287-300.

www.americanpharmaceuticalreview.com | | 21
 DRUG DELIVERY
Kolliphor® HS 15 - An Enabler for
Parenteral and Oral Formulations
Shaukat Ali and Karl Kolter
BASF Corporation and BASF SE
Shaukat Ali has worked in the pharma industry for
over 24 years including 14 years at BASF. His areas of
expertise include the controlled release, solid dispersions,
lipid based emulsifying systems, liposome drug delivery,
and film development technology. He received his PhD
in chemistry from the City University of New York, and
postdoctoral training from the University of Minnesota
and Cornell University. He is the editor-in-chief of
Journal Analytical and Pharmaceutical Research and
serves as a member of the editorial advisory board of
several pharmaceutical journals including the American
Pharmaceutical Reviews. He also serves as a panel
of expert in the USP expert committees for General
Chapters-Physical Analysis, Continuous Manufacturing
and Excipient Performance <1059>. He has published
over 45 articles in the scientific journals and is the
inventor of 14 US patents.
After having worked for 7 years at Knoll AG in
Ludwigshafen, Karl Kolter joined BASF AG in 1993,
where he has been responsible for R&D activities in
pharmaceutical excipients, drug formulations and the
application technology of vitamins and carotenoids
for pharma and food. Dr. Kolter’s current work is the
development of innovative excipients mainly for solid
oral dosage forms, which has already resulted in various
new products in the Kollicoat® and Kollidon® range
(Kollicoat® MAE 30 D, 100P, SR 30 D, IR, IR White, Protect,
Kollidon® SR, CL-F, CL-SF, Ludipress® LCE). He obtained
his Ph. D. in pharmaceutical chemistry at the University
of Mainz, Germany. He has published more than 100
articles and posters, and is the inventor of 90 patents.
American Pharmaceutical Review | January/February 2019
Abstract
Kolliphor® HS 15 is an exceptional solubilizer with attributes suited for parenteral and
oral formulation of poorly soluble molecules. As a consequence, it is primarily used
in screening of new chemical entities (NCEs) and also used as a vehicle in delivery
of molecules encapsulated in micelles or micro-emulsions. Its physicochemical and
application relevant characteristics are attributed to a unique structure comprised
of 12-hydroxystearic acid (lipophilic moiety) and polyethylene glycol (hydrophilic
moiety). As a solubilizer with exceptional safety and toxicity profiles, and regulatory
perspectives, it offers unique advantages over many other solubilizers of its class for
injectable (IV, infusion, im, sc) formulations, in self-emulsifying drug delivery systems
(SEDDS/SMEDDS) and lipid based nanoparticulates.
Keywords: Kolliphor® HS15, Solutol® HS 15, polyoxyl 15 hydroxystearate, macrogol 660
hydroxystearate, solubilizer, self-emulsifying systems; SEDDS and SMEDDS, critical
micelle concentration, lipid nanocapsules, parenteral excipient, oral drug delivery,
histamine release, Pgp inhibition, leachable and extractable.
Introduction
Kolliphor® HS 15 (formerly regarded as Solutol® HS 15) a non-ionic surfactant, has been
used as a solubilizer in parenteral and oral formulations for over several decades.1
Solubilizers like Kolliphor® HS 15 and others have been a subject of continued interest
in the industry since the number of poorly soluble new chemical entities (NCEs) is
constantly increasing. Thus, the effort continues to bring these molecules to pipeline
for development in oral and parenteral formulations.2 Kolliphor® HS 15 outperforms
in its class of many lipid based solubilizers, and hence, is frequently used in in vitro
screening and in vivo efficacy studies of compounds.3,4 Kolliphor® HS 15 bears the
hallmarks of an exceptional solubilizer with inherent amphiphilicity stemming from
its combined lipophilic and hydrophilic characteristics derived by reacting 1 mole of
12-hydroxystearic acid with 15 mole of ethylene oxide. It has the hydrophilic lipophilic
balance (HLB) of 16 and a critical micelle concentration (CMC) of 0.005%-0.02%. Due
to its unique solubilization characteristics and safety profiles, this excipient has also
been used in ophthalmic, suppository, macromolecule and protein formulations.5,6
Kolliphor® HS 15, a monographed excipient comprised of about 65%-70% of
ethoxylated mono- and di-ester components, where both the carboxylic group
as well as the hydroxy group can be ethoxylated, and about 30%-35% of free
PEGs, has a unique composition that does not only act as a solubilizer but also as
a co-solvent for poorly soluble molecules.7 In addition, it offers advantages over
highly polymeric solubilizers because it spontaneously self-assembles into micellar
structures consisting of a large number of molecules. Kolliphor® HS 15, a non-ionic
compound in nature, is pH independent and forms micelles typically ranging 10 - 15
nm. It enables an efficient drug loading into its hydrophobic interiors and generates a
22

thermodynamically stable, highly concentrated system, a pre-requisite
for longer systemic circulation, faster absorption and enhancement of
bioavailability.
Approved by FDA in injectable and ophthalmic drugs, and by other
regulatory agencies globally in many other drug products for humans
and animals, Kolliphor® HS 15 has created an enormous interest
particularly for the formulation of poorly soluble drugs and vitamins.
There are innumerable publications describing its use in different
drug formulations and technologies. This review will focus mainly on
chemistry and applications in oral and parenteral drug formulations.
More specifically, this article will shed light on the utilities of Kolliphor®
HS 15 in design and development of self-emulsifying systems,
lipid nanoparticles in oral and parenteral applications, and create
an understanding of its underlying mechanisms pertaining to its
interaction with Pgp with respect to other solubilizers. The regulatory
and toxicological information with future perspectives will also be
discussed in this review.
It is to be noted that the name Kolliphor® HS 15 is kept in the body of
the manuscript while the name Solutol® HS 15 has been left unchanged
where indicated in the references.
Chemistry and Physicochemical Properties
As shown in Figure 1, Kolliphor® HS 15 is synthesized by reacting
12-hydroxystearic acid (I) with ethylene oxide (II) in presence of an
alkaline catalyst to yield the major components, polyethoxylated
derivatives III and V, which represent mono- and diesters. The minor
components VII (free polyethylene glycol), IV and VI are also formed.
Quantification of Kolliphor® HS 15
Physicochemical attributes of Kolliphor® HS 15 follow both the USP and
Pharm. Eur. compendia. The test methods have been developed and
validated per the monographs’ requirements. Though these methods
have been used for qualifying the excipient itself the challenges remain
to quantify Kolliphor® HS 15 in drug formulations. This is due, in part,
to the structural complexity of ethoxylated fatty acid derivatives and
Figure 1. Synthesis flow chart of Kolliphor® HS 15
23
American Pharmaceutical Review | January/February 2019
DRUG DELIVERY
Table 1. Chemistry and physicochemical properties of Kolliphor®
HS 15
CAS # 70142-34-6
Molecular weight 960 - 1900 g/mol
Solidification point: 25°C – 30°C
Hydroxyl value: 90 - 110
Acid value: ≤1
Water <0.5%
pH (10% in water) 6-7%
Density (25°C) 1.03 g/ml
Viscosity (30% in water, 25°C) ~12 mPa s
Solubility,
Water >12 g/10 g
Ethanol 5 g/10 g
Isopropyl alcohol 4.5 g/10 g
Hexane Insoluble
Free ethylene oxide < 1 ppm
because of challenges to separate and identify each of the individual
components in the product itself and even more complex in drug
formulations due to presence the of many other excipients together.
The distribution of these hydrophilic and lipophilic components in
Kolliphor® HS 15 can lead to a greater impact on the stability and the
performance of drug products in general. To address this, Janet et al.
and Hammond et al. developed an advanced analytical technique using
matrix-assisted laser desorption/ionization (MALDI) technique coupled
with an ion mobility device to separate and quantify the individual
components of Kolliphor® HS 15 and used it as a tool to control the
product’s quality attributes.8,9 Zhang and co-workers, developed an
analytical method for simultaneous quantification of Kolliphor® HS
15 (lipophilic fatty acid esters, and hydrophilic PEG fragments) in a
drug formulation prepared with another excipient like Miglyol® 812.10
With the exception of quantification of only free PEG (ca. ~30%) by
HPLC, the lack of compendial test methods, makes it challenging to
quantify the main mono- and di-ester moieties in the formulation. The
authors developed an UPLC method coupled with a NQAD nebulizer
probe for simultaneously quantifying the lipophilic moieties as well
as the hydrophilic free PEGs in the formulation.10 The UHPLC coupled
NQAD method is unique and capable to separate and quantify both
the lipophilic and hydrophilic components without derivatization. In
brief, utilizing the UPLC/NQAD technique, the authors quantified the
lipophilic component amounts to 67.8% eluted at retention time of
2 min; while the other three hydrophilic PEG components amount to
14.7%, 10.7% and 6.9% eluted at the retention time of approx. 6 min,
7 min and 8.6 min, respectively. The total free PEG component was
estimated to be 32.3%, which was in agreement with the compendial
acceptance criteria of 27.0% to 39.0% for free PEG (USP). The lipophilic
and hydrophilic components were also analyzed by gel permeation
chromatography (GPC), a method that used an isocratic condition to
estimate the major and minor components by Coon et.al.11 The authors
quantified the lipophilic component to be 70% at retention time of
7 min and the hydrophilic component to be about 30% free PEGs at
retention time of 12 min, again supporting that the composition of
 DRUG DELIVERY
Kolliphor® HS 15 is highly complex but the results are aligned with
the manufacturer’s specifications. The GPC method, however, lacked
the quantitative determination of the minor pegylated fatty acid
components. It can be taken to suggest that the quantitative analysis
of Kolliphor® HS 15 might be challenging, especially, if only smaller
amounts of Kolliphor® HS 15 are present in the plasma following oral
administration where the bioavailability remains very low (ca. 14%).
Bhaskar and coworkers used LC-MS/MS method with an ion suppression
effect to quantify the Kolliphor® HS 15 in the plasma samples from rats
collected following oral and parenteral administration.12,13 Using this
method, the authors identified a total of twelve oligomers with the
most abundant ions corresponding to m/z 481, 525, 569, 613, 657, 701,
745, 789, 833, 877, 921, 965 by electrospray ionization, and estimated
the concentration by combining all the peak areas for each oligomer.
The liquid chromatography/tandem mass spectrometry method thus
quantified the lipophilic and hydrophilic components in Kolliphor®
HS 15, which was found to be 63.3% and 36.7%, respectively. The
authors were also able to establish the pharmacokinetic parameters
of Kolliphor® HS 15 as well as of 12 PEG oligomers following oral and
intravenous administrations in Sprague Dawley rats by using this state
of the art technique.
Solubilization Capabilities
Kolliphor® HS 15 is soluble in aqueous media and polar organic
solvents but insoluble in apolar solvents. In aqueous solutions,
however, it spontaneously forms micelles which helps encapsulation
and increases solubility, an attribute dependent on the solubilizer
concentration. Figure 2 illustrates that the solubility of poorly soluble
drugs increases with increasing amounts of Kolliphor® HS 15. For
example, with increasing concentrations of Kolliphor® HS 15 from 1%,
5% to 10%, the solubility increases almost linearly and the micellar size
changes only slightly as more drug molecules get encapsulated into
the interior core of the micelles.1,14,15
The linear increase at lower concentrations is typical for this kind
of solubilizer as polysorbate 80, Kolliphor® EL or Kolliphor® RH 40
also show a similar behavior.15 A further increase of the solubilizer
concentration can change the micelle structure and subsequently
results in a deviation from the linear profile.
Figure 2. Solubilization capability of Kolliphor® HS 15
for different drugs
24
American Pharmaceutical Review January/February 2019
Figure 3 exhibits the solubilization capacity of Kolliphor® HS 15 for a
number of model drugs and compares it with other commonly used
solubilizers. The data suggests that Kolliphor® HS 15 is an equally
effective solubilizer as Kolliphor® RH 40 and polysorbate 80. Out of 10
drugs screened, it was observed that four drugs were best solubilized
by Soluplus®, but Kolliphor® HS 15, Kolliphor® RH 40 and polysorbate
80 were found to outperform the others. The ranking of poloxamer 407
(Kolliphor® 407) was not as good as the other solubilizers tested which
can be attributed to the much lower amphiphilicity of poloxamers.
The difference in hydrophobicity between a fatty acid and the PEG
moiety is much larger than between a polypropylene glycol and a
polyethylene glycol moiety.
Application Relevant Characteristics
Kolliphor® HS 15, one of the few excellent solubilizers, has widely been
used in recent times to improve the solubility of many poorly soluble
compounds.4 In comparison with other known solubilizers including
poloxamers, polysorbates, and castor oil based surfactants, it shows an
exceptional solubilization capability for many insoluble compounds as
shown in Figures 2 and 3.
The physicochemical properties of Kolliphor® HS 15 as a solubilizer have
been studied extensively.7 With its profound safety and toxicological
profile relatively better than polysorbate 80 and/or Kolliphor® EL, it is an
excellent choice for screening and pre-clinical pharmacokinetic studies
of cytotoxic and oncology drugs among many other indications. Being
versatile and highly functional, it offers unique benefits due to its
compatibility in in vitro testing as well as in drug formulations as self-
emulsifying/micro-emulsifying systems, lipid nanoparticles for oral,
parenteral, topical, biologics, nasal, and gene delivery applications.16
Furthermore, its unique structure with availability of terminal free
hydroxyl moieties can be modified to a copolymer for safe application
in gene delivery. For example, Yin et al. used a Kolliphor® HS 15 grafted
copolymer with polyethyleneimine (PEI with Mw 25 kD) in gene
delivery to study the cytotoxicity (IC50) against different cell lines, and
found that transfection efficiency of graft copolymer (Solutol-g-PEI)
was significantly improved owing to its higher complexing ability with
DNA compared to pure PEI polymer.17
Figure 3. Solubilization capabilities of Kolliphor® HS 15
and other solubilizers

Table 2A. Light scattering measurements of Kolliphor® HS 15 micelles (placebo) in aqueous
solutions and phosphate buffer pH 7.0
Water
Attributes
2 g/l 20 g/l
Micelle diameter, nm 11.5 • 11.7
MW, Dalton 163,000 • 154,000
Molecules/micelles 163 • 154
Table 2B. Light scattering measurements of Kolliphor® HS 15 micelles loaded with drugs in
phosphate buffer pH 7.0
7.5% Tocopherol
Attributes 2% PHB ester
acetate
MW, Dalton 473 180.2
Micelle diameter, nm 24.3 13.6
Micelle MW, Dalton 2.6 x 106 2.3 x 105
Drug molecules/micelle 2061 127
The micellar structure of Kolliphor® HS
15 helps solubilize the compounds in
the micellar core almost independent
of pH or biorelevant media. The critical
micelle concentration is 0.06-0.1 mM and
hydrodynamic radius is 11-15 nm.18 The
micelles are stable upon steam autoclaving at
121°C/20 min, making it compatible to heat
sterilization procedures, a hallmark of stability
of the molecule under harsh conditions.
Thysen examined the physicochemical
attributes of Kolliphor® HS 15 with and
without vitamin E acetate before and after
sterilization by autoclave.14 The author did
not observe any changes in the particle size
of the micelles and/or saponification and pH
values of placebo or vitamin formulation. In
another study, for example, 1% propofol was
effectively solubilized with 8% Kolliphor®
HS 15, and the formulation was stable and
particle size remain unchanged at 40°C
for over 8 weeks.19 In an earlier study, it
was also observed that solubility of many
drugs improved dramatically and drug
loaded micelles were also stable for several
weeks without any change in the particle
size.1 The authors used a CONTIN software
program to calculate the size, distribution
and composition of the micellar diameter,
and measured the molecular weight of the
micellar structure. The average MW of a 12 nm
Kolliphor® HS 15 micelle comprised of about
170 molecules was determined to be 170
k Dalton. Depending on the type of poorly
soluble actives and their concentration the
PBS pH 7.0
2 g/l 2 g/l
• 12.7 • 12.2
• 172,000 • 153,000
• 172 • 153
0.5% 3%
Sulfathiazole Acetaminophen
255 151
13.0 12.0
2.2 x 105 1.9 x105
22 185
number of active molecules per micelle
varied between 22 and 2061, as shown in
Table 2. This study provides a deeper insight
into the structure of non-loaded and drug-
loaded micelles.
The amphiphilic characteristics of Kolliphor®
HS 15 are also important to form tiny oil
droplets from a self-emulsifying drug
delivery system (SEDDS/SMEDDS) to carry
the maximum payload and effectively deliver
the drug molecules.
Special Dosage Forms and
Applications
Self-emulsifying drug delivery systems
(SEDDS)
Self-emulsifying drug delivery systems
(SEDDS) have been a subject of continued
interest, due, in part, to faster development of
new chemical entities to market as compared
to other solid dispersion and micronization
technologies.20 Thus, the norm of developing
a self-emulsifying formulation requiring
solubilizers like non-ionic Kolliphor® HS 15 is
highly sought after due to its compatibility
with both weakly acidic and alkaline drug
molecules (unpublished data). A relatively
large percentage of lipophilic (PEG-ylated
fatty acids) and substantial amount of
hydrophilic or free polyethylene glycol
components in Kolliphor® HS 15, play a crucial
role in encapsulation and self-emulsification
processes with other lipids/oils. For example,
Kolliphor® HS 15 in combination with
25
American Pharmaceutical Review | January/February 2019
DRUG DELIVERY
desired amounts of co-surfactants and/
or oils enables simultaneously dispersed
droplets and maintains supersaturation,
that could yield indiscriminate permeation
and absorption of drugs through GI tract.
The smaller particles created by using the
larger amount of the lipid surfactants and co-
surfactants in turn help the rapid absorption
and enhancement of bioavailability. Patel
et al., investigated lumefantrine (LF) SEDDS
prepared with the surfactant Kolliphor® EL
and oleic acid (OA). The faster dissolution of
lumefantrine was attributed to a reduction in
droplet size and a greater surface area of the
self nano-emulsifying LF-OA ionic complex.21
The faster dissolution was due to significant
reduction in diffusion layer thickness caused
by nanosizing of particles.22
Selection of the appropriate solubilizers, co-
solubilizers and oils is important to design
an effective oral drug delivery system with
controlled particle size, polydispersity,
and zeta potential.23 The authors detailed
the identification of lipids and lipid-based
excipients for formulations to optimize
the oral delivery of lipophilic drugs via
SEDDS/SMEDDS. Li and Zhao and Sapra et
al. proposed the strategies for early phase
screening of SEDDS formulations involving
the solubilizers like Kolliphor® HS 15 among
others.24,25 The authors also described a range
of important routes of administration and the
respective excipients/solubilizers because of
their abilities to self-emulsify APIs to achieve
the desired particle size for required doses,
efficacy and stability. Sadurni et al. designed
nano-emulsions, each comprised of binary
mixtures of Kolliphor® HS 15/Miglyol® 812,
Kolliphor® HS 15/soybean oil and Kolliphor®
EL/Miglyol® 812, and characterized them
by the small angle x-ray scattering and
dynamic light scattering techniques.26 The
authors noted that the increasing amounts
of surfactants between 60% and 90% in
SEDDS led to smaller particles (ca. < 50
nm), yielding the distinct phase behavior
changes in hexagonal phase (HII) and
lamellar crystalline phase (Lα). Heshmati et al.
investigated the emulsifying characteristics
and pharmacokinetics of four indirubin
E083 loaded self-emulsifying systems, each
comprised of Kolliphor® HS 15 with varied
amounts of medium chain mono- and di-
 DRUG DELIVERY
glyceride oils, and a cosolvent PEG 400 or ethanol.27 The authors
concluded that the higher amounts of the surfactant Kolliphor® HS
15 in SNEDDS, showed a significantly greater bioavailability with
respect to SEDDS, which comprised of lower amount of Kolliphor®
HS 15, suggesting further that smaller particles are critical to faster
absorption and improved bioavailability. Zhou et al. used Kolliphor®
HS 15 in design and development of brusatol loaded SEDDS consisting
of a medium chain triglyceride (MCT) and polyethylene glycol 400
(PEG 400) as a solvent, and evaluated against dextran sodium sulfate-
induced ulcerative colitis.28 The authors observed that the therapeutic
efficacy of drug loaded SEDDS improved and bioavailability increased
>2-fold as compared to suspension formulation in mice due to the
particle size differences.
Particle size reduction is important in aiding the absorption and
bioavailability but it also improves the safety of APIs as well as
minimizes the adverse effects by avoiding excessive dosing. Benbrook
et al. used Kolliphor® HS 15 in the chemoprotective activities of SHetA2
in self-emulsifying systems because of its excellent safety profile and
the lack of toxicity when administered by oral gavage to rats.29 The
studies concluded that the SHetA2 administered orally at 750 ppm
in Kolliphor® HS 15 SEDDS for over 6 weeks, achieved the desired
absorption of drug levels without causing any toxicity in mice. At
higher dose, however, it accumulated in the lower GI tract resulting in
the reduced systemic absorption or poor bioavailability. Menon et al.
also used Kolliphor® HS 15 in nanoprobes for cellular and molecular
imaging analysis by employing Nile red doped hexamethyldisiloxane
(HMDSO) nano-emulsions prepared by Kolliphor® HS 15-lecithin binary
combination. The nano-probes had the average particle size of 71±39
nm and were stable in human plasma much longer than 24 hours that
enabled to successfully measure the pO2 changes in cells by magnetic
resonance imaging.30
Solid Self-emulsifying Drug Delivery Systems (s-SEDDS)
As the interest in self-emulsifying systems continues to grow, so is
the interest in solid-SEDDS to counter the shelf life and mitigate the
food effects and/or concerns of stability with liquid SEDDS.31 Thus,
the efforts continue to develop stable solid-SEDDS for oral dosages
to effectively deliver the drugs in pellets and tablets to overcome the
precipitation and enhance the stability of drugs in the formulations.
Abdalla and Mader used Kolliphor® HS 15 with glyceryl monostearate
(GMS) and microcrystalline cellulose (MCC) for stable SEDDS pellets
and studied the release profile of the model drug diazepam from the
pellets.32 The authors concluded that the diazepam loaded MCC pellets
with Kolliphor® HS 15, formed a self-emulsified system upon contact
with aqueous media, and drug release was expedited dramatically
as compared to those lacking Kolliphor® HS 15. These pellets showed
a sluggish release and did not perform well. This study also sheds
light on how the solubilizers like Kolliphor® HS 15 embedded in the
solid MCC matrix had the ability to quickly self-emulsify and strongly
improve the delivery of poorly soluble molecules. Other groups also
used solid matrices like Neusilin US2 and mesoporous silica, and
developed the s-SEDDS with aims to increase solubility and enhance
bioavailability of drugs.33,34 Sri et al. evaluated the s-SEDDS formulation
26
American Pharmaceutical Review | January/February 2019
of valstaran comprised of Kolliphor® HS 15, PEG 400 and Capmul®
MCM adsorbed on Neusilin® US2, and investigated the dissolution and
pharmacokinetics of the drug from a solid SEDDS adsorbed matrix. The
bioavailability of valsartan in rats was improved 1.6-fold as compared
to a suspension.35 Cerpnjak et al., on the other hand, used adsorbing
polymeric matrices each composed of HPMC or maltodextrin with
Kolliphor® HS 15 as solubilizer with another co-surfactant/solvent in
design of self-emulsifying systems for naproxen prepared by a spray
drying process.36 The authors observed that maltodextrin based
s-SMEDDSs were granular smooth-surface microspheres, while the
HPMC based s-SMEDDS were irregularly shaped microparticles. The
dissolution profiles of maltodextrin based particles showed a faster
release, very similar to liquid SMEDDS, but the HPMC based s-SMEDDS
showed an incomplete and slightly longer dissolution profile due to
gelling characteristics of this polymeric carrier. Other solid matrices
have also been evaluated in lyophilized matrices. For example, Li et al.
evaluated the sucrose based lyophilized solid SEDDS formulation of
Lornoxicam in rats.37 The solid formulation was prepared by adsorbing
a liquid SEDDS consisting of Kolliphor® HS 15, Labrafil® M 1944 CS and
Transcutol® HP. The pharmacokinetic parameters were assessed and
the bioavailability of the drug improved over 1.5-fold with respect to
the commercially available tablets, suggesting that Kolliphor® HS
15 is a an effective solubilizer and suitable for lyophilization with
sugars in the development of SEDDS tablets. Kolliphor® HS 15 is
compatible with sugars and oleochemicals, it is also compatible with
cyclodextrins. For instance, the liquid efavirenz dispersions prepared
with Kolliphor® HS 15 and β-CD (2:0.05) increased the absorption
rate ~5-fold and the AUC by ~ 2-fold as compared to pure drug.
In vitro dissolution of efavirenz increased about 4-fold, while the
bioavailability enhanced 17-fold when Kolliphor® HS 15 and β-CD
were used with povidone K30 in a ternary mixture as opposed to a
binary mixture of β-CD and PVP K30.38
Varshosaz and Ghassami studied fenofibrate in spray dried binary
mixtures, each prepared separately with Eudragit® E100 and Kolliphor®
HS 15 and HPC as the carriers.39 Colloidal silica was used as an anti-
tacking agent. Fenofibrate was dissolved in acetone with Eudragit®
E100 or Kolliphor® HS 15 or hydroxypropyl cellulose (HPC) at 1:1 and
1:5 ratios (drug/excipient) and spray dried with silica as an anti-tacking
agent. The dissolution efficiency varied for each binary formulation and
was dependent upon the ratios of API and polymeric carrier. The angle
of repose or flowability was slightly lower with a binary mixture of
Kolliphor® HS 15 and fenofibrate as compared to Eudragit®/fenofibrate
binary blend. This difference might be related to the waxy nature of
the Kolliphor® HS 15 and its stickiness that decreased flowability and
dissolution efficiency as compared to Eudragit® and HPC polymers. The
dissolution enhancement rate of the drug with increased flowability
was presumably due to particle size reduction of binary powder blends
as compared to untreated drug.
Gonzales and co-workers investigated the self-emulsifying properties
and pharmacokinetic profiles of cyclosporine A (CyA) in a formulation
comprised of Kolliphor® HS 15, Labrafil® M2125CS and oleic acid
(/7:2:1 (v/v/v)).40 The oral bioavailability of these SEDDS in rats was

about twofold greater than the micronized suspension lacking
Kolliphor® HS 15 (70% vs. 36%). The improved oral bioavailability was
caused by marked improvement in the solubility of CyA in micro-
emulsion as compared to micronized suspension (ca. 136 μg/ml vs.
23.2 μg/ml in human gastric juice, and 133 μg/ml vs. 10.8 μg/ml in
simulated intestinal juice). It also holds true for many other drugs
in SEDDS/SMEDDS. For example, Cui et al et al. investigated in vivo
efficacy of a drug in rat of a formulation containing 50% Kolliphor® HS
15 as a surfactant and 35% Transcutol® as a co-surfactant and 15% of
an oil consisting of ethyl oleate, Maisine® 35-1, and Gelucire® 44/14.
The bioavailability of the drug in SEDDS improved > twofold over the
tablet formulation.41
Lipid nanocapsules, solid lipid nanoparticles and nanostructure
lipid carriers
Kolliphor® HS 15 has been investigated for in vitro hemolytic activity
of a micro-emulsion formulation containing oleic acid and soybean
oil.42 It was found that the Kolliphor® HS 15 based micellar nano-
particles were stable over 3 months and were far less hemolytic
(<0.2%), making it most suitable to parenteral formulations without
any additional cosolvents. Chauvet et al. found that Kolliphor® HS 15
based lipid nanocapsules (LNCs) bearing the particle size of 25 nm,
influenced cellular calcium signaling in the brain through activation
of neuronal channels, presumably by interaction of cholesterol rich
membrane lipids with lipophilic/fatty acid chains of the solubilizer.43
Such interactions could have important clinical implications as these
LNCs are able to penetrate deeper into the membrane, allowing
to endocytose the drug into the cells, and affecting the biological
activities.44 Dulieu and Bazile developed the lipid nanocapsules (LNCs)
from freeze dried formulations containing Kolliphor® HS 15 and Lipoid®
S100 in trehalose solution.45 The resulting particles were stable due to a
lipid protective coating around the emulsion droplets, and surrounding
Kolliphor® HS 15 on the outer core of the particles, providing a stealth
protection. The rigidity and stability of the core increased as the ratios
of lipid/Kolliphor® HS 15 increased at the outer coating surface. This
stealth protection is important for longer systemic circulation of a drug
as it gets carried through for delivery into the specific tissues or site.46
Pitorre and co-workers studied the lipid nanocapsules (LNC) derived
from Kolliphor® HS 15, Labrafac® W and Span® 80 to target the lymph
nodes by subcutaneous administration of a model compound.47 The
authors demonstrated that when the fluorescent labeled compound
was administered in rats subcutaneously (sc) in neck versus the IV in tail
vein, it was highly selective to only specific lymph nodes but was less
selective and poorly distributed in all other lymph nodes. The particle
size of LNC (ca. 50 - 100 nm) was apparently important in distinguishing
the bio-distribution and absorption of these nanocarriers by the
tissues or organs.48 Drug loaded LNCs, and their escape by RES and
being recognized for uptake by the cancer cells to help deliver
the drugs at the specific target sites by subcutaneous injection as
opposed to intravenous route, provide an alternative and effective
delivery of drug while reducing the adverse effects. Paclitaxel-
loaded LNCs comprised of Kolliphor® HS 15, cholesterol, Labrafac®,
and Lipoid® S100 were used in vitro and in vivo efficacy studies of
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American Pharmaceutical Review | January/February 2019
DRUG DELIVERY
drugs against multidrug resistance (MDR) glioma tumors and found
that the Kolliphor® HS 15 containing LNC loaded with paclitaxel
demonstrated superb activities against commercially available Taxol®
formulation containing Kolliphor® EL and ethanol.44 These differences
in bio-efficacy were due to tumor targeting through intracellular
compartmentalization of paclitaxel in the multi-drug resistance
(MDR) cells caused by interaction and inhibition of membrane
associated glycoprotein (Pgp) efflux pump, leading to cell death. LNC
loaded paclitaxel suspension comprised of Kolliphor® HS 15, Lipoid®
S100, and Captex® 8000 have also been evaluated for increasing the
oral bioavailability of paclitaxel.49 In this study, the authors observed
that LNCs were highly effective in enhancing the bioavailability to
21%, about a 3-fold increase as compared to Kolliphor® EL based
Taxol® formulation (approx. 7%). Like LNCs, the self-emulsifying
loaded paclitaxel formulations, s-SEDDS and s-SMEDDS, were equally
effective and showed, respectively, 5-fold and 2-fold increases in
the oral bioavailability as compared to Kolliphor® EL based Taxol®
formulation.50,51
Improving the bioavailability of paclitaxel loaded LNC with Kolliphor®
HS 15, offers a unique opportunity to further explore the structural
components and packing of lipids in these assemblies. To shed
light on the understanding of physio-chemical and morphological
characteristics of lipid nanoparticles derived from glyceryl tristearate/
castor oil and prepared by combining each with a solubilizer like
Kolliphor® HS 15 or poloxamer 188, Dora et al used the dynamic
light scattering (DLS), differential scanning calorimetry (DSC), wide-
angle X-ray scattering (WAXS), atomic force microscopy (AFM) and
transmission electron microscopy (TEM and cryo-TEM have been
used to elucidate the structures of these assemblies.52 The oil/water
(o/w) nano-emulsion (NE) and solid lipid nanoparticle (SLN) were
derived from the solubilizer with castor oil or tristearin. Lipid-based
nanocarriers (NLCs) were prepared by hot solvent diffusion method by
dissolving castor oil, tristearin, and lecithin in organic solvents followed
by homogenization of solution containing 0.1% Kolliphor® HS 15 (Tm
24.3 °C; ΔH 78.3 g/J) or 1% poloxamer 188 (Tm 54.3 oC, ΔH 129 g/J).
In a separate study, the authors observed that quercetin loaded SLNs
each with poloxamer 188 and Kolliphor® HS 15 solubilizers exhibited
45% and 70% release, respectively; while the NLC did not control the
release because of the castor oil was not internalized and surrounded
by the solid lipid.53 Joshi et al. investigated the delivery of a poorly
soluble anti-malarial drug artemether (ARM) in the nanostructured
lipid carriers (NLC) as the Nanoject for intravenous delivery in rats,
and observed an extended release of drug over 20 hours as compared
to 6 hours with the conventional micro-emulsion formulation.54
The Nanoject’s extended characteristic was due to presence of the
saturated glyceryl dilaurate lipid that controlled the API’s release
and improved the adverse effect and safety. As a consequence, the
survival rate with NLC IV administration improved as compared to
marketed oily intramuscular formulation (Larither®). The data also
suggests that the Nanoject, an extended NLC formulation, significantly
improved the ARM’s antimalarial activity lasting over 20 days as
compared to intramuscular (IM) dose formulation. Furthermore, the
biocompatibility and microstructure formation with excipients like
 DRUG DELIVERY
Kolliphor® HS 15 in the Nanoject rendered the reduction in pain upon
intravenous injection. Because of their excellent safety in oral and
injectable formulations, and mildness to skin, the SLN and NLC have
also been investigated in the topical drug delivery.55,56
Liquid vs. Solid dispersion formulations
Lipid based self-emulsifying and solid dispersion technologies are
competing technologies for increasing solubility and enhancing
bioavailability of the same poorly soluble molecules. The choice
of the excipients though is different for each technology, and may
vary depending upon the formulation and dosage requirements,
and the API per se. El-Laithy and co-workers evaluated two poorly
soluble molecules biphenyl dimethyl dicarboxylate and silymarin
in two different formulations, one based on solid dispersions (SD)
prepared with poloxamer 188 by melting and the other by co-
precipitation with deoxycholate, and compared the two amorphous
solid dispersions (ASDs) with respect to a lipid based self-emulsifying
SMEDDS comprised of 40% polysorbate 80, 10% Miglyol® 812 and 50%
Transcutol®.57 The authors observed that both formulations, SMEDDS
as well as ASD, were equally effective as the commercially available
product against the hepatic fibrosis in Albino rats. Examples like these
in the commercial world are limited; products like lipid based ritonavir
(Norvir®) and ritonavir and lopinavir (Kaletra®), launched originally as
soft gel capsules (SEDDS) and substituted later on by amorphous solid
dispersions (ASD) tablets because of much higher stability and active
contents. Each dosage though requires different excipient compositions
in the individual formulations and technologies, both SMEDDS and
ASD dosages possess similar pharmacokinetics profiles.58 For example,
ritonavir’s liquid SEDDS contains polyoxyl 40 hydrogenated castor
oil (Kolliphor® RH 40) and ritonavir/lopinavir’s liquid SEDDS contains
polyoxyl 35 castor oil (Kolliphor® EL), both having a similar HLB value
as Kolliphor® HS 15. Pharmacokinetic profiles and bio-distribution of
model compounds in liquid formulations containing Kolliphor® HS
15 and other solubilizers have been investigated following single IV
dosing.59 Scheller et al. investigated the delivery of galanin-receptor 3
selective antagonist, SNAP 37889, and achieved the desired solubility
in 30% Kolliphor® HS 15 micro-emulsion injectable solution by
solubilizing the drug in a paste form and diluting it in phosphate buffer.60
The authors used this IV formulation for evaluation of the galanin
antagonist in neuropharmacology of rats and mice. Other applications
of Kolliphor® HS 15 include the development of cyclosporine
ophthalmic sterile liquid solution with castor oil and glycerol to yield
formulations with the excellent stability and efficacy with no apparent
toxicity, incompatibility and/or ocular irritation.61 Zhao et al. used a
phospholipid based IV micro-emulsions composed of Kolliphor® HS 15,
Miglyol® 812, soy lipid, PEG 400 and ethanol in the parenteral delivery
of ibuprofen eugenol ester (IEE).62 Solubility of the ibuprofen derivative
improved over 21000-fold (64 mg/ml) as compared to placebo, and
the ibuprofen based IEE micro-emulsions exhibited prolonged acting
time, attributed due to the stealth protection of droplet surface by
Kolliphor® HS 15 from being recognized by opsonins, and taken up by
the cells of reticuloendothelial systems (RES).63,64
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American Pharmaceutical Review | January/February 2019
Parenteral Propofol IV formulation
Propofol (2,6-diisopropylphenol) has been formulated in Kolliphor®
EL aqueous solution but it brings risk associated with anaphylactoid
reactions and high incidence of pain associated with injection.65 To
alleviate these side effects, an alternative o/w based 1% propofol
emulsion, preferentially formulated with soy oil, egg phospholipids
and glycerol, has been developed but due to unforeseeable challenges
including the elevated serum triglycerides level and impediment
in fat metabolism due to high lipid content in the formulation, it
warrants additional studies to develop a much safer IV micro-emulsion
formulation. To alleviate much of the safety concerns and mitigate
the adverse effects, Kolliphor® HS 15 is an alternative surfactant for
the development of micro-emulsion IV formulations.66 The authors
used Kolliphor® HS 15 with PEG 400, propylene glycol, glycofurol in
a 1% propofol IV emulsion formulation. Ryoo et al. also formulated a
1% propofol IV micro-emulsion with 8% Kolliphor® HS 15 in ethanol
and found that it qualified the desired criteria; was stable and safe
without being hemolytic to red blood cells. These findings led to
the development and marketing of Aquafol®.67 Lee et al. further
investigated the safety of propofol IV micro-emulsion formulation and
modified the composition containing only 0.7% of Kolliphor® HS 15
mixed with 10% purified poloxamer 188 in 1% glycerin as a cosolvent.68
The re-formulation dramatically improved the safety with fewer
adverse effects as compared to the previously marketed Aquafol®
formulation containing 8% Kolliphor® HS 15 with 5% glycofural as
a cosolvent.67 The adverse effect like pain following injection was
evaluated by quantifying the free propofol in propofol IV emulsions.
Date and Nagarsenker evaluated the propofol IV emulsion and found
that a binary mixture of Kolliphor® HS 15 and polysorbate 80 combined
in 1:1 ratio, was more effective as compared to those consisting of co-
solvents like glycofurol and/or polypropylene glycol.69 Furthermore,
the Kolliphor® HS 15/polysorbate 80 binary combination of propofol
IV emulsion drastically improved safety and potentially reduced pain
on injection. It also provided a better content uniformity without
compromising the pharmacodynamic activity and physicochemical
stability with respect to commercially available Propovan® emulsions.
It is also worth noting that the free propofol, if left non-encapsulated
in micro-emulsion caused bearable pain following IV injection but its
severity was wide spread in twice larger population as compared to
medium-chain or large-chain triglyceride based propofol treated IV
micro-emulsion formulation.70
A few other propofol IV micro-emulsions are in clinical stages of
development. In a comparative clinical study, propofol IV Kolliphor®
HS 15 formulation was evaluated with Kolliphor® HS 15 in combination
with soy lipid based nano-emulsion and found that both formulations
performed equally good with superb safety and efficacy with fewer
side effects in patients.71 Rittes et al. also investigated the Kolliphor®
HS 15 in propofol IV nano-emulsion and compared it with soy lecithin
IV emulsion formulation in patients undergoing gynecological
procedures. The patients were monitored for heart rate, systolic and
diastolic blood pressures and peripheral oxygen saturation.72 The
authors concluded that the groups with 1% or 2% propofol in soy
 DRUG DELIVERY

lipid based and Kolliphor® HS 15 surfactant based IV nano-emulsion Table 2. Chemistry and physicochemical properties of Kolliphor® HS 15
formulations elicited pain on injection but the intensity of pain was
Drug/Excipient Conc. μg/ml* Normalized IC50 (saline/excipient)**
less severe with Kolliphor® HS 15 IV, and both formulations showed
Cyclosporine A 5 12.6
neither hemodynamic changes nor any adverse effects of clinical
Kolliphor® HS 15 8 4.2
relevance. It was also consistent with the findings that the adverse
Kolliphor® RH40 31 5.3
reaction was not related to Kolliphor® HS 15 but due to free propofol
if left un-encapsulated.73 In a clinical study, the non-lipidic Cleofol® Kolliphor® EL 31 6.7

IV formulation showed much greater incidence of severe pain as Polysorbate 80 31 7.7

compared with medium chain triglyceride based Lipuro® IV formulation Vitamin E TPGS 8 6.8

and long chain triglycerides based 1% propofol IV emulsion available as
Diprivan®. This adverse effect was attributed to free propofol in Cleofol®
IV formulation which lacked the medium or long chain triglycerides.74
To alleviate the adverse effect associated apparently with free propofol
in IV micro-emulsions, Morey et al. used sodium salt of fatty acids (C8,
C10, C12) in combination with purified poloxamer 188 for complete
encapsulation of drug to yield a transparent micro-emulsion solution
with particle size < 50 nm.75
Inhibition of Pgp efflux pump
P-glycoprotein (Pgp) (MW 170 kD), is a large integral transport protein
in the membrane when overexpressed pumps out the undesired
molecules entering and/or accumulating into the cells. Thus, the
overexpression of Pgp provides a protective function in blood
brain barrier, colon, pancreas, kidneys, and gastrointestinal tract,
more specifically, it prevents the accumulation of xenobiotics and
metabolites in the brain. In addition, the overexpression of Pgp is
highly prevalent in the cancer patients undergoing chemotherapy.76
There is a range of chemicals and therapeutic agents which act as
chemosensitizers or substrates that affect the function of Pgp pump.77
For instance, surface active agents such as poloxamers have shown
different activities on the Pgp efflux depending upon the structure
of the molecules.78 Batrakova et al. classified the poloxamer into 4
groups and those with longer hydrophobic polypropylene oxide (PPO)
moieties penetrate deeper into the blood brain barrier (BBB) membrane
leading to endocytosis and accumulation of drug into the cytoplasm.79
Constantinides and Wasan also investigated the Pgp inhibition by lipid
based excipients such as Kolliphor® EL, Kolliphor® HS 15, poloxamers
among others with poorly soluble drugs and found a significantly
improvement in intestinal absorption of these molecules due to Pgp
inhibition, which probably led to acceleration of drug transport across
the cell membrane.80
Wang and co-workers studied the mechanism of Pgp activities on
mouse fibroblast cell line NIH 3T3 of a number of surfactant/lipid based
solubilizers acceptable to intravenous (iv), intramuscular (im) and
oral (p.o.) formulations.81 The Pgp inhibitory activities data normalized
against 0.9% NaCl solution, is summarized in Table 2.
The data clearly indicates that the solubilizers tested had a very different
impact on Pgp inhibition ranging from no effect to a strong one. For
instance, Kolliphor® HS 15 resulted already at a very low concentration
of 8 µg/ml in a normalized IC50 of 4.2 which was only exceeded by
TPGS with a value of 6.8. In contrast most of the poloxamers except
poloxamer 124 did not exert a strong action, even not at higher
Poloxamer 124 125 5.7
Poloxamer 188 31 1.1
Poloxamer 237 125 2.8
Poloxamer 338 500 1.3
Poloxamer 407 500 1.5
0.9% Saline - 0.96
*Concentrations were based on toxicity screens and were the maximum excipient
concentrations for this cell line without directly affecting cell viability.
** IC50 values are based on paclitaxel and its effect on cell growth. Normalized IC50 denotes
the paclitaxel IC50 saline/excipient ratio. A high value relates to a strong P gp-inhibition of the
excipient at the given excipient concentration.
concentrations. In general, the lipid based surfactants were more
potent regarding Pgp inhibition than poloxamers. With reference to
cyclosporine A, the activities of the best solubilizers were still below
the drug’s Pgp inhibitory property.81
The mechanism by which the lipid based solubilizers like Kolliphor®
HS 15 act as chemosensitizers that influences the Pgp inhibition is
poorly understood. Such inhibitory activity is apparently critical for
explaining the permeation of drugs via endocytosis and, hence, the
efficacy thereafter. Kolliphor® HS 15 based CriticalSorb® was used as
an absorption enhancer in the epithelial cell cultures (e.g. A549, Caco-
2 and Calu-3), with an aim to deliver or transport the biologic drugs
via mucous membrane.18 The authors concluded that Kolliphor® HS
15 promotes transcellular apical to basolateral transport of biologics
through Calu-3. Furthermore, Kolliphor® HS 15 when tested against
the epithelial cell cultures showed a relatively high IC50 (ca. 6.5–10.2
mM) as compared with other absorption enhancers or surfactants
such as sodium cholate (2.04–4.94 mM) and polysorbate 80 (5.41–
6.49 mM), suggesting much milder in vitro activities of Kolliphor® HS
15 than other solubilizers. Huo et al. evaluated the mixed micelles of
Kolliphor® HS 15 and poloxamer 407 (4:1) for delivery of the oncology
drug icariside II (IS) in rats, and found that the mixed micelles improved
the entrapped efficiency to 95% and increased >2-fold bioavailability,
presumably caused by increasing permeability across cell membrane
due to inhibition of Pgp.82 In a study, Kommuru and co-workers
demonstrated that Kolliphor® HS 15, when used as a surfactant carrier,
may increase the permeability and intracellular concentration and
residence time of CoQ10 via paracellular transport by inhibition of Pgp
and/or CYP450 enzymes.83 These findings have been further supported
by enhanced bioavailability of other drugs that influence the inhibition
of Pgp and cytochrome P450 (CYP) 3A.84 Though the specific molecular
changes to Pgp are not yet fully understood, the influence of Kolliphor®

29
American Pharmaceutical Review | January/February 2019
 DRUG DELIVERY
HS 15 on the Pgp could be nonspecific conformational changes due to
surfactant molecules penetrating deep into the plasma membrane.
Like Kolliphor® HS 15, other lipid solubilizers such as vitamin E-TPGS,
Kolliphor® EL, Kolliphor RH 40, and polysorbate 80, are well-known
chemosensitizer that may temporarily inhibit the Pgp pump, which
could then trigger the transport of drugs across the intestinal
mucosal membrane, leading to faster absorption and improved oral
bioavailability preferably by lymphatic uptake.85
Biological activities of Kolliphor® HS 15
Shubber and co-workers investigated the mechanism of in vitro mucosal
permeability of Kolliphor® HS 15 based CriticalSorb® as absorption
enhancer in the epithelial cell cultures of A549, Caco-2 and Calu-3 cell
lines, with aims to deliver the biologic drugs via mucous membrane by
epithelial cellular transport, a novel route to overcome the parenteral
route for administration of macromolecules such as insulin, hGH,
albumin and IgG.18 The authors demonstrate that increasing the MW
of macromolecules slowed down the permeability of these molecules,
while the permeation of Kolliphor® HS 15 was apparently faster with
low MW macromolecules. The permeation of drug molecules through
Calu-3 cell lines decreased in the order: Insulin > hGH > Albumin >
IgG. In another unrelated study, Lin et al. demonstrated that the fatty
acid methyl esters of palmitic (C16:0) and stearic (C18:0) acids exhibit
the potent vasodilatory properties which could be important in
neuroprotection following cerebral ischemia.86 The authors compared
the biological activities of Kolliphor® HS15 with the structurally similar
fatty acid methyl esters having an ester linkage with C16 and/or C18
fatty acids. The authors demonstrated the neuroprotective effect by
calculating the number of survived hippocampal neurons by individual
expression of enhanced levels of Kolliphor® HS 15, palmitic acid methyl
ester, and stearic methyl ester in treated rats as compared to controls.
The data suggests that with appropriate dosage of Kolliphor® HS 15
and/or C16 and C18 methyl esters, the levels of each component was
overexpressed in neurons with all showing the increase of about 68-
69%, which was an effective level against cerebral ischemia treatment.
In a study Bartels et al., investigated the protective effect of antioxidative
vitamins against lipid peroxidation by parenteral administration of
Kolliphor® HS 15 formulations comprised of α-tocopherol, ascorbic
acid, and β-carotene on the extent of ischemia and reperfusion liver
injury.87 In another study, Bartels and co-workers used the soybean
lipid in combination with Kolliphor® HS 15 in parenteral infusion of
lipid soluble anti-oxidative vitamins to reduce the adverse effects by
monitoring the toxicity by the elevated liver enzymes caused by the
residual solvents.88 Coon et al. investigated the multidrug resistance
of Kolliphor® HS 15 in mice, and found that like verapamil, Kolliphor®
HS 15 reduced the efflux of rhodamine through KB 8-5-11 cells but it
Table 3. Hemolytic activity and serum histamine levels following iv administration of Kolliphor® HS15 and polysorbate 80 in beagle dogs
Hemolytic activity (% hemolysis)
Solubilizer
0.1% 1%
Kolliphor® HS15 0 0
Polysorbate 80 0 0
American Pharmaceutical Review | January/February 2019
did not affect the transport of alanine or glucose into the cells, and
claimed that its effect on membrane transport activity was highly
non-selective. The IC50 of Kolliphor® HS 15, however, was 140 µm as
versus 65 µm of verapamil against the KB cell line.11 Buckingham et
al. investigated the PEG-fatty acids esters in vitro performance against
KB cell lines. The optimized oleic acid ester with 20 mole ethylene
oxide (or PEG), e.g. CRL 1337, was over 8‐fold as potent as Kolliphor®
HS 15 and over 19‐fold as potent as Kolliphor® EL in vitro, as evident
by accumulation of rhodamine 123 pigment in multidrug‐resistant
KB 8–5–11 cells.89 The authors concluded that the hydrophobic fatty
acid components of surfactant and its hydrophilic‐lipophilic balance
are critical in determining the potency of a surfactant. Woodburn and
co-workers investigated the binding of lipoproteins (LDL and HDL) and
porphyrin based photosensitizers (C8KC and MP) with Kolliphor® HS 15
and compared the binding assay with a synthetic solubilizer/emulsifier
CRM, comprised of poly-(oxyethyleneglycerol) triricinoleate bearing
the structurally similar hydrophobic and hydrophilic components
as the Kolliphor® HS 15.90 The authors observed that Kolliphor® HS
15 and CRM when used as vehicles showed similar interactions with
C8KC and MP in the in vitro electrophoretic assay. Furthermore, higher
concentrations of Kolliphor® HS 15 and CRM (>0.06%) led to a decrease
in electrophoretic mobility of human LDL and HDL in vitro. In mouse,
both Kolliphor® HS 15 and C8KC showed a similar half-life (t1/2) of 1.7
hours, and were cleared out from mouse plasma in vivo over 6 hours.
It can further be taken to suggest that vehicles like Kolliphor® HS 15
were able to circulate for much longer period alongside the drug in the
plasma without being taken up by RES.64
Compatibility with Plastic Containers
Kolliphor® HS 15 is an exceptional solubilizer for use in parenteral
formulations due to its controlled manufacturing particularly regarding
microbial and endotoxin contamination. The storage of Kolliphor® HS
15 in plastic containers or bags may lead to degradation or erosion of
the storage reservoir caused by extraction of low molecular weight
components. Thus, the attempts have been to minimize the leaching
and extraction of additives from the package liner.91 Only limited
information is available to demonstrate the suitability of the aqueous
micro-emulsion Kolliphor® HS 15 formulation in such containers.
Cheung et al. investigated the leachable and extractable properties
of commonly used solubilizers from iv-bags and iv-infusion lines
derived from polyvinyl chloride (PVC) plasticized with 30-40% of bis-
(2-ethylhexyl) phthalate (DEHP) as an additive. The PVC/DEHP IV bags
each contained Kolliphor® HS 15 (5 mg/ml), polysorbate 80 (3 mg/ml),
polysorbate 20 (3 mg/ml), or poloxamer 188 (3 mg/ml) at pH 7.92 The
authors observed that the leaching effect was insignificant or none
with poloxamer 188 but all other solubilizers including PS 80, PS 20
Serum histamine levels (nM)
10% t = 0 min t = 15 min t = 60 min
8.7 5 220 8
11.1 3 >50,000 247
30

and Kolliphor® HS 15 resulted in leaching of DEHP. The leaching ability
increased linearly as the solutions in the IV bags were exposed to longer
periods at similar concentrations and usage conditions. A similar trend
was also observed with PVC IV bag/infusion tube plasticized with
tri-2-ethylhexyl trimellitate (TOTM) as an additive. Like PVC/DEHP IV
bags, with PVC/TOTM IV bags the leaching ability decreased linearly
in the order: Kolliphor® HS 15 >> polysorbate 80 > polysorbate 20
>> poloxamer 188, suggesting that poloxamer 188 outperformed in
comparison with other solubilizers such as polysorbate 80, polysorbate
20 and Kolliphor® HS 15. This is not surprising since poloxamer 188 has
a much lower solubilization capacity which counts also for extraction
of lipophilic plasticizers. This study also sheds light that Kolliphor®
HS 15 may not be compatible with the PVC IV bags containing DEHP
and TOTM as additives. Since polysorbate 80 and polysorbate 20 are
commonly used to stabilize the proteins and large molecules, the
leaching abilities of the polysorbates could be a disadvantage for
anticipated use in the biologics’ IV formulations, however, the typical
concentrations used here are much lower. In this regard poloxamer
188 in general due to its compatibility with the plasticizers in PVC
IV bags, should be more suitable which may accelerate the usage of
poloxamer 188 as a stabilizer downstream in injectable IV and infusion
formulations, and as a shear protector in the upstream cell culture.93
Regulatory and Toxicological Perspectives of
Kolliphor® HS 15
Kolliphor® HS 15 is monographed in the United States Pharmacopeia
by Polyoxyl 15 hydroxystearate, and the European pharmacopeia by
Macrogol 660 hydroxystearate. It is also listed in the inactive ingredient
database (IID).94
Safety and toxicology of Kolliphor® HS 15 have been evaluated in non-
clinical studies and the results have been reported.95 These studies aid
in the development of formulations with the guidance on usage levels
for certain routes of administration. Several drug products containing
Kolliphor® HS 15 have been approved globally to date.
Briefly, Kolliphor® HS 15 does not show significant signs of acute
toxicity in rats, mice, dogs and rabbits as shown by studies with oral and
parenteral administration. The oral LD50 is >20 g/kg in rats and mice.
The acute intraperitoneal LD50 is >8 g/kg for mice, and the intravenous
LD50 for rats and rabbits is >1 g/kg. In beagle dogs, the intravenous LD50
is >3 g/kg, however, symptoms of histamine release were observed in
a dose dependent manner, but this sign was reversible within a few
hours without causing any mortality or death. Histamine release in
dogs as the most sensitive animal is a general side-effect of solubilizers
with a high amphiphilicity and solubilization capacity. Thus, it occurs
also with polysorbate 80 and Kolliphor® ELP, typically to a much larger
extent.95
In sub-chronic tox studies, the oral dosing of Kolliphor® HS 15 in dogs
continued for 4 and 13 weeks respectively at 1g kg body weight, did
not cause any specific toxicity and histopathological changes or organ
toxicity or any signs of histamine release (NOAEL >1g/kg). Similar
studies were performed in rats resulting in NOAELs of 2g/kg and >1.5g/
kg. Repeated intravenous administration continued for 13 weeks at 750
31
American Pharmaceutical Review | January/February 2019
DRUG DELIVERY
mg/kg in rats, also did not show any specific sign of toxicity; the caused
lipid deposition in liver and spleen tissues was considered not to be an
adverse effect. Other studies lasting 2 and 4 weeks revealed a NOAEL of
>200 mg/kg, the highest dose tested. Dogs are much more sensitive to
parenterally applied compounds and thus a dose of 100 mg/kg caused
pseudo-allergic reactions associated with histamine release, however
no other signs of substance specific toxicity or histopathological
changes were observed. No pseudo-allergic reactions occurred at a
dose of 25 mg/kg. In comparison, Kolliphor® EL at 5 mg/kg caused the
allergic reaction and the effect was equivalent to 100 mg/kg dosing
of Kolliphor® HS 15, suggesting that the latter has a tremendous
benefit in this regard. Kolliphor® HS 15 was compared in another i.v
study in dogs at 100 mg/kg with polysorbate 80 resulting in much
lower histamine levels, as shown in Table 3.96 Taking these results into
account, Kolliphor® HS 15 represents the safest excipient in the field of
strong solubilizers.7
Kolliphor® HS 15 did not show any sign of genotoxicity either
in Salmonella typhimurium reverse mutation assay (Ames test),
chromosome aberration test, HPRT test or mouse micronucleus test.
The reproductive and developmental toxicity in rats at a maximum
intravenous dose level of 464 mg/kg did not reveal any substance-
related influence on the prenatal development of the embryo or fetus,
or postnatal growth.
From drug formulation perspective, Kolliphor® HS 15 has been
evaluated for cancer preventative activity of a new chemical entity
SR13668 in dogs and monkeys.97 SR13668 formulation did not show
any pathological changes including hematology, clinical chemistry
and coagulation effects. The bioavailability of drug following dosing
p.o. at 90 mg/kg, showed an increase to about 15% and 7% in dogs
ad monkeys, respectively. These findings were critical in assessing the
clinical studies of SR13668 in humans. Reid et al. investigated the Akt
inhibitor SR13668 in Phase 0 clinical chemoprevention trial in healthy
volunteers, and observed that formulation containing Kolliphor® HS 15
with TPGS yielded the maximum oral bioavailability as compared to PEG
400/Labrasol® formulation.98 From the pharmacokinetic findings, the
authors concluded that the Phase 0 data could be used as a guidance for
the initiation of Phase 1 clinical studies. Dulin evaluated the Kolliphor®
HS 15 and other solubilizers in early phases of development in oral self-
emulsifying system (SEDDS) and found that Kolliphor® HS 15 formulation
outperformed and improved the solubility and bioavailability of a new
chemical entity (NCE); which necessitated the advancement of a lead
candidate in the clinical development.99 This study showed that the
oral bioavailability improved several folds with Kolliphor® HS 15 as
compared with phospholipid formulation due to much slower rate of
absorption with longer Cmax in early stages of screening with lipids in
animal models. Consequently, the NCE in Kolliphor® HS 15 formulation
further advanced to the first-in-human Phase 2 studies after successful
Phase 1. In all 12 subjects studied, Kolliphor® HS 15 formulation was
safe and caused no adverse effects after first dosing at 150 mg/capsule
and subsequently dosing to maximum tolerated dose (MTD) of 1500
mg/10 capsules, demonstrating an excellent tolerability and safety
of Kolliphor® HS 15 in the formulation.100 Stokes et al. conducted
 DRUG DELIVERY
separately toxicological studies of Kolliphor® HS 15 and Kolliphor®
RH 40 formulations each with polyethylene glycol 400 as a co-solvent
in rats and dogs at different dose volumes, at 10 mL/kg for rats and
at 2 mL/kg or 5 mL/kg for dogs.101 The authors observed that neither
vehicle, regardless of dose volume, nor formulation caused any toxicity
either in rats following 91 days of dosing or in dogs following 8 days of
dosing. Kolliphor® HS 15 formulation produced only a fewer adverse
effects in rats notably at much higher dose volumes, causing only slight
dehydration and hemoconcentration. In lower dose volumes, however,
both Kolliphor® HS 15 and Kolliphor® RH 40 formulations were safe
given orally and minimized the severity or incidence of adverse effects.
Conclusion
Kolliphor® HS 15 is an excellent solubilizer with unparalleled attributes
leading to a safe administration of oral and injectable drug molecules.
The data presented in the review article and reported independently
by many other authors from earlier publications, clearly demonstrates
that Kolliphor® HS 15 is highly versatile and is an exceptional excipient/
solubilizer for screening of NCEs in the early phases and building
platform to provide a good start in leading the way to select the right
formulation technologies for development of lead candidates. It also
demonstrates that the safety of this excipient is well studied which
had helped attain the recognition for freely usages in many approved
marketed drug products globally for both in humans and animals as
well. With the recent launch of parenteral and ophthalmic drugs in
the US, Kolliphor® HS 15 is no longer a novel excipient. As a result,
the global acceptance of Kolliphor® HS 15 in future drug candidates
will continue to rise. The compliance to the compendial monographs
in the USP and European pharmacopeia, further ease the acceptance
in the solid and liquid oral SEDDSs/SMEDDS as well as in parenteral
formulations. Other applications also continue to take heed such as in
biologics by virtues of its structural compatibility with a wide range
of lipid and non-lipid based surfactants and solubilizers and polymers
alike and with macromolecule APIs such as peptides, nucleic acids and
biologics. With an increase in the number of NCEs from the discovery,
Kolliphor® HS 15 will continue to play an important role as a solubilizer
for the molecules either small or large with difficult-to-dissolve
attributes and pave the way in the industry for development of drug
candidates by non-conventional novel formulation technologies. The
NCEs will dictate the technologies requiring Kolliphor® HS 15, either
in liquid oral versus parenteral self-emulsifying and LNC or solid oral
based s-SEDDS formulations and efficient delivery of low, medium or
high dose drugs.
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» MICROBIOLOGY »

Excluding Burkholderia Introduction

In non-sterile drug product manufacturing, low microbial counts are

cepacia complex from tolerated and the final drug product does not necessarily need to
be free of microorganisms to be released to the market. The recom-
mended microbiological quality requirements for different dosage
Aqueous, Non-Sterile forms may found in USP <1111> Microbiological Examination Of Non-
sterile Products: Acceptance Criteria for Pharmaceutical Preparations and
Substances For Pharmaceutical Use. The microbial limits and absence
Drug Products of specified microorganism requirements related to the invasiveness
of the dosage form were established in USP <1111> and some micro-
organisms, if found in a particular product, are considered objection-
able in that they can adversely affect the appearance, physicochemical
attributes or therapeutic effects of a non-sterile drug product or due
to their numbers and/or pathogenicity, may cause infection, allergic
response or even toxemia in patients receiving the product. Recent
U.S. recall surveys have found that the presence of objectionable mi-
Tony Cundell, PhD croorganisms, and not microbial numbers, represent the vast majority
Principal Consultant, of microbiologically related FDA recalls of non-sterile drug products.
Microbiological Consulting, LLC This review article will discuss the definition of an objectionable
Scarsdale, New York microorganism, the prevalence of members of the Burkholderia
cepacia complex (BCC) in U.S. product recall and nosocomial infection
outbreaks, why BCC members are serious opportunistic pathogens,
the screening and identification methods for members of the
complex, and how the risk of microbial contamination of non-sterile
drug products can be mitigated.

What is an Objectionable
Microorganism?
It is notable that the U.S. Federal Good Manufacturing Regulations
21 CFR 211.113 Control of microbiological contamination requires
that objectionable microorganisms be excluded from non-sterile
drug products, but contains no actual definition of an objectionable

36 | | January/February 2019

microorganism and certainly does not provide a list of microorgan-
isms to be excluded. However, FDA microbiologists attending industry
meetings have stated that they plan to publish a guidance document
in the next 1-2 years addressing non-sterile drug products. The as-
signment of the responsibility is to the pharmaceutical manufacturer
who must develop a written program to exclude objectionable micro-
organisms from their drug products was, in the author’s opinion, the
right decision. Not only does the manufacturer bear the responsibility
for the safety of their products but alone has the complete range of
knowledge of the pharmaceutical ingredients, formulation, manufac-
turing processes, product attributes, and intended patient population
to make these critical judgments.
So what is an objectionable microorganism? The PDA Technical Report
No. 67 Exclusion of Objectionable Microorganisms from Non-sterile
Pharmaceutical and OTC Drug Products, Medical Devices and Cosmetics
defined objectionable microorganisms as follows:
1. Microorganisms that can proliferate in a product adversely
affecting the chemical, physical, functional and therapeutic
attributes of that pharmaceutical product.
2. Microorganisms that due to their numbers in the product
and their pathogenicity can cause infection in the patient
via the route of administration when treated with that
pharmaceutical product.
The points to note in this definition are what is objectionable is drug
product specific and the objectionable microorganism may either
cause an infection in the patient or affect the functionality of the drug
product.
Why are Members of the Burkholderia
cepacia Complex Objectionable
Microorganisms?
Why do we consider that the Gram-negative bacterium Burkholderia
cepacia and many other members of the BCC are objectionable
microorganisms? The reasons include:
• Prominence of B. cepacia in U.S. drug product recalls
• Recent high visibility in recent infection outbreaks for
B. cepacia due to bacterial contamination of non-sterile
drug products and consumer care products
• Role as an opportunistic pathogen in compromised hospital
patients
• Unique ability to overcome antimicrobial preservative
systems
• Resistance of BCC members to many widely used antibiotics
B. cepacia is an aerobic, Gram-negative, oxidase-positive, rod-shaped
bacterium that is an opportunistic pathogen, with a reputation of
overcoming antimicrobial preservative systems and antiseptics and
growing in multiple-use preserved oral liquids, topical products and
nasal sprays. It a member of a group of closely related species in the B.
« MICROBIOLOGY »
cepacia complex with a high metabolic versatility, wide environmental
distribution, and variable virulence due in part to its large genomic
size (7-8 mbp). Furthermore, members of B. cepacia complex have the
ability to form biofilms in pharmaceutical water systems making it
more difficult to eliminate from the system.
BCC members cause serious infections in individuals with cystitis
fibrosis (CF) and chronic granulomatous disease. Two BCC members
B. cenocepacia and B. multivorans account for greater than 85% of
the CF infections (Reik et al, 2005). It is an opportunistic pathogen in
mechanically ventilated patients, the immunosuppressed, infants, the
elderly and those with serious underlying disease.
U.S. Drug Product Recalls
A 2012 survey analyzed 144 reported recalls of non-sterile pharmaceu-
tical drug products (5%), over-the-counter drug products (42%), cos-
metics (31%), medical devices (14%) and dietary supplements (8% of
the total recalls) for microbiologically related issues for the 7-year pe-
riod from 2004 through 2011. This represents an average of 20 recalls
annually. The publication highlighted that the majority of these recalls
(72%) were associated with objectionable microorganisms and not for
exceeding microbial enumeration limits (Sutton and Jimenez, 2012).
The objectionable organisms associated with the recalls by microbial
type were:
• Burkholderia cepacia – 34%
• Yeast/molds – 21%
• Other Pseudomonads – 15%
• Members of the family Enterobacteriaceae – 11%
• Bacillus cereus – 9%
• Chryseobacterium spp.– 5%
• Staphylococcus spp. – 4%
• Achromobacter spp. – 1%
A hierarchy of microbial infection risk can be established simply based
solely on the invasiveness of the pharmaceutical dosage form. The
increasing invasiveness would range from solid oral dosage forms to
inhalation products. In addition, the medical status of the recipients
will influence the risk. For example, a recent survey published in a
leading medical journal suggests 4% of U.S. adults are believed to
be immunosuppressed (Harpaz et al, 2016). Note: Sterile injectable
products have the greatest risk of microbial infection due to their
parenteral administration.
Prominent Recall Case Histories
On May 2008, Hydrox Labs, Elgin, IL issued a voluntary recall of labeled
alcohol-free mouthwash they manufacture. As a result of this recall,
Cardinal Health initiated a voluntary recall of a lot of alcohol-free
mouthwash. The unopened and used mouthwash containers were
www.americanpharmaceuticalreview.com | | 37
» MICROBIOLOGY »
tested, and certain samples were found positive for B. cepacia strongly
suggesting intrinsic microbial contamination. The product was
distributed to hospitals, medical centers, and long-term care facilities
nationwide but not for retail sale. Most troubling, the mouthwash
may be found in certain Personal Hygiene Hospital Admission Kits
distributed to patients.
It is widely recognized that B. cepacia poses little medical risk to healthy
people. However, people who have certain health problems such
as weakened immune systems or chronic lung diseases, particularly
cystic fibrosis (CF), infants or the elderly or on mechanical ventilators,
may be more susceptible to infections with B. cepacia. This pattern
was observed in this outbreak, as the patient infection was limited to
those ventilators in intensive care units and not the general patient
population who also received the mouthwash in admission kits.
Adding to the risk, B. cepacia is often resistant to common antibiotics,
so they are more difficult to treat.
A recent review article (Abdallah et al, 2018) found by searching
PubMed using the terms “Burkholderia”, “outbreak” and “intensive care
unit” between January 1994 and December 2017 thirty outbreaks of
BCC infection or colonization in non-CF patient in intensive care units
with many outbreaks associated with contaminated medical products.
Intrinsic contamination was associated with liquid laxative, skin
antiseptics, alcohol-free mouthwash, ultrasound gel, and moisturizing
body cream with resulting high mortality rates.
Another case history that is informative is the infection outbreak
and the nationwide recall of an oral laxative (Marquez et al, 2017).
On July 16, 2016 the FDA announced a voluntary nationwide recall
of all non-expired lots of oral liquid docusate sodium manufactured
by PharmaTech LLC, Davie, Florida in one pint (473 mL) bottles and
distributed by Rugby Laboratories for contamination with B. cepacia
and linked to a five state outbreak. Later laboratory evidence linked
the B. cepacia to the company’s purified water system.
In an August 10, 2016 update, the CDC confirmed 60 cases from an
eight state outbreak was caused by the same strain of B. cepacia using
molecular typing and recommended that clinicians and their patients
not use any brand of liquid docusate sodium as a stool softener or
other medical reason. Liquid laxatives like Docusate™ are widely used
to treat infants and the elderly that have difficulties swallowing and
are typically higher risk groups for bacterial infection.
The FDA was sufficiently concerned in 2017 to issue an advisory notice
of the dangers of BCC contamination of aqueous, non-sterile drug
products (FDA, 2017)
What Defines Membership in the
B. cepacia Complex?
The bacterium B. cepacia was originally viewed as a pseudomonad,
first isolated from rotting onions. The genus Pseudomonas is a
large and complex heterogeneous group of organisms belonging
to the family Pseudomonadaceae that originally contained 211
38 | | January/February 2019
validly described species but 56 of which have been reclassified
to other genera. They are constantly undergoing continuous
taxonomic revision due to improvements in methodologies of
species classification. Organisms previously classified within the
genus Pseudomonas (rRNA homology groups I-V) are now divided
among the ten genera Pseudomonas, Burkholderia, Ralstonia,
Comamonas, Acidovorax, Delftia, Hyrodenophaga, Brevundimonas,
Stenotrophomonas and Xanthomonas.
The genus Burkholderia was created by Yabuuchi et al (1992) to
accommodate the former rRNA Group II pseudomonads excluding
P. pickettii and P. solonacearum, which were transferred to the genus
Ralstonia. Traditionally, Burkholderia species are known as plant
pathogens and soil bacteria with the exception of P. mallei and P.
pseudomallei human and animal pathogens.
More recent research (Coenye et al, 2001) has resulted in a number
of changes to the taxonomy of Burkholderia cepacia complex (BCC).
The BCC currently comprises 17 species which exhibit a high degree of
16S rRNA (98–100%) and recA (94–95%) gene sequence similarity, and
moderate levels of DNA–DNA hybridization (30–50%).
Characteristics that Make
Members of B. cepacia Complex
Opportunistic Pathogens
B. cepacia is a well-known nosocomial pathogen that is intrinsically
resistant to aminoglycosides and first- and second-generation
cephalosporins. Members of the BCC are widely found in wet
locations in the environment and hospital settings. Their ecological
and metabolic versatility and resistance to a wide range of antibiotics
and antiseptics may be partly explained by their large genomic size
(7-8 mbp). In December 1995, an outbreak involving B. cepacia in
respiratory cultures from patients without cystic fibrosis was traced
to intrinsically contaminated alcohol-free mouthwash (Bernstein et
al, 1996). An investigation by the FDA suggested an association with
the deionization procedure of the water used to prepare the product.
Mechanically ventilated patients are vulnerable to pathogens in their
mouths and upper airways because of their inability to maintain the
mucociliary and cough mechanisms that normally protect the lower
respiratory tract (Beck-Sague et al, 1996). These outbreaks of B. cepacia
related to mouthwash highlight the increased risk for respiratory
colonization and infection among patients on ventilators.
Testing for the Absence of
B. cepacia Complex
The pharmaceutical industry needs a standardized test for screening
non-sterile, aqueous, drug products for members of the BCC. In clinical
microbiology, primary culture for BCC should be performed on a
selective agar to avoid overgrowth by other bacteria and to readily

identify the pathogen. Examples of selective solid media include
Burkholderia cepacia selective agar (BCSA), Burkholderia cepacia agar
(BCA) (formerly known as Pseudomonas cepacia agar or PCA), and
Oxidation-Fermentation Polymyxin Bacitracin Lactose agar (OFPBL).
Recent evaluations suggest that BCSA is more selective and grows BCC
colonies more rapidly than the other media. All media contain multiple
antibiotics to maintain selectively. B. cepacia selective agar (BCSA)
contains 1% lactose and 1% sucrose in an enriched base of casein and
yeast extract with 600 U of polymyxin B per ml, 10 μg of gentamicin
per ml, and 2.5 μg of vancomycin per ml (Henry et al, 1997). Incubation
at 30-35°C for 48-72 hours is recommended.
In response to stakeholder requests, a proposed USP test for the
absence of Burkholderia cepacia complex was published as an in-
process revision in the September-October 2018 Pharmacopieal
Forum. A 90-day comment period was available for stakeholders
to review and comment on the proposed general test chapter
<60> Microbiological Examination of Non-sterile Products: Tests for
Burkholderia cepacia complex.
The proposed USP chapter briefing states: Because no standard
method currently exists for the detection of Burkholderia cepacia
complex (BCC), a new chapter is being proposed. BCC species are
gram-negative, rod-shaped bacteria that include opportunistic
pathogens. Many BCC have the potential for overcoming antimicrobial
preservative systems and antiseptics, and grow in preserved aqueous
oral liquids and topical products. BCC may cause serious infections in
individuals with cystic fibrosis and chronic granulomatous disease,
mechanically ventilated patients, immune-suppressed people, and
those with serious underlying disease. This chapter proposal contains
test procedures and media formulations for the detection of BCC.
To conduct the test, 10 g of drug product would be added to 90 mL of
USP Fluid A and a 10 mL aliquot transferred to soybean-casein digest
broth with Lecithin and Polysorbate 80 and incubated at 30-35°C
for 48 to 72 hours. After the incubation period, the broth would be
streaked out with a sterile inoculation loop onto BCSA and incubated
at 30-35°C for 24-48 hours. BCC typically grows as yellow colonies with
a pink to yellow zone in the medium. If no colonies are isolated or the
colonies are not subsequently identified as a BCC member, the test
material passes the test.
As with related tests for specified microorganisms as found in USP <62>,
the BCC screening test is subjected to growth promotion and method
suitability testing. Microorganisms used are: Growth- Promoting
and Indicative - Burkholderia cepacia, Burkholderia cenocepacia, or
Burkholderia multivorans and inhibitory - Pseudomonas aeruginosa. The
three members of the BCC selected for suitability testing are the most
significant in CF infection. More than one BBC member was selected as
more representative of the 17 members of the complex.
Sandle (2018) has published a review article discussing the relative
merits of the proposed USP <60> BCC screening test.
« MICROBIOLOGY »
Identification of Members of the
B. cepacia Complex
Basic commercial microbial identification systems may be limited in
their ability to identify accurately non–fermenters and these organisms
can be very time consuming to identify by phenotypic tests due to
the necessity for subculture and low reactivity in key biochemical
tests. Differentiation of species within the B. cepacia complex can be
particularly problematic, even with an extended panel of biochemical
tests, as they are phenotypically very similar and most commercial
bacterial identification systems cannot reliably distinguish between
them (See Manual of Clinical Microbiology. 10th ed. Washington DC:
ASM Press; 2011).
Polymerase Chain Reaction (PCR) amplification and base sequencing
is usually considered to be a good method for bacterial identification,
as it is simple, rapid, sensitive and specific. The basis for PCR diagnostic
applications in microbiology is the detection of infectious agents and
the discrimination of non-pathogenic from pathogenic strains by
virtue of specific gene sequences. However, it does have limitations.
Although the 16S rRNA gene is generally targeted, identification is
difficult when the 400-500 base sequences of the homologous genes
have high similarity in BCC members.
The emerging identification technology Matrix-assisted laser desorp-
tion ionization-time-of-flight mass spectrometry (MADLI-TOF MS),
which analyzes the riboprotein composition of bacterial cells, is use-
ful to identify member of the BCC. Despite the initial capital cost of
the instrumentation, the advantage of MALDI-TOF MS is it can identify
bacteria in minutes compared to days using traditional methods and
the cost per test is less than a dollar. However, the database must in-
clude mass-charge fingerprints for all of the BCC members to be fully
successful. The good news is that these databases are continuously
being updated to include more microorganisms so they will improve
over time.
Other molecular techniques include Pulsed Field Gel Electrophoresis
(PFGE), Multi - Locus Sequence Typing (MLST), Multiple-Locus Variable-
Number Tandem-Repeat Analysis (MVLA), repetitive extragenic
palindromic sequence-based PCR (rep-PCR), and Whole Genome
Sequencing (WGS) have been applied to microbial identification
and strain typing especially in epidemiology studies in response
to infection outbreaks. All of these approaches enable subtyping of
related strains, but do so with different accuracy, discriminatory power,
and reproducibility.
MLST has been successfully used with the closely related members of
the B. cepacia complex. The technique uses seven housekeeping gene
compared to the single target with 16S rRNA gene sequencing. The
ability of this technique to carry out both strain differentiation and
species identification in a single approach represents a major advance
that should greatly aid the clinical diagnosis of B. cepacia complex
infection (Baldwin et al, 2005). However, the equipment and expertise
to conduct MLST is limited specialized clinical microbiology and CF
clinics, contact identification laboratories and not pharmaceutical QC
microbiology laboratories.
www.americanpharmaceuticalreview.com | | 39
» MICROBIOLOGY »
The BCC MLST schema uses fragments of these seven housekeeping
genes:
• ATP synthase beta chain (atpD)
• Glutamate synthase large subunit (gltB)
• DNA gyrase subunit B (gyrB)
• Recombinase A (recA)
• GTP binding protein (lepA)
• Acetoacetyl-CoA reductase (phaC)
• Trptophan synthase subunit B (tryB)
At present, 17 current members of the BCC are recognized and include
B. cepacia, B. multivorans, B. stabilis, B. vietnamiensis, B. ambifaria,
B. athina, B. pyrrocinia, B. latens, B. diffusa, B. arboris, B. seminalis, B.
cenocepacia, B. contaminans, B. dolosa, B. lata, B. ubonensis and B.
metallica (Vandamme and Dawyndt, 2011). With the future application
of WGS this number could change either up or down.
Risk Mitigation Steps to Exclude
B. cepacia from Non-Sterile Drug
Products
Risk mitigation steps to exclude B. cepacia complex include:
• Pharmaceutical ingredients selection
• Product formulation including robust antimicrobial
preservative system
• Management of pharmaceutical water systems
• Equipment cleaning and sanitization
• Manufacturing processes
• Risk-based microbial testing programs
When selecting pharmaceutical ingredients the microbiological
quality of these ingredients may be important depending on the
dosage form. Keep in mind the quantity of the ingredient used in
the product, the manufacturing process of the ingredient, physical
attributes of the product especially water activity, the antimicrobial
effectiveness of the formulation, and the intended use of the product.
There is a hierarchy of risk for microbial contamination with chemically
synthesized ingredients having the lowest risk and animal-derived
ingredients the highest risk. The hierarchy is animal-derived > plant-
derived > mineral-derived > semi-synthetic > synthetic ingredients.
This generalized sequence can be modified by the extent of processing
during manufacturing (Cundell, 2005).
Multiple-use non-sterile products must be resistant to microbial
contamination and growth due to their low water activity, i.e.,
<0.6, inherent antimicrobial activity or to the effectiveness of their
antimicrobial preservative system. During product development,
candidate formulations are assessed using the methods described in
USP <51> Antimicrobial Effectiveness Testing. As recommended in the
USP chapter, additional challenge organisms, including members of
40 | | January/February 2019
the BCC, can be added to the test to ensure the formulation has the
most robust preservative system.
Investigations of BCC infection outbreaks have consistently identified
the pharmaceutical-grade water system at the manufacturing site to
be the probable cause of the product contamination. Manufacturers
of aqueous non-sterile drug products should install a well-designed
water system, and well maintain and manage the system aggressively.
Process equipment including tanks, pumps, and filling lines, especially
their product contact surface must be adequately cleaned and stored
dry to avoid product contamination. Microbial surface monitoring
must be included within standard equipment cleaning protocols and
periodic cleaning verification implemented.
For each dosage form, the unit manufacturing operational steps can
be analyzed for potential microbial contamination risk. For example,
for an oral liquid the manufacturing steps are ingredient procurement,
equipment cleaning, weighing, blending, mixing, container-closure
preparation, and filling. Ingredient water and processing equipment
cleaning may be viewed as areas of greatest risk for microbial
contamination. Another factor may be the order of addition of the
preservative, as they must be present in the aqueous phase of an
oil-water suspension. For a comprehensive discussion of microbial
contamination of non-sterile drug products, the reader is referred to
the USP General Informational Chapter <1115> Bioburden Control of
Non-sterile Drug Substances and Products.
When USP <60> becomes official, whether a microbiological
specification for the absence of B. cepacia complex will be added to
any drug product monographs will remain to be seen. A risk-based
microbial testing program would involve more frequent testing of
aqueous drug products than non-aqueous products. For example,
based on a comprehensive risk assessment and a testing history, a
compressed tablet may not have a microbiological specification and
would not be subjected to release testing, whereas each batch of a
topical cream would be subject to full testing as recommended in USP
<1111>
Conclusions
Although microbial contamination of drug products results in
few patient infections, the pharmaceutical industry must not be
complacent working to eliminate this risk. The author hopes that this
review article contributes to this goal through a timely discussion of
the role that BCC plays in microbial contamination and how it can be
detected.
References
1. Abdallah, M. et al, 2018 Burkholderia cepacia complex outbreaks amongst non-cystic
fibrosis patients in the intensive care units: a review of adult and pediatric literature. Le
Infez. in Med. 4:299-307
2. Baldwin, A., E. Mahenthiralingam et al 2005 Multi-locus sequence typing scheme that
provides both species and strain differentiation for the Burkholderia cepacia complex. J.
Clin. Microbiol. 43(9):4665-73.
 « MICROBIOLOGY »

3. Beck-Sague C.M., R. L. Sinkowitz et al. 1996 Risk factors for ventilator-associated
pneumonia in surgical intensive care unit patients. Infect Control Hospital Epidemiol.
17:374-6.
4. Bernstein B, Dineen T, Kehl S, Wilson P, Sohnle P. 1996 Outbreak of Burkholderia cepacia
colonization and infection related to contaminated oral mouthwash {Abstract}. In:
Program and Abstracts of the 34th Annual Meeting of the Infectious Diseases Society of
America, New Orleans, Louisiana, September 1996.
5. Coenye, T., P. Vandamme et al, 2001 Taxonomy and Identification of the Burkholderia
cepacia Complex J. Clin. Microbiol. 39(10): 3427–3436
6. Cundell. A. M. 2005 Managing the Microbiological Quality of Pharmaceutical Excipients.
PDA J. Pharm. Sci & Technol. 59(6): 381-395
7. FDA 2017 FDA advises drug manufacturers that Burkholderia cepacia complex poses
a contamination risk in non-sterile, water-based drug products, U.S. Food and Drug
Administration, U.S. Department of Health and Human Services, at: https://www.fda.gov/
Drugs/DrugSafety/ucm559508.htm
8. Harpaz R, R.M. Dahl and K.L. Dooling 2016 Prevalence of Immunosuppression Among US
12. Sandle, T. 2018 Burkholderia cepacia complex: Review of origins, risks and methodologies
www.europeanpharmaceuticalreview.com/article/80557/burkholderia-cepacia-
complex-review-of-origins-risks-and-test-methodologies/
13. Sutton, S and L. Jimenez, 2012 A Review of Reported Recalls Involving Microbiological
Control 2004-2011 with Emphasis on FDA Considerations of “Objectionable Organisms”
Posted January 1 2012 Amer. Pharm. Rev.
14. USP <60> Microbiological Examination of Non-sterile Products: Tests for Burkholderia
cepacia complex Pharmacopeial Forum 46(5) Sept.-Oct. 2018
15. Vandamme, P and P. Dawyndt, 2011 Classification and identification of the Burkholderia
cepacia complex: Past, present and future Syst Appl Microbiol. 34(2):87-95
16. Yabuuchi, E. et al, 1992 Proposal of Burkholderia gen. nov. and transfer of seven species
of the genus Pseudomonas homology group II to the new genus, with the type species
Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol.
36(12):1251-75.

9.
Adults, 2013. JAMA. 316(23):2547-2548
Henry D. A., M.E. Campbell , J.J. LiPuma, and D.P. Speert 1997 Identification of Burkholderia
Author Biography
cepacia isolates from patients with cystic fibrosis and use of a simple new selective
Tony Cundell received a PhD in microbiology from Lincoln University,
medium. J. Clin. Microbiol. 35:614–619
New Zealand and had a long career as a microbiologist working in
10. Marquez, L., K. N. Jones et al 2017 An outbreak of Burkholderia cepacia Complex infections
quality and product development in the pharmaceutical industry.
associated with contaminated liquid Docusate. Infect. Control & Hosp. Epidemiol. 38(5):
567-573 Currently he works as an independent consultant specializing in
11. Reik R, T. Spilker, and J.J. LiPuma 2005 Distribution of Burkholderia cepacia complex
microbial testing, contamination risk assessment, and regulatory issues.
species among isolates recovered from persons with or without cystic fibrosis. J Clin He is a member of the 2015-2020 USP Microbiology Expert Committee.
Microbiol 43:2926–2928 Telephone: 914 725-3947

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www.americanpharmaceuticalreview.com | | 41
» FACILITY TOUR »
FACILITY TOUR
Eurofins Lancaster Laboratories
Lancaster, PA
For bio/pharmaceutical companies to bring a new product to market,
many things have to go right.
From the time a new, potentially therapeutic substance is discovered
to the time it is available to consumers, the clock is ticking. Bio/
Pharmaceutical companies need to launch new products as quickly as
possible, yet still maintain the highest levels of quality - a quality that is
demanded by regulatory agencies and the public.
Therefore, suppliers and service providers to the industry must
continually fine-tune their capabilities, adding innovative and
differentiating services, honing staff expertise, and constructing new
facilities to help their clients win the race to market.
Eurofins Lancaster Laboratories, a premier global supplier of analytical
testing services to the bio/pharmaceutical and medical device
industries, has embarked on a strategy that offers their clients cutting-
edge services and expertise, state-of-the-art facilities, quality data, and
scientific solutions they can trust.
The company’s ambitious growth plans have encompassed many
upgrades and 13 building expansions to their campus in Lancaster,
Pennsylvania, since its founding in 1961. Recent enhancements
include best-in-class laboratory information management systems,
implementation of LEAN strategies, and upgraded instrumentation
and laboratory facilities.
Perhaps the company’s crowning achievement, however, is the
completion of their latest 168,000 square foot, five-story building
addition designed to incorporate the latest LEAN technologies to
42 | | January/February 2019
streamline processes and workflows to enhance the customer service
experience and ensure speed-to-market remains a top priority.
Meeting Client Needs
Neal Salerno, President, Eurofins Lancaster Laboratories, details the
business and market decisions that led to the construction of the
new facility.
“One of the benefits of a partnership with Eurofins Lancaster
Laboratories is that we are a “one-stop” facility where a bio/
pharmaceutical company can come here and get any testing they
might possibly need--from starting materials through process
intermediates, bulk delivery, drug products, and packaging--the entire
value chain for every drug development program.”
Salerno continues, “Offering these services in one location is a
huge value to our customers. And our new building is a continued
enhancement to ensure that as new product technology comes on-
line, we are ready and able to help facilitate its testing and help our
clients get to market faster.”
In addition, Salerno mentions the changing dynamics and regulations
within the industry as another reason for the construction of its
new building expansion, “Between data integrity requirements
and the increased importance of testing throughout the entire
pharmaceutical life cycle value chain, we saw that as an opportunity
 « FACILITY TOUR »

to extend our capabilities and our processes, and this significant

addition is a culmination of our desire to serve the industry and help
our partners succeed.”

Supporting Research and

Emerging Technology
“The bio/pharmaceutical industry is the most research intensive
industry in the word," Salerno explains. “We are fortunate to support
some of the most innovative, creative, and scientifically advanced
companies in our industry. When you work on the cutting edge, the
pace of change is staggering. Constant investment to keep up with
our client’s needs has become part of our company culture. One
emerging technology is gene therapy, and we support most leaders
to rooms and areas benefit from free-flowing hallways. Sample prep and
in the field. To better support these clients, we have significantly
storage rooms are near the labs. All of this was done to decrease waste,
expanded our BSL2 capacity. We can support all testing required
improve quality and speed data delivery to clients—all of paramount
for release of gene therapy products, quickly and efficiently, at our
importance to biopharmaceutical companies.
Lancaster campus. A second emerging technology is Antibody
Drug Conjugates (ADCs). In a quest to better treat cancer patients, “Every part of the design of this building has really had some kind of
biopharma companies are using vectors (antibodies) that target LEAN assessment and design from a LEAN specification.”
and release a cytotoxic payload directly into a tumor cell. Classical Salerno continues, “How do you get testing and other tasks done
chemotherapy treats patients systemically, making the patient very as quickly as possible? We believe the more we can shrink the time
sick in an attempt to eradicate the cancer. ADC technology isolates frame to help get that product to market, the better. We’ve designed
the cytotoxin to the tumor cell. To test these drugs requires handling everything in our new building around LEAN processes to ensure that
of highly potent/cytotoxic materials. Our campus investments our experienced scientists have the wherewithal and the bandwidth
include special laboratories and equipment to support this promising to focus on the science while making this a great place to work.”
technology while protecting our employees.”
“It really is a very contemporary facility that has completely redesigned
our flow to be able to get testing results out the door quickly, efficiently
with the highest level of quality,” says Salerno.
The New Building’s Features
and Benefits
As constructed, the new building adds 50% more capacity to the
Campus Culture
company’s existing 330,000 square foot facility for a total of more than A new, state-of the-art bio/pharmaceutical testing facility can only
1/2 million square feet. function as designed if the people working within it are given the best
Along with the investments in its microbiology services, the company environment possible.
has enhanced its other service offerings, including an expansion The company believes their staff is what sets them apart from other
of its Extractables and Leachables Testing group for testing both companies in this segment of the industry.
pharmaceutical and medical device products. The company also has
“It’s really about our scientists and our supporting staff and how
a new area dedicated to method development and testing for small
they’re working with our clients and getting regulatory compliant
molecule products.
data to them most proficiently. We’re very well known in the industry
One of the biggest features of the expansion is the inclusion of a massive for our stellar quality and from the partnerships we foster with our
cleanroom. Designed for the company’s Microbiology Testing Services, customers. That doesn’t happen without a phenomenal team.”
the cleanroom was designed as a response to market changes. As
Employing an outstanding team means attracting and retaining top
Salerno explains, “One of the things that we’re starting to see, especially
talent. The company has always cultivated a great work environment,
for the Medical Device business, are products that are much larger in
but, with the latest addition to the Lancaster campus, the company now
nature and a little bit more challenging to handle. We needed to have
has taken the work environment to another level for its talented staff.
the capability to take some of these unique products, handle them,
and test them accordingly.” And, as a modern building, one of the key “There’s always the challenge of how do you attract and keep talent,”
features is that its design was optimized to streamline processes. As you says Salerno. “We really focus quite a bit on that. Not only do we think
walk through the completed building, Salerno explains, departments that this is a wonderful company to work for, it’s just a great local area
that work together are located in close proximity to each other. Access where you have a very rural and close-knit community with a cool

www.americanpharmaceuticalreview.com | | 43
» FACILITY TOUR »

small city, so we really are trying to work on some of the amenities for
our folks that will continue to move us forward and to make this the
optimal environment to grow a career, balance work/family life, and be
a fun place to call home.”
True to the old adage that the way to someone’s heart is through
their stomach, the new building features a brand-new cafeteria and
gourmet food service. It also features numerous open areas where
employees can work or take a break and also offers an outdoor seating
area with an expansive patio and panoramic views the countryside.
“Our new building has a great feel to it; it’s a very sleek, open and
bright building,” says Salerno. “And it will also serve as the foundation
for how we modernize all the other buildings on our campus. We have
begun modifications and renovations to infuse LEAN initiatives as well
as give the facility one look and feel. I believe we have designed and “Our system is world-class and one that you won’t find elsewhere in
constructed a very impressive facility that people are happy to work in.” the industry. The facility is designed really around that, which is how I
The company also sees their growth and success tied directly to the can, for example, be in a chamber and have wireless connection, how
opportunities they offer to employees. I can scan a product in and out versus hand typing it, and how I make
sure that I have positive control over everything that I’m doing.”
“As we’ve grown, we have given our folks the resources to expand both
professionally and personally, and also to really take part in developing
cutting edge technology. We have some of the greatest scientists in
the world here, and they do amazing things supporting our clients’ Future Campus Enhancements
quest to better the human condition,” says Salerno.
While most companies might take a break after designing, constructing,
outfitting, and staffing a new facility, Eurofins Lancaster Laboratories is
already looking at the next steps in its journey to continue to offer its
Local and Global Network current and future clients the best services possible.

Eurofins Lancaster Laboratories is part of Eurofins BioPharma Product
Testing, the largest network of harmonized GMP product testing
laboratories worldwide. The company boasts four facilities in the
U.S. and 30 throughout Europe and Asia. This network of facilities
is very well integrated – all facilities have the same LEAN systems;
customized, proprietary Laboratory Information Management System
(LIMS); and harmonized quality systems in place. The sites all work
collaboratively to ensure customers have the right solutions for the
project – regardless of the location.
“It’s actually a very close connection between all of the sites,” says
Salerno. “One of the things that we’re very proud of is the fact that
the service level that you get here, is the same service level you get
at all of our sites. We help from a risk mitigation standpoint so that
our customers are not necessarily tied to just one facility, but there’s
multiple facilities that may have your methods if that’s what the
customer needs.”
The company has also invested heavily in its IT infrastructure, basically
moving away from paper to a fully electronic data system.
“You can’t walk around this facility without seeing somebody with
a laptop or their electronic notebook acquiring real-time data and
performing instant data review. We’ve become very flexible as to how
we’re doing our work, where we’re doing our work.”
IT infrastructure upgrades have allowed the company to tie all
electronic notebooks to their LEAN systems and allow their LEAN
systems to securely tie in with the customer’s systems, creating a
seamless platform.
As Salerno explains, a lot of thought and planning went into the
design of the building, with future expansions in mind. “We’ve put
the infrastructure in place for additional expansions in the future. For
example, the land development and zoning was completed to include
an additional 300,000 square feet of future facilities. We also accounted
for future expansion when installing the wiring, such as fiber optics,
switch lines, etc. On top of that, really, it’s a very advanced building.”
The recent completion of this expansion, the campus-wide
improvements in IT infrastructure, the implementation of Lean
strategies to improve workflow processes and time to market, the
strength and breadth of the Eurofins network all culminate in a
company that is keen on ensuring their clients’ success.
“All of our investments to our physical campus, combined with
our dedicated staff have been done to ensure that we can handle
anything and everything that our customers need in order to
succeed,” Salerno concludes.
2425 New Holland Pike
Lancaster, PA 17601
Tel: 717-656-2300
pha@eurofinsUS.com, www.EurofinsLancasterLabs.com

44 | | January/February 2019

Application of a Small EF Hand Affinity Tag for
Expression, Purification and Biophysical Studies of G
Protein-Coupled Membrane Receptors
Alexei A. Yeliseev1 and David J. O’Connell2
1
National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health,
Bethesda, MD
2
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin,
Dublin D04 V1W8, Ireland
Abstract
Heptahelical G protein-coupled receptors (GPCR) comprise a large
family of integral membrane proteins involved in a wide array of
cell signaling pathways. For high resolution structural studies of
these receptors, multi-milligram quantities of pure and structurally
unperturbed proteins are required. Purification of recombinant GPCRs
typically involves their solubilization into detergent micelles followed
by chromatographic purification. Because of relatively low expression
levels of these recombinant receptors, it is challenging to design an
efficient strategy for selective and efficient purification with high yield.
Here, we describe a recently introduced purification system employing
a high affinity molecular switch based on fragment complementation,
with a calcium dependent capture and EDTA mediated chelation
elution. This technique was successfully applied to the purification of
the recombinant cannabinoid receptor CB2, a promising target for the
development of drugs for inflammation, immunological disorders and
pain. It is feasible that similar strategies can be successfully employed
for expression and purification of other membrane protein targets.
Introduction
G protein-coupled receptors (GPCR) comprise a large family of integral
membrane proteins involved in a wide array of cell signaling pathways.
The high resolution structural studies require large quantities of pure,
homogenous and stable proteins. Purification of GPCRs typically re-
quires solubilization of these receptors from cell membranes into deter-
gent micelles, followed by one or more steps of affinity purification. This
is notoriously challenging, because of the relatively low expression lev-
els of GPCR in membranes and their instability in detergents. In recent
years, significant improvements have been made in designing efficient
protocols for expression of GPCRs in different cell types, their stabiliza-
tion, and purification by affinity chromatography. 1-5
« SEPARATION PURIFICATION »
Human cannabinoid receptor CB2 is primarily expressed in cells of
immune origin, and is an attractive target for the development of
drugs for management of inflammation, immunological disorders
and pain.6-8 Structural studies can provide critical contributions to the
rational design of novel, specific drugs targeting this receptor.
Purification of a recombinant GPCR typically involves one or more
steps of affinity chromatography, relying on engineered tag(s) fused
to the expressed protein. Poly-histidine (immobilized-metal affinity
chromatography, IMAC) and FLAG tags have been successfully used. 3, 9-15
However, IMAC is not always efficient for purification of GPCRs, especially
for those targets that are expressed at low levels, because of the low
affinity of interaction between the His-tag and the resin in detergent-
containing buffers. The antibody-epitope interaction exploited by such
systems as FLAG-tag/ FLAG resin may produce a high purity of the target
protein, even in the presence of detergents.3,16 However, the application
of FLAG resin for preparation of multi-milligram quantities of GPCR is
expensive, especially in the case of commercial preparation of affinity
resins. Several other affinity tag/matrix pairs have been described in
the literature, but very few of them have been applied successfully to
preparative-scale production of GPCR.17
When choosing the tag/affinity matrix pair for purification of
membrane proteins for structural studies, the following parameters
should be considered:
• High affinity in the presence of detergents (ideally in the
picomolar or low nanomolar range)
• Ability to elute the captured protein in mild buffer
conditions
• High binding capacity
• Ability to regenerate/re-use the matrix
• Low cost
• Small size of the affinity tag
www.americanpharmaceuticalreview.com | | 45
» SEPARATION PURIFICATION »

Currently, there only a few examples of commercial kits that satisfy

these requirements. One feasible alternative to the antibody/
epitope interaction is a Strep-tag/StrepTactin system that relies on
interaction between a small eight amino acid-long epitope (Strep-tag
and a modified Streptavidin (StrepTactin). The recently introduced
modification of the system StrepTactin XT affords pico- to nano-molar
affinities between the Strep-tag and a resin, even in the presence
of detergents.18 This approach has been successfully applied to
purification of both soluble and membrane receptors.19 The limitation
of the system is that the protein captured on the resin is eluted with
high concentrations of biotin, and the elution kinetics are quite slow.
Because of its amphiphilic properties, biotin incorporates readily into
detergent micelles, which may interfere with downstream applications Figure 1. Fragment complementation between EF hand domains
of the purified protein. of calbindin D9k as a basis for affinity purification (A) Ribbon
diagram of calbindin D9k based on X-ray structure coordinates24
An alternative strategy utilized in this work relies on a calcium- (PDB entry 4icb), with calcium ions shown as silver spheres (B)
dependent molecular switch based on the high affinity fragment Affinity purification of EF1-tagged protein depends on calcium
complementation between the EF1 and EF2 domains of the Ca2+ dependent structural organization of the EF-hands, high-
affinity complementation between the EF1 peptide tag and
-binding protein calbindin D9k.20-23 The high affinity binding of the EF2-GGC affinity ligand immobilized on agarose beads (grey)
two EF domains is based on interaction between hydrophobic core and the dissociation of the bound complex upon chelation of
residues located in the α helices and loops of these fragments (Figure calcium releasing pure protein.
1 A). The interaction between these domains can be broken by simply
chelating the Ca2+ ions resulting in the loss of the helical secondary
structure. Therefore, the elution step for EF-based purification of the
recombinant protein can be performed at very mild conditions, at
physiological pH, with addition of EDTA (Figure 1 B).
We tested the application of such Ca2+-dependent complementation
in the purification of cannabinoid receptor CB2.25 The procedure takes
advantage of the binding of calcium ions by the EF1 epitope fused to
the recombinant receptor, and the EF2 affinity ligand coupled to the
agarose matrix. In the presence of calcium, the helix-loop-helix motif
on both EF1 and EF2 fragments is restored, facilitating a high-affinity
binding of the target receptor to the resin.
Figure 2 A, Constructs for expression of CB2 receptor in E. coli.
Several constructs with the EF1 domain and other expression partners 2B. Expression levels of EF1-CB2 constructs in E. coli membranes
(determined by Western blot, probed with anti-CB2 antibody).
fused in various positions relative to the CB2 receptor were expressed
in E. coli cells (Figure 2 A). The sequence of the DNA fragment encoding
the 43 amino acid-long EF1 tag was optimized for codon usage of E. coli the C-terminus of the fusion (constructs 353, 354) did not have any
cells. Confirming our earlier observations for expression of CB2 receptor negative effects on expression of these proteins.
in bacterial cells,26 we determined that the presence of maltose-
binding protein (MBP) of E. coli as the N-terminal fusion partner was We further examined the expression of EF1-CB2 fusion proteins in
required for expression of CB2, and for its accumulation in bacterial other expression hosts besides E. coli. Figure 3 depicts the results of
cell membranes (Figure 2 B). When expressed with its signal sequence the expression of these constructs in suspension culture of Expi293F
intact, the MBP is transported to the periplasmic space of E. coli, and is HEK cells. In these mammalian cells the constructs were expressed
believed to act as a chaperone directing the downstream part of the without the MBP fusion partner. The sequences of the 43 amino
fusion (CB2) to the cytoplasmic membranes.26 The periplasmic location acid- EF1 tag, CB2, and a small linker were optimized for expression
also facilitates the formation of a disulfide bridge in the extracellular in mammalian cells. The expression levels of both CB2-352 (EF1 in
loop 2 of CB2, which is essential for stability of this receptor.27 the N-terminal position) and CB2-353 (EF1 in the C-terminal position)
Consequently, only the fusion constructs that harbor the N-terminal were approximately equivalent. Both constructs express at levels
MBP fused to CB2 were expressed in E. coli cells; the construct CB2-351 slightly lower than the control construct CB2-370 that harbors a twin-
(comprising the EF1-CB2 without an N-terminal MBP) did not appear to Strep-tag (22 amino acids including linker sequence) upstream of
express as determined by Western blot with anti-CB2 antibody (Figure CB2. The expression levels of MBP-CB2-130 fusion construct in E. coli
2 B). Another significant observation is that the location of EF1 tag (normalized per milligram of membrane protein) were the highest
upstream of CB2 appears to have a negative effect on the expression among the construct and expression hosts tested. These findings
levels of this fusion protein (construct 352). The location of EF1 tag at underscore the importantance of the nature, size and the relative

46 | | January/February 2019

Figure 3. Expression levels of CB2-EF constructs in Expi293F cells vs E. coli cells.
position of the affinity tag on the expression
levels of the target protein. Naturally, the
effects of the affinity tags on expresison
levels of the target protein also depend on
a host cell. Our data show a potential utility
of the EF1 tag for expression of integral
membrane GPCR not only in a bacterial but
also in a mammalian host.
The functionality of the recombinant receptor
can be assessed by a ligand binding assay
(typically, using a radioisotope-labeled
ligand) and by measuring the activation
of G protein on the receptor in response
Figure 4. Functional activity of EF1-CB2 constructs expressed in E. coli. A, saturation
ligand binding on membrane preparation from E. coli BL21(DE3) expressing EF1-CB2; B,
G protein activation assay on membrane preparations of EF1-CB2.
to ligand binding.27,28 Both assays are
typically performed on preparations of cell
membranes expressing the recombinant
receptor. Figure 4A shows the saturation
binding of the tritium-labeled high affinity
agonist CP-55,940 on the membranes
expressing construct CB2-354. The binding
of the ligand is specific, with the Kd~ 1.4 nM,
similar to the high affinity binding of this
synthetic cannabinoid ligand to other EF1-
CB2 expression constructs.25
Another informative functional test quantifies
the rates of activation of G protein on CB2
www.americanpharmaceuticalreview.com
« SEPARATION PURIFICATION »
receptor in response to cannabinoid agonist
binding. The assay can be performed with
either bacterial membranes expressing
CB2 protein or with purified receptor
reconstituted into liposomes. Briefly, the
receptor preparation is incubated with
saturating concentrations of the activating
ligand and is supplemented with purified
subunits of G protein (in this case, Gαi1 and
Gβ1γ2), in the presence of GDP. The reaction
is initiated by the addition of radiolabeled
analog of GTP, 35S-γ-GTP. This nucleotide
analog is poorly hydrolyzible, and upon G
protein activation on the receptor it tightly
binds to the Gαi subunit. Thus, the complex of
the labeled GTP with G protein accumulates
for the duration of the reaction and provides
a quantitative assessment of the amount of
functional CB2 receptor. Figure 4B illustrates
this concept by comparing membranes
expressing different CB2 constructs. It reveals
that the N-terminal (relative to CB2) location
of EF1 tag is detrimental for the expression,
while the C-terminal location of the tag results
in higher expression levels. Furthermore, the
placement of a small thioredoxin epitope
between the C-terminus of CB2 and EF1 tag
has a positive effect on expression levels
of construct CB2-354. To summarize, these
data confirm the correct folding and full
functionality of EF1-CB2 constructs expressed
in E. coli.
The purification of CB2 receptor performed
using the EF1-EF2 complementation strategy
with affinity resin prepared by thiol coupling
of the EF2-GGC peptide to SulfoLink coupling
agarose resin as described earlier.25 The
recombinant fusion protein is solubilized
from cell membranes in detergent micelles
comprised of zwitterionic detergent
CHAPS and non-ionic n-dodecyl-β-D-
maltopyranoside (DDM). For stabilization
of the receptor, cholesteryl hemiscuccinate
(CHS) and a high affinity ligand (CP-55,940)
are added, and all purification buffers are
supplemented with 15-30% (v/v) glycerol.
All steps of purification are performed at 4°C
or on ice to preserve the correct fold of the
recombinant protein.
The detergent extracts with solubilized
receptor are mixed with EF2-agarose, and
the binding is typically performed as a batch
purification overnight, in the presence of
Ca2+. The batch mode helps to maximize the
| | 47
» SEPARATION PURIFICATION »

binding capacity of the resin, which can be

as high as 0.5 mg of protein per mL of resin.
The resin is then washed extensively with
Ca2+ -containing buffers to ensure that the
high affinity interaction between EF1 and EF2
epitopes is maintained. No significant loss
of the captured protein is detected during
this step. The elution is performed by the
addition of EDTA that chelates Ca2+ ions. The
protein bound to the affinity matrix is released
gradually; typically, 4-5 column volumes of
EDTA-containing elution buffer are necessary
to complete the process (Fig. 5). Both the Figure 5. Purification of EF1-CB2 constructs on EF2 agarose resin. SDS-PAGE stained
C-terminal location of the EF1 tag (constructs with Instant Blue. A, construct CB2-352. B, Construct CB2-354. L, load (crude solubilized
CB2-353, CB2-354) and mid-fusion location protein extract); W, wash fractions; E, elution fractions; CE, combined, concentrated
elution fractions. The position of MBP-EF1-CB2 fusion protein and the cleaved EF1-CB2 are
(construct CB2-352) are suitable for purification indicated with arrows.
using this strategy, although in the case of
mid-fusion EF1 tag, the binding capacity of
the resin appears to decrease, possibly due
to steric hindrance effects from the large MBP
fusion partner. Also, in the case of construct
CB2-352 some spontaneous cleavage of the
fusion construct can occur during purification
that results in the removal of MBP and release
of the EF1-CB2. Importantly, a significant purity
of the protein preparation can be attained
after just one chromatography step, with an
enrichment factor for the EF1-CB2 protein of at
least 1000-fold.
Both the capture and elution steps are
performed at neutral pH values. The mild
condition of the purification preserve the
functional structure of the receptor, as
confirmed by activity tests of the purified
products as described earlier.25
We next determined the binding kinetics
between EF1-CB2 proteins and immobilized
EF2-GGC in the detergent and glycerol rich
solubilization buffer used to purify EF1-CB2
at 25, 10 and 4 degrees Celsius. EF2-GGC
that was thiol coupled to the carboxymethyl
dextran surface of a BiaCore CM5 sensor chip Figure 6. SPR analysis of the interaction between EF1-CB2 fusion proteins and EF2-GGC
and purified EF1-CB2-352 and EF1-CB2-354 functionalised surface in solubilization buffer. (A) Sensorgrams showing the relative
response recorded during injection of CB2-352 (black), CB2-354 (red) and EF1 peptide
fusions were injected over the surface for (yellow) at a concentration of 100 nM, followed by buffer flow, over the EF2-GGC
30 minutes followed by buffer flow for 60 surface. (B) Fitting to (i) the association phase in a concentration series of 100 nM (red),
minutes. EF1 wild type peptide was injected 50 nM (orange), 25 nM (green) and 12.5 nM (blue) CB2-354, and to (ii) the dissociation
phase after injecting 100 nM CB2-354. The equilibrium dissociation constant, KD, for the
as a positive control (Figure 6A).25 The
CB2-354 – EF2 interaction was estimated from the ratio of the fitted rate constants as KD
equilibrium dissociation constant, KD, for the = koff / kon = 3.10-9 M.
CB2-354 – EF2 interaction was estimated from
the ratio of the fitted rate constants as KD =
koff / kon = 3.10-9 M (Figure 6B) and 5.10-9 M for location of the EF1-tag in this fusion. The method of elution, highlights the potential to
EF1-CB2-352, that had an apparently faster off maintenance of the high affinity of interaction optimize this system further in the capture,
rate than that of EF1-CB2-354, which while between EF1 and EF2 sequences in this purification and analysis of functional integral
not significant, may be due to the N terminal buffer at low temperature, with the simple membrane proteins.

48 | | January/February 2019

To summarize, the use of EF1 tag for recombinant production of CB2
and affinity purification on EF2 matrix facilitates the isolation of the
fully functional protein in just one chromatography step. The yield of
the recombinant protein may be further improved by optimizing the
expression conditions and purification parameters. The technology
of preparation of the recombinant CB2 can be adapted to expression
and purification of other GPCR and recombinant receptors and open
a path for a meaningful biophysical characterization of these proteins.
Acknowledgements
This work was supported by the Intramural Research Program of the
NIAAA, NIH and the Science Foundation Ireland Investigator Project
Grant 12-IP-1620. Authors gratefully acknowledge the contribution
by Dr. N.N. Mhurchu and Gavin McGauran (University College Dublin),
Professor Sara Linse (Lund University), valuable technical assistance
of Mr. K. Hines and Ms. L. Zoubak and continuous support by Dr. K.
Gawrisch (NIAAA, NIH). Expression in Expi293 cells was performed by
the group of Dr. J. Zmuda (ThermoFisher Scientific).
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“what’s hot”, Frontiers in Bioscience-Landmark 14, 944-957.
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www.americanpharmaceuticalreview.com | | 49
» BIOPHARM PROCESSING »

Facility Design for Continuous Bioprocessing and

Smart Manufacturing
Attributes for Success
Jeff Odum, CPIP
NCBiosource

The “traditional” fed-batch biomanufacturing facility and its equipment

Setting the Stage have some common attributes.

• Complex manufacturing and utility systems

In testimony given 2013 during a congressional modernization
hearing, FDA Director Janet Woodcock clearly outlined the regulatory • Process designed for lower average yields and cell
impetus for the industry’s continued drive to implement continuous densities resulting in inefficiencies
manufacturing platforms.1 She challenged the biomanufacturing
• Cleaning and contamination control for each batch are
organizations to take advantage of advances in process and facility
expensive and often difficult
design to help achieve improved manufacturing reliability, increased
process robustness, and lowering of manufacturing costs. • Complex, large, fixed stainless steel-based equipment
Move forward to 2017 when CDER finalized the guidance, “Advance- platforms
ment of Emerging Technology, Applications for Pharmaceutical In- • High operation and maintenance costs
novation and Modernization ”2 Again, the driving focus of regulatory
• Resource and maintenance intensive
policy is to promote technology that can produce a more robust man-
ufacturing process with fewer interruptions in production and ensure • Inflexible, process dedicated designs
that facility assets can support these advanced platforms.
• Extensive stainless steel piping systems
Yet continuous biomanufacturing is not a recent phenomenon.
• Inflexible equipment configuration
Perfusion-based bioreactor operations have been in use for over 25
years. The focus use was on high-value and labile proteins from low
yield expression systems. These systems had many production and
regulatory challenges. But with new platform technologies, advanced
equipment designs, and advances in automation/control systems,
continuous manufacturing is rapidly gaining acceptance across a
wider spectrum of manufacturing operations.

Traditional State vs. Future State

As the industry sees production costs rise in an expanding global mar-
ket forecast to surpass $200 billion (US)3 there is a focus on optimiz-
ing the product-process-facility relationship. Current advancements in
manufacturing science are resulting in increasing titers which translates
to shrinking bioreactor capacity for the same throughput. Perfusion bio- Figure 1.
reactor operations can provide much higher capacity utilization allow-
ing smaller equipment for the same throughput.

50 | | January/February 2019

• High operational costs and
resourcing requirements
• Increased risk in numerous
complex in-process material
transfers
• Complex stainless steel CIP/SIP
cleaning systems
• Risks due to temperatures,
chemicals, loads, and
automation control
If you focus more on the specific process
design attributes in traditional upstream
and downstream manufacturing, addressing
the need for large volumes of expensive
complex media, microbial control concerns,
complicated change-over protocols for large
chromatography columns, and complex
validation execution are normal.
The traditional fed-batch facility requires sig-
nificant amounts of physical and controlled
manufacturing space (Figure 1) when com-
pared to a continuous facility approach for
similar production output. The reduction in
space is because continuous systems have a
much higher utilization rate of expensive re-
sources than fed-batch processes that have
lower average titers and significant overhead
associated with batch changes.
Facility layout is driven by a complex set
of relationships between unit operations,
equipment design and configuration, flows
and segregation and control strategies.
When companies embark on continu-
ous manufacturing implementation, there
should have been a decision process similar
to that shown in Figure 2.4
From such an analysis, key attributes of
what will become the facility design will
begin to evolve.
For continuous manufacturing these attri-
butes would include:
• Design to support a 24/7
operational approach
• A synchronized process flow
through all unit operations
• Equipment/unit operations that
support high cell densities, high
yield expression systems
• Facility design focused around a
“scale-out” approach
• Elimination of hold steps
• Process control platforms
implementing PAT-driven active
control strategy
• Flexible equipment platforms
that support significant cleaning
reductions and changeover,
particularly this associate with
single-use technologies.
Figure 2.
Figure 3.
www.americanpharmaceuticalreview.com
« BIOPHARM PROCESSING »
The Look of Continuous
Manufacturing
There are some key success metrics around
implementing continuous versus fed-batch
manufacturing operation that impact facility
design. In the three reference studies shown
in Figure 3, there are three downstream
chromatography operations implemented;
the actual number of columns may vary
depending on purification strategy.5
From a process design view, there are
multiple options around continuous process
implementation. For a typical MAb cell
platform, these would be as follows.6 (Figures
4, 5, 6)
Each approach will have design strategies
that impact facility design related to layout,
orientation, and segregation strategy. For
option 1, high density cell banking in bags
and the implementation of an intense closed
system design approach will significantly
reduce upstream footprint. In option
2, high throughput and multi-column
| | 51
» BIOPHARM PROCESSING »

Figure 4. Option 1

Figure 7.

Figure 5. Option 2

Figure 8.

Figure 6. Option 3

chromatography permits a more efficient use of resins and a reduction

in buffer consumption per gram of product. It may also permit
the use of pre-packed SUS columns. For option 3 where the entire
process becomes a continuous operation, in-line dilution layered
with multi-column chromatography operations will further reduce
buffer volumes. And for this option, multi-column chromatography
purification coupled with perfusion bioreactors supports an efficient,
validated continuous manufacturing platform.

Multi-column chromatography to support continuous manufacturing

allows for the use of smaller columns with reduced bed heights which
are easier to load and operate and can be run at high cycle rates or
in simulated moving bed configuration. This simultaneous processing
allows for the same processing time while using less resin, up to a
50% reduction in resin volume.7 In one business case, moving from
a traditional fed-batch chromatography operation to a multi-column
rotating chromatography process resulted in a 275% increase in
productivity (g/Lresin hr) and resin cost reductions of over $750,000
at 2000L scale.8

Taking a closer look at one of the key design approaches shows the
significance of the facility impact. The traditional batch approach for
Figure 9.
buffer prep/hold in large vessels shown in Figure 7 requires significant
space, and utility and operational support.9

52 | | January/February 2019

In-line dilution of buffers allows for process buffers to be produced
“just in time” and at the “point of use”. It is a simple approach for
mixing buffers using one (or more) concentrated stock solutions with
water added as needed to optimize large volume buffer production
and eliminate hold requirements. Implementing an in-line dilution
scheme (Figure 8) can move the platform from stainless to single-
use quite easily and can lead to a significant reduction in (30 – 70 %)
buffer vessel size, as well as increased flexibility in meeting future
volume requirements.10
The challenges of this approach must also be addressed in the facility
design. These include the fact that some required concentrated
buffers might be more corrosive resulting in higher safety and material
specifications. Temperature and pH changes during the mixing
operations must be controlled on a continuous basis with high-level
automation control. By design, these systems are more complex
system to operate, also leading to a greater level of control automation.
Results
How does all of this translate into manufacturing optimization? There
are many examples of data available. The data examples shown below
gives some stark contrasts between the traditional fed-batch and
continuous operation results.11
Continuous manufacturing also has a number of quality advantages
that are possible during the facility design development.
• Shorter residence time at higher titers; reductions from
14 days down to 3 days reduces equipment sizing and
space requirements
• Protein/resin interaction will now be measured in hours vs.
minutes which results in shorter processing time
• Shorter processing time will lead to less/shorter
intermediate hold times
• Real time process control means that there will be faster
feedback control response time to reduce process drifting
and deviations, improving quality and reducing risk
« BIOPHARM PROCESSING »
• Generation of large amounts of on-line data will require
advanced PAT-based control platforms that will increase
process understanding through increased process control
using QbD concepts
• It opens the door for the option for real time release
testing from building in quality rather than relying on
testing in quality
• Increased reproducibility and on-line control, targeting a
state of “in control” rather just maintaining “steady-state”
conditions which is an enabler defined by the FDA for an
active control strategy.12
What’s Next?
Both industry and the FDA are supporting the paradigm shift to
continuous manufacturing.13 The continued movement to integrate
both drug substance upstream and downstream operations can be
seen by the number of companies moving into this operational space.
The benefits of this shift will be:
• Promising business case improvements in productivity,
resulting in lower CAPEX, OPEX requirements
• Facilities that will be smaller with less work-in-progress
materials
• Improve product Quality & Safety
• Enabling faster response to market demand changes
References
1. Federal Record, Congressional testimony, December 12, 2013; Janet Woodcock, M.D.,
CDER Center Director
2. US FDA, available at http://www.fda.gov/downloads/Drugs/
GuidanceComplianceRegulatoryInformation/Guidance/UCM478821.pdf
3. Wairikoo, V , 2017 Biopharmaceutical Trends: Bioprocess online; 2012
4. J. Odum, et. al, “Do’s and Don’ts in Continuous Manufacturing Facility Design” Workshop at
2nd Annual CCP Summit, Boston, 2018
5. Study 1: Pollock, J., et al., Biotechnology & Bioengineering, Volume 110, 2013, 206-219
Study 2: Walther, J., et al., J of Biotechnol. Volume 213, 2015, 3-12
Study 3: Godawat, R., et al., J. Biotechnol. Volume 213, 2015, 13–19
6. J. Odum, et. al, “Do’s and Don’ts in Continuous Manufacturing Facility Design” Workshop at
2nd Annual CCP Summit, Boston, 2018
7. R. Lu, “Multicolumn Chromatography: A First Step Toward Continuous Purification”, ISPE
Biomanufacturing Conference, December 2018.
8. ibid
9. J. Odum, et. al, “Do’s and Don’ts in Continuous Manufacturing Facility Design” Workshop at
2nd Annual CCP Summit, Boston, 2018
10. ibid
11. ibid
12. L. Lee, “Regulatory Initiatives for Supporting Innovation in Pharmaceutical
Manufacturing” PDA/FDA Joint Regulatory Conference, September 2015
13. FDA Voice posted by Lawrence Yu, http://blogs.fda.gov/fdavoice/index.php/2016/04/
continuous-manufacturing-has-a-strong-impact-on-drug-quality
www.americanpharmaceuticalreview.com | | 53
» FORMULATION AND DEVELOPMENT »
Modeling in Drug Metabolism for Drug Discovery
and Development
Kristen Cardinal
Project Coordinator, Drug Metabolism, Covance
Hao Sun, PhD
Principal Pharmacokineticist, Translational Sciences, Seattle Genetics
You wake up in the morning. Amazon Alexa tells you that today
is mostly cloudy but will rain at noon. She also reminds you to take
your daily medications and to wish your sister happy birthday. You
scroll through your Facebook feed and an ad appears that tells you a
restaurant is now serving your favorite gingerbread pancakes. You start
your car engine, and as you being to drive to work, Google Maps tells
you an alternative route should save you 20 minutes. You arrive at your
office and your boss asks you to shop for a 3D printer for a “delicious”
project, printing real cakes. You find a good-ranking model at Amazon,
which also automatically recommends all of the cake-printing supplies
you need. This type of thing is happening every day to most of us. It
is artificial intelligence (AI), or at least, the start of an AI era. Alexa, or
Apple’s Siri, uses speech recognition designed to understand a user’s
request and return a response. Facebook’s AI knows who you are from
photos and videos you’ve uploaded and conversations with friends,
and decides which news feeds, friend recommendations, and ads
you receive. By definition, artificial intelligence is a computer-aided
modeling process that solves cognitive problems such as learning and
pattern recognition, which are aspects of human intelligence.
Artificial intelligence comes from sophisticated computer model-
based “machine learning” and “deep learning”. Microsoft and IBM
provide AI infrastructure using modeling tools. Farmers are able
to develop intelligent planting, irrigating and harvesting systems
to increase yields to feed the expanding population of our planet.
Algorithms and data, often “big data” (defined as data with large
volume, high velocity and complex variety), are the core of modeling
and learning. Wall Street quants build complex mathematical models
to predict stock prices for maximized profits and minimized risks. J. P.
Morgan develops “trading bots” to execute trades, which model and
make decisions using your data from the moment you type a buy/sell
price from your cell phone. Big data feed machine-learning algorithms
to acquire artificial intelligence.
54 | | January/February 2019
In drug discovery and development, modeling and simulation have
also been explored and gradually applied in many areas recently. The
Human Genome Project was successfully completed in 2003, which
has provided an enormous amount of genetic data for deep learning
about a disease or target for drug discovery. Over 15 years later, drug
discovery has made significant improvements because of the deeper
understanding of drug targets. However, many diseases, especially
most types of cancer, still have no cure. At best, we can maintain the
disease condition to extend life for months or even years. The human
body is astronomically complex. Diseases, if not genetic, are generally
either life-style-related such as cardiovascular diseases, diabetes and
obesity, or age-related such as Alzheimer’s and rheumatoid arthritis.
Cancer can be both. Many signal pathways are involved with cancer;
these combinations make it extremely difficult to find a cure. The role
of the immune system makes it even more complicated. Modeling
aspects of the human body such as the immune system requires more
advanced physiology-based modeling and AI.
Drug Metabolism
Alexa reminded you this morning to take a 10 mg atorvastatin
tablet for your slightly elevated blood cholesterol level. What
happened to the tablet in your body after you swallowed it is the
science of drug metabolism, or more accurately, drug metabolism
and pharmacokinetics (DMPK). DMPK studies the absorption,
distribution, metabolism and excretion (ADME) of a drug. The birth of
drug metabolism as a distinct branch of science began in the 1960s,
particularly after the discovery of the role of cytochrome P450s in
metabolism. Atorvastatin is metabolized by a specific P450 called
CYP3A4 into several more polar metabolites, and they leave the body
mainly by biliary excretion with a tiny portion in urine. While the drug
is available in other dosage levels such as 20, 40 or 80 mg tablets,

you may only need to take 10 mg once daily,
as Alexa reminds you every morning. All
these details are related to drug metabolism,
which determines the efficacy and safety
of atorvastatin.
Alexa also reminded you this morning not
to drink grapefruit juice with atorvastatin.
This is a part of drug metabolism as well,
or drug-drug interactions (DDI). A special
ingredient in grapefruit called bergamottin
inactivates CYP3A4 via formation of
covalently bound electrophilic metabolites.
Thus, the net effect of eating two grapefruits
with a 10 mg tablet may actually be like
taking a 20 mg tablet, a possible overdose.
A crazier example of overdosing by drug-
drug interaction is combining cocaine and
alcohol. The combination is popular among
drug users for more intense highs, which
also causes increased incidence of sudden
death. When cocaine and alcohol coexist
in the blood, drug-metabolizing enzymes
catalyze the formation of a toxic metabolite
called cocaethylene.
Drug metabolism is a tiny but exciting area
in the pharmaceutical industry. For the
last 10 years, drug metabolism assays and
animal studies have mainly been outsourced.
Actually, small companies are mostly virtual
in DMPK. DMPK scientists that remain
these days in pharma and biotech mainly
monitor outsourced studies and integrate
data for project teams. Data integration,
Figure 1. Multiple-dose pharmacokinetic simulation using single-dose profiles
which includes data analysis, modeling and
prediction, is a key process to aide in decision
making. Creativity is required more than ever
these days for drug metabolism. Modeling,
simulation, high-throughput data analysis
and “smarter” data integration are key to keep
drug metabolism as a unique core function in
today’s drug discovery.
Modeling in Drug
Metabolism
Thousands of years ago, people already
knew how to use models to improve ev-
eryday life. For example, Egyptian sundi-
als, Hindu-Arabic numerals, Babylonian
sexagesimal numerals, and Chinese abacus
are prototype modeling systems. People
use models to explain phenomena and/or
make predictions for communication and
decision-making. This applies to modeling in
drug metabolism as well. Several categories
of modeling in drug metabolism are ADME
data modeling, pharmacokinetic (PK) mod-
eling, data mining, structure-based model-
ing, and physiologically-based pharmacoki-
netic (PB/PK) modeling.
Data modeling. The simplest modeling in
drug metabolism is to model enzyme kinetics
with linear or nonlinear equations. Examples
are Michalis-Mention kinetics of P450-
catalyzed reactions and 4-parameter logistic
www.americanpharmaceuticalreview.com
« FORMULATION AND DEVELOPMENT »
modeling of P450 inhibition/induction. Major
CROs and pharmaceutical companies auto-
mate and customize computer applications
to handle routine data analysis and model-
ing on a large scale. With the development
of computer algorithms, thousands of data
analysis procedures can be accomplished in
seconds, with high-quality tables and figures
generated instantly. A SAS-based applica-
tion called AIR Binder (ADME Instant Report
Binder) was recently developed that automat-
ically performs data analysis/modeling, dy-
namic visualization and reporting of preclini-
cal and clinical ADME assay data.1,2 Based on
our experience, productivity was significantly
enhanced with the use of AIR Binder, which
is attributed to its use of object-oriented pro-
gramming concepts to organize SAS macros,
together with SAS’ powerful statistical analy-
sis capacity and ODS graphics.
PK modeling. Pharmacokinetic modeling
with non-compartment analysis (NCA), one-
compartment, or multiple compartments is
routinely used in drug metabolism to explain
time-course drug concentration data for both
small molecules and biologics. These models
are derived from exponential functions and
differential equations. Multiple-dose model-
ing simulated by using single-dose data can
help the design of efficacy and safety studies,
which is a “smarter” quantitative approach
for drug metabolism. On the platform of AIR
Binder, we built a PK simulator called ψ (PSI,
Pharmacokinetic Simulation Interface), which
| | 55
» FORMULATION AND DEVELOPMENT »

models and simulates the user-defined dose and dosing schedules Structure-based modeling. X-Ray crystal structures of the drug
instantly (Figure 1). PK/PD modeling has been widely implemented target proteins have been widely used for drug design and discovery,
to correlate the exposure of a drug with its efficacy/safety - the phar- which is a structure-based rational drug design approach. X-ray
macodynamic (PD) properties of a drug. In addition, the starting dose crystallography is a century old. The father and son team Sir William
for first-in-human clinical trials have been modeled with the NOAEL- Henry Bragg and William Lawrence Bragg won the Nobel Prize in
based approach (no observed adverse effect levels), or more recently, physics in 1915 for their work in X-ray crystal structures. Rosalind
the MABEL-based approach (minimal anticipated biological effect Franklin’s X-ray diffraction image (so-called “Photo 51”) was crucial data
level), which is another example of PK/PD modeling. that led to the development of a double helix DNA model by James
Watson and Francis Crick in 1953 (and their Nobel Prize in physiology/
Data mining. When you stop your car at an empty traffic light after
medicine in 1962). Advancements have been made over the last 20
midnight, have you wondered why it takes only a couple seconds
years to crystalize drug-metabolizing enzymes. Currently, all major
to turn green in big cities like Seattle but may take two minutes in
human P450 crystal structures have been determined at a very good
a small Wisconsin town? It is likely attributed to IoT, or internet of
resolution, often co-crystalized with probe substrates or inhibitors.
things. IoT is a system of chips or devices connected to the Internet
These crystal structures, along with structure-based modeling such
that can send and receive data without requiring human interaction.
as molecule docking, molecular dynamics, and the hybrid quantum
An adaptive IoT transportation management system (installed in
mechanics/molecular mechanics (QM/MM) modeling, have been
most big cities) enables traffic lights to adapt to changing road
used in metabolism-derived drug design to solve metabolism-related
and weather conditions. The data processing performed with IoT
problems such as regioselectivity, stereoselectivity, reactive metabolite
is the simplest type of data mining. High-throughput screening has formation, metabolic switch, mechanism-based inactivation, and
given large pharma a gigantic database from which they can mine drug-drug interactions.6-12
useful information for metabolism-derived drug design and lead
optimization. Large datasets from metabolic stability, passive/active Most non-P450 drug metabolizing enzymes have also been crystalized
and modeled for drug design and lead optimization. Aldehyde oxidase
permeability, and P450 inhibition assays have been routinely mined to
(AO) catalyzes the metabolism of various azaheterocycle-containing
build QSAR (Quantitative Structure-Activity Relationship) models. For
drug molecules. A series of structural analogs of zoniporide were
example, from the database of P450 inhibition by pyridine-containing
modeled for metabolic clearance prediction by altering their molecular
compounds, QSAR models have been built from the 2-, 3-, or 4-pyridyl
interactions with the active site residues of AO.13 Carboxylesterase is
inhibition patterns on CYP1A2, 2C9, 2D6, and 3A4 enzymes.3
the enzyme responsible for the metabolism of cocaine via a hydrolysis
Instead of mining quantitative data, another application of data reaction. Carboxylesterase-catalyzed reactions were modeled to
mining is on patterned qualitative data, such as those from mass design efficient ester prodrugs.14 Several major classes of glutathione
spectrometry. Mass spectrometer has a long history. In the Manhattan S-transferases (GSTs) were also crystalized; such enzymes catalyze
Project, it was used to separate isotopes of uranium. In 1976, NASA sent glutathione conjugation of reactive metabolites or covalent-binding
mass spectrometers to Mars to analyze Martian atmosphere and soil. drugs such as acalabrutinib, a novel BTK inhibitor for the treatment
Since John Fenn and Koichi Tanaka’s development of soft desorption of mantle cell lymphoma.15 To facilitate the application of various
ionization methods in 1980s (Nobel Prize in chemistry in 2002), mass third-party modeling tools, we developed a Python-based application,
spectrometry has gradually become the core of drug metabolism MARS (Molecular Modeling and Rendering Solver), to automate
research for bioanalysis and metabolite identification. Metabolite the structure-based modeling process for improved efficiency,
identification and characterization using mass spectrometry is a interpretation and visualization of modeling results (Figure 2).
process of pattern recognition. Molecular ions are selected by the
PBPK modeling. Thirty or forty years ago, and perhaps still today in
mass spectrometer, with high-energy collision breaking the molecule
some countries/places, the term “pediatric drug” did not exist. If a
into fragments. The resulting fragmentation patterns are fingerprints
for structural elucidation, which traditionally need highly trained
personnel to manually explore. The patterns of small molecules are
relatively simple, but today’s drug discovery has diverse chemical
moieties such as peptides, oligonucleotides, and antibody-drug
conjugates, which make metabolite characterization more complicated
than ever. Recently, we put together a Python-based application, LEO
(Linearization, Extension and Oxidation), to generate solutions for
relatively challenging molecules.4,5 The algorithm was built to search
theoretical metabolic pathways: cleavage reactions (“linearization”),
conjugation reactions (“extension”), and oxidation-reduction reactions
(“oxidation”). Due to the difficulties of manually determining all Figure 2. Atorvastatin molecule was modeled to show
possible combinations of these reactions, an advantage of computer interactions with CYP3A4 active site residues for the formation
modeling is that it improves efficiency and shortens the turnaround of major active aromatic hydroxylation metabolites
time of metabolite identification.

56 | | January/February 2019

5-year old is getting sick, the parent may just give him/her a half or
a third portion of the adult dosage. Although it is still challenging
to get clinical trials for certain pediatric population today, PBPK
modeling helps predict the right dose level and schedule based on
general population PK and the unique physiological parameters of a
child. PBPK modeling applies physiologically realistic compartmental
structure by combining sets of differential equations to simulate
pharmacokinetic profiles in a special population and/or situation, such
as pediatrics, drug-drug interactions, hepatic/renal impairment, food/
juice effect (such as grapefruit juice), and pharmacogenetics.
In addition to PK parameters (such as dose, route and frequency
of administration) and physicochemical properties of a drug
molecule (related to permeability, partitioning to tissues, plasma
protein binding, etc.), physiological parameters such as age, weight,
height, and genetic features are also integrated in PBPK models.
These physiological parameters can be very difficult to collect and
complicated to generalize for a model, which is why intelligent
algorithms are required. For the last few years, regulatory agencies
such as the FDA and EMA have encouraged drug companies to
simulate untested scenarios based on key clinical data. For example,
when evaluating the DDI potential of a drug candidate as a “victim”
(substrate of an enzyme/transporter), a strong inhibitor will normally
be selected in clinical trials. Weak inhibitor scenarios can be simulated
with PBPK models that have been refined with derived strong inhibitor
clinical data, which is a useful process to address information gaps,
enhance benefit/risk assessment and properly inform prescription
drug labeling.
Future Modeling in Drug Metabolism
There are no perfect models. We are using models to improve
communication, help decision making, and ultimately to achieve
artificial intelligence, hopefully. For modeling in drug metabolism,
sometimes we have too many assumptions and parameters for a
model but not enough data collected, which makes the model less
robust or “over-fit”. Many factors need to be considered for model
optimization, such as covariance of parameters for statistic models,
appropriate descriptors to choose, or time-scale dynamic change of
protein 3-D structures. The human body is a dynamic system that can
adapt to many changing things, good or bad. The lack of adequate
and reliable system data, which can be attributed in part to changing
physiological parameters with age and disease status, presents major
challenges for PBPK modeling. IoT may help in the future. For example,
an ingestible pill-sized sensor may be used, which gets activated upon
swallowing and starts gathering data from patients. Another factor
that presents challenges for modeling is the placebo effect. Love,
hope, care, and sprit all can change conditions of the human body;
sometimes, the placebo effect is huge.
With the start of an era of AI, big data mining, and deep learning,
the gap to realistic drug metabolism models is narrowing. “Smarter”
modeling in drug metabolism and discovery is on the near horizon.
Every industry is working hard now on modeling and developing
artificial intelligence. Self-driving cars are in final testing for
« FORMULATION AND DEVELOPMENT »
production. Ford’s Argo and GM’s Cruise are making progress. People
are even talking about Google’s flying cars. SpaceX has identified its
first space tourist, and Blue Origin expects to begin selling tickets for
space travel this year. Elon Musk says he is on track to launch people to
Mars within six years. This is an era of great technology advancement.
In the not too distant future, smart modeling and artificial intelligence
will be fully integrated into drug discovery and development, and also
into our tiny but exciting drug metabolism field.
References
1. Sun H, Cardinal K, Voorman R. AIR Binder 2.0: a dynamic visualization, data analysis and
reporting SAS application for preclinical and clinical ADME assays, pharmacokinetics,
metabolite profiling and identification. MWSUG 2017: PH04; 1-26.
2. Sun H, Cardinal K, Kirby C, Voorman R. AIR Binder: an automatic reporting and data analysis
SAS application for cytochrome P450 inhibition assay to investigate DDI. PharmaSUG 2017:
PO09; 1-8.
3. Sun H, Scott DO. Using crystal structures of drug metabolizing enzymes in mechanism-
based modeling for drug design. In: Renaud J-P, ed. Structural Biology in Drug Discovery:
Wiley, 2019 (in press).
4. Cardinal K, Sun H. Metabolite identification and characterization by mining mass
spectrometry data with SAS and Python. PharmaSUG 2018: AD34; 1-5.
5. Sun H, Cardinal K. Mining metabolites of peptides and antibody-drug conjugates from
mass spectrometry data using SAS and Python. SASGF 2018: 2510; 1-6.
6. Sun H, Bessire AJ, Vaz A. Dirlotapide as a model substrate to refine structure-based
drug design strategies on CYP3A4-catalyzed metabolism. Bioorg Med Chem Lett
2012;22:371-376.
7. Sharma R, Sun H, Piotrowski DW, et al. Metabolism, excretion, and pharmacokinetics of
((3,3-difluoropyrrolidin-1-yl)((2S,4S)-4-(4-(pyrimidin-2-yl)piperazin-1-yl)pyrrol idin-2-
yl)methanone, a dipeptidyl peptidase inhibitor, in rat, dog and human. Drug Metab Dispos
2012;40:2143-2161.
8. Miao Z, Sun H, Liras J, Prakash C. Excretion, metabolism, and pharmacokinetics of
1-(8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl)-4-(ethylamino)piperidine-
4-carboxamide, a selective cannabinoid receptor antagonist, in healthy male volunteers.
Drug Metab Dispos 2012;40:568-578.
9. Sun H, Scott DO. Metabolism of 4-aminopiperidine drugs by cytochrome P450s: molecular
and quantum mechanical insights into drug design. ACS Med Chem Lett 2011;2:638-643.
10. Sun H, Scott DO. Structure-based drug metabolism predictions for drug design. Chem Biol
Drug Des 2010;75:3-17.
11. Sun H, Sharma R, Bauman J, et al. Differences in CYP3A4 catalyzed bioactivation of
5-aminooxindole and 5-aminobenzsultam scaffolds in proline-rich tyrosine kinase 2
(PYK2) inhibitors: retrospective analysis by CYP3A4 molecular docking, quantum chemical
calculations and glutathione adduct detection using linear ion trap/orbitrap mass
spectrometry. Bioorg Med Chem Lett 2009;19:3177-3182.
12. Sun H, Piotrowski DW, Orr STM, et al. Deuterium isotope effects in drug pharmacokinetics
II: Substrate-dependence of the reaction mechanism influences outcome for cytochrome
P450 cleared drugs. PLoS One 2018;13:e0206279.
13. Dalvie D, Sun H, Xiang C, Hu Q, Jiang Y, Kang P. Effect of structural variation on aldehyde
oxidase-catalyzed oxidation of zoniporide. Drug Metab Dispos 2012;40:1575-1587.
14. Sun H. Capture hydrolysis signals in the microsomal stability assay: molecular mechanisms
of the alkyl ester drug and prodrug metabolism. Bioorg Med Chem Lett 2012;22:989-995.
15. Podoll T, Pearson PG, Evarts J, et al. Bioavailability, biotransformation, and excretion of the
covalent BTK inhibitor acalabrutinib in rats, dogs, and humans. Drug Metab Dispos 2019;
47:155-163
www.americanpharmaceuticalreview.com | | 57
An Interview With...
Crystal Mersh
President and CEO
Quality Executive Partners, Inc.
In general, what are some current issues facing
pharmaceutical companies in regards to
pharmaceutical technician/employee training?
Having led global quality organizations for many years, I had the
opportunity to see first-hand the many challenges of ensuring that
employees had the appropriate level of knowledge; and that their
knowledge then translated to skills, motivation and commitment to
do the right thing when faced with inevitable issues in pharmaceutical
manufacturing and analysis. Unfortunately, over time, our industry
has regressed from educating our employees on the technical
and scientific implications of why we work in the manner we do,
to very specific and limited transactional training associated with
execution. A highly skilled employee requires both and deserves
both: education and training. When people understand “the why”
behind the requirements of a process, behavioral psychology data
shows us that they are increasingly committed and motivated with a
sense of responsibility in performing their functions correctly. In our
industry, skill and motivation are both critical to a high performing
organization and to the corporate bottom line.
Another challenge in today’s highly competitive market is to deliver
efficient and impactful learning models that will provide the rapidly
changing skillsets demanded by advances in emerging therapies and
manufacturing technologies. At the same time, existing demands for
standardization across global landscapes continue to pressure existing
practices. Our industry’s outdated approach of focusing on transactional,
procedure-based training only expands the need for investment in
resources as unfortunately the industry has not kept pace with the ever-
increasing need and demand.
I would also say that we have all too often simply accepted the costs
associated with deviations, recalls, batch rejections and other poor quality
events, as simply a cost of doing business and that there is little that can
be done on a human-factor level to move that needle. Other industries
do not accept this predisposed position and neither should pharma.
Continuous improvements, lead by a highly educated workforce, can
and will reduce these costs, benefiting the company’s bottom line as well
as benefiting the patient with a more consistent flow of safe medications.
Continuous improvement only begins with continuous learning.
How can technology in general help pharmaceutical
companies better train their employees?
Our world has significantly changed due to technology; we can see
this everywhere in our personal lives. Frankly it is time for us to catch
up in the life sciences sector and benefit from these advancements.
58 | | January/February 2019
»
When Quality Executive Partners (QxP) began to look at pursuing the
development of an educational platform with broad impact, we quickly
recognized that technology was central to achieving this goal. People-
based educational solutions have lost the critical mass necessary to
meet these needs at the demanded scale. Industry internal subject
matter experts are already overloaded with the tasks of bringing new
products and processes to market and resolving the issues of the day to
maintain daily operations of their respective organizations. Computer-
based learning can be effective and certainly plays a role, but again
this often takes the highly valuable SME away from other critical tasks
as a significant amount of time is needed to develop and maintain the
content internally.
So, as we look across the industry today and talk with key leaders, we
consistently hear that the industry is too reliant on, and has a false
security in, the “read and understood” learning model. This approach is
utilized far too often and rarely delivers consistent and comprehensive
understanding, much less positively affecting skills and behaviors.
The key in advancing learning, which has a positive impact on
patients and a profitable impact on the business, lies in providing
on-demand high-quality technical material, which is engaging and
fun, immediately relevant, and scalable across the entire corporate
enterprise. This requires technology and expertise in both content
and the technologies deployed.
Virtual Reality (VR) has been making in-roads
into many industries. How is it applied to the
pharmaceutical industry? Are there specific
challenges to making VR work and be applicable
to the industry?
Virtual Reality is a term that is getting a lot of play in the life sciences
sector, and there is a very broad understanding, and often times a
misunderstanding, of the various types of VR and their intended
application. We regularly see that companies are interested in
innovation projects associated with VR, almost seemingly to be able
to say that they employ VR technology without a clear understanding
of how the technology can, and should, be applied to improve their
businesses. The two most often utilized technologies in pharma are the
“see what I see” applications, which is really not VR, and VR simulators,
which are not interactive or immersive. Both could have application, but
are very limited when compared with fully interactive and immersive
Virtual Reality.
The two biggest challenges to making VR work for an organization,
and moreover ensuring that it has a positive impact, lies in the level
and application of the technology utilized, and the capability and
pharmaceutical technical knowledge of the development team.
Interactive and immersive VR is the highest level of VR capability and
offers the greatest benefit in building skills and retention.
The most critical factor in VR development, as in most revolutionary
innovations, is the development team. Our Virtuosi team of pharma
technical experts and our VR developers are first and foremost
engineers and scientists who understand the critical learning
objectives of pharmaceutical processes, as well as what mistakes are
commonly made in manufacturing and laboratory environments. Our
highly skilled VR engineers build upon this foundation to develop
realistic graphics, interactions, and feedback loops that allow learners
to practice their skills in a fully interactive environment that provides
real-time coaching and consistent performance feedback. This
combination of skills and industry expertise has allowed us to develop
a truly unique educational product that is the only comprehensive
product of its kind available anywhere in the world.
Can you describe the Virtuosi platform and its
features and benefits? How is the Virtuosi approach
to pharmaceutical learning/training different than
other training systems?
Virtuosi is a fully integrated educational platform leveraging the
power of interactive, immersive VR. Our first educational series focuses
on Sterile Product Manufacturing and Microbiology. It has been
thoughtfully designed with clear learning objectives based upon our
expansive view of the topics and understanding of the critical learning
parameters necessary to develop deep understanding, practical skill
and confidence in performance.
This series consists of over 80 hours of education and skill
development broken down into 31 educational modules covering all
aspects of sterile product manufacturing and microbiology laboratory
operations. Modules are organized into comprehensive curricula,
which are customizable to the needs of the learner and progressive
in technical difficulty to build knowledge and skill over time. Within
these modules are 16 interactive, immersive VR experiences where the
learner can apply their knowledge, experience success, and build skills
to defined proficiency levels.
As an on-demand solution, learners progress at their own pace with
the ability to repeat topics and experiences or to use as a reference for
situations such as deviation investigations.
The most unique aspect of the Virtuosi experience is that it provides
a safe space where mistakes can be made, removing potential for
embarrassment and fear of being wrong, as well as individual and
private real-time virtual coaching, which is unique to their actions
within the virtual environment. Experiencing failure engages learning,
which further embeds comprehensive understanding of “the why”.
Studies have shown that interactive, immersive VR substantially
improves learning and knowledge recall as these interactions and
experiences leverage the brain’s pre-wired capability to encode
and store spatial and visual inputs more efficiently. This is a key
differentiator from any other learning system, approach or application
available today.
Specifically, can you detail the benefits a
pharmaceutical company might realize by
employing this platform?
Virtuosi is a business solution. Driving top and bottom line growth is
the objective of the platform and Virtuosi was designed specifically for
these purposes.
Specific benefits of employing Virtuosi are vast based upon the
objective of the company and deployment methodology. These
opportunities include:
• Reduction of human error leading to batch rejections
and deviations; reducing direct costs for lost time
and materials, indirect costs in the labor necessary to
investigate and correct them, as well as equipment
down time and lost production opportunity;
• Support of organizational scale-up and growth where
time to competency is critical;
• Promotes education as desirable, fun and important;
• Creates a personalized on-demand learning
environment that allows the learner to take control of
his/her own growth and development;
• Limits and protects the operational environment
allowing for reduced equipment usage for training and
risks of environmental contamination; and
• Standardizes expectations across an enterprise and
allows for self-inspection of operational practices aiding
in continuous improvement.
Looking ahead, how will QxP continue to offer its
current and future customers the best available
platforms and training materials?
It has taken an extraordinary effort of 25 very talented and driven
professionals over two years to deliver Virtuosi, and we are committed
to maintaining the content and technology as these evolve. Quality
Executive Partners participates in many forums as thought leaders
and influencers on technical and regulatory content, as well as VR
technologies. As regulations and industry practices evolve, our product
managers will constantly review our content and technology leading to
updates so that our clients have the latest education in procedures and
techniques. We are deeply entrenched in the VR technology community
and are constantly evaluating new components and technologies
prior to their release into the mainstream. Additionally, we will host
User Communities with different areas of focus to gain insights and
recommendations for content changes and continuous improvement.
Further series are under development, including biotechnology, solid
dose manufacturing and chemistry laboratory operations.
www.americanpharmaceuticalreview.com | | 59
FORMULATION AND DEVELOPMENT
Liquid-Fill Capsules – Benefits for
Highly Potent API Formulation and
Scale-Up
Alyn McNaughton
Lonza Pharma & Biotech
Alyn McNaughton is technical director
at Lonza Pharma & Biotech. He is based in
Edinburgh, United Kingdom.
It is currently estimated that over 25% of all drug products in development have highly potent
or highly toxic active pharmaceutical ingredients (API) and require some form of specialized
handling. In oncology this figure is likely closer to 70%.1 These types of products represent
a growing sector of investment in the pharmaceutical industry, with the market value from
existing and new product launches expected to double between 2018 and 2025.2
New therapeutic treatments are frequently targeted to act locally at the site of therapy,
which can reduce side effects and result in significantly lower dosages required. While these
treatments offer huge benefits to the patient, the pharmaceutical industry faces the challenge
of developing these API into safe and effective low-dose products – including consistently
producing very low-dose products, e.g., microgram doses – while maintaining safety for
operators during processing. Encapsulated liquid-based products offer a proven approach to
overcome many of the challenges associated with the development, scale-up and commercial
manufacturing for these highly potent API (HPAPI), from safer handling to producing more
accurately dosed and homogeneous formulations for low-dose products.3
Liquid-Filled Capsules
Liquid-fill technology is not new, and it benefits from a long history of continued innovation
and market precedence. The original softgel patent dates from 1834, with the current process
evolving from that created by RP Scherer in the 1930s. Hard-shell liquid-fill capsules were a
further innovation from Cuine et al of the University of Strasbourg, producing several papers on
the potential advantages of this technology in the 1970s. In the early 1980s, several additional
publications from other authors presented the potential advantages for content uniformity
and reduction of airborne contamination.4
While commercially available products in the consumer health and nutrition sector were already
utilizing softgel technology in the 1980s, key milestones for the technology in both hard and
soft capsules in the pharmaceutical segment happened later that decade with the commercial
approval and commercialisation of Vancocin® capsules. This product demonstrated the potential
to produce an oral dosage form with a sensitive molecule that had been challenging for more
traditional product approaches. Sandimmune’s® position as the first commercially successful
lipid-based bioavailability-enhanced formulation further demonstrated the potential benefits
of liquid-based technology (the product, containing the active ingredient cyclosporine, also
represented a breakthrough in transplant management). A number of new products have
since been launched using lipid-based formulations with softgel or liquid-filled hard capsule
technology to address the continuing challenge of poor solubility.
Although liquid-fill capsule products continue to be seen principally as a mechanism to increase
bioavailability for poorly soluble drugs;5 they are also increasingly evaluated to address low-
dose HPAPI challenges.4 The technology allows the incorporation of an API powder into a liquid
60
American Pharmaceutical Review | January/February 2019

formulation as either a solution or as a suspended solid, which then
removes the risk of subsequent airborne powder during manufacture
and can also improve homogeneity for low-dose products. Market
precedence is firmly established, as is the technology required for
developing, scaling and manufacturing liquid-based capsule products
with challenging, potent or toxic API. A few examples of marketed
liquid-filled products with these challenging compounds in both hard
capsule and softgel formats include hormones or related compounds,
like promestriene, progesterone or dutasteride, vitamin A analogues
such as isotretinoin and Vitamin D analogues like colecalciferol and
ergocalciferol. Currently, it is estimated that 40% of liquid-filled hard
capsule products in development utilize HPAPI.6
Benefits of Liquid-Fill Capsules
Liquid-fill capsules can provide benefits in terms of quickly scaling
up a product to commercial manufacturing volumes, as the four-step
process of liquid filling – dispense, mix, fill and seal – can be scaled
up more rapidly than other types of formulation processes, primarily
due to most of the process being independent of scale. While liquid
filling is not without its challenges during scale-up, it provides an
option for companies looking to generate high-potency oral products
with a relatively straightforward process of reaching commercial scale.
As such, the technology is utilized as a rapid product development
and screening tool for promising compounds with poor solubility,
specialized handling requirements and other formulation challenges.
Liquid-fill capsules offer a number of specific benefits for manufacturers
developing and scaling up low-dose HPAPI formulations.7 First, liquid
filling can provide perfect homogeneity for low-dose products,
through the generation of a solution. Where occasionally this is not
possible, enhanced homogeneity can still often be achieved, as the
high shear mixers utilized are efficient at rapidly de-agglomerating and
dispersing powder into a homogenous dispersion. Further, particle size
reduction can be achieved as part of an in situ process, integrating a
bead mill recirculating through the mixing vessel.
The liquid-fill process can also offer safer and more efficient handling –
by incorporating the powder API into the liquid excipients, the risk from
exposure of powder handling for further manipulation and processing
is removed. This approach ensures that operators are under no risk of
airborne powder exposure through the majority of the manufacturing
process. The liquids can also be readily pumped between processes,
ensuring efficient handling is retained.
A third benefit is that liquid-fill capsule products do not require
reformulation due to changes in the production scale, as the
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American Pharmaceutical Review | January/February 2019
FORMULATION AND DEVELOPMENT
mechanisms employed during manufacture of development batches
are almost identical to those at full scale. Parallel filling heads and
automated capsule manipulation provide the increased speed
required to produce commercial volumes without the need for product
or process change.
Finally, the four-step process for liquid filling contains a minimal
number of processing steps to support rapid development and
scale-up compared to equivalent solid oral technologies, such as an
eight-step wet granulation process: dispense, dry blend, wet mass,
sieve, dry, screen, granulate, compress. Streamlined processing is
increasingly critical for meeting the industry’s need for simplified
compound screening, rapid first-in-human studies and accelerated
timelines to market.
The Four-Step Hard Capsule Liquid-Filling Process
Step One: Dispense
The first step of developing and commercializing a liquid-fill product
with a highly potent API formulation is to isolate and dispense the
active ingredient in appropriate amounts. Handling highly potent or
toxic powder is a challenge at any scale, and in development there
are many ways to contain small quantities of API while they are being
evaluated. As the arriving container starts to get larger during scale-up,
control may become less standard and it often becomes necessary to
ensure that adaptable containment can be used. During development
this may be as simple as having off-the-shelf isolators that the small
containers and equipment can be moved into for the operations to
take place.
However, during scale-up, the weight of materials for manual handling
and physical constraints of entry for larger containers prevent usage
of this type of isolator. Unless an appropriate isolator is available that
already matches the containers and scale, it is not cost-effective to
have a fixed isolator for irregular manufacturing. Two ways to approach
this challenge include:
1. The design and implementation of a permanent hard isolator
that can cope with many different container sizes and types;
2. The use of flexible isolators, allowing a huge range of options to
deal with containers and processes, which are not routine, at a
fraction of the cost of building or modifying a hard isolator.
Both approaches can be qualified to similar levels for short-term
occupational exposure, but the hard isolator must be verifiably cleaned
before re-use. In contrast, the flexible isolator is disposed of at the end
of use, with its relatively short lifespan offsetting the need to both
clean and verify cleanliness.
Step Two: Mix
The next stage of the process requires transferring the dispensed
material into the mixing vessel and ensuring it is homogeneously
mixed. This mixing depends on whether the API is solubilized in the
 FORMULATION AND DEVELOPMENT
excipients or is suspended in them, and during scale-up the ability to
transfer the powder into the mixer becomes a significant control factor
alongside the creation and maintenance of homogeneity.
When a solution is generated it is critical to understand if time and
energy are required to dissolve the API. For a suspension formulation,
homogeneous dispersion and stability of the dispersion are important
to characterize. During small-scale manufacturing and development,
this process can be fully contained within an isolator, at least for
the first stage of wetting down any powder materials with liquid
excipients. Often this is conducted as part of the same process as
dispensing. Subsequent dilution for these small-scale processes can
then be conducted in a less contained environment, as the initial liquid
addition and mixing has removed the risk of dust generation. Visual
assessment of solubilization and dispersion usually provides the first
check of homogeneity, followed by analytical assessment over a period
of time to check how quickly homogeneity is achieved and how readily
it is maintained.
During scale-up to commercialization, the liquid-fill formulation
provides several advantages for powder transference. Generally, the
process starts with some, or all, of the liquid excipient in the mixing
vessel. All liquids are degassed under vacuum to prevent bubbles
from generating during the filling process. This vacuum over the
liquid presents a means of transferring the powder from a dispensing
isolator, which has a valve pipe connected directly to the underside
of the mixer so it can be directly sucked into the underside of the
liquid while mixing. This prevents any form of “blooming,” where the
API would be dispersed over the surface of the vessel; instead the API
is entrapped within the liquid and quickly wet. On the rare occasion
when the powder properties preclude this type of transfer there
are alternatives that can be used, such as direct powder pumps or
pre-wetting the powder with liquid excipient and transferring the
concentrated solution or slurry.
Once compounding in the mixer, several variables can affect the
process, including homogeneity of solution/suspension, particle size
of a suspended powder, viscosity of the mixture, moisture content and
thermal degradation rate of the components. Liquid-fill products often
utilize jacketed vessels as mixers, with heating and cooling, which are
appropriate for vacuum degassing and contain both high shear mixer
heads and paddle type agitator stirrers. If oxidation is a risk, nitrogen
can be introduced into the mixing vessel at the end of the vacuum
application, rather than air, to bring it back to ambient pressure.
Temperature can play a role. Elevated temperatures may be used
during mixing to aid in the dissolution of the API if all the components
are sufficiently thermally stable, but temperatures beyond 70°C may
damage the capsules. The temperature may also be altered to control
viscosity of the mixture: reduced viscosity to aid mixing and filling or
increased viscosity to maintain homogeneity. Due to the broad range
of rheological properties the mixers can handle, tight control limits are
not usually required. The homogenizer (high shear mixer) is used to
put energy into the system to aid dissolution or to disperse powder
to a homogeneous suspension. It quickly creates heat in the system
but also provides rapid dispersion and de-agglomeration without
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American Pharmaceutical Review | January/February 2019
significant particle size reduction taking place. The homogenizer speed
and continuous, or intermittent, time of application can also affect the
outcome. The agitator is generally employed after the homogenizer is
complete to maintain an even temperature throughout the bulk liquid
and sometimes to assist with maintaining homogeneity. While these
controls are required the window of control is usually not too tight.
Step Three: Filling
Once compounded, the mixture is pumped into the filling machine.
At this stage, the high-potency risks from airborne powder have
been completely removed and the operation can be conducted in
a controlled but less contained environment. Production risks do
remain: Fill accuracy and uniformity, prevention of external capsule
contamination and minimizing pressurization are the key attributes to
assess while filling the compounded mixture into hard capsules.
The accuracy of fill is achieved relatively easily, as precision pumps
provide an accurate dosing capability over a broad range of viscosities,
but it does entail two key challenges:
1. The first challenge is to ensure the mixture is cleanly filled into
the shells at as fast a rate as possible without it “squirting” out
the other side, dripping, splashing or “tailing,” where the fill
stream is not broken between capsules. This is achieved through
a balance of nozzle selection, pump timing and drawback
settings, designed to break the fill stream and set filling speed
and temperature/viscosity control. Cleanly filling into capsules
is critical to ensure that the external capsule does not become
contaminated. Contamination on the outside of capsules could
prevent sealing, and high-potency material on the outside of
the shell presents a contamination risk to healthcare workers,
patients and their families, in addition to a cosmetic impact.
2. The second risk is pressurization of the two-piece capsule shells
as the product is scaled up. Both the cap and body components
of the capsule contain air, which is compressed as the capsule is
closed at high speed. Impacts on the pressurization within the
capsule at this stage include:
• the design of the capsule shell, to vent this air;
• the temperature of the fill mixture, which may further heat this
air; and
• the quantity of fill material in the capsule body, which affects the
distribution of the pressure.
In a worst-case scenario due to over-pressurization, the capsule can
self-open prior to being sealed, or distort during the sealing process.
Even some time later, an over-pressurized capsule could split as it
re-equilibrates from the physical and chemical stresses applied to it
during manufacture. Appropriate expertise and attention are required
to ensure pressure is minimized to the point no longer presenting a
risk to the product, and the result matches the sealing technique used.
Scale-up of the filling process is straightforward, contingent upon
the potential risks having been worked out of the product during
development. Small-scale filling equipment works on exactly the same
mechanisms as larger scale equipment, with multiple pump heads

and automated capsule handling providing most of the speed gain.
Some refinement, with additional controls to adapt to the impacts
of high speed filling, is available on commercial-scale equipment.
Unfortunately, many companies rely on inexperienced vendors who
do not have familiarity of commercial processing to conduct their early
work, and this is the stage of the process where those design errors
can have the biggest impact. As a result, some scale-up operations
of products transferred from an organization inexperienced with all
scales of manufacture may require the correction of prior mistakes
before the scale-up can take place.
Step Four: Sealing
Sealing of the capsules takes place when the formulation is already
enclosed within the shells and presents little risk from potent material.
Sealing prevents leakage from liquid-fill products, or thermo-softening
materials that may be exposed to sufficient temperature to melt the
components. In the case of banding, this seal also provides tamper
evidence for individual capsules, which is increasingly required for
certain compound classes. The main goals in the sealing process are
to ensure the capsules have no leaks, bubbles or cosmetic problems.
When the previous scale-up filling development has been appropriately
completed, ensuring the internal pressure is appropriate and the
external capsule is not easily contaminated, then the sealing process
is generally straightforward. Fusion sealing and banding are the two
standard approaches to sealing capsules. Fusion sealing applies the
seal between the body and cap using a micro-spray and banding
applies a band of seal solution over the cap and body joint. The choice
between the two is generally not for technical reasons but will impact
the selection of capsule type being used.
For banding activities, the process of scaling up to commercialization
rarely requires product-specific setup other than the shell selection
already mentioned. To ensure robust sealing, the process must optimize
several variables at commercial scale: banding solution composition
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American Pharmaceutical Review | January/February 2019
FORMULATION AND DEVELOPMENT
and viscosity, banding speed, disc type and speed, disc height, band
solution quantity applied, drying temperature and airflow.
Conclusion
In summary, liquid-fill capsule technology has long-established
market precedence and a range of specialized applications – including
providing a versatile solution for safe and effective handling of highly
potent and other complex API. Scale-up is straightforward, contingent
upon development that has been conducted appropriately, due to
the consistency of liquid handling mechanisms. Powder containment
measures are only required in the first few steps of the manufacturing
process. Specialized CDMO partners exist in the marketplace with the
prerequisite infrastructure, expertise and processing equipment in
place to support effective and agile liquid-filled product development
from feasibility studies through clinical trials and commercial
manufacture.
References
1. HPAPIs and Cytotoxic Drugs Manufacturing Market, Roots Analysis, August 2014,
http://www.rootsanalysis.com/reports/view_document/hpapis-and-cytotoxic-drugs-
manufacturing-market/64.html
2. Grand View Research, High Potency Active Pharmaceutical Ingredients (APIs) Market
Analysis By Product (Synthetic, Biotech), Manufacturer (In-house, Outsourced), Drug Type,
Therapeutic Application (Oncology, Hormonal, Glaucoma, Others), And Segment Forecasts,
2018 – 2025
3. S. Brown. “Why high potency drugs require a specialised approach.” European
Pharmaceutical Manufacturer
4. Geoff Rowley, “Filling of Liquids and Semi-solids into two piece hard capsules”
Pharmaceutical Capsules, Pharmaceutical Press, 2004, pp. 169-194
5. Feeney et al.,50 years of oral lipid-based formulations: provenance, progress and future
perspectives, Adv. Drug Deliv. Rev., 101 (2016), pp. 167-194
6. Lonza Pharma & Biotech, Internal Market Analysis, 2018.
7. Matt Richardson and Sven Stegemann, Filling two-piece hard gelatin capsules with liquids,
Tablets and Capsules, January 2007
An Interview With...

Danielle Clay
Director of Global Strategic Marketing and Business
Development for Injectable Drug Delivery, Evonik

In general, what are some current issues facing
pharmaceutical companies in regards to
formulating effective drug delivery systems?
A series of disciplines in the field of specialized parenterals including
personalized medicine, nucleic acid APIs and gene editing are
driving demand for advanced drug delivery technologies. These
technologies must not only be safe and efficacious, but simple to
customize and efficient to manufacture. Other common formulation
challenges include the effective penetration of target cells, and
ensuring extended release occurs reliably for either systematic or
local delivery over days, weeks or months. Technologies such as lipid
nanoparticles (LNPs), which can safely encapsulate the API to protect
it against degradation while enhancing biodistribution and solubility
characteristics, are well positioned to address such challenges.
Many pharmaceutical companies are seeking to partner with CDMOs
that have established core competencies and a proven record for
performance with specific drug delivery technologies such as LNPs.
These strategic partnerships, which can span formulation and
process development as well as manufacturing, must ensure the
target delivery system is compatible with both the API and excipient.
Specialized equipment, such as LIPEX® extruders, and manufacturing
facilities capable of both aseptic processing and flammable solvent
handling, can also be required to bring these products to market.
The agreement we signed last year with Precision Nanosystems to
develop and manufacture high-quality nanomedicines is an excellent
example of how it can also be beneficial to collaborate to address
other unmet market needs.
»
and lipophilic drugs. That is a key reason why they are now the de
facto standard to deliver nucleic acid-based vaccinations and other
therapies, where the payload must be protected until such time as
it can be delivered to the site to silence targeted genes or express
therapeutic proteins. They can also be designed to exhibit specific
physicochemical properties such as particle size, surface charge and
surface function to satisfy a variety of performance requirements.
Can you tell us how Evonik is advancing lipid
nanoparticle-based drug delivery systems?
Evonik continues to consolidate its position as a leader in the
development of lipid nanoparticles, as well as other related
technology areas including polymeric microparticles and implants
that are required to deliver specialized parenterals. At our Vancouver
site in Canada, our team has been supporting customers in the
development and clinical manufacturing of lipid nanoparticle-based
drug delivery systems since the 1990s. We provide the formulation
and process development support to select a lead candidate, quickly
advance the product into the clinic, and then support its scale-up
and commercial manufacturing at our established U.S. facilities. We
also provide LIPEX® extruders ranging from benchtop to commercial
production scale.
To further encourage market growth within this technology area, we
are making selective investments in nanomedicine research, and also

Why are lipid-based products emerging as a way

to better formulate specific products? What are
the advantages?
There has been a resurgence of interest in lipid-nanoparticle
technologies over the last decade, given their well-established
record of safety and proven ability to encapsulate APIs that would
otherwise degrade before reaching the target site. We expect that
this market segment will continue to grow as therapeutic targets
become more specific, and demand increases for highly potent and
gene-based drugs.
One of the main advantages of LNP-based technologies is that
they can reliably encapsulate high payloads for both hydrophilic

64 | | January/February 2019
developing new process technologies and biomaterials that will be
required for certain personalized, RNA or gene-based therapies. We
also continue to expand industry collaborations between Evonik and
many of the world’s foremost scientific experts within the market.
What types of products benefit most from
lipid-based drug delivery systems? Is Evonik
working to expand this list?
Lipid-based drug delivery systems have been traditionally used
for highly potent molecules such as anti-cancer agents, antibiotics
or antifungals and oligonucleotides that require intravenous
administration. While the market for nucleic acid-based vaccines
and therapies is yet to reach critical mass, there are hundreds of
personalized medicines in development that are expected to utilize
these technologies moving forward.
Evonik is working with various customers to help ensure their RNA
molecules do not degrade before they reach the target site so that
delivery effectiveness increases by an order of magnitude. Other
projects have helped to improve the solubility of lipophilic APIs, reduce
systemic toxicity, and enhance biodistribution as well as cellular and
tissue uptake. Demand for these specialized delivery technologies
is also being driven by demand from patient groups seeking more
effective therapies, and payers transitioning to performance-based
reimbursement strategies.
What is your process for evaluating or
recommending a product in development for a
lipid-based drug delivery system?
Typically, pharmaceutical and biotechnology customers come to
us with a defined target product profile that requires a complex
drug delivery solution. We help them to not only match the fit of
their molecule to the right lipid-based drug delivery system, but
also ensure the process is readily scalable into a commercially viable
manufacturing process. We can also help determine if a customer’s
drug might be better suited to other formulation technologies such
as such as extended-release microparticles and implants. Following
recent investments and strategic acquisitions across core technology
areas, we believe that Evonik is uniquely positioned to serve our
customers in the development and manufacturing of specialized
parenteral drug products.
Looking ahead, how will Evonik continue to offer
its current and future customers the best available
lipid nanoparticle-based drug delivery systems and
development capabilities?
Advanced drug delivery technologies are helping to enable and
enhance the next generation of specialized parenteral medicines. That
is why this market has become a core growth area for Evonik, where
we are allocating significant resources to support new and existing
customers in the development, scale-up and production of highly
complex drug products.
We recently completed a significant expansion of our Evonik
Birmingham Laboratories site in the U.S. state of Alabama, where
we manufacture our own bioresorbable excipients and customer’s
specialized parenterals at clinical and commercial scale. A state-of-
the-art aseptic filling line is also about to come online in Birmingham
for the commercial filling of liquids, powders and suspensions
into vials. In addition, we are investing in a major expansion of our
R&D and CGMP manufacturing capabilities for lipid-based drug
delivery systems at our Vancouver site in Canada. Such investments
will further accelerate our ability to serve as a strategic partner
with the necessary core competencies in design, development
and production to help customers bring new, high-performance
parenteral medicines to market.
www.americanpharmaceuticalreview.com | | 65
» BIOPHARM DEVELOPMENT »

Self-Cleaving Tags Based Introduction

Reliable, consistent, predictable and cost-effective protein purification

on Split Inteins: platforms, with the smallest possible number of steps, are highly
desirable for protein research and manufacturing. This is one of the
reasons why intervening protein (intein)-based protein purifications

Increased Reliability have become a focal point among other intein applications.
Knowledge of inteins has expanded exponentially over the last three
decades, resulting in many new patents and publications, as well as

Enabling Higher the identification of inteins in archaea (47%), bacteria (25%), phages,
viruses, and even single and multi-celled eukaryotes.1 Additionally,
inteins have now been artificially modified and fused to different

Throughput Applications functional protein domains to improve their utility in protein

purification systems. Existing intein-based purification systems
currently use three distinct types of inteins: full-length inteins that
contain a splicing domain and an endonuclease domain, mini-inteins
that contain a splicing domain but lack an endonuclease domain, and
split-inteins that consist of two short intein segments (the N-terminal
intein, IN, and C-terminal intein, IC), which must reassemble in order
to splice. To develop these systems, inteins have been engineered
with increasing functionality over the last several decades, leading
Izabela Gierach
to increasingly useful purification platforms. Early inteins possessed
Co-founder, Protein Capture Science, Dublin OH simple mutations to interrupt the native splicing reaction, which led
to isolated cleaving activities, but they were slow and unreliable, and
David W. Wood required problematic reagents. Later designs increased the cleavage
The Ohio State University, Department of Chemical & Biomolecular Engineering, Columbus, OH rates and control, but were then plagued with premature cleaving
and Co-founder, Protein Capture Science, Dublin OH and a persistent lack of reliability. A most recent design seems to
have solved these problems; providing highly effective control and
reliability, along with simple and inexpensive reagents. The major
steps in developing these systems, along with some of the early efforts
are herein described.

Importance of Intein/Extein Junction Mutations for

Generating Isolated Cleaving Activities
Early work established that intein-mediated protein splicing is a
spontaneous process, where the intein excises itself from between
two flanking extein segments in a self-catalyzed splicing reaction.
Key to the reaction are a highly conserved N-terminal Cysteine,
Serine or Threonine, along with a C-terminal Asparagine or Glutamine
on the intein sequence, which are directly involved in the bond
rearrangements of the canonical splicing reaction.2-5 The only required
residue external to the intein is a conserved Serine or Cysteine at the

66 | | January/February 2019
 « BIOPHARM DEVELOPMENT »

N-terminus of the C-extein (although this
residue is not required for cleaving). During
the splicing process, the intein/extein peptide
bonds flanking the intein are cleaved as the
N- and C-exteins are ligated, resulting in
ejection of intein and formation of the mature
host protein consisting of the ligated exteins.
Over time it was discovered that the intein/
extein junction residues could be modified
by a single Cysteine to Alanine or Asparagine
to Alanine mutation, which would suppress
the intein splicing activity while allowing
residual C- or N- terminal self-cleavage side
reactions, respectively. At the same time,
research showed that the resulting cleavage
rates could be controlled simply by changing
the pH and/or temperature, or by adding of
thiol-containing compounds.6-9
degradation and yield loss. Additionally, this
bulky intein was often larger than the target
proteins, and exhibited variable cleavage
rates in the laboratory.
A pH Sensitive Intein Development
as a Thiol Replacement
For broader industrial and laboratory appli-
cations, additional inteins were developed
where the cleavage reaction was controlled
with a small pH shift. These inteins were spe-
cifically designed to be benign to proteins
with disulfide bonds, and be cost-effective
through the use of simple buffers. An early
example was the ΔI-CM mini-intein (referring
to a Cleavage Mutant minimal intein), which
was developed from the Mycobacterium tu-
berculosis RecA intein. To develop this intein,
the native endonuclease domain was first
deleted from the full-length parent intein,
then the initial intein Cysteine was mutated
to Alanine to suppress splicing, and finally
an internal Aspartic Acid to Glycine mutation
was identified to accelerate cleaving.8 These
mutations provided accelerated C-terminal
cleavage at pH 6.2, but suppressed cleav-
age at pH 8.5, when compared to both the
initially designed mini-intein and full-length
parent (see Figure 1). This intein is highly ef-
fective for most simple proteins expressed in
E. coli, but often exhibits premature cleaving
of targets during expression. Critically, pre-
mature cleaving by this intein has effectively
limited its used to proteins expressed in the
E. coli cytoplasm, thus necessitating a new
intein for mammalian and other secreted
protein systems.

Thiol and Disulfide (S-S) Bond

Breakage
The first commercially available intein-based
protein purification systems were known
as the IMPACT TM and IMPACT-CNTM systems
(Intein Mediated Purification with an Affinity
Chitin-binding Tag). These systems relied on
a modified full-length intein (454 amino acid
residues) derived from the Saccharomyces
cerevisiae VMA1 gene. This system is still
in use, although the cleavage reaction
requires a high concentration of thiol (e.g.
1, 4-dithiothreitol, β-mercaptoethanol or
cysteine), which limits the scalability of the
process. Perhaps most importantly, the
required thiol (or other reducing agent) Figure 1. Examples of improvement of the inteins over time.
affects disulfide bonds in the target proteins,
which often results in unwanted target

www.americanpharmaceuticalreview.com | | 67
» BIOPHARM DEVELOPMENT »

increasing pH sensitivity in the absence of appended to an expressed target protein

Development of a zinc (US Patent #9,796,967).11,12 The resulting (US Patent #10,066,027, issued in September,

Split-Intein Protein intein exhibits highly pH sensitive cleaving,

while resolving premature cleaving and
2018).13 Once commercialized, the user
would simply tag their target protein with
Purification Platform providing rapid cleaving after purification. the 36 amino acid C-terminal intein segment,
Importantly, additional research on this express the resulting fusion in the host of
intein has provided guidance on designing their choice, bind the tagged target to the
A Split Intein as a Possible reliable or at least predictable performance capture resin, wash away impurities with a
Solution to Premature of the cleaving reaction with various target pH 8.5 buffer, and then cleave the target from
Cleavage Problems proteins, effectively eliminating the need the immobilized tag with a buffer shift to pH
for native extein residues to be added to the 6.2. Importantly, stripping the cleaved tag in
During early mechanistic studies, contiguous
target protein. These advances ultimately led a subsequent process can then be done to
inteins were sometimes artificially split into
to the development of a new purification regenerate the resin. A simplified illustration
two segments to control splicing and cleaving
platform, consisting of a capture resin with of this protein purification method is
reactions. Although this potentially provided
an immobilized N-terminal segment of described in Figures 2 and 3.
a new means for suppressing premature
cleaving, the split intein segments were often a modified Npu DnaE split intein, which On a side note, it is interesting that even
difficult to express and reassemble without captures the small C-terminal intein segment though the pH-sensitive inteins described
a refolding step. Soon thereafter were found
examples of naturally split inteins, where two
intein segments fused to their respective
exteins are expressed, assemble and splice
inside their host cell in a process referred
to as trans-splicing. The naturally occurring
split inteins expressed and folded well, and
were quickly adopted for protein purification
methods. Although no premature cleaving
was observed, control of the cleaving
reaction in the assembled intein was still
lacking. In one early system, thiol was shown
to induce rapid cleavage, but reliability
and the effects on disulfide bonds was still
a concern.10 Further, this and other inteins
often required three amino acids to be added
to the terminus of the desired target protein
in order to guarantee reliable cleaving, which
was unacceptable in cases where a truly
Figure 2. Product development pathway from I-CM C-terminal cleaving intein to modi
native target was required. ed NpuDnaE split intein.

A Breakthrough in Split Intein

Development
Led by previous work in developing
the contiguous ∆I-CM intein (US Patent
#6,933,362), highly specific mutations were
rationally introduced into the naturally split
Npu DnaE intein. Although the resulting
intein resolves premature cleavage issues
and provides somewhat accelerated cleaving,
the ability to control cleaving via pH required
additional development. This advance was
provided through a rationally designed
sensitivity-enhancing domain, which was Figure 3. Single column puri cation using Npu DnaE split intein.
originally intended to increase sensitivity
to zinc, but had the additional effect of

68 | | January/February 2019

here are derived from different species (the ΔI-CM mini-intein was
originally derived from the Mycobacterium tuberculosis RecA intein,
while the split intein was derived from the Nostoc punctiforme DnaE
intein), the gene mutations were “transferable” in terms of their
functional impact on behavior of the intein.10 This newly patented split
Npu DnaE intein performs similarly to the earlier ∆I-CM intein in terms
of pH sensitivity and general cleaving rate, where both purifications
take place at pH 8.5 followed by a pH 6.2 cleaving reaction. The key
difference is the suppression of premature cleaving by splitting the
intein during expression, and the now predictable performance of the
cleaving reaction with different target proteins. In practice, the system
is comparable to the classic FLAG Tag affinity method in terms of
achieving a high-purity target protein, but with the added advantages
of a tagless target and more affordable purchase and operating costs.
Conclusions
Over the last 15 years, intein purification systems have been tested
with proteins expressed in a wide variety of host cells, including
bacteria, yeast, plants and mammalian cells, as well as with different
conventional and non-chromatographic purification tags (e.g., Elastin-
Like Peptide, polyhydroxybutyrate-binding phasins and the self-ag-
gregating peptide fusion tags 18A and ELK16).8,14-22 The advantage of
these methods is that the target protein can be released from effec-
tively any purification tag without the additional step of proteolytic
tag removal. In the split intein system, the resin effectively becomes
part of the intein cleaving process, providing a strong and selective
purification with tag removal functionality in a single operation. This
unique technology potentially provides a completely new and disrup-
tive method for producing therapeutic proteins at research and manu-
facturing scale, and it is projected that it will ultimately constitute a
core approach for industry.
It is predicted that the introduction of this technology to the market
will have a great impact on how biopharmaceuticals are discovered,
developed and manufactured, and will introduce new flexibility and
simplicity compared to the existing complex, conventional methods.
This technology also brings a truly groundbreaking opportunity to
simplify and accelerate biopharmaceutical research, which equates to
more rapid development of therapeutics that can save and prolong
patient lives.
Author Biographies
Izabela Gierach, PhD, MBA, MS is a co-founder, CEO of Protein Capture
Science, with over 17 years of professional experience in scientific research
(including research on intein systems, non-clinical and clinical research),
business project management, and products development.
David Wood, PhD is a Professor of Chemical and Biomolecular
Engineering at The Ohio State University and co-founder, CSO of Protein
Capture Science, with biopharmaceutical experience at Amgen. He has
developed the intein technology described herein.
« BIOPHARM DEVELOPMENT »
References
1. Lennon CW, Belfort M. Inteins. Curr Biol. 2017;27(6):R204-R206.
2. Li Y. Split-inteins and their bioapplications. Biotechnol Lett. 2015;37(11):2121-2137.
3. Cheriyan M, Perler FB. Protein splicing: A versatile tool for drug discovery. Adv Drug Deliv
Rev. 2009;61(11):899-907.
4. Cooper AA, Stevens TH. Protein splicing: self-splicing of genetically mobile elements at the
protein level. Trends Biochem Sci. 1995;20(9):351-356.
5. Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y. Molecular structure
of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine
triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem.
1990;265(12):6726-6733.
6. Shah NH, Muir TW. Inteins: Nature’s Gift to Protein Chemists. Chem Sci. 2014;5(1):446-461.
7. Southworth MW, Amaya K, Evans TC, Xu MQ, Perler FB. Purification of proteins fused to
either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein.
Biotechniques. 1999;27(1):110-114, 116, 118-120.
8. Wood DW, Wu W, Belfort G, Derbyshire V, Belfort M. A genetic system yields self-cleaving
inteins for bioseparations. Nature biotechnology. 1999;17(9):889-892.
9. Lahiry A, Fan Y, Stimple SD, Raith M, Wood DW. Inteins as tools for tagless and traceless protein
purification. Journal of Chemical Technology and Biotechnology. 2017;93(7):1827-1835.
10. Guan D, Ramirez M, Chen Z. Split intein mediated ultra-rapid purification of tagless protein
(SIRP). Biotechnology and bioengineering. 2013;110(9):2471-2481.
11. Belfort M, Belfort G, Derbyshire V, Wood DW, Wu W, Inventors; Rensselaer Polytechnic
Institute, assignee. Genetic system and self-cleaving inteins derived therefrom,
bioseparations and protein purification employing same, and methods for determining
critical, generalizable amino acid residues for varying intein activity. US patent 6,933,362.
August 24, 2005.
12. Ma B, Nellis DF, Zhu JS, Wood DW, Inventors. Compositions related to controllable
intervening protein sequences (CIPS) comprising reversible zinc-binding motifs and
inteins. US patent 9,796,967. October 24, 2017.
13. Wood DW, Shi C, Inventors; Ohio State Innovation Foundation, assignee. Protein Production
Systems and Methods Thereof. US patent 10,066,027. September 4, 2018.
14. Fong BA, Gillies AR, Ghazi I, et al. Purification of Escherichia coli RNA polymerase using
a self-cleaving elastin-like polypeptide tag. Protein science : a publication of the Protein
Society. 2010;19(6):1243-1252.
15. Fong BA, Wood DW. Expression and purification of ELP-intein-tagged target proteins in
high cell density E. coli fermentation. Microbial cell factories. 2010;9:77.
16. Gillies AR, Hsii JF, Oak S, Wood DW. Rapid cloning and purification of proteins: gateway
vectors for protein purification by self-cleaving tags. Biotechnology and bioengineering.
2008;101(2):229-240.
17. Wu WY, Gillies AR, Hsii JF, et al. Self-cleaving purification tags re-engineered for rapid
Topo(R) cloning. Biotechnology progress. 2010;26(5):1205-1212.
18. Wu WY, Miller KD, Coolbaugh M, Wood DW. Intein-mediated one-step purification of
Escherichia coli secreted human antibody fragments. Protein expression and purification.
2011;76(2):221-228.
19. Shi C, Meng Q, Wood DW. A dual ELP-tagged split intein system for non-chromatographic
recombinant protein purification. Applied microbiology and biotechnology.
2013;97(2):829-835.
20. Warren TD, Coolbaugh MJ, Wood DW. Ligation-independent cloning and self-cleaving
intein as a tool for high-throughput protein purification. Protein expression and
purification. 2013;91(2):169-174.
21. Fan Y, Miozzi JM, Stimple SD, Han T-C, Wood DW. Column-Free Purification Methods
for Recombinant Proteins Using Self-Cleaving Aggregating Tags. Polymers.
2018;10(5):468/461-468/412.
22. Shi C, Han TC, Wood DW. Purification of Microbially Expressed Recombinant Proteins via a
Dual ELP Split Intein System. Methods Mol Biol. 2017;1495:13-25.
www.americanpharmaceuticalreview.com | | 69
» SPECTROSCOPY »

Assessing Aluminum Abstract

The filling level, sedimentation, and resuspension of aluminum

Vaccine Adjuvant Filling, adjuvants (alum) in sealed vials can be quantitatively assessed in situ
using the water proton NMR (wNMR) technology. wNMR demonstrates
high sensitivity and high throughput capacity (10-40 sec per vial).

Sedimentation, and wNMR makes it possible to quantify alum filling and suspension in
every vial in a batch before product release by vaccine makers and
before injection by end-users.

Resuspension in Sealed
Introduction
Vials using Water Aluminum salts are the most widely used vaccine adjuvants,
appearing in over twenty marketed vaccine products.1 Aluminum salts
Proton NMR are covalent hydroxocomplexes between Al(III) and anions, such as
OH-, PO43- and SO42-. These complexes form insoluble nanometersized
primary particles which then agglomerate into irregular-shaped
micrometer-sized particles.2 Aluminum-adjuvanted vaccines are
formulated as aqueous suspensions. Aluminum salt particles, with
or without adsorbed antigens, are heavier than water and thereby
tend to sediment. This tendency to sediment creates potential quality
problems for aluminum-adjuvanted vaccines, both before and after
Marc B. Taraban1, Christopher B. Fox2,3 and product release.
Yihua Bruce Yu1 Sedimentation of the suspended particles during the fill-finish step of
1
Department of Pharmaceutical Sciences, and Bio- and Nano-Technology Center, University of manufacturing may cause uneven filling of vials (here, the vials refer to
Maryland School of Pharmacy, Baltimore, MD any form of containers). Uneven filling, if severe enough, may lead to
2
Infectious Disease Research Institute, Seattle, WA mis-dosing. One such example is Novomix 30®, where a manufacturing
3
Department of Global Health, University of Washington, Seattle, WA error in 2013 caused up to 50% over- or under-filling of the vial with
insulin, i.e., insulin concentration deviates from the specified value by
up to ±50%.3 A patient collapsed in a hypoglycemic coma state after
taking this product in September, 2013.4 One month later, 33 batches
of Novomix 30® were recalled.3 Like aluminum-adjuvanted vaccines,
Novomix 30® is an aqueous suspension containing insoluble insulin
particles. In general, suspensions have more complex flow properties
than solutions, making uneven filling of the vials more likely. For some
vaccines, 50% over-filling of aluminum salts will exceed the regulatory-
approved limit of the aluminum content - 0.85 mg of Al(III) per dose in
US and 1.25 mg Al(III) per dose in Europe.5 Under-filling, on the other
hand, might result in subpar efficacy of vaccines.
After product release, aluminum-adjuvanted vaccines sediment to the
bottom of the vial during transportation and storage. Package inserts
typically instruct rigorous shaking immediately before use. Incomplete

70 | | January/February 2019

resuspension may lead to product recall. For example, in April 2010,
the World Health Organization recommended recall and destruction
of all lots of SHAN5 vaccine that contained particles that were not fully
re-suspended by shaking.6 Shipping stresses may make re-suspension
difficult, with significant vial-to-vial variability.7
Pre-release uneven filling and post-release shipping stress may cause
defects at the drug product (DP) level that are not detectable by
prefilling testing of the drug substance (DS), no matter how extensive
the testing is. Such DP-level defects may display vial-to-vial variability
and thereby pose stiff challenges to quality control (QC); unless every
vial in a batch is quantitatively inspected, serious DP-level defects may
escape detection.
In the context of aluminum-adjuvanted vaccines, current analytical
technologies include atomic adsorption spectroscopy (AAS)8
and 27Al NMR spectroscopy9 to assess aluminum filling level, UV
absorption spectroscopy,7 optical scanning analysis,10 microCT,11
and visual observation (the WHO shake test)12 to assess aluminum
particle sedimentation and resuspension. All these technologies,
except microCT and visual observations, are ex situ, i.e., they require
taking the DS out of the vial for analysis, thereby compromising DP
integrity. MicroCT and visual inspection are in situ methods and do
not compromise DP integrity. However, microCT involves ionizing
radiation, expensive equipment and complex and time-consuming
procedures. Visual observation is subjective and might be difficult to
execute, depending on vial size and labelling (a vial containing 0.5
mL whitish suspension in a sealed and labeled vial is challenging for
visual inspection).
When an analytical procedure compromises DP integrity, it can only
be applied to a small percentage of the finished vials in each batch
(statistical sampling). This feature makes it hard to catch pre-release
DP defects caused by manufacturing errors if the defect rate is low.
For example, in the aforementioned Novomix 30® case, the defect
rate was 0.14%.3 Also, the complexity and cost of current analytical
procedures restrict their use to highly trained personnel at the
manufacturing plant. This feature precludes their implementation at
the point-of-care by end-users to detect post-release defects caused
by shipping stresses.
To catch DP-defects, whether they occurred pre- or post-release, in
situ analytical technologies that maintain DP integrity are needed.
Here, we report a technology that can quantitatively assess aluminum
adjuvant filling level, sedimentation, and resuspension in sealed and
labelled vials. It builds on the water proton NMR (wNMR) technology
we previously presented in this journal.13
Suspended, Sedimented, and
Resuspended Aluminum Adjuvants
Aqueous suspensions of two aluminum vaccine adjuvants - aluminum
oxyhydroxide [Alhydrogel®85] and aluminum phosphate [Adju-Phos®]
(both manufactured by Brenntag Biosector and distributed by Sergeant
Adjuvants)—were prepared in sealed vials (4 mL total volume). The
original commercial suspensions were diluted using water to give a
« SPECTROSCOPY »
concentration range of 0.1 to 5 mg/mL (here, weight refers to that of
elemental (Al(III)). The goal is to determine the response of wNMR to
aluminum salt filling concentration; the sensitivity of such responses
forms the basis to detect potential filling errors during manufacturing.
The same samples were also used to verify the sensitivity of wNMR
towards aluminum salt sedimentation and resuspension. To this
end, prior to the analysis, samples were allowed to sediment in an
undisturbed environment for 2-3 weeks at 4°C. The fully sedimented
aluminum salts were resuspended by vigorous shaking. wNMR data
were collected on the fully sedimented and resuspended samples.
Figure 1 shows photos of suspended and sedimented aluminum
adjuvants in sealed vials.
Figure 1. (left) Suspended and sedimented Alhydrogel® in
sealed vials. (right) Suspended and sedimented Adju-Phos® in
sealed vials.
wNMR measurements were performed in sealed and labelled vials,
which were loaded into the NMR instrument using a plastic sample
holder (Figure 2). The same parameters and experimental settings
were used to measure suspended, sedimented, and resuspended
samples. The water proton transverse relaxation rate, R2(1H2O), was
measured using standard CPMG pulse sequence14 with 2 transients.
The instrument was a low-field (0.56 T, 24 MHz) wide-bore (26 mm)
benchtop NMR instrument. Duration of a single measurement for
the fully suspended samples, depending on the aluminum adjuvant
concentration, varied from 10 to 40 seconds. Duration of a single
measurement of the fully sedimented samples within all concentration
ranges was 40 seconds. For each vial, the measurement was repeated
thrice. Alhydrogel® and AdjuPhos® were stored at 4°C respectively for
21 and 14 days for sedimentation of the aluminum salt to evaluate
whether storage time has impact on the extent of resuspension.
Water Proton NMR Sensitivity
toward Filling, Sedimentation
and Resuspension of Aluminum
Adjuvants
For both Alhydrogel® and Adju-Phos®, in the fully suspended states,
wNMR demonstrated high sensitivity towards the changes in the
www.americanpharmaceuticalreview.com | | 71
» SPECTROSCOPY »
Figure 2. A sealed vial of Alhydrogel® in a holder before being
loaded into in the probe of the benchtop NMR for
data collection.
concentration of aluminum salt. As seen from Figure 3, for both
adjuvants, the dependences of R2(1H2O) vs. Al(III) concentration were
perfectly linear with extremely high response effectiveness of 1-2 s-1
per mg/mL (cf. with < 0.1 s-1 per mg/mL, e.g., proteins).13
Figure 3 also shows about 70% larger response effectiveness observed
for Adju-Phos® compared to Alhydrogel®. Alhydrogel® and AdjuPhos®
diff er in charge, exchangeable protons per Al(III), and proton exchange
rates, which may all contribute to the observed differences in response.
For example, Alhydrogel® particles are positively charged, while Adju-
Phos® particles are negatively charged.15 Negatively charged Adju-
Phos® particles likely exchange proton with water molecules more
efficiently than positively charged Alhydrogel®, resulting in a higher
water proton relaxation rate R2(1H2O). Also, differences in R2(1H2O)
values may in part be the result of differences in average hydrodynamic
diameters between Adju-Phos® particles and Alhydrogel® particles.16
Figure 3. Comparison of the response of R2(1H2O) to the
concentration of Al (III) in each suspended adjuvant. Inset shows
the lower Al(III) concentration range.
72 | | January/February 2019
Figure 4. R2(1H2O) vs. aluminum salt concentration observed
for suspended, sedimented and resuspended Alhydrogel®
(left) and Adju-Phos® (right). In each panel, solid and dashed
lines respectively represent suspended and sedimented
aluminum salt.
Once fully sedimented, both Alhydrogel® and Adju-Phos®,
demonstrated a sharp drop in the observed R2(1H2O) values within
the whole range of the concentrations under study (Figure 4). In
fact, R2(1H2O) in sedimented samples displayed little change over the
experimental concentration range for both adjuvants. And, as seen
from Figure 4, for both adjuvants, resuspension of the aluminum salt
particles brings the R2(1H2O) values back to the levels observed for the
original suspensions. This forms the basis for using R2(1H2O) to evaluate
the extent and uniformity of vial filling in each batch.
Of note, our results demonstrate high sensitivity of wNMR to aluminum
adjuvant concentration in the fully suspended state. Insets in Figure 3
show the response effectiveness in the very low concentration range,
from 0.0 to 0.1 mg/mL of Al(III). The aluminum concentration range
used in this work (0.1 to 5 mg/mL) likely encompasses any realistic
adjuvant concentration variation caused by filling errors. Therefore,
the resulting R2(1H2O) response effectiveness could be used to
estimate the wNMR sensitivity to potential filling errors in vaccine DPs
containing aluminum oxyhydroxide (such as Alhydrogel®) or aluminum
phosphate (such as Adju-Phos®). Table 1 shows the estimated R2(1H2O)
changes in response to various levels of filling errors for two vaccine
products, Pediarix® containing 1.70 mg/ mL of Al(III) in the form of
aluminum oxyhydroxide, and TDVAXTM containing 1.06 mg/mL of Al
(III) in the form of aluminum phosphate.
Thus, the high sensitivity of wNMR as shown in Figure 3 supports its
application as a noninvasive analytical tool for accurate quantification
of the filling level, sedimentation, and resuspension of aluminum-
adjuvanted vaccines.
Comparison of wNMR with
Other Techniques
Compared with other analytical techniques, such as atomic
adsorption spectroscopy, 27Al NMR spectroscopy, UV and fluorescence
spectroscopy, and optical scanning analysis, the foremost advantage
of wNMR is that it is an in situ technology, capable of quantitative

Table 1. wNMR response to filling error of aluminum oxyhydroxide-
and aluminum phosphate-adjuvanted vaccines.a
ΔR2(1H2O), s-1 ΔR2(1H2O), s-1
Filling Error, % Pediarix® (aluminum TDVAXTM (aluminum
oxyhydroxide) phosphate)
0 0 • 0
±5 ±0.091 (±10-3) • ±0.091 (±10-3)
±10 ±0.181 (±10-3) • ±0.181 (±10-3)
±25 ±0.453 (±10-3) • ±0.454 (±10-3)
±50 ±0.908 (±10-3) • ±0.909 (±10-3)
a
Estimates were based on the data presented in Figure 3 and the slopes of linear dependences
(for aluminum oxyhydroxide, 1.067 s-1 per mg/mL; and for aluminum phosphate, 1.715 s-1 per
mg/mL). Errors in the bracket are based on the SD of three consecutive measurements. The
value of ΔR2(1H2O) is based on measurements conducted on the adjuvant alone. For each
vaccine, the actual ΔR2(1H2O) value might be modulated by antigen-adjuvant interactions.
inspection of sealed vials. As opposed to other techniques, wNMR does
not require opening the sealed vial, and thus does not compromise
the DP integrity. Secondary advantages including simplicity,
affordability, high throughput potential and the ability to quantify,
make wNMR more preferable than micro-CT or visual inspection for
in situ inspection. These advantages arise because wNMR detects not
the DS, but water, which is by far the most abundant component of all
aluminum-adjuvanted vaccines.
The disadvantage of wNMR is that it is an indirect technology; it does
not monitor aluminum adjuvants directly. To convert R2(1H2O) of a
vial into absolute Al(III) content, the vaccine maker needs to establish
a calibration curve for each product, as shown in Figure 3. wNMR is
more suited to detect inconsistency, change and anomaly at the DP
level rather than ascertain the absolute chemical composition of the
aluminum adjuvants at the DS level; the latter can be accomplished by
invasive analytics before the fillfinish step of vaccine manufacturing.
Conclusion
wNMR is a noninvasive analytic based on parameters of the water
proton NMR signal, such as the transverse relaxation rate, R2(1H2O).
For aluminum adjuvants, wNMR can assess filling, sedimentation and
resuspension of aluminum adjuvants in sealed vials in an in situ fashion.
As such, it opens the possibility to quantitatively inspect every vial in
a batch of vaccines both at the point-of-release by manufacturers and
the point-of-care by end-users.
Acknowledgement
Work at the University of Maryland was supported in part by the FDA
through the Maryland Center of Excellence in Regulatory Science and
Innovation (1U01FD005946).
« SPECTROSCOPY »
Author Biographies
Marc Taraban, PhD, is assistant professor (research) in the Department
of Pharmaceutical Sciences at the University of Maryland School of
Pharmacy. He was actively involved in research in the areas of structural
biology, biophysics of enzymatic reactions and structure-properties
relationships in soft matter biomaterials. Currently, he is exploring
dynamic NMR applications to study biologics and nanoparticle products.
Christopher Fox, PhD, is the Vice President of Formulations at IDRI. He
has developed multiple vaccine adjuvant formulations for a variety of
infectious disease indications, including cGMP manufacturing, clinical
testing, and technology transfer to developing country manufacturers. He
currently leads efforts to develop a thermostable tuberculosis vaccine as
well as scale up adjuvant manufacturing capacity for pandemic influenza
preparedness.
Yihua Bruce Yu, PhD, is professor of pharmaceutical sciences at the
University of Maryland School of Pharmacy and the director of its Bio-
and Nano-Technology Center. He has conducted research on protein
biophysics, biomaterials and imaging agents. His current research is
on analytical technologies for complex drugs, including biologics and
nanomedicines.
References
1. FDA (2018) https://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/
ucm093833.htm
2. HogenEsch, H., O’Hagan, D.T., Fox, C.B. npj Vaccines 3, 51 (2018).
3. European Medicines Agency (EMA). (25 October 2013) Batches of the Insulin Medicine
NovoMix 30 FlexPen and Penfi ll to be recalled, (EMA/ 657469/ 2013). http://www.ema.
europa.eu/docs/ en_GB/document_library/Press_release/2013/10/WC500153147.pdf
4. FDA (2013) MAUDE Adverse Event Report: Novo Nordisk A/S Novopen Insulin Delivery
Device. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfmaude/detail.cfm?mdrfoi
__id=3621493
5. Vecchi, S. et al., J. Pharm. Sci. 101, 17-20 (2012).
6. WHO (2010). WHO recommends recall and destruction of lots of SHAN5 vaccine as a
precautionary measure. https://www.who.int/immunization_standards/vaccine_
quality/who_unicef_joint_statement_Shan5_26apr10.pdf
7. Guo, J. et al., J. Pharm. Sci. 105, 2009-2013 (2016).
8. Mishra, A. et al., Biologicals 35, 277-284 (2007).
9. Khatun, R. et al., J. Pharm. Biomed. Anal. 159, 166-172 (2018).
10. Muthurania, K. et. al., J. Pharm. Sci. 104, 3770-3781 (2015).
11. Lewis, L.M. et al., J. Pharm. Sci. 106, 2163-2167 (2017).
12. Kartoglu, U. et al.Bull. World Health Organ. 88, 624-631 (2010).
13. Yu, Y.B., Feng, Y., Taraban, M. Am. Pharm. Rev. 20, 34-39 (2017).
14. Meiboom, S., Gill, D. Rev. Sci. Instrum. 29, 688−691 (1958).
15. InvivoGen. https://www.invivogen.com/Adju-Phos
16. Morefi eld, G. L. et al., Vaccine 23, 1588–1595 (2005).
www.americanpharmaceuticalreview.com | | 73
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ICH Q12 as an Enabler for PAT and
the Digital Revolution
Chair: Christine Moore, Merck
BioSimilars
Chairs: Antonio Moreira, University of
Maryland-Baltimore County and Nadine
M. Ritter, Global Biotech Experts
Particle Characterization &
Engineering - I - Focus on
Characterization
Chairs: Nicolas Abatzoglou, University
of Sherbrook, Joep Timmermans,
Pfizer, and Michael Puppolo, Eli Lilly
Enhanced Approaches to Analytical
Methods
Chairs: Yubing Tang, FDA and Kimber
Barnett, Pfizer
IIoT, Analytics and Control
Chairs: Tom O'Connor, FDA and Jun
Huang, Pfizer
Data Infrastructure and Knowledge
Chairs: Christian Airiau, Sanofi,
Juergen von Frese, DA SOL and Brian
Rohrback, Infometrix
Using New Sampling & Process
Sensor Developments to Incorporate
PAT and QbD in Process
Optimization
Chair: Mel Koch, CPAC, University of
Washington
1100 E. Washington St. Suite 103
Grayslake, IL 60030 USA
Tel: 847-543-6800 Fax: 847-548-1811
info@ifpacnet.org
Copyright 2019 IFPAC, Grayslake, IL 60030
Tuesday, March 5th - PM
Advancing Pharmaceutical Science
in Generic Industry - II
Chairs: Darby Kozak, FDA and
Geoffrey Wu, FDA
News from the Front: Lessons
Learned from Continuous
Manufacturing Practitioners
Chairs: Lawrence De Belder, Johnson
& Johnson and Ahmad Almaya, Eli Lilly
Bridging the Gap: Digital Design to
Digital Operation
Chairs: TBA, FDA and Industry
Representative
Analytical Methodologies &
Innovative Techniques
Chair: Gert Thurau, Roche
Pharmaceuticals
Advanced Biomanufacturing
Technology & Innovation
Chair: Seongkyu Yoon, University of
Massachusetts-Lowell
Particle Characterization &
Engineering II - Focus on
Engineering
Chairs: Joep Timmermans, Nicolas
Abatzoglou and Michael Puppolo
Synergy of Real Time Analytics in
Small vs. Large Molecules
Chairs: Sameer Talwar, GSK, Michael
Dotlich, Eli Lilly, Zhenqi (Pete) Shi, Eli
Lilly and Chikkathur Madhavarao, FDA
Data Integrity: Ensuring End-to-End
Data Quality with the Emergence of
Cloud, IoT and Industry 4.0
Chairs: Krishnakali Ghosh, FDA and
Jon Dieringer, Eli Lilly
Plus... Chemometrics (COPA &
Calibration), Mass Spec/Process
Spectroscopy and Poster Session
Tuesday Evening - Bring Innovation
to Life in Large Organizations with
the Start-Up Method
Chair: Xiaoyu Zhang, Eli Lilly
IFPAC
Wednesday, March 6th - AM
Performance Based Models
Chairs: Sharmista Chatterjee, FDA and
Sal Garcia, Eli Lilly
Recent Developments to Realizing
Continuous Manufacturing - II
Chairs: Theodora Kourti, McMaster
University and Rapti Madurawe, FDA
Process Analytical Measurements -
New Frontiers of Research and
Beyond
Chairs: David Myers, Eli Lilly, Bryan
Castle, Eli Lilly and Rodolfo
Romañach, University of Puerto Rico-
Mayaguez
Excipients
Chairs: Brian Carlin, DFE Pharma and
Chris Moreton, FinnBrit
Manufacturing Technologies for Cell
& Gene Therapies
Chairs: Antonio Moreira, UMBC,
Seongkyu Yoon, University of
Massachusetts-Lowell and Vaneeta
Grover, GlaxoSmithKline
Particles in Biologic Drug Products
Chair: Ashwinkumar Bhirde, FDA
Exhibition Schedule:
Join your fellow exhibitors at IFPAC-19:
• Agilent Technologies
• Applied Materials
• ASTM
• Brimrose Corporation
• Bruker Corporation
• B&W Tek
• CAMO Software Inc.
• Canty
• CPAC, Center for Process Analysis &
Control
• CPACT
• DRS Daylight Solutions, Inc
• Duquesne University/SPCTECH
• Emerson Automation Solutions
• Endress + Hauser, Inc.
• Eurofins
• ExpoPharma Engineering Services
• Extrel
• FIAlab Instruments
• 4-Tune Engineering
Product and Process Robustness Continuous Process Verification
Chairs: Celia Cruz, FDA and Sonja S. (CPV) - the Ultimate PV Frontier and
Sekulic, Pfizer Continual Improvement Enabler:
Practices, Challenges and
Emerging Technologies for Product Opportunities
Design and Delivery Chairs: Ranjit Deshmukh, MedImmune
Chairs: Tom O'Connor, FDA, Angela and Vibhakar Shah, FDA
Liu, Pfizer and Xiaoyu Zhang, Eli Lilly
Cell & Gene Therapy
Plus... Spectroscopic Solutions for Chairs: Vaneeta Grover, GSK, Antonio
Complex Systems/Portable, Handheld Moreira, UMBC and Seongkyu Yoon
& Small Footprint Instrumentation and UM-L
Smart Data - Design of Experiments
Smart Sensors for Bio-Processing
NEW- Student Poster Session & Chairs: Jagdish Tewari, Sanofi and
Competition on Wednesday AM Cenk Undey, Amgen
Wednesday, March 6th - PM PAT Implementation/CM
Chairs: Martin Warman, Consultant
API for Process Understanding of and Mel Koch, CPAC, University of
Drug Substance in Small Molecules Washington
(SMDS)
Chair: Manoharan Ramasamy, Merck Process Analysis & Control
Chair: Walter Henslee, JPAC
The Challenges and Opportunities
on the use of Multivariate Statistical Plus... OnSite Analysis and Process
Process Control (MSPC) for Process Analytics with TDL Panel Discussion
Monitoring and Control
Chair: Zhenqi (Pete) Shi, Eli Lilly REGISTER TODAY!
New Session Coming Soon! View more details at:
Chair: Ali Afnan, Step Change Pharma www.IFPACglobal.org
Monday, March 4th ......................2:00 - 7:00 PM
Grand Opening ..................2:00 PM
Tuesday, March 5th ......................10:00-4:00 PM
Wednesday, March 6th ................9:00-12:00 Noon
• GEA North America • Sentronic, GmbH
• GRANUTOOLS • Siemens
• IFPAC • tec5USA
• INDATECH • Tech4Imaging LLC
• Innopharma Technology • Thermo Fisher Scientific
• InProcess-LSP • Tornado Spectral Systems
• IQ-International Consortium • Viavi Solutions
• Kaiser Optical Systems • Waters Corporation
• Keit Spectrometers • Wyatt Technology Corporation
• MicroSaic Systems Plc
• Minitab Don’t miss this opportunity! Full exhibition serv-
• Nova Biomedical, Inc. ices will be provided during the three day exhib-
• ONDAX, Inc. it. Companies interested in exhibiting are invit-
• Optimal Industrial Technologies, Ltd. ed to contact exhibition management as soon
• Optimization Firm LLC as possible. Email: info@ifpacnet.org; Tel: 847-
• Photon Systems Inc. 543-6800; Web: www.IFPACglobal.org
• Powrex Corporation
PAT
• RedStart Instrumentation Ltd.
• Rutgers University. ERC
• Sartorius Stedim Biotech GmbH
• Sartorius Stedim Data Analytics Process Analytical Technology
www.americanpharmaceuticalreview.com | | 77
 BY THE
NUMBERS

MARCH 18-21, 2019

NEW YORK CITY

DCAT Week is the premier business development event for

the pharmaceutical development & manufacturing industry.

Business Development
Opportunities for high-level strategic meetings in
private Business Meeting Spaces with key decision
makers managing spend in the $1.3 trillion global
pharmaceutical industry.
Networking
Multiple receptions, learning sessions and lounge
spaces offer ideal opportunities for making valuable
business connections.

Industry Education
Education programs that bring together leading
industry experts who provide timely market analysis,
best practices, and key insights on the issues shaping
the pharma and biopharma industries.
 700+
companies
represented

50+
countries
represented

1 in 4
attendees are
C-suite executives

92%
10k+ of attendees
are high-level
high-level industry decision makers
professionals

Learn more at DCATWeek.org

» TRADESHOW PREVIEW »
INTERPHEX 2019
April 2-4, 2019 — New York, NY
Experience the Full Product Development Life Cycle, as the industry
looks to reduce costs, increase productivity and maximize efficiency,
INTERPHEX is perfectly positioned to address and deliver the solutions for
these ongoing challenges by providing the latest in state-of-the-art tech-
nologies and access to industry expertise from subject matter experts.
Celebrating 40 Years of Delivering State-of-the-Art Technology
and Innovation!
INTERPHEX succeeds in bringing together inspired professionals with
key industry leaders to share knowledge and best practices, as well as
see the latest cutting-edge technologies needed to cost-effectively de-
velop and manufacture quality products.
• Combination of Technical Conference, Exhibits,
Demonstrations and Business Opportunities
• Access to 625+ Leading Suppliers Exhibiting Cutting-Edge
Technologies and Value-Added Services
• No Cost Technical Conference Featuring Hot Topics Presented
by Industry Experts
• INTERPHEX Exhibitor Award Winners! (below are 2018
winners)
• Best in Show: Varnx Pharmasystems – Microcell Vial Filler
• Best New Product/Service: Nymi – Nymi Authentication Solution
• Editor’s Choice (sponsored by Pharmaceutical Processing):
LifeAire Systems – The LifeAire System
• Best Technologies Innovation: MilliporeSigma – Millistak+®
HC Pro
• INTERPHEX Efficiency Champion: Steris Life Sciences – Steris
VHP DC-A
• Biotech Innovation: groninger USA – FlexPro50
INTERPHEX Live! Our Live Technical Talk Show features a moder-
ated panel discussion with Industry SMEs
• “Changing the drug development approach in the age of
technology and need for Acceleration”
• “Biosimilars 5.0”
• “An Innovative Approach to Cell/Gene Therapy Facility Design”
• “Collaboration and Proven Results: Where Do We Go from
Here? SUPAC? QbD?”
• “Pharma Intelligence: 2019 State of Investment”
• “Oral Administration and Therapy”
• “Advanced & Highly Adaptive Automation Technology
Integrated with Industry 4.0 Trends”
• “Adding Intelligence to Legacy Assets Through Artificial
Intelligence and Domain Expertise for Operational Excellence”
80 | | January/February 2019
• “A Path Towards Better Freeze Drying Product and Process
Using Mass Spectrometry”
• “Car-T Cell Advancements”
• “Leveraging Technologies into the Smart Supply Chain”
• “Regulatory Review: What’s New?”
Session Tracks
• Strategies for Flexible Manufacturing Efficiencies & Improvements
• Optimizing Facilities through Innovation & Technologies
• Managing the Interface with Risk Management and
Quality Systems
• Biotech and the Acceleration to Market
• Alignment of Facility, Suitability with Formulation Capability
and Stability Across Supply Chain
• Leveraging Partnerships with CMO/CDMO Capability to
Advance Process Innovation
NEW for 2019: INNOPHEX, in collaboration with IPS-Integrated Project
Services, featuring Cell Processing, Gene Therapy and Pharma 4.0
NEW for 2019: Technology-focused Networking events – contract ser-
vices, Pharma 4.0, Packaging & Processing
Expanded Contract Services, More CMO, CDMO and CPO than ever! PBOA
sponsored section dedicated to contract development and manufacturing
NEW for 2019: Dedicated stage exclusive to cell processing, gene ther-
apy and Pharma 4.0 sessions
NEW for 2019: MG America’s Processing & Packaging Summit, learn
from an industry leader
And much, much more…
Featured Speakers
Michael E. Williams, M.D., University of Virginia Health System,
Emily Couric Clinical Cancer Center
Joe McGinnis, Senior Regulatory Compliance Advisor, Johnson & Johnson
Rick Morris, Senior Vice President R&D, Pall Biotech
Fernando Muzzio, Distinguished Professor Chemical and Biochemical
Engineering, Rutgers University
Exhibit Hall Hours
Tuesday, April 2 - 10:00am - 5:00pm
Wednesday, April 3 - 10:00am - 5:00pm
Thursday, April 4 - 10:00am - 3:00pm
Technical Conference Hours
Tuesday, April 2 - 10:30am - 5:00pm
Wednesday, April 3 - 10:30am - 5:00pm
Thursday, April 4 - 10:30am – 12:15pm
 Premier
Association
Spon sor
INNOVATION INNOPHEX
STAGE
Booth 1281 Booth 3941
9:45am – 10:00am
10:00am – 5:00pm
10:30am – 11:15am BioSpherix Medical
Keynote
11:30am – 12:15pm Satorious Stedium
1:15pm – 2:00pm Keynote Integrity Bio Inc.
2:15pm – 3:00pm Keynote Esco Technologies
PAT-based Linear
pH Gradient as an
Alternative Platform
3:15pm – 4:00pm TerumoBCT
to Downstream
Process Monoclonal
Antibodies
INNOPHEX Reception
Sponsored by
Smart Evolution:
Planning Your
4:15pm – 5:00pm
First Laboratory or
Manufacturing Facility
4:00pm - 5:00pm
INNOVATION
STAGE
Booth 1281 Booth 3941
10:00am – 3:00pm
10:00am – 12:00pm
10:00am – 10:45am
11:00am – 11:45am Keynote
1:00pm - 3:00pm
1:15pm – 2:00pm Keynote
2:15pm – 3:00pm Keynote
A QbD Approach
Towards
Manufacturing of
3:15pm – 4:00pm
Parenteral Packaging
Components with
First Line USA
Cleaning
Chromatography
Resin Residues
from Surfaces:
4:15pm – 5:00pm evaluation of cleaning
process design
for non-dedicated
chromatography
columns
INNOVATION
STAGE
Booth 1281 Booth 3941
10:00am – 3:00pm
Using Simulation
for Optimization
10:00am – 10:45am throughout the Facility
Lifecycle
Stopping Infections -
How Hospital Flooring
11:00am – 11:45pm Factors Into Life or
Death Solutions
INTERPHEX
STAGE LIVE
Crystal Palace Lobby
Catalent Pharma
Biosimilars 5.0
An Innovative Approach
to Cell/Gene Therapy Piramal Pharma
Facility Design
Collaboration & Proven
Results: Where we go
Confab Laboratories
from here? SUPAC?
QbD?
Pharma Intelligence:
2019 State of CMIC CMO USA
Investment
Oral Administration and
Therapure Biopharma
Therapy
INNOPHEX INTERPHEX
STAGE LIVE
Crystal Palace Lobby
Advanced & Highly
Adaptive Automation
Technology Integrated
with Industry 4.0 Trends
Adding Intelligence to
Legacy Assets Through
NanoCool Artificial Intelligence & LSNE Contract Mfg.
Domain Expertise for
Operational Excellence
A Path Towards a Better
Freeze Drying Product
Avid Bioservices, Inc.
& Process using Mass
Spectrometry
Car-T Cell
Ajinomoto Bio-Pharma
Advancements
Leveraging
Technologies into a
Smart Supply Chain
Regulatory Review:
Berkshire Sterile Mfg.
What's New?
INNOPHEX INTERPHEX
STAGE LIVE
Crystal Palace Lobby
PDA Round-table
Hikma Pharmaceuticals
INTERPHEX NO COST TECHNICAL CONFERENCE
April 2-4, 2019 | JAVITS CENTER | NYC
TUESDAY, APRIL 2, 2019
CMO/CDMO STAGE 1 STAGE 2 STAGE 3 STAGE 4 STAGE 5
STAGE VENDOR PRESENTATIONS
Booth 1053 Booth 1076 Booth 1577 Booth 1877 Booth 2276 Booth 2477
Exhibit Hall Grand Opening & Exhibitor Awards
Show Floor Open & IPX/BPI Poster Hall
A Transformational
Living Cell Environment:
A Paradigm Shift in Real-time measurement
The Evolution of
Personalized Medicine and controlling of
PDA Round-table EBR:Augmented Batch
that Provides the Most hydrogen peroxide bio-
Records
Comprehensive Protection decontamination cycles
Across All Cell Culture/
Processing Operations
Single use centrifuge
U2k for large scale FDA and EU GMP Annex 1 Flexible and Efficient S.T.A.R. (Systematic
separation and differences of cleanroom Manufacturing Facilities: Transition to Quality and
Trillium
collection of high specification. Is it not time A holistic approach to the Risk) WARS: May the
density mammalian cells to eliminate them? design and implementation Interface be With You!
and mAbs
Tech Lunch & Learn
Scaling with Single-Use
Fermentation creates
challenges to equipment
Continuous Aseptic
design but brings economic
Manufacturing using Spray MillaporeSigma
and productivity benefits... Four Steps to Safer, More
CRB Tech Tank Freeze Drying: Equipment,
Applying 45 years of Reliable Biomanufacturing
Process and Product 1:00pm - 1:30pm
fermenter engineering
Considerations
optimization to design
and scaling of Single-Use
Fermenters.
Single-Use System Supports
a Freeze/Thaw Platform
Single-Use Filling Systems SP Scientific/Pentech
In-Line Monitoring and Real- Capable of Providing an
CRB Tech Tank Maximize Filling Line
Time-Release End-to-End Solution for Fluid
Flexibility and Efficiency 2:00pm - 2:30pm
Drug Substance Inter-Facility
Storage and Delivery
Innovative RAPA : ‘Risk
Analysis & Preventive BOSCH Packaging
Aseptic Processing: Regulations For Submitting Actions’ approach. Case Technology
CRB Tech Tank Achieving Advanced While Tableting & Encapsulating Study of OSD High Potent
Staying Flexible Machine Transactions Online (OEL1 to OEL5) Facilities
3:00pm - 3:30pm
Design meeting cGMP & HSE
requirements.
Innovative GAPA: ‘Gap
Analysis & Preventive
Actions’ design Risk Management
approach. Case Study: Strategies to achieve A risk based approach for Antares Vision North
Exploring a Blockchain
cGMP Sterile Facilities Containment Performance cleaning and disinfection
Enabled Supply Chain
Design new trends Targets for ADC program 4:00pm - 4:30pm
involving Continuous Applications
Process Validation
Guidelines (FDA2011).
WEDNESDAY, APRIL 3, 2019
CMO/CDMO STAGE 4 STAGE 5
STAGE 1 STAGE 2 STAGE 3
STAGE VENDOR PRESENTATIONS
Booth 1053 Booth 1076 Booth 1577 Booth 1877 Booth 2276 Booth 2477
Show Floor Open & IPX/BPI Poster Hall
IPS Technology Tours (Registration Required)
Requirements of Walking through a practical
Facility Focus: Single 2019 Edition of NFPA energy conservation
How to Increase Efficiency
Use BioManufacturing: 652 Standard on program (ISO50001) using
Teligent Inc. and Flexibility with Single
Designing the Facility of Combustible Dust - On an IoT technology for
Use Fermentors
the Future your Way Towards compressed air usage at a
Compliance? large pharma facility
It’s the Climb: Cyber Resilience: Ensuring
Scaling the Digital your infrastructure can Contect Inc.
How Are You Driving CFD for clean environment:
Transformation withstand adverse events
Speed to Market? Application examples
mountain one step at and attacks without 11:00pm - 11:30pm
a time jeopardizing productivity
Tech Lunch & Learn
IPS Technology Tours (Registration Required)
Facility Focus: Priming The New Horizons
Taking Advantage of
Projects for Success: in the Cleanroom Rethinking cleaning
Your Manufacturing Data:
Planning and Capital Monitoring After the validation for API
BioPhorum’s Big Data to
Appropriation for GMP Issuance of ISO 14644- manufacturing
Smart Data Initiative
Facilitie 2:2015
Case study: un-optimized Facility Intensification
BOSCH Packaging
procedures and practices and Cost Reduction Cleaning Cycle
Technology
PDA Round-table leading to recurring Opportunities using Development for Parts
Microbial Contamination in an Integrated Buffer Washers
2:00pm - 2:30pm
grade A (ISO 5) Delivery Platform
Next-Generation, Integrated
Grade B or not to B:
Single-use pH, Dissolved Combining Industry 4.0 Qure Medical
Starting vial break and Quality Systems - It’s a
Grand River Oxygen, and Pressure and Big Data to Reduce
other open cell culture ‘Risky’ Business
Instrumentation for Manufacturing Costs 3:00pm - 3:30pm
processes.
Bioprocessing
Computational Fluid
Flexibility and Predictability: Critical Utilities Support of
Dynamics - A Modeling
How to Achieve both in New Continuous Manufacturing -
Tool for Clean Room
Facilities The Keys to Success
Airflow
THURSDAY, APRIL 4, 2019
CMO/CDMO STAGE 4 STAGE 5
STAGE 1 STAGE 2 STAGE 3
STAGE VENDOR PRESENTATIONS
Booth 1053 Booth 1076 Booth 1577 Booth 1877 Booth 2276 Booth 2477
Show Floor Open & IPX/BPI Poster Hall
Reducing Business Risk
and Improving Data A More Strategic Approach Design Aspects for Technology Solutions for
Integrity Through cGMP to Tactical Planning In Facilities Employing Challenges in Cold Chain
Meeting FDA ALCOA(+) Pharma Manufacturing Single Use Technology Storage and Transportation
Requirements
Viral Safety of Cleaned Digital Transformation:
Surfaces Using a Risk Based Getting Fit for the
Approach Future
*As of January 18, 2019. Schedule subject to change.
REGISTER FOR YOUR TECHNICAL CONFERENCE AND EXHIBIT HALL PASS AT:
WWW.INTERPHEX.COM/REGISTER
www.americanpharmaceuticalreview.com | | 81
» »
SECTION TITLE

Pharmaceutical

P .N.
P.I N Methods of Treating Cancer of the

Points
Points
Patent Innovation News
Central Nervous System;
V.I. Teichberg, and A. Ruban; Yeda
Research & Development, USA;
U.S. Patent # 10,159,717, December 25, 2018.
In the last few years, an ever-increasing body of data have suggested
that glutamate (Glu), the major excitatory neurotransmitter in the
brain, plays a crucial role in the growth of malignant gliomas, their
The purpose of this column is to highlight

Article Title
invasiveness and ability to destroy neighboring brain tissue. The
and summarize recent key patents in the present invention provides the method of administering to the
pharmaceutical arena issued by the US
Introduction
subject a therapeutically effective amount of an agent, which
reduces the blood glutamate levels. Thus, it enhances brain to
Patent Office in November-December 2018. Paragrapgh Text
blood glutamate efflux and thereby treats the cancer of the central
nervous system. The patent claims a chemotherapeutic agent for
treating glioma which can cross the blood brain barrier and at
Anvit Vasavada, M.S., Sunny Christian least one glutamate modifying enzyme capable of reducing blood
M.S.R.A., Amitkumar Lad, Ph.D. and glutamate levels and enhancing brain to blood glutamate efflux.
Hemant N. Joshi, Ph.D., MBA* The said glutamate modifying enzyme is lyophilized, emulsified,
Tara Innovations, LLC encapsulate or formulated as a tablet, pill, dragee or capsule.
www.tarainnovations.com
*hemantjoshi@tarainnovations.com

Stabilized Formulations of
CNSName
Author Compounds;
R.K. Chang, M.L. Vieira, L. Liang,
Author Title
Author Company
P.P. Bhatt, A.B. Huang, and. S.V. Patel;
Supernus Pharmaceuticals, USA,
U.S. Patent # 10,149,853;
December 11, 2018.
This patent provides a formulation of molindone having
superior stability. The immediate release dosage form of
molindone is marketed as Moban®, which extensively and
rapidly metabolized with an oral dose plasma elimination
half-life of about two hours. It is taken three to four times
daily with a typical maintenance dose. The patent claims the
pharmaceutical formulation comprising molindone as single
active pharmaceutical ingredient and having modified release
comprising at least one release-controlling polymer and at
least one stabilizer. Optionally an additional formulation was
developed comprising molindone in an immediate release,
extended release or delayed release formulation for once a day
or twice a day administration.
Gold Complexes of
Alkylated Phosphines;
S.S. Al-Jaroudi, A. Alhoshani, M. Altaf,
and A.A. Isab; King Fahd University of
Petroleum and Minerals, Saudi Arabia;
U.S. Patent # 10,144,749;
December 4, 2018.
Present invention discloses a pharmaceutical composition and a
method of treating cancer. The pharmaceutical composition uses
complexes of gold and chemotherapeutic agents and inactive
carriers. The method disclosed here to treat cancer involves the
delivery of the above stated pharmaceutical composition. The
effect of the composition is measured by analyzing mutation
before and after the administration. This mutation is calculated
by measuring the concentration of substances produced in the
body by ELISA assay. These substances are known as biomarkers.
A decrease or increase in responses generated by biomarkers may
indicate that a disease is being treated. Examples of biomarkers
used to detect the presence of a disease or any other pathological
condition in the body include but are not limited to BRCA1, BRCA-
2 and Ki67.

82 | | January/February
November/December20192011

Multi-Ported Drug Delivery
Device Having Multi-Reservoir
Cartridge System;
F. Amirouche and M.L. Cantwell;
Picolife Technologies, LLC; U.S. Patent
# 10,130,759; November 20, 2018.
The present invention relates to a multi-ported drug delivery
device having a cartridge system with multiple interconnected
reservoirs that are independently actuated for delivery of
medicament(s). The device has a pump driver system, a cartridge
system, a cannula and an insertion mechanism, and a plurality of
conduits. Different reservoirs contain different medicaments. For
example, to treat diabetes, the reservoirs may contain insulin and
glucagon. The delivery of medicaments can be at a controlled
and continuous rate for a pre-determined or user-defined period
of time. Alternatively, the delivery of medicament can also be at
a programmable rate that is regulated by the patient. The device
can deliver micro-doses of medicaments.
Drug Device Configured for
Wireless Communication;
R.L. Altschul, N.D. Theise, R.A. Ene, M.
Rapkin, and R. O’Brien; Pop Test Abuse
Deterrent Technology, USA, U.S. Patent
# 10,137,288; November 27, 2018.
The inventors of the device describe it as a perfected “Smart Pill”,
which is able to prevent deaths by overdose of medicines. It is
also claimed to help decrease misuse and abuse of harmful drugs.
The smart pills disclosed in the patent are capsules which have
electrical sensors. These sensors send signals to the pharmacist
or the prescribing doctor. Through these signals it monitors
the time and duration of the medicine taken by patient. If the
patient accidently takes another capsule, the first capsule inside
patient will sense the presence of another capsule and built-in
sensor will not allow release of drug from the second. Through
this technology it may avoid unintentional overdosing. The
smart pills can have a variety of sensors like electronic, biological,
chemical, harmonic and digital. Through these sensors, smart
pills can be programmed to perform tasks such as preventing
drug-drug interactions, drugs of abuse, dose dumping by alcohol
consumption and teratogenic drugs during pregnancy.
« SECTION TITLE »
Child-Resistant Blister Package;
K.F. Trombley, K.L.T. Chan, I. Lafosse-Marin,
J.R. Morosey, Jr., K.M. Sanchez, and K.L.
Schmeichel; The Procter & Gamble Company,
USA; U.S. Patent
# 10,130,557; November 20, 2018.
Medicines are generally packaged in bottles, cartons, blister packages,
or other suitable packaging prior to use. Child-resistant features can be
added to reduce the risk of a small child accessing and ingesting the
medication. This patent is particularly directed to child-resistant blister
packages. Child-resistant features require a combination of dexterity
(special hand skill), strength, and intellect to operate. The child-resistant
blister package has a protection layer with a top face, a bottom face and
a periphery. It has a blister layer with one or more cavities and an access
layer with a top face, a bottom face, and a periphery. The bottom face of
the access layer has a line of weakness that allows the unit dose to be
removed from the cavity in one-step by applying a force to the top of
the cavity and pressing the unit dose through the line of weakness. The
bottom face of the protection layer and the top face of the access layer are
permanently joined along the entire periphery of the package. The blister
package can also contain a tear resistant layer.
Volatile Organic Compound Formulations
Having Antimicrobial Activity;
G.A. Strobel and B. Blatt; Ecoplanet
Environmental LLC, USA; U.S. Patent
# 10,117,841; November 6, 2018.
This patent invention includes novel chemical formulations having
antimicrobial activity and their methods of use. The fraction of human,
animal and industrial waste that is untreated gives rise to bacterial and
microbial growth, which poses a threat to health. Safer and effective
means for treating microbe-laden surfaces in medical or hospital
environments and for treating agricultural crops for unwanted microbial
growth are needed, in addition, reducing the unwanted odors produced
in the breakdown of fecal matter in industrial farming operations is
of utmost importance. The antimicrobial formulation invented in the
patent is made of propanoic acid, iso-butyric acid, isoamyl hexanoates
and a carrier selected from the group consisting of bentonite, zeolite
and perlite. This formulation effectively reduces microorganisms and
the effects of microbial outgrowth in a wide range of industrial settings,
as well as in methods of human and animal waste treatment.
www.americanpharmaceuticalreview.com | | 83
» ADVERTISER'S INDEX »

INTERPHEX 2019
Javits Center, NYC
April 2-4, 2019
CALL FOR EDITORIAL
American Pharmaceutical Review will be publishing an INTERPHEX Innovations guide which will be
included in the March 2019 issue.
If you are exhibiting at the show and introducing a new product or service, we would like to tell our
readers about your newest innovations for the pharmaceutical industry.

Please submit the following: Please send material to:

1. A press release of approximately 150 Michael Auerbach
words describing the new product or Editor in Chief
service along with a digital photo of at American Pharmaceutical Review Journal
least 300 dpi. Please include your Booth mauerbach@comparenetworks.com
Number and company website. If you have any other questions please
2. Contact information to include company contact the Editor at the above email address.
name, address and contact person.
(Contact person information will not be Deadline for all information is:
published but used for reference in case February 22nd, 2019
we need to contact you).

Company Page # Web Address

Ajinomoto Althea, Inc CV2 www.AjiBio-Pharma.com

Associates of Cape Cod 5 www.acciusa.com

B&W Tek 19 www.bwtek.com/NewQTRam

Catalent (Orientation) CV4 www.catalent.com

CPhI Japan CV3 www.cphi.com/japan

Eurofins Lancaster Labs CV2 www.eurofins.com/biopharma

Excipient World Conference & Expo 35 www.ExcipientWorld.org

MilliporeSigma 10, 11 www.EMDMillipore.com/pyromat

Prefilled Syringes East Coast 17 www.pfsamericas.com

PTI 13 www.ptiusa.com

SUEZ 2 www.sieversinstruments.com

WITec 21 www.witec.de

The contact directory is for information purposes only. While every effort has been made to ensure it is accurate and complete, any errors or omissions are not the responsibility of the publisher.

84 | | January/February 2019
 18-20 March 2019 | Tokyo, Japan

Japan’s pharma market awaits you

Grow your business in Japan

3 days of exhibition
Learn
More than 200 seminars
and conferences!
8 zones covering the entire supply chain CPhI Japan hosts a wide range of FREE
and paid seminars and conferences
from the government, industry
20,000+ 550+ 65+ associations and other key players
attendees exhibitors countries
“Attending CPhI Japan was a
PLUS: FREE ACCESS to good chance to get in contact
with Japanese companies and to
Japan Life Science Week understand the Japanese pharma
A week full of events alongside CPhI Japan industry and trends. We are
as the seedbed of the growing Japanese looking forward to attending this
healthcare and pharma industry. wonderful exhibition again and
to exchange our experience with
NEW local Japanese companies.”
ZON
for 2 ES ANDREA SALVADORI, Sales Area Manager, PQE
019

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