Volume 25 Issue 4 | May/June 2022

SHOW ISSUE

CRS 2022 Annual Meeting

Microscopy &
Microanalysis 2022

Optimizing a
Viral Testing Strategy
Assuring Quality of
Oligonucleotide APIs and DPs
A Discourse on
Pharmaceutical cGMP
FDA Form 483 Trends
www.americanpharmaceuticalreview.com
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 May/June 2022 | Volume 25, Issue 4

COVER FEATURES
46 MICROBIOLOGY
Optimizing a Viral Testing Strategy
Dr. Tim Sandle, PhD
Head of GxP Compliance and Quality Risk Management
Bio Products Laboratory Limited

52 BIOPHARMACEUTICAL
Assuring Quality of Oligonucleotide APIs and DPs
Carla Luciani
Engineering and Material Sciences
Vertex Pharmaceuticals

72 MANUFACTURING
A Discourse on Pharmaceutical cGMP FDA Form 483 Trends: Why are We Re-Living the Same Issues
Over the Last 23 Years?
Steven J. Lynn, MS
Executive Vice President, Pharmaceuticals
Regulatory Compliance Associates Inc.

www.americanpharmaceuticalreview.com | | 3
IN THIS ISSUE »
10 BIOPHARMACEUTICALS
QC CORNER
Long-Acting Injectable Suspensions
28 Affinity Approaches to Selective, Sensitive MS Assays
René Holm
Sponsored Content from MilliporeSigma
University of Southern Denmark
Department of Physics, Chemistry and Pharmacy
18 ROUNDTABLE
VENDOR VIEWPOINTS
Controlled Release
63 Combining the Best of Both Worlds with
Semi-Targeted Metabolomics
30 FORMULATION AND DEVELOPMENT
Bashar Amer, Metabolomics Application Development
The Promise and Challenges of mRNA Vaccine Development Thermo Fisher Scientific
Robert Dream 66 Method Changes for Bacterial Endotoxins Testing (BET): Steps to
HDR Company LLC Follow for a Straightforward Process
Hayden Skalski
36 DRUG DELIVERY
SUEZ Water Technologies
Introduction to mRNA-LNPs, Their Manufacture and Future Perspectives
Dr. Philip M E Probert, Head of Technical
Dr. Juliana M Haggerty, Head of LNP Centre of Excellence REGULAR FEATURES
Dr. John M Liddell, Chief Technologist
Mr. Graham Worrall, Chief Technologist 6 Message from the Editor
Centre for Process Innovation, UK
7 Editorial Advisory Board

42 BIOPHARMACEUTICALS
An Interview with Tony Saavedra
68
A Next-Generation Workforce for Next- Generation Therapies Preparing Sievers DataShare Elite Stoftware
for Cell and Gene Therapies
70 Editor's Top Tech
Emily Moran, Vice President, Viral Vector Manufacturing
The Center for Breakthrough Medicines (CBM) 79 Equipment Focus

60 FACILITY TOUR 86 P.I.N. Points

Growing to Serve: Eurofins’ Portage, Michigan, Facility Expands to Offer
88 Advertiser's Index
New Capabilities and Services

80 MICROBIOLOGY SPECIAL FEATURES

Cleaning and Disinfection: An Important Pillar of Contamination Control 8 CN Perspectives

Ratul Saha, PhD, Director, Contamination Control 9 Social Media Connections
Seres Therapeutics

4 | | May/June 2022
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 » Message from the Editor »
Monsters Among Us
If you have kids, or grandkids, know any kids, or are a kid at heart and haven’t seen Monster’s Inc. you
owe it to that kid in your life to watch this movie with them.

The CG animated movie is based in an alternate reality (of course) where monsters use bottled children’s
screams to power their society. Monster’s Inc. is the name of the company that bottles the children’s
screams. John Goodman voices the character of Sulley one of the best “scarers” and Billy Crystal is Mike
Wazowski, Sulley’s “handler” and best friend. Monster’s Inc. serves up children’s closet doors, through
which the “scarers” go into the kids’ bedrooms while they are asleep and scares them. The scream gets
collected, the scarer leaves quickly – and the process repeats itself.

The twist in all this (among others) is that the monsters are just as afraid of the kids they scare as the kids
are afraid of the monsters. Of course, one of the kids gets into the monster’s world and Sulley and Mike
try to figure out how to get her back, leading to this classic exchange:

Mike: I think I have a plan here: using mainly spoons, we dig a tunnel under the city and release it
into the wild.
Sulley: Spoons?
Mike: That's it, I'm out of ideas. We're closed. Hot air balloon? Too expensive. Giant slingshot? Too
conspicuous. Enormous wooden horse? Too Greek.

Thinking of this movie quote led me to wonder: are we out of ideas? Have the things that scare us won?

The COVID-19 pandemic is well into its third year. And, while the vaccines and treatments are showing
there is hope, the variants that continue to reveal themselves cause great worry. Not only because we
have to determine if the current vaccines are still effective, but perhaps more troubling and scarier, is
that it seems most people have moved on. Mask wearing is way down, I’m sure vaccination rates are way
down too. I feel another surge is right around the corner. If not in full swing right now.

Should we talk about Monkeypox? Or climate change? The war in Ukraine? Mass shootings?

Monsters – every one of them.

Have we, as a society, had scary times in the past? Of course. Have we overcome them? Yes.

Can we do it this time around? I sure hope so.

As I write this, summer is right around the corner. Usually, summer time means a slowing down, a time
of respite, a time to enjoy the nice weather. Maybe time to take a vacation.

I truly hope this season gives us a respite from the monsters we have been dealing with. And also gives
us a chance to recharge, to come up with new ideas to fight what makes us fearful.

As Mike Wazowski found out, you can’t use spoons to fix what frightens you. Bigger tools are needed.

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

6 | | May/June 2022
 « Editorial Advisory Board »
Shaukat Ali, Ph.D. John Finkbohner, Ph.D. Shane R. Needham, Ph.D.
Technical Support Manager Director, Regulatory Affairs Laboratory Director
BASF Corporation MedImmune Alturas Analytics, Inc.

Ghulam Shabir Arain, Ph.D., CSci, CChem, Adam S. Goldstein Daniel L. Norwood, MSPH, Ph.D.
FRSC, FCQI Senior Manager, Clinical Purification, Operations/Development Distinguished Research Fellow
Managing Director
DGS Pharma Consulting Ltd., UK Genentech Boehringer Ingelheim Pharmaceuticals, Inc.

Katherine Bakeev, Ph.D. Davy Guillarme, Ph.D. David Radspinner

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

Walter Dziki, Ph.D. Ronald W. Miller, Ph.D., MBA Wayne K. Way, Ph.D.
Associate Research Fellow President , Technology Consultant Analytical Business Marketing Manager
Abbott Laboratories Miller Pharmaceutical MilliporeSigma

Stuart Farquharson, Ph.D. Ganapathy Mohan, Ph.D. Larry Wigman, Ph.D.

President & CEO Head of Global CMC Regulatory Affairs Principal Scientific Manager
Real-Time Analyzers, Inc. Merck, Sharp and Dohme Corp. Genentech

www.americanpharmaceuticalreview.com | | 7
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 resources:
Pharmaceutical Outsourcing, Biocompare, Labcompare, and Tablets & Capsules.

Data Utilization for Life Sciences: Looking Forward and Back to

Shape What’s Ahead
Over the course of the last two decades, the healthcare industry has witnessed the digitization and consolidation of data to improve scientific
research. In the past, information was siloed, disparate, and focused on health claims data. Today, digital health systems are turning to
representative, quality, and timely data to improve patient care in the increasingly digital world. This data trove is revolutionary, and the vision
is clear. Any health care system that has a vision that’s worthwhile has to ultimately have a goal of improving patient outcomes. Whether
it’s a health system designed to put patients at the center, the EHR for the caregivers, or if it’s a way of connecting to the companies that are
innovating therapies that could solve some of these diseases, the goal must be in improving the quality of life or the life expectancy of these
patients – all aspects where data plays a crucial role.
https://bit.ly/3M4ZjAD

PCR-Based Diagnostics: Beyond and Because of COVID

Nowadays, everyone is familiar with molecular diagnostics, or knows what a PCR test is, because of COVID. However important advances
were taking place in molecular diagnostics even before COVID.
Most molecular diagnostic assays are PCR-based. Which means they rely on amplification of specific genetic material where primers,
polymerase, and probes are added. According to a recently published report about molecular testing trends on ResearchAndMarkets.com,
the global molecular diagnostic market is predicted to grow from $19 to $30 billion in worth from 2021 to 2027.
Advances in infectious disease and cancer diagnostics, the main applications of molecular diagnosis, can be grouped as: (1) tests that provide
more answers (2) whole-genome sequencing/detection (3) point-of-care molecular testing. Clinicians and researchers interviewed concur
that although many of the innovations occurred pre-COVID, they were all accelerated because of COVID.
https://bit.ly/3M1t4Co

Achieving Energy-Efficiency in Academic Labs

Scientific research consumes a lot of energy. That’s a fact. From the experimental equipment to the analysis machines, to the need to maintain
airflow in a lab, to the general working environment of the lab, a lot of energy is used—especially in academic labs where the research
experiment can be performed over long timeframes, sometimes days. This doesn’t include the energy required to create the multitude of
chemicals found in a lab. As it stands, research labs are the second largest consumers of energy after data centers.
Academic labs have been pushing for greener solutions and reduced energy consumption over the years. As the impacts of excess energy
consumption have become more apparent, alongside the increase in energy costs year on year, the time to act on producing greener labs has
never been more urgent.
https://bit.ly/3MY8xjj

Raman Spectroscopy: Multiple Techniques Can Analyze

Pharmaceutical Tablets and Capsules
Raman spectroscopy provides a number of great features for pharmaceutical analysis. Depending on the specific Raman technique applied
and instrument employed, it offers a rapid and nondestructive full chemical analysis, as the Raman spectrum contains molecular signatures of
all the species in the sample (e.g. the pharmaceutically active ingredient as well as all the excipients). The general appearance of the spectrum
enables a qualitative analysis. Taking a closer look at the relative peak intensities allows the concentrations to be extracted. Utilizing appropriate
training data facilitates evaluation of individual spectra or even entire data sets in a fast and reproducible fashion.
https://bit.ly/3wVSDzb

8 | | May/June 2022
AMERICAN PHARMACEUTICAL REVIEW
Social Media Connections
FDA Drug Information
@FDA_Drug_Info
FDA recently revised the Emergency Use Authorization
(EUA) for Evusheld (tixagevimab co-packaged w/ cil-
gavimab) to include new info on hypersensitivity reac-
tions & the risk of cross-hypersensitivity w/ #COVID19
vaccines & related clinical recommendations:
https://go.usa.gov/xJrhq
U.S. Pharmacopeia
@USPharmacopeia
New analysis: Price drives shortage risk for #anticrobial
medicines in the US: http://ow.ly/vr4o50JlQMl
#supplychain
PhRMA
@PhRMA
Millions of Americans require life-long treatment due
to blood diseases such as Leukemia, anemia, and sickle
cell disease. We’re working on new medicines to help
improve their lives. Learn more about the threat of
blood diseases and the latest progress:
http://onphr.ma/3wBpsRJ
Health Canada and PHAC
@GovCanHealth
Canada is proud to begin a 3-year term on the
@WHO’s Executive Board where we will champion
action on WHO governance, emergency prevention,
preparedness, and response, along with health equity,
gender equality, and health determinants.
http://ow.ly/7Jhj50JjLbl
Connect with American Pharmaceutical Review at:
facebook.com/AmericanPharmaceuticalReview
twitter.com/ampharmrev
linkedin.com/groups/American-Pharmaceutical-Review-3889659
CDC
@CDCgov
Monkeypox doesn’t spread as easily as common
illnesses like COVID-19 or the flu. People can only catch
#monkeypox if they have close contact with someone
who is infected. Know what to look for.
https://bit.ly/3Gt4SaT
Pfizer Inc.
@pfizer
Today, we were joined by Rwanda, Ghana, Senegal, Malawi
& Uganda as we launched a new initiative to help close
the global health equity gap & enable sustained access
to medicines and vaccines in 45 lower-income countries.
More here: https://on.pfizer.com/3afNuKx #HealthEquity
#WEF22
U.S. Pharmacopeia
@USPharmacopeia
Simply put, #standards save lives. Learn how in a new piece
by USP’s Lawrence Evans III, Senior Director, Global Health
Technical Programs. #globalhealth #maternalmortality
#WSWeek http://ow.ly/VZ0G50Jem6J
ECDC
@ECDC_EU
What will be the future #COVID19 health impact? We have
just launched the European COVID-19 Scenario Hub, that
will present modelling projections on how the COVID-19
#pandemic may evolve in terms of cases, hospitalisations
and deaths.
www.americanpharmaceuticalreview.com | | 9
» BIOPHARMACEUTICALS »

Long-Acting Injectable
Long Acting Injectables (LAIs) are a drug formulation option that
provide a slow and sustained release of the Active Pharmaceutical
Ingredient (API) after administration.1 LAI formulations have several

Suspensions
advantages relative to classical oral formulations, including reduced
frequency of administration, enhanced therapy adherence and patient
compliance, and potentially also a lower level of adverse effects, when
these are associated with peak plasma concentrations. Experience
from the antipsychotic area have shown that these attributes of
the LAIs often lead to improved therapeutic effect,2 whereby the
formulation approach provides improved quality of life for the patients
using them.3-7 The first LAI products were approved by the Food
and Drug Administration (FDA) in the 1950s, including formulations
René Holma* based on oil solutions and aqueous suspensions. Some products have
a
University of Southern Denmark, Department of Physics, Chemistry and Pharmacy, been approved since, but from 2000 and onwards a larger amount of
Campusvej 55, 5230 Odense, Denmark LAI products have been approved in multiple different therapeutic
*corresponding email address: reho@sdu.dk areas. LAIs are particularly relevant in areas with longer or chronic
treatment, such as schizophrenia, hormone replacement therapies,
immunodeficiency virus (HIV), and tuberculosis.8-11 There is no agreed
classification of the different LAIs in the academical literature, however,
they can roughly be categorized into four main formulation classes as
depicted in Figure 1, with some special systems not falling into any of
these categories.
The field of LAI formulations is a broad and very interesting
field, however, in the present article the focus will be on aqueous
suspensions, which is the most frequently used formulation
strategy for the marketed LAIs. A suspension is a dispersed multi-
phased heterogenous system of insoluble particles – for parenteral
applications primarily intended for intramuscular or subcutaneous
injection. Suspensions are inherently thermodynamic unstable,
and it is one of the most difficult parenteral formulations to both
formulate and also to manufacture. An aqueous suspension for
parenteral injection contains up to seven different component types,

Figure 1. Graphical representation of the main

formulation types used as long acting injectables.

10 | | May/June 2022
 OPTIMIZED DRUG SOLUBILITY AND STABILITY
Captisol is the trade name for Ligand’s solvent-free processed modified cyclodextrin preparation.
Captisol is a patent-protected mixture of chemically modified cyclodextrins with a modifying
structure to optimize drug solubility and stability. Captisol was invented and developed
by scientists at the University of Kansas’ Higuchi Biosciences Center specifically for drug
development and formulation.

Captisol overcomes solubility and stability hurdles faced during each phase of development.
Captisol can make a substance more soluble and an agent more stable. Captisol can convert
a solid to a liquid or an oil to an aqueous solution. Combinatorial chemistry, high throughput
screening (HTS), and molecular genetics have led to an increase in the number of insoluble and
unstable molecules, peptides, and proteins being investigated for their therapeutic activity. There are
currently more than 50 Captisol-enabled products in clinical development. This unique technology
has enabled several FDA-approved products, including Amgen’s KYPROLIS®, Baxter International’s
Captisol, NEXTERONE®, Gilead’s VEKLURY®, Acrotech Biopharma L.L.C.’s and CASI Pharmaceuticals’
A Ligand EVOMELA®, Melinta Therapeutics’ BAXDELA™ and Sage Therapeutics’ ZULRESSO™. There are

Technology many Captisol-enabled products currently in various stages of development.

3911 Sorrento Valley Road SEAMLESS TRANSITION TO CLINICAL TRIALS

Suite 110 Captisol may increase systemic exposure for toxicology studies of investigative compounds
San Diego, CA 92121 and has a proven clinical safety record. In early development, Captisol formulation can lead
877-575-5593 to a seamless transition from nonclinical safety to clinical trials. Captisol-enabled products are
cdinfo@captisol.com approved in more than 60 countries.

MULTIPLE ADMINISTRATION ROUTES ENSURE

TARGETED DELIVERY
Captisol’s chemical structure was designed to create new products by improving solubility,
stability, bioavailability, and dosing of active pharmaceutical ingredients. Routes of
administration investigated include parenteral, oral, ophthalmic, nasal, topical, and inhalation
products. Once inside the body, Captisol releases the drug agent, which then travels to its
target. The interaction between Captisol and the agent is not permanent, and Captisol is safely
expressed from the kidneys.

PATENTED AND VALIDATED MANUFACTURING

Of all modified cyclodextrins, Captisol is an ingredient in the most approved products in the U.S.
Manufactured under cGMP, at multiple locations, using a patented and validated all-aqueous
process, annual manufacturing capacity is being increased to 500 MT. Captisol is supplied in ultra-
low endotoxin, ultra-low bioburden, low-chloride forms in 100g, 1kg, 5kg and 20kg packages for
R&D use. Commercial pack sizes include 1kg, 5kg, and 20kg, with the ability to fill metric-ton orders.
» BIOPHARMACEUTICALS »
see Table 1, which should be defined as a part of the formulation
work, as appropriate.
Table 1. Formulation components of an
aqueous suspension for parenteral injection,
examples taken from commercial LAI suspensions.
Component Example
Active pharmaceutical ingredient (API) Aripiprazole
Vehicle/dispersing media Water for injection
Stabilizer Polysorbate 20
Resuspending agent PVP K17
Buffer Phosphate buffer
Isotonicity agent NaCl
Cryoprotectant Mannitol
Compounds Suited for an
Aqueous Suspension
For a compound to be formulated into an aqueous suspension one
of the critical properties to ensure physical stability and to obtain
the desired release rate is lack of solubility in the dispersing media,
i.e., the compound should be relatively insoluble in both water and
the micelles a potential stabilizer may form. The compounds used
in current commercial LAI products are for the majority compounds
that have been used orally for a period of time and then at a later
stage been used in a LAI formulation. These compounds have clearly
lacked the right physical chemical properties for a suspension, as
most have been adjusted by conversion of the drug into a different
solid form than used in the tablet or a prodrug. Esterification of an
alcohol group with fatty acids have been the general approach. Esters
can be hydrolyzed in vivo by the various esterases present at the
administration site. The in vivo drug release is thereby driven by the
slow dissolution rate of the crystalline prodrug, which is subsequently
rapidly hydrolyzed. This provides a sustained plasma concentration
profile of the active drug, but with negligible concentrations of the
prodrug in the systemic circulation.12-14 The atypical antipsychotic
paliperidone (PAL) prodrug, has been developed based on this
principle. The compound is esterified with palmitoyl ester and used
in the aqueous LAI suspension. Similar examples exist of aripiprazole,
where an N-methyl-ester have been formed to get a two months
release relative to the one-month release for the hydrate of the parent
drug molecule. Another way to decrease the aqueous solubility of a
drug is by salt of solvate formation. Alternative new solid forms can be
considered, which have been done in the olanzapine and aripiprazole
LAI formulation.
Formulating an Aqueous Suspension
A critical formulation parameter for LAI suspensions is the particle
size, as this defines the dissolution rate. Reducing the drug particle
size results in an increased surface area, which leads to an increased
dissolution rate, as described in the modified Noyes-Whitney equation,
12 | | May/June 2022
i.e., Nernst-Brunner equation, see equation 1. where dC/dT is the drug
dissolution rate, D the diffusion coefficient, S the surface area, V the
volume of dissolution medium, h the diffusion layer thickness, Cs the
saturation concentration, C the concentration in the solvent, and t
time. The size needs to be adjusted to the individual compound and to
the release duration aimed for, e.g., three months.
Eq. 1
This size has to be defined experimentally as the Nernst-Brunner
equation can only be used conceptually to explain the concept, not
accurately to calculate the particle size that would match the needed
release rate. The particle size will vary as a function of both the release
rate and the compound. For instance, the size of medroxyprogesterone
acetate in the epo-Provera formulation have a mean particle size (Dv50)
of around 10 μm, whereas cabotegravir in the Vocabria formulation is
around 200 nm.
As mentioned above, suspensions are inherently thermodynamically
unstable, hence they can potentially be subject to a number of
physical instability phenomenon’s, see Figure 2. To prevent this
a stabilizer/surfactant is added to the formulation. The main
physical instabilities include particle interaction which may lead to
flocculation, agglomeration or Ostwald Ripening dependent upon
the way the change occurs. Some of these changes are reversible, e.g.,
sedimentation, others irreversible, e.g., Ostwald ripening. Stabilizers
help prevent flocculation, coalescence and Ostwald ripening, which
sometimes can be sufficient to stabilize the suspension. If the stabilizer
is not sufficient it is possible to lower the chemical activity in the system
by increasing the viscosity, i.e., adding a viscosity inducing agent such
as sodium carboxymethylcellulose (sodium CMC) and if not sufficient
the addition of a thixotropic excipient as Avicel CL-611 may help and
as a last resort lyophilization can be considered. In short, a thixotropic
system, is a gel when not exposed to extensive physical stress, e.g.,
shaking, but when exposed to physical stress it becomes like a liquid.
This means that the suspension would be almost physically locked
when on the shelf but can be activated into a liquid by the user or
health care provider just before administration. Temperature is also
an important parameter to consider for dispersed systems, when
selecting stabilizers and when defining the storage temperature.
As described above, stabilizers, needs to be defined to e.g., prevent
particle size growth in the suspension during shelf-life and to
prevent caking, which would complicate resuspendability before
administration. To support this, properties such as zeta potential,
rheology, are particle size distribution are important parameters that
need to be followed also as a part of the formal stability study. The
stabilizers used should preferably, be well tolerated, and approved
for parenteral use, however, as only a limited number of stabilizing
excipients have been approved for parenteral use it may be needed
to look beyond this point for a specific compound. Approved
stabilizers include polymers such as cellulose derivatives (e.g.,
HPMC, HPC), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) and
polyethylene glycols (PEG) and surfactants such as polysorbate 20
and 80, poloxamers 188, sorbitan monooleate (Span 80), and D-alpha-
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Figure 2. Schematic representation of potential physical instability phenomenons that disperse systems, including suspensions, may
undergo. Outer images are aged dispersions and the equation the current mathematical understanding of the conversion process.
tocopheryl polyethylene glycol 1000 succinate (TPGS), and lecithin.
Combination of these is obviously a possibility, which provide a lot
of potential options to investigate. The stabilizing excipient selection
can be challenging due to the lack of a fundamental understanding of
their interactions within suspensions, where the DLVO theory forms
the fundamental understandings. The stabilizers are first chosen for
its ability to stabilize the suspensions from physical instability, but
important for the formulator to consider is the potential impact the
stabilizer may also have on the in vivo performance of the suspension,
which may be influenced by a surfactant.15,16 The drug to stabilizer ratio
seen in literature has been described to vary from 1:3 to 50:1,17,18 but
specific compounds and stabilizer combinations may not necessarily
fall inside this range.
The concentration of the solid (active pharmaceutical ingredient) in
the suspension can vary significantly, from single digit %w/v up to
50% w/v, but as a good rule of thumb, achievable concentrations for
most compounds will be in the range of 200-300 mg/mL. Beyond the
stabilizer and viscosity increasing excipient isotonic agents, buffers,
antioxidants etc. should also be considered.
Manufacturing an
Aqueous Suspension
Reduction of particle size for suspensions for injection can be
produced either by the top-down or the bottom-up methods. The
production method will influence particle characteristics, such as
particle size, crystallinity and surface properties and potentially
14 | | May/June 2022
also the selection of stabilizer. Therefore, the chosen method of
production may play a role on drug suspensions stability and
composition. The bottom-up approach includes precipitation of the
compound into the desired particle range, which may be obtained
by the anti-solvent method. Here the compound is solubilized in
an organic solvent, when introduced into an aqueous media it will
crystallize out. While no commercial products are currently produced
by this approach, developments in crystallization technology may
change this in the future.
The top-down approach is the current state-of-the-art method to
obtain particles of reduced size and all current marketed parenteral
suspension products are manufactured using top-down methods,
which produce the desired particle sizes from bigger sized particles.
The most used top-down techniques are wet and jet milling, and
homogenization using rotor stator or high-pressure homogenization
(HPH).19,20 In the case of HPH, the suspension goes through either
a jet-stream homogenizer or a piston gap homogenizer. Wet ball
milling is obtained by milling the suspension with beads, where the
grinding is obtained by rotation or other methods of mechanical
motion of the bulk.
For parenteral suspensions sterility should be considered, i.e., should
the compound be isolated sterile or should it be gamma-radiated after
crystallization. The vehicle that goes into the formulation should be
sterile as well, which can be obtained by several methods.
However, dispersion systems are in general challenging as terminal
heat sterilization is normally not an option, as it would destabilize the
formulation physically and thereby reduce the quality of the product.
» BIOPHARMACEUTICALS »
Defining a sterile process for a disperse formulation is therefore a
special case and needs to be truly considered both in small and large
scale, i.e., what can be autoclaved, where can incubators be used, etc.
The LAI Development Process –
the Project Management Part
Developing a formulation that releases the compound for a longer
period of time needs to be done based upon diligent project planning,
which would look very different when compared to plans used for
development of more conventional formulations, where some of the
considerations are mentioned below. It is beyond the scope of the
present article to go into the detailed considerations for every step
of the development process, hence this section below should be
considered as a list for inspiration that is not exhaustive. Some of the
important elements to consider include:
• Is there a clinical rational for a LAI and is the compound suited
in a LAI?
• Compound or compound solid form
• Defining a formulation and how to obtain sterility
• In vitro dissolution studies and nonclinical studies to support the
formulation development
Any project requires a clinical rational – as does an LAI project. If there
are no benefits for patients, caretakers, healthcare professions or soci-
ety then there is obviously not a project. However, as mentioned in the
introduction experience from the antipsychotic space teaches that the
patients will get a more effective treatment with LAIs, so there is reason
to believe that the same experience could be obtained in other thera-
peutic areas. Having said this, there will of course be cases where these
arguments do not apply, hence it makes sense to consider the potential
benefits for all new LAIs before initiating the development work.
The compound is for most LAI projects preselected, as in most cases
the compound has already been approved for oral usage. This means
that information exists of the dose and clearance. A LAI formulation
induces flip-flop pharmacokinetics; hence it can be evaluated in silico
if there is a likelihood of using the compound in a LAI formulation
reaching the clinically relevant plasma concentrations, see e.g., Rajoli
et al.3 that have evaluated the possibility of converting four approved
tuberculosis compounds into a LAI. It must be stressed that this
evaluation cannot be translated into a formulation description, but
only a risk evaluation for the project.
While the compound may be predefined, the physical form of
the molecule used in the oral formulation may not be suited for a
suspension, i.e., it may be too soluble which would lead to a fast
release and a physically unstable suspension. The physical properties
of the active compound may hence need to be adjusted by selection
of a hydrate or a less soluble salt and if this does not work as a last
resort a prodrug modification. As discussed above, this has been done
on multiple compounds used in antipsychotic treatments, where both
an oral and a LAI option is available, e.g., olanzapine and aripiprazole.
16 | | May/June 2022
These activities need to be diligently planned and linked up to some
formulation feasibility work to select the right form or prodrug.
Formulation development of suspensions is largely described above,
however, it is associated with a lot of experience-based decisions as
there is a lot of scientific fundamental gaps in the field, e.g., defining a
physically stable suspension and the suspension particle size to obtain
the defined release rate is highly empirical.
The selection of the formulation needs support from both in vitro
dissolution studies and nonclinical evaluations. With respect to the
in vitro dissolution method, there is not much available support in
the literature to develop the method, but there may be available
organizational experience to support this approach if not it will be an
important part of the development work – to develop discriminating
in vitro dissolution methods for qualitative purposes. If a level A in vitro
in vivo correlation can be obtained it would lead huge benefits for the
project during development and after potential marketing.
Besides the in vitro work extensive in vivo work in nonclinical models
should be considered – the advice would be to investigate as many
factors as possible in the development phase to clarify the critical
formulation parameters before entering clinical trials. Changing
the formulation after start of clinical trials is not an easy task and it
would take a considerable amount of time to do so due to the very
extended-release profile. It is also highly advised to plan for more than
one formulation in the initial human pharmacokinetic studies to risk
deviate the project.
While a larger nonclinical package may be collected, the translatability
into a human pharmacokinetic profile is not guaranteed and due
to the inherent lack of scientific understanding of a suspensions
physical stability this may also at a later timepoint make it needed
to reconsider the selected formulation. Altogether from the start
of the development to first-in-man it can easily take more than two
years dependent upon the number of factors that needs clarification;
however, it is the authors clear opinion that undertaking the effort
will have a large and beneficial impact on the patient’s life that we
serve as formulators and industry.
References
1. Kim YC, Min KA, Jang DJ, et al. Practical approaches on the long-acting injections. J Pharm
Investig. 2020(50)147–57.
2. Okoli CTC, Kappi A, Wang T, Makowski A, Cooley AT. The effect of long-acting injectable
antipsychotic medications compared with oral antipsychotic medications among people
with schizophrenia: A systematic review and meta-analysis. Int J Ment Health Nurs.
2022;31(3):469-535.
3. Ascher-Svanum H, Faries DE, Zhu B, Ernst FR, Swartz MS, Swanson JW. Medication
adherence and long-term functional outcomes in the treatment of schizophrenia in usual
care. J Clin Psychiatry. 2006;67:453-460.
4. Nikam KR, Pawar MG, Jadhav SP, Bairagi VA. Novel trends in parenteral drug delivery
system. Int J Pharm Technol. 2013;5:2549–2577.
5. Kovarova M, Benhabbour SR, Massud I, et al. Ultra-long-acting removable drug delivery
system for HIV treatment and prevention. Nat Commun. 2018;9:4156.
6. Marmora L, Casas CP, Grubb I, McClure C. Long-acting technologies for infectious diseases
in LMICs. Lancet. 2018;392:1610–1611.
 « BIOPHARMACEUTICALS »

7. Nachman S, Townsend CL, Abrams EJ, et al. Long-acting or extended-release antiretroviral 16. Chamanza R, Darville N, van Heerden M, De Jonghe S. Comparison of the local tolerability
products for HIV treatment and prevention in infants, children, adolescents, and to 5 long-acting drug nanosuspensions with different stabilizing excipients, following a
pregnant and breastfeeding women: knowledge gaps and research priorities. Lancet HIV. single intramuscular administration in the rat. Toxicol Pathol. 2018;46(1):85-100.
2019;6:e552–558.
17. Patravale VB, Date AA, Kulkarni RM. Nanosuspensions: a promising drug delivery strategy.
8. Hirsch SR, Gaind R, Rohde PD, Stevens BC, Wing JK. Outpatient maintenance of chronic J Pharm Pharmacol. 2004;56(7):827-840.
schizophrenic patients with long-acting fluphenazine: double-blind placebo trial. Report
18. Van Eerdenbrugh B, Van den Mooter G, Augustijns P. Top-down production of drug
to the Medical Research Council Committee on Clinical Trials in Psychiatry. Br Med J.
nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid
1973;1:633–637.
products. Int J Pharm. 2008;364(1):64-75.
9. Kaunitz AM. Injectable long-acting contraceptives. Clin Obstet Gynecol. 2001;44:73-91.
19. Keck CM, Müller RH. Drug nanocrystals of poorly soluble drugs produced by high pressure
10. Remenar JF. Making the leap from daily oral dosing to long-acting injectables: lessons from homogenisation. Eur J Pharm Biopharm. 2006;62(1):3-16.
the antipsychotics. Mol Pharm. 2014;11:1739–1749.
20. Patravale VB, Date AA, Kulkarni RM. Nanosuspensions: a promising drug delivery strategy.
11. Swindells S, Siccardi M, Barrett SE, et al. Long-acting formulations for the treatment J Pharm Pharmacol. 2004;56(7):827-840.
of latent tuberculous infection: opportunities and challenges. Int J Tuberc Lung Dis.
2018;22:125–132.
12. Remenar JF. Making the leap from daily oral dosing to long-acting injectables: lessons from
the antipsychotics. Mol Pharm. 2014;11(6):1739-49. Author Biography
13. Cleton A, Rossenu S, Crauwels H, et al. A single-dose, open-label, parallel, randomized,
dose-proportionality study of paliperidone after intramuscular injections of paliperidone Dr. René Holm is a professor in pharmaceutical physical chemistry at
palmitate in the deltoid or gluteal muscle in patients with schizophrenia. J Clin Pharmacol. the University of Southern Denmark, a position he has held since April
2014;54(9):1048-1057.
2021. Before that he worked for more than 20 years in the pharmaceutical
14. Rossenu S, Cleton A, Hough D, et al. Pharmacokinetic profile after multiple deltoid or
industry. Professor Holm’s research is in the field of long acting injectables
gluteal intramuscular injections of paliperidone palmitate in patients with schizophrenia.
Clin Pharmacol Drug Dev. 2015;4(4):270-278. (LAI) for different modalities with a focus on the science behind the

15. Wu L, Zhang J, Watanabe W. Physical and chemical stability of drug nanoparticles. Adv formulation systems, which will help de-risk the development process of
Drug Deliv Rev. 2011;63(6):456-469. LAI projects.

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» ROUNDTABLE »

Controlled Release
John Tillotson Shannon Kelly
Pharmaceutical Technical Business Director Vice President Business Development and Formulation Technology
ABITEC Corporation Colorcon

Nathan Dormer Bikash Chatterjee

Director of Drug Product Development CEO
Adare Pharma Solutions Pharmatech Associates—a USP company

Robert J. Bloder Dr. Ali Rajabi-Siahboomi

Chief Business Officer Vice President - Chief Innovation Officer
Ascendia Pharmaceuticals Colorcon

Sandip Tiwari, PhD Jim Huang, PhD

Head of Technical Services- NA CEO
BASF Pharma Solutions Ascendia Pharmaceuticals

Brent Moody Hayden Skalski

Director Science and Technology Lead Product Application Specialist – Biodetection
Catalent Sievers Analytical Instruments

Tom Quinci
Director of New Business Development and Partnering
Celanese

Commercial products with more predictable manufacturing

Looking back at the past year, how has the pandemic
and associated supply chain issues affected the
development of controlled release products?
John Tillotson, Pharmaceutical Technical Business Director,
ABITEC Corporation: Primarily, the pandemic has challenged supply
chain logistics and increased the manufacturing costs of numerous
excipients including controlled-release excipients. This includes the
cost of developing controlled release products.
Nathan Dormer, Director of Drug Product Development, Adare
Pharma Solutions: The pandemic has invariably affected every
industry’s supply chain and Adare Pharma Solutions has been
no exception.
campaigns and backup raw material vendors have generally fared
better than new research and development programs, since long-term
planning and risk mitigation is integral to marketed products.
For new formulations undergoing prototyping, where excipients are
less defined and constantly evolving, procuring samples or meeting
minimum quantities has come with occasional delays. Overall, our
equipment and material vendors have been very accommodating,
and we are grateful for efforts to maintain availability of their products.
Robert J. Bloder, Chief Business Officer, Ascendia Pharmaceuticals:
Ascendia Pharmaceuticals has responded to the supply chain
disruptions in a number of ways to protect our clients and projects.
We have enhanced the communication and timing pertaining to our
component suppliers and set-up processes to expedite and ascertain

18 | | May/June 2022
» ROUNDTABLE »
long-lead time items. We have also forged lasting relationships with
qualified vendors and continue to build redundancies/back-up
suppliers as needed. Ascendia champions transparency and openness
with our client partners and suppliers.
Sandip Tiwari, Ph.D., Head of Technical Services- NA, BASF Pharma
Solutions: The COVID-19 pandemic has caused severe disruptions to
the global economy including the pharmaceutical industry, which
exhibited a decline in overall activity including development during
the lockdown because of the shutdown of manufacturing and R&D
facilities, acute shortage in the supply of raw materials/packaging
components and absence of potential manpower including forced
remote working, lack of effective disinfection and personal protection
equipment strategies for the new viral infection.
Though supply chain activities have experienced challenges during
the pandemic, the pharmaceutical industry is projected to gradually
recover post-COVID-19, which will present attractive opportunities
for development of novel treatment modalities and controlled release
products beyond small molecules including nucleic acids, peptides,
proteins and antibodies.
Brent Moody, Director Science and Technology, Catalent:
Unfortunately, supply chain challenges have become a way of life across
all industries, and pharmaceutical development is no exception. Partly,
this has been driven by demand. From the earliest days of the pandemic,
Catalent observed a marked increase in the repurposing of old drugs
(or drugs originally intended for other indications) against COVID-19
as well as other emergency, chronic, and general health care needs.
Often, this has required modifying the pharmacokinetic profile through
controlled release formulation. This led to a corresponding increase
in demand for raw materials used in controlled release formulations,
processing equipment and maintenance capabilities, and not least
of all, for formulation development and manufacturing know-how.
Fortunately, Catalent has been able to mitigate some of the impact
through a robust and well-established network of vendors. Redundancy
in qualified vendors, libraries of equivalent materials, and an agile global
warehousing network have allowed us to meet the increasing demand
and supply chain pressures. We have also relied on our global network
of formulation scientists to meet the timeline pressures associated with
increased demand for modified release product expertise.
Tom Quinci, Director of New Business Development and
Partnering, Celanese: Development of controlled release products
was directly impacted due to reduced staff working in formulation and
analytical laboratories, while companies shifted focus to address the
pressing issues of meeting their own supply demands. Accordingly,
projects have been reprioritized and are taking longer in development,
with some programs placed on hold. I have seen more development
outsourced to CROs and CDMOs to keep projects moving forward.
CROs and CDMOs have stepped in as a solution to help pharmaceutical
companies extend their development activities without accruing
additional fixed overhead. Many large companies are doing what
start-ups have been doing for years—conducting development
outside of a brick-and-mortar infrastructure. Especially in the areas
of early development and early proof of concept work, third party
providers can accelerate capacity to explore new formulations, new
technologies, and new drug delivery platforms. Celanese announced
20 | | May/June 2022
an expansion of our Feasibility & Development Lab to support
preclinical R&D, and we anticipate that demand for early development
support will continue to grow as the model proves to provide benefit.
Shannon Kelly, Vice President Business Development and
Formulation Technology, Colorcon: The pandemic has highlighted
vulnerabilities through the supply chain, from the starting materials
through the entire logistics chain to the patient. The impact is not
limited to materials and the supply chain as companies have struggled
to keep R&D functioning due to personnel being impacted through
lockdown and travel restrictions.
Companies are now looking for reliable supply closer to manufacturing
locations and limiting the movement of products for further steps such
as packaging and distribution to reduce risk. They are paying more
attention to sustainability and the impact of materials, technology,
and manufacturing locations.
Colorcon was in a unique position to continue to service the market
through the early adoption of virtual platforms. Throughout the
pandemic we utilized digital technology to collaborate and continue
to support our customers through their formulation and development
from lab to scale-up.
In general, what are some notable industry trends
regarding controlled release product development
that you have noticed over the past year?
Tillotson: The trends are towards advanced technologies in controlled
release, such as combined release modalities including magnetic
controlled release and light controlled release with nanoparticles.
Additionally, targeted controlled release with lipid nanoparticles is an
area that continues to be of interest.
Dormer: From the perspective of a technology driven CDMO,
Adare Pharma Solutions is experiencing growing interest and de-
mand for developing patient centric medicines by leveraging our
solution portfolio.
Focus is on formulation development for vulnerable pediatric and
elderly patients, but dysphagic patients likewise require a tailored
formulation strategy.
In fact, multi-particulate formulations combining controlled release
properties and eases of swallowing require the application of
specialized, compatible technologies.
Bloder: Over the past year, Ascendia has noted an increased demand
for controlled release expertise for sterile and oral product dosage
forms. We are fortunate in that our technologies cover dosing regimens
that range from bi-monthly to once a year for Long-Acting Injectables
for CNS and cancer treatments. Additionally, we are experiencing an
increase in demand for lipid nanoparticles for use in sustained release
of small molecules and biologicals.
Tiwari: The rationale for development of a controlled release
product of a drug is to enhance its therapeutic benefits, minimizing
side effects, enhanced patient compliance while improving the
management of the diseased condition and product life cycle. Given
the benefits of the controlled release products and emerging need of
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» ROUNDTABLE »
repurposing existing drugs for the treatment of various viral diseases
post COVID-19, several novel drug delivery systems and devices are
likely to be developed and commercialized in the near future both for
oral and parenteral applications.
Development of efficient therapies for chronic and non-
communicable diseases such as cancer, diabetes, and hypertension
are expected to boost the growth of controlled release products.
The rising aging population globally is one of the major reasons
contributing to the potential growth in this segment, largely due to
non-adherence to the medication regimen. Patient compliance to
medication is a challenge in the geriatric population and reducing
the dosing frequency is the key to compliance and management of
the diseased condition. Hence, the demand for controlled release
drug delivery systems is expected to grow with the rising patient
population with these diseases.
Moody: The pandemic has laid bare the need to better treat and
control chronic conditions which may lead to adverse outcomes
for patients. For example, a once-daily dosing option could
offer greater convenience, potentially optimize compliance, and
improve therapeutic outcomes for patients with chronic conditions.
Unfortunately, some of these are quite widespread, which has placed a
corresponding emphasis on scalability and continuous manufacturing
techniques to meet large-scale global demand. For example,
continuous processes such as Hot Melt Extrusion (HME) and twin-
screw granulation have been increasingly utilized in the production of
modified-release tablets. We have also witnessed increased demand
for modified release products for pediatric patients, who present
their own set of challenges, many of which vary with age. Finally, the
pressure to get promising new modified release therapies to patients
in an increasingly short time frame has led to an emphasis on the use
of in silico tools such as physiologically-based pharmacokinetic (PBPK)
modeling, to streamline product development.
Bikash Chatterjee, CEO, Pharmatech Associates—a USP
company: Patient centricity has emerged as a central component
of many drug sponsors’ development portfolio, translating to a
resurgence of interest in the benefits of controlled and modified-
release dosage forms. Patient centric strategies encompass many
solutions according to what facet of the patient interaction you are
trying to address or optimize but in general the intent is to improve
the convenience and safety of administration, patient compliance
with treatment, and general patient engagement. Because of this
new strategic interest, controlled- and modified-release drug design
has moved from a patent extension strategy to one that is central to
marketplace advantage. Pediatric and geriatric markets alike have
specific challenges with treatment compliance considering taste and
swallowability considerations. For treatment regimens that require
treatment multiple times a day, a controlled release solution can
dramatically reduce compliance issues. Patient centricity in a drug
sponsors portfolio development will increase in importance in the
foreseeable future with controlled release design and technology
solutions at the center of these strategies.
Dr. Ali Rajabi-Siahboomi, Vice President - Chief Innovation Officer,
Colorcon: Many technologies that are used for oral controlled release
formulations have been around for a long time, especially matrix
tablets, multiparticulate formulations and osmotic technologies.
22 | | May/June 2022
These are still widely used, as companies have gained knowledge and
experience with their formulation and manufacturing processes.
Developments have been seen through line extensions and
reformulations that allow patent extension and provide different
release profiles to improve patient acceptability, dosing, and overall
therapeutic goals.
The use of extended-release technologies for Fixed-Dose Combinations
(FDC) has allowed a significant number of new drug applications
to be made under the 505(b)2 scheme with the US Food and Drug
Administration (FDA). Some of these may be a combination of
extended and immediate release in the same dosage form, like bi-layer
tablets, or a mix of barrier membrane-coated multiparticulates that
are loaded into a capsule. The FDC technology improves the patient’s
experience by reducing the pill burden (taking one dose rather than
two or more) and enables synergistic treatment of a condition with a
single dosage formulation.
The application of a barrier membrane coating, such as Surelease®,
ethylcellulose aqueous dispersion can be used to modify release
for both tablet and multiparticulate formulations. This top-coat
overcomes the so-called initial burst release for highly soluble drugs
which is important to maintaining controlled release of the API (Active
Pharmaceutical Ingredients). When applied for taste-masking, where
dissolution and taste profile contribute to the acceptability criteria,
the coating provides a barrier preventing release of an unacceptable
tasting medicine until the API has left the mouth.
Are pharmaceutical companies that are developing
controlled release products looking for more data
from suppliers/service providers? If so, what are
the reasons and what kind of data are they most
interested in?
Tillotson: The most interesting data is applications data. Formulators
have a need to know how a given controlled-release excipient will
perform under differing conditions, such as location of the proposed
controlled-release, performance in differing release media, and to
what speed and extent the release from a given excipient occurs.
Additionally, formulators are interested in novel release controlling
excipients with multi-modal benefits, such as solid lipid nanoparticles
which can control release as well as provide for specific therapeutic
targeting, or solid SEDDS systems that can provide for increased
bioavailability, controlled release, and ease of manufacturing. In
all cases, the applications data and excipient performance under
varying and relevant conditions is the information of most value. This
allows for the evaluation of how a respective excipient will perform in
identified applications.
Dormer: During development of specialty-controlled release
products, we often evaluate non-standard uses of excipients to
achieve the desired product profile. In those instances, there is a need
for basic and advanced characterization of the formulation materials
in combination with each other and during stability studies. Our lab
appreciates any time a material vendor can support quick technical
questions or provide “cutting edge” data on their own products. Of
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course, new products or excipients that reduce or eliminate legacy
limitations are always welcome.
Jim Huang, PhD, CEO, Ascendia Pharmaceuticals: Pharmaceutical
companies developing Controlled Release (CR) products want to
increase the success rate of CR at the preclinical and clinical stages,
as well as during commercialization and life cycle management.
Formulation robustness is important in-vitro and in-vivo but also to
establish IVIVR/IVIVC to reduce the biostudy burden in case of minor
changes in formulation, process and other SUPAC changes. They are
also looking to partner with a CDMO who can help create IP to further
strengthen their marketing position and life cycle management.
Tiwari: Drug products including controlled release products are
produced in a manner that ensures their identity, safety, strength,
quality and purity. This requires consideration of the quality of all
ingredients in a drug product, including excipients. Excipients in a
drug product can impact drug substance solubility and permeability,
stability, release rate, and absorption. Variability in excipients,
especially controlled release excipients, therefore need to be part
of the formulation risk assessment. Working with excipient supplier
can facilitate the understanding of the impact of excipient variability
on drug product performance and identification of critical material
attributes for defining subsequent control strategy. Collaboration
with Active Pharmaceutical Ingredient (API) and excipient suppliers
is the key to avoid future challenges with material supply as the
specifications for incoming raw materials need to be set based on
the manufacturing capabilities and control strategies used by the
supplier’s operations teams and not based on the sole assessment of
pharmaceutical companies developing the products.
More recently US FDA has been mandating nitrosamine impurities risk
assessment of all marketed products and those products with pending
applications both for APIs and drug products. Such a risk assessment
necessitates collaboration with suppliers of API, excipients, and
packaging components so that information can be collected, and risk
analysis performed for the finished product and if the risk is identified
then control strategies are put in place to avoid costly recall scenarios.
BASF as an innovator excipient company had taken an early lead in this
area and made nitrosamine risk assessments available for its excipient
products for use by the pharmaceutical companies.
Moody: Modern pharmaceutical companies are looking for more than
a ‘pair of hands’ when it comes to development and manufacturing
of modified release products. As a leading development partner for
such companies, Catalent has, over many years, encountered a wide
range of product challenges and has acquired significant knowledge
in how to overcome them. Pharmaceutical company customers rightly
expect to be able to leverage that experience to streamline their
own product development programs. We also recognize that in the
current environment, where redundancy of supply is so important,
the modified release products that we develop at one site may have
to be manufactured at multiple other locations around the world.
So increasingly, our customers are looking for product and process
design data for a robust package as well as information pertaining
to raw materials and excipients that may impact product quality and
manufacturability. Finally, there is an increasing expectation among
pharmaceutical companies that their development partner use PBPK
modeling to guide modified release product development decisions.
24 | | May/June 2022
This often starts with in silico feasibility analysis to inform whether
modified release is the right strategy. Once established, a robust model
can be used to set specifications, analyze prototypes, and streamline
final form selection.
Chatterjee: One area where there is sustained interest in controlled
release solutions is the area of complex generics. Complex generics
must demonstrate bioequivalence with the innovator product, just as
any generic must do, but for drug formulators this is easier said than
done. Small changes in pharmaco-chemical characteristics can mani-
fest differently therapeutically. Complicating the picture is the need
to do a bridging study for any modified or controlled release drug for-
mulation since you cannot compare systemic bioequivalency directly.
These types of studies are larger and more costly than standard bio-
equivalency studies. So, it is paramount that the drug sponsor have
confidence that their formulation will be comparable based upon
characterization and testing before embarking upon a costly and po-
tentially lengthy bridging study. Suppliers have not historically shared
the details of their full formulations or manufacturing processes, leav-
ing drug sponsors to design their processes based upon a very limited
level window of the possible material variation they could encounter.
As the industry begins to seriously consider the merits of continuous
manufacturing for finished drug products a supplier’s ability to pro-
vide information relating to their formulation, raw material character-
ization and in-process tests will be essential to drug sponsors’ ability to
consistently manufacture conforming product.
Kelly: Excipients with consistent quality and physical characteristics
play an integral role in drug release from controlled release products.
Drug release profiles may be affected by several factors including
polymer type and level, drug particle size, dose and solubility, ratio of
polymer to the drug, filler type and level, and ratio of polymer to filler.
To ensure reproducible batch-to-batch consistency it is important to
understand how any variability in these factors and the manufacturing
process could influence the release performance if inconsistency
occurs. A Quality by Design (QbD) approach is typically used to
identify and characterize the impact of varying process parameters
and determine the Critical Material Attributes (CMA) for the excipients
and rate-controlling polymers.
Using the right technology and choice of excipients, it is possible to
develop a stable formulation with reduced complexity and excellent
reproducibility. Partnership with suppliers of ingredients and
equipment is critical during the development phase to ensure that
the formulation and process parameters used during development
are transferable to commercial-scale equipment. Similarly, to avoid
future supply issues, formulators should discuss with suppliers before
putting in place any limiting specifications for ingredients.
Hayden Skalski, Lead Product Application Specialist – Biodetection,
Sievers Analytical Instruments: Companies developing controlled
release products, along with parenteral drug companies, will often
request data from suppliers or service providers to help them make
a decision on whether or not they will choose that particular supplier.
These companies want to make sure the product from the provider
works, that it is safe to use, and most importantly that it is compliant.
From my experience, the data that these companies want to see is data
to demonstrate how their specific product will work on the supplier/
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» ROUNDTABLE »
service provider’s product, especially instrumentation and products for
quality control laboratories. Controlled release product manufacturers
and those who make APIs can have complex product matrixes, which
often require different treatments in order to be successfully tested
in the lab prior to release. Therefore, having the assurance that their
products will work is a big reason why they want to see that data.
As the industry is global in nature – how do
suppliers of controlled release products/technology
meet the requirements to maintain consistent
quality and regulatory compliance worldwide?
Tillotson: During the development of excipients, a Quality by Design
approach should be employed to optimize towards a respective
controlled release excipient’s quality target product profile. After
development, c-GMP manufacturing is required and adherence to six
sigma protocols also guarantee manufacturing reproducibility and
consistent quality. Additionally good distribution practices should be
observed to guarantee shipping and storage conditions are suitable
to maintain the controlled release excipients Critical Quality Attributes
(specifications) are maintained throughout the product supply chain.
Overall, the manufacturing and supply must guarantee that the
controlled release excipient provides highly consistent performance.
Dormer: Maintaining quality consistency in controlled release
products versus simple tablets or capsules is not fundamentally
different but does require added layers of process checks to monitor
Critical Quality Attributes affected by complex manufacturing,
specialized excipients or multiple intermediates. Process checks for
controlled release products must satisfy the safety and regulatory
requirements in the countries of distribution, like for any product.
It often seems like a simple process on paper, but the complexity of
controlled release products combined with supply chain variables
can result in inconsistencies that are not readily apparent. Quality risk
management should always be a core focus for organizations.
Huang: Ascendia has established quality and regulatory compliance
systems that meet the requirements of major markets, including the
U.S. and EU. When developing products, particular attention is given
to using excipients, color, and final product image that are generally
acceptable globally. We also conduct ICH stability studies, including
different climatic zones by ICH, in the world for stability conditions.
Examples are temperate, mediterranean/subtropical, Hot dry, Hot
humid/tropical and higher humidity.
Tiwari: The suppliers of controlled release products use the best
practice guidelines and appropriate current Good Manufacturing
Practices (cGMP). Adherence to the cGMP assures the identity,
strength, quality, and purity of drug products by requiring that
manufacturers of products adequately control manufacturing
operations. This includes establishing strong quality management
systems, obtaining appropriate quality raw materials, establishing
robust operating procedures, detecting and investigating product
quality deviations, and maintaining reliable testing laboratories. This
formal system of controls at a pharmaceutical company or a supplier
of a raw material, if adequately put into practice, helps to prevent
instances of contamination, mix-ups, deviations, failures, and errors.
This assures that drug products meet their quality standards.
26 | | May/June 2022
Moody: While our industry is united in the goal of delivering quality
life-saving therapies to patients, exactly how each company does that
– or more specifically, how we demonstrate that – may vary somewhat
across regions and regulatory authorities. As a development and
manufacturing partner, it helps to have global regulatory and quality
teams with experience and insight into the specific guidance and
regulations of various authorities. These groups can provide critical
guidance to development and manufacturing sites in areas such as
qualifying raw materials to ensure consistency across batch and grade,
developing validation protocols, appropriate sampling and testing,
changes in vendors, equipment, process, etc. And increasingly, our
customers look to our global network of experts for guidance on
importing and exporting between various regions.
Kelly: The pharmaceutical supply chain is a complex network
that covers inbound materials to manufacturing plants, inter-
company supply chains, packaging, and onward distribution to end-
user customers.
One of the biggest challenges with pharmaceutical excipients,
including controlled release technologies, is that most are made by
chemical companies, which make materials for wider industrial use.
Excipients for pharmaceutical use make up only a fraction of customers
for the chemicals industry – and yet the pharma industry has specific
GMP (Good Manufacturing Practice) regulations for excipients because
of how they will be used.
To maintain consistent quality and regulatory compliance suppliers of
technologies and materials need to have a deep understanding and
knowledge of the regulatory needs of the pharmaceutical market
around the world and ensure materials meet these requirements.
Globalization and increased outsourcing activities have led to some
extraordinarily complex supply chains for pharma manufacturers.
Companies need good insight into sources and channels for materials
and a robust supply network is a critical consideration as companies
plan to de-risk the threat of interruptions across the entire supply chain.
For Colorcon, we continue to ensure our film coating manufacturing
plants operate with the same raw materials, the same equipment, and
the same processes; plus, a considerable amount of work has been
done to validate the interchangeability of products from each of the
sites, enabling continuity of supply.
Skalski: Global companies must test their product batches
independently for specific laboratory assays in order to meet safety
requirements prior to products being released to the market.
These tests, also known as critical release tests, are a crucial step in
examining the purity of products to ensure that patient safety will
not be compromised. These can include Total Organic Carbon (TOC),
Bacterial Endotoxins Testing (BET), and bioburden. These quality tests
go through rigorous review upon completion, and results are analyzed
by secondary lab analysts and Quality Assurance (QA). Products and
results are often trended over time to examine any Out Of Trend (OOT)
or Out Of Specification (OOS) results, which in turn will help identify
any outlier in the manufacturing process or laboratory and can be
investigated. Pharmaceutical companies have internal processes
that must be adhered to in order to safely sign off on product release
and also investigate any abnormal results prior to being released.
These processes closely align with FDA and EMA regulations so that
companies are being compliant and not risking any harm to patients.

Considering the last two years and looking ahead,
what has the pharmaceutical industry learned and
how will it apply these lessons in the future?
Tillotson: Certainly, given the COVID-19 pandemic, the pharmaceutical
industry has learned a great deal over the past two years. Initially, the
industry became quite agile formulating novel vaccines and delivery
technologies to meet the challenge. Subsequently, the industry has
had to adapt to certain outcomes resultant from the pandemic, such
as supply chain issues, rising raw material costs, and supply scarcity.
Overall, the industry has learned the value of agility and planning
in the face of every-changing conditions, to ensure supply of the
pharmaceutical goods and services that public health requires.
Dormer: Adherence to therapy is a driver for drug efficacy and should
be facilitated by designing products unencumbered by complex ad-
ministration procedures and cumbersome administration schedules.
The COVID pandemic further demonstrated the need and the value of
drugs that are simple and easy to take.
Social distancing and isolation are significant obstacles to therapy
adherence, especially for more vulnerable patients. It is therefore
crucial to design and develop products, such as controlled and
modified release medicines, that provide a simplified dosing schedule
to reduce dosing errors and enhance lifestyle compatibility for the
target patient population.
Bloder: I believe among the many learnings is that the pharma
industry has recognized the importance of supply chain functionality
and the profound impact it can have on the everyday lives, from life-
saving vaccines to baby formula. We live in a fragile ecosystem and we
all can contribute each day to achieve its success.
Tiwari: While the pandemic in the last two years posed several
new challenges to the pharmaceutical industry, it also gave an
opportunity to reassess the status quo and innovate for providing
high-quality medicines with an unprecedented need and demand.
Besides strengthening the supply chain, other key learnings from the
pandemic for the pharmaceutical industry are:
Collaboration: The industry worked relentlessly to find novel treatment
modalities for prevention and treatment of COVID-19 by collaborating
and sharing information. Pfizer and BioNTech is a good example of
collaboration that brought mRNA vaccine. Collaboration and strategic
partnerships are expected to grow exponentially to address the needs
of the future pandemic.
Digital transformation and machine learning tools are getting
integrated into pharmaceutical manufacturing and research and
development for faster drug product development, control of
manufacturing processes, supply chain traceability and direct data
submission to regulatory bodies. BASF’s virtual pharma assistant
tools for drug product development, regulatory product information,
interactive sustainability tool for its products and e-commerce are
good examples of digital transformation.
Innovation: In the area of drug delivery, classical drug delivery
approaches are being applied to improve the emerging therapeutic
modalities such as controlled release (initially developed for small
« ROUNDTABLE »
molecules) across the therapeutic spectrum. While technologies and
drug delivery strategies for new therapeutic modalities are being
adapted to improve the delivery of older drugs with repurposing
applications. Use of polyethylene glycol conjugation with proteins
first and then for improving delivery of small molecules latter is a good
example of innovation in drug delivery space.
Moody: If the past two years have taught us anything, it is that the
most important organizational resource is people. Well-qualified and
experienced people can adjust and adapt to changing paradigms in
communication, workspace, travel, and lifestyle in general. Resiliency
and flexibility are the new norms. Within the highly regulated
framework that we understand, companies must find innovative
ways to allow employees to do their jobs better and faster in a post-
pandemic world. This includes using tools such as remote learning,
automation, enhanced communication platforms, and a general
openness to innovative ideas and change. As the pandemic recedes,
we all look forward to returning to something resembling normalcy.
But given our technological capabilities and what we have learned, it is
unlikely that things will ever go back to exactly the way they were. The
successful companies of tomorrow will be the ones that quickly adapt
and put the best tools in place to support their people.
Quinci: With the rapid development and commercialization of the
COVID vaccine, the pharmaceutical industry has learned how fast
innovation can happen. Moderna proved a relatively new entrant
can disrupt large pharmaceutical companies and bring innovation to
market quickly. BioNTech brought an entrepreneurial drive to their
partnership with Pfizer, helping to speed time to market with their
version of the vaccine. I believe the success of the COVID vaccines
raises the bar on what is an acceptable time to market and proves what
can happen when industry, regulatory agencies, and governments are
motivated and united behind a common cause.
More than ever, innovation is needed to develop improved drug
modalities and overcome longstanding therapeutic hurdles. For
example, novel controlled release technologies offer an opportunity
to address effectiveness and safety issues encountered with traditional
treatments. The overly risk-adverse mindset of the past doesn’t have
to be the norm moving forward and long timelines that were once
accepted can be questioned as the industry takes on the challenges of
the future.
Siahboomi: The rapid increases in computing power and the
emergence of new capabilities in AI (Artificial Intelligence), virtual
modelling, automation, and data analytics, are accelerating the pace
of innovation not only for new chemical entity (discovery, but also for
drug dosage design. 3D printing technologies to manufacture solid
dosage forms is gaining momentum, allowing for complex designs
with regards to flexible shapes and precise release profiles. This
technology is still in development with some success in the nutritional
market and limited approvals in pharma.
The use of AI is already starting to have an impact through drug
discovery and bio-simulation that enables the pharmaceutical
industry to narrow down the most efficacious compounds, de-risk R&D
decisions, predict preclinical outcomes before human trials, reduce
animal testing, and therefore facilitate speed to market at a lower cost.
www.americanpharmaceutical.com | | 27
 QC CORNER
Affinity Approaches to Selective,
Sensitive MS Assays
Geoffrey Rule, Ph.D.
Principal Scientist
MilliporeSigma, Bellefonte, PA
A business of Merck KGaA, Darmstadt, Germany
Use of antibodies for detection of very low analyte
concentrations has been evolving since Yalow and Berson
developed the radioimmunoassay in the late 1950’s1.
Despite the tremendous advancements immunoassays
provided in clinical science, some immunoassays were
prone to interferences from cross-reactivity and erroneous
results. Later, the direct combination of immunoaffinity
techniques with LC-MS would be realized as exquisitely
powerful2. The extreme selectivity of antibodies makes
them indispensable when preparing complex samples. In
combination with the sensitivity of MS this can lead to low
limits of quantitation for important analytes.
A multitude of papers have been published on use of
immunoaffinity techniques for determination not only of
small molecules but for a variety of pharmaceutically and
clinically relevant proteins by MS. Neubert et al.3 published
a special report on the latter topics.
Reagents and materials for affinity techniques are widely
available making this approach worth considering for
clinical and therapeutic applications. The combination
of affinity purifications, either during sample preparation
or chromatography, makes a powerful technique for
enrichment and analysis of low-level proteins.
Strategies for Proteins
For protein determination there are several strategies
for target enrichment and analysis. For LC-MS analysis, a
bottom-up approach to sample preparation is commonly
used where the protein is digested enzymatically and one
or more of the resulting peptides are used as surrogate
markers of the protein. Immuno (affinity) enrichment is
conducted either before or after the digestion or, in some
cases, both.
28 | | May/June 2022
Cory Muraco
Biomolecule Workflows Manager
MilliporeSigma, Bellefonte, PA
A business of Merck KGaA, Darmstadt, Germany
For targeting of IgG or mAbs, a Protein A or Protein G
enrichment can selectivity pull out antibodies by the Fc, or
tail region, from a culture broth or serum sample. Where
an antibody directed towards a target protein is available,
either in polyclonal or monoclonal form, the anti-analyte
antibody can selectively enrich the analyte while removing
undesired matrix components.
In the ideal situation, a Stable Isotope Labeled Internal
Standard (SIL-IS) version of the target protein is available
and added to the sample prior to the capture step.
This approach may be more practical in pharma where
development of SIL-IS protein can be carried out using
existing methods and heavy labelled amino acids. After
capture of target and SIL-IS proteins, digestion provides
peptides stemming only from the target protein and SIL-IS.
An alternative is to submit the entire protein sample to
digestion, adding a SIL-IS peptide either before or after this
step. Antibodies toward one or more target peptides are
then used to enrich these markers of the protein of interest,
as well as corresponding SIL-peptides. A challenge of this
approach is creating antibodies to the target peptides
as this can entail time and expense. On the other hand,
SIL-peptides are more readily obtained than SIL-proteins.
In both cases, peptides from digestion are subjected to
LC-MS(/MS) analysis for quantitation.
Affinity-based enrichment technologies
Several formats are available for affinity enrichment of
target analytes including magnetic bead-based, column-
based, and in pipette tip or filter plate devices.
Beads are available in a variety of types and chemistries
ranging from agarose beads to silica, organic polymer,
and magnetic particles. Beads are adaptable to a range of
 What if biologics
characterization
felt easier?
uses from individual tubes for manual preparation, or 96-well plate formats for ease in
automation. High-throughput, automated sample preparation, is facilitated by robotic
workstations providing either magnetic retention of beads or use of pressure to force Trust our analytical products
solutions through the affinity medium. and expertise in every stage of
your biologics development.
Column-based approaches can use particle-based or monolithic structures as the
affinity support, for example when using the Chromolith WP 300 Protein A column. This • Product characterization
monolithic column features 2 µm macropores and 300 Å mesopores, enabling rapid • Method development
transport of mAbs through the column without a concomitant increase in backpressure • Quality control
or loss of efficiency. In a simple approach to antibody quantitation, the Protein A column • Release
is used to retain mAbs from a culture broth. Unwanted culture medium components are
washed away prior to mAb elution, with a low pH elution buffer, UV detection, and
quantitation.
More complex methods utilize coupled-column systems of pumps, columns, and Visit us
SigmaAldrich.com/BiologicsQC
switching valves. For example, an affinity resin is prepared as a first enrichment column
onto which the sample is delivered. After a period of washing, retained peptides or
protein are delivered to an analytical column for separation and detection.

Chemistries to bind proteins

A variety of covalent or noncovalent attachment chemistries are available that bind
proteins to solid supports. In addition to Protein A and G, noncovalent attachments are
possible with other proteins (e.g., streptavidin), oligonucleotide aptamers, antibodies
to a “FLAG” sequence coded into the terminal ends of recombinant proteins, and
immobilized metal affinity chromatography (IMAC) resins. The latter bind and retain
recombinant proteins with a histidine tail, or “His-Tag”.
For covalent attachment, the available chemistries differ with respect to the resin or
solid support. Some use an epoxy group that attaches to carboxy, thiol, hydroxyl, and
amine functional groups on the protein, depending on the pH of the solution. Others
also exist including N-hydroxysuccinimide (NHS), and carboxy linkages.

1. Yalow, R.S. and Berson, S.A., Immunoassay of endogenous plasma insulin in man. J Clin Inves, 1960, 39(7):
1157-1175.
2. Rule, G.S. and Henion J.D., Determination of drugs from urine by on-line immunoaffinity chromatography-
high-performance liquid chromatography-mass spectrometry. J Chrom, 1992, 582: 103-112.

3. Neubert, H., et al., Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem

Mass Spectrometry: Current State and Future Vision. Clin Chem, 2020, 66(2): 282-301.

Find out more at http://sigmaaldrich.com/LMhplc

© 2022 Merck KGaA, Darmstadt, Germany

and/or its affiliates. 40230 03/2022

www.americanpharmaceuticalreview.com | | 29
» FORMULATION AND DEVELOPMENT »

The Promise and

Decades of research led to the development of the first mRNA
vaccine that has changed the course of the Coronavirus Disease
2019 (COVID-19) Pandemic. Looking ahead to the potential of mRNA

Challenges of mRNA whether that is starting or building out capacity for producing
mRNA vaccines or other mRNA-based therapeutics, the possibilities
are endless.

Vaccine Development Current trends and perspectives of the applications, potential, and
benefits of mRNA therapies for infectious diseases, oncology, and
other therapeutics indicates a promise of entering into a new era of
patient hope and resolve.

The COVID pandemic brought mRNA vaccines to the forefront of

innovation and progress in therapeutical medicine with the rapid
release of highly efficacious (93–95%) vaccines by BioNTech/Pfizer
Robert Dream and Moderna. Once the sequence of SARS-CoV-2 was published,
HDR Company LLC Moderna had its first candidates available in only 28 days.6 Full phase
1–3 trials and release of millions of doses were completed in months,
much shorter than the years typically required for vaccines discovery,
development and commercial launch.7

In addition to the shortened development time and high efficacy,

there are other advantages in the use of mRNA for both prophylactic
and therapeutic vaccines.8 One is the safety profile, which includes
that the antigen is typically expressed for only a matter of days and
can be modulated by the design of the mRNA. mRNA vaccines are
also much more controllable than attenuated and vector vaccines.
Unlike DNA-based approaches, mRNA vaccines do not require nuclear
entry, so there is less risk of genomic integration and mutagenesis.
mRNA vaccines enable robust development of cellular and antibody
responses, these can be targeted to some degree by the design of the
mRNA, the choice of delivery method, or other approaches.

The core principle behind mRNA as a technology for vaccination is to

deliver the transcript of interest, encoding one or more immunogen(s),
into the host cell cytoplasm where expression generates translated
protein(s) to be within the membrane, secreted or intracellularly

30 | | May/June 2022
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» FORMULATION AND DEVELOPMENT »
located. Two categories of mRNA constructs are being actively
evaluated; non-replicating mRNA (NRM) and self-amplifying mRNA
(SAM) constructs. See Figure 1.
Figure 1. Two categories of mRNA constructs are being
actively evaluated.12
There are also advantages in the production method, as synthesis
is based on well-established in vitro transcription processes in a
cell-free system. The cell-free system helps reduce cost, time, and
manufacturing footprint. The plasmid DNA (pDNA) template for mRNA
vaccines requires a cell-based fermentation step, but this is not a highly
costly or time-consuming step. mRNA offers flexibility, as vaccines
for variants or multivalent vaccines can typically be manufactured
without a significant change of production process.
mRNA is transcribed from a linearized pDNA template then captured,
concentrated, encapsulated, and purified for delivery to patients.
mRNA therapeutics is an exciting emerging therapeutic modality.
There are opportunities for improvement and maturation, especially
in the areas of manufacturing, administration, and supply chain.
Due to its negative charge, mRNA does not easily enter the cells. It
can be rapidly degraded by nucleases such as the enzyme RNase.
Lipid nanoparticle (LNP) encapsulation, which is used in the current
mRNA based COVID vaccines, helps mitigate this problem, as do other
methods involving substitution of modified bases or design of the
mRNA. Alternatively, physical methods such as electroporation can be
employed. This approach has gained traction in ex-vivo–administered
therapeutic cancer vaccines but is not highly efficient.
Distribution of vaccine is another issue, as current mRNA vaccines
require frozen storage. Alternative methods, such as lyophilization,
are under study.9 Manufacturing also presents issues, one of the main
challenges in mRNA processing is the lack of dedicated equipment
and consumables fit for the relatively small volumes and large size
of mRNA molecules, compared to traditional recombinant proteins.
There is also room in technology development to improve scalability
and process consistency.
32 | | May/June 2022
Figure 2. mRNA-based approach for a COVID-19 vaccine
(Courtesy BioNTech13)
mRNA Industry Perspectives
With the quick spread of the COVID pandemic, mRNA for prophylactic
vaccines took the public spotlight due to an urgent need. These
vaccines demonstrated the promise of mRNA therapeutics through
their quick developmental timeline and high efficacy.
While COVID vaccines are notable, most mRNA vaccines investigated
to date have been focused on cancer therapy (e.g.; iNeST, BioNTech-
Genentech/Roche), and dozens of clinical trials have been completed
or are ongoing. Many of the ongoing trials are for personalized
therapeutic cancer vaccines and should be completed in the next
one to four years. Promising results in this area could further advance
the mRNA industry.
Many therapeutics are in early development, across diverse areas that
will have high impact if successful. Success in mRNA therapies could
potentially displace less effective therapeutic approaches, such as
vaccines for influenza, tuberculosis, or other applications.
Types of mRNA-Based Therapies
The COVID vaccines are prophylactic vaccines for infectious disease.
Other prophylactic vaccines are in development against influenza,
Zika, dengue, rabies, and Venezuelan equine encephalitis viruses, as
well as bacterial infections such as Staphylococcus and tuberculosis. 9,11
Unique approaches include expression of a neutralizing monoclonal
antibody for chikungunya virus.9
mRNA vaccines have gained traction as a therapeutic approach for
cancer. mRNA can elicit immune responses to mutated oncogenes or
regulatory cancer genes such as TP53, which are shared across many
cancers, in a therapeutic pan-cancer approach.
Other approaches for cancer include personalized therapy, where
vaccines are developed for a person’s individual mutations. In this
regard, a patient’s mutanome would be identified by next generation
sequencing, and a handful of custom mRNA vaccines would be
developed targeting the individual’s particular neoantigens.1
 « FORMULATION AND DEVELOPMENT »

Oncology
1. Fixed combination of shared cancer antigen

2. iNeST - patient specific cancer antigen therapy

3. Intratumoral immunotherapy

4. mRNA-encoded antibodies
Figure 3. Validated patient-centric bioinformatic process.
5. mRNA-encoded cytokines

to name a few and others as well.

Therapeutic cancer vaccines are advancing quickly in development,

with over 70 clinical trials completed and more results expected in
the next two to three years.10 Techniques under evaluation include
the direct stimulation of antigen-presenting cells (APCs) via ex
vivo electroporation of mRNA. Other approaches include direct
intra-tumor injection, whole body approaches, and targeted organ
Figure 4. A typical mRNA vaccines production process line.
approaches. Currently large number of clinical trials using mRNA
focuses on the treatment of melanomas, prostate and brain cancer.10
While numerous applications of mRNA vaccines are in various stages
of development, targeting specific organs, tissues, and cells with It’s all about proteins - An mRNA can teach the body how to make a
LNPs is still under research. specific protein that can help the immune system to prevent or treat
certain diseases.
The use of mRNA could grow new blood vessels after a heart attack. Or
mRNA Technologies fight off an aggressive cancer.
What is mRNA? Messenger RNA–or mRNA–exists in all of the cells in
The structural elements of the mRNA have an impact on its
the body. It is an essential component of all living organisms
performance. This includes potential immunogenicity, efficacy of
translation and stability of the molecule. Developers/manufacturers A mRNA drug product needs to be appropriately formulated in order
to be protected from degradation by extracellular RNAses. The right
leverage experience to design, synthesize, manufacture and formulate
formulation is critical to ensure the appropriate delivery of the RNA to
therapeutics mRNA, and adapt its composition to suit the desired
the intended site of action.
application. Examples of commercial applications and/or in clinical
To employ multiple mRNA delivery formulations, each should be
stage are as follows:
designed for different functions and optimized for therapeutic
product needs based on the intended application and route of
Infectious Disease delivery. An optimized mRNA provides superior immunogenicity.
1. Covid (Coronavirus)

2. Influenza (mod mRNA)

Encapsulation and
3. Influenza (as mRNA)
Delivery Technologies
4. Shingles
The use of nanostructures, such as LNPs, is common in mRNA
5. Malaria
therapeutics. These generally deliver higher efficiency than naked
6. Tuberculosis mRNA and allow for a broad variety of administration routes. A
challenge with nanostructure technology is that, it is complex by
7. HSV - herpes simplex virus
nature, and involves many potential ingredients with many possible
8. HIV - human immunodeficiency virus clinical outcomes. There is incomplete understanding in this regard.
9. VZV - Varicella-Zoster Virus Nanostructure properties are critically important to clinical outcomes
and include protection of nucleic acids, controlled release of RNA
10. EBV - Epstein–Barr virus
inside the cell, cell and tissue selectivity, translation efficiency, toxicity,
11. CMV - Cytomegalovirus and long-term stability.7

www.americanpharmaceuticalreview.com | | 33
» FORMULATION AND DEVELOPMENT »
Nanostructures are sophisticated and may be composed of several
components, such as common lipids, polymers, proteins, cholesterol,
or custom proprietary components such as ionizable lipids. 8,9
Often,
conjugates such as polyethylene glycol–lipids are used. Each of
these components influences the structural properties. For example,
polymer content can control particle size and affect efficiency and
cell tropism. Structural lipids, such as cholesterol, can affect particle
stability. Empty nanoparticles without payload can form if not mixed
correctly. Thus, nanostructure composition and formation are critical
for desired clinical effect.7 LNPs are the leading non-viral delivery
system for many systems, including gene therapy.7
There are other delivery methods under study and development.
Exosomes are thought to use a receptor, and may offer more efficient
uptake, greater specificity, and fewer side effects.2 This is a promising
early area of research.
Naked mRNA has been evaluated for cancer therapy by approaches
including direct injection to the tumor. Generally, naked RNA is
considered less efficient than other methods, but it is easy to prepare,
as it requires only a buffer.5,9 In some applications, the intrinsic high
immunogenicity of naked mRNA may provide benefit via boosted
adjuvant activity.5
Trends in mRNA-Based Therapeutics
The potential of mRNA vaccines gained scientific attention in 1990
after the in-vivo expression of a protein was observed after injection
of naked mRNA into the skeletal muscle of a mouse. Since then, the
10
industry has seen rapid development and expansion. Today, more than
140 clinical trials have looked at mRNA to address infectious disease,
cancer, and a variety of other application areas.
Two forms of mRNA structure are currently being developed:
conventional non-replicating mRNA and self-amplifying mRNA. Non-
replicating mRNA vaccines have the conventional mRNA form, and
do not have replication capability built into the mRNA sequence. The
Figure 5. Non-replicating mRNA (Courtesy BioNTech13).
34 | | May/June 2022
sequence of the antigen is flanked by untranslated (UTR) regions, a 3’
poly (6) tail, and a 5’ cap. The cap, UTR, Open Reading Frame (ORF),
and tail can be designed to up- or down-regulate expression, or to
modulate immune response.9
Modified nucleotides such as pseudouridine and 5-methylcytidine
can be used to lessen undesirable innate immune system responses
and to increase translation efficiency.7,9 Thus, there are many aspects
of the clinical response that can be modulated simply by the design
of the mRNA.
Non-replicating mRNA vaccines are transient by nature and typically
express antigen for a few hours or days (the cellular half-life of the
BioNTech/Pfizer and Moderna vaccines is estimated to be eight to
ten hours). For some applications this can be beneficial, however for
others such as systemic protein therapies, extended expression of a
protein would be beneficial.
Self-amplifying mRNA (saRNA) approaches, which enable the mRNA
to replicate, are under development. This, in turn, can extend the
expression window to weeks (four to six weeks). Typically, saRNA
is based on the addition of viral replicase genes, in cis or trans
configuration, from alphavirus, flavivirus, or picornavirus. These
strategies can either increase expression level or lower mRNA dose
requirements 10 to 100-fold. saRNA could potentially expand mRNA
technology across many applications while lowering manufacturing
demand. There are many areas of mRNA technology that are under
development, optimization and mRNA design are important aspects
of current efforts.
Other RNA therapies outside of mRNA are being developed or
have been approved. Among these are antisense oligonucleotides,
which modify gene expression; small interfering RNA (siRNA),
which also modifies gene expression via a different mechanism;
aptamers, which can bind other ligands, including RNA; and guide
RNAs, used for CRISPR targeting. Many of these RNA therapeutics
share overlapping technology with mRNA vaccines. An example is
Alnylam’s approved siRNA therapeutic Onpattro©, which uses LNP
technology.7,8 Thus, RNA therapeutics overall is advancing rapidly in
addition to mRNA vaccines.
What is the Difference Between an
mRNA and a Viral Vector Vaccine?
Both mRNA and viral vector vaccines contain instructions that teach
our cells how to create “spike proteins”, which is the protein found on
the surface of the virus that causes COVID-19. Once your cells produce
COVID-19 spike proteins, your immune system recognizes that those
proteins don’t belong in your body and creates antibodies to stop the
virus from spreading and causing damage when you are exposed to it.
Neither vaccine contains the virus that causes COVID-19.

The instructions in the mRNA vaccines are messenger RNA (mRNA),
the genetic material that tells your cells how to make proteins. The
mRNA is surrounded by tiny lipids (fatty molecules) which help mRNA
enter directly into your cells. Once your cells create the spike proteins,
your body breaks down the mRNA.
In viral vector vaccines, spike protein DNA is placed inside a modified
version of a different virus that doesn’t cause illness. This non-
harmful virus delivers the DNA instructions to your cells. This virus
is called the vector.
Transformation
It is a credit to decades of research and innovation that led to the mRNA
vaccine through technology and science to come to fruition. With the
disruption of the COVID pandemic, this technology got its moment
and has proven to be extremely safe and effective. Pfizer’s COVID-19
vaccine is the first mRNA product to achieve full FDA approval in the U.S.
Vaccine manufacturers are progressing in developing mRNA vaccines
to protect against other respiratory viruses such as the flu. Moderna
is exploring applications of the technology to protect against HIV. It’s
a new era for vaccine technology and production, and a testament
to scientific progress and decades of research. mRNA research was
under study and development for decades, the global pandemic has
redefined “business as usual” across countless industries, creating an
array of opportunities and roadblocks for today’s professionals and
organizations. Scientist and others are tackling some of the biggest
challenges anticipated by post-pandemic world from transformational
trends in bioprocessing to the technologies that are reimagining
biomanufacturing for the digital age.
The convergence of science, technology, regulatory, relevant
equipment manufactured to suit and facility design will be a paradigm
shift that is required to accelerate drug substance and product
manufacture and delivery to the patient. By localizing the operation
site in an optimized closed system, units can be implemented in a
local setting eliminating the complicated and costly supply chain. This
is a key to business success and reaching the patient. Emphasizing
the employee experience, innovation experts know that the most
successful, cutting-edge products are designed around the end-user/
patient experience. Human-centered design strategies can also benefit
the workplace. With a deep understanding of employees’ functional
and emotional needs, business leaders can design a workplace that
not only enables productivity, but also recognizes workers’ humanity,
and brings balance to the forefront/operation.
Exploring the trends transforming bioprocessing, fueled in part by
challenges resulting from the pandemic, a new work paradigm is
emerging in the field of biopharmaceutical manufacturing. To stay
ahead in this “new normal,” industry players must embrace a range
of trends, from building flexibility in supply chains to incorporating
AI and machine learning strategies to prioritizing employee health
and safety.
« FORMULATION AND DEVELOPMENT »
Building a more equitable world and understanding the potential
of participatory design and collaborative approaches to global
challenges are informed by participatory design, a groundbreaking
concept in which end users are actively involved in the innovation
process. Through participatory design, we learn how to connect
more meaningfully with people of diverse perspectives, and to make
decisions with them collectively and quickly. This can lead to more
desirable and sustainable solutions.
How will cities transform in a post-pandemic world? The built
environment is constantly evolving, but every so often, major changes
redefine the trajectory of city development. The global pandemic and
rapid development of new technologies has brought us to the cusp
of another transformative moment in the history of urban landscapes.
The built environment is constantly evolving, but every so often,
major changes redefine the trajectory of development. The global
pandemic and rapid development of new technologies has brought
us to the cusp of another transformative moment in the history of
urban landscapes.
References
1. Vormehr M, Schrörs B, Boegel S, Löwer M, Türeci Ö, Sahin U. Mutanome engineered
RNA immunotherapy: towards patient-centered tumor vaccination. J Immunol Res.
2015(2015);595363. doi: 10.1155/2015/595363.
2. Tsai S-J et al. Exosome-mediated mRNA delivery for SARS-CoV-2 vaccination. Preprint at
bioRxiv 2020 (Nov 6). doi: 10.1101/2020.11.06.371419.
3. Springer AD, Dowdy SF. GalNAc-siRNA conjugates: leading the way for delivery of RNAi
therapeutics. Nucleic Acid Ther. 2018 Jun;28(3):109-118. doi: 10.1089/nat.2018.0736.
4. Lou B, De Koker S, Lau CYJ, Hennink WE, Mastrobattista E. mRNA polyplexes with
post-conjugated GALA peptides efficiently target, transfect, and activate antigen
presenting cells. Bioconjug Chem. 2019 Feb 20;30(2):461-475. doi: 10.1021/acs.
bioconjchem.8b00524.
5. Zeng C, Zhang C, Walker PG, Dong Y. Formulation and delivery technologies for mRNA
vaccines. Curr Top Microbiol Immunol. 2020 (Jun 2); 10.1007/82_2020_217. doi:
10.1007/82_2020_217.
6. Teo SP. Review of COVID-19 mRNA vaccines: BNT162b2 and mRNA-1273. J Pharm Pract.
2021(Apr 12);8971900211009650. doi:10.1177/08971900211009650.
7. Kim J, Eygeris Y, Gupta M, Sahay G. Self-assembled mRNA vaccines. Adv Drug Deliv Rev.
2021 (Mar); 170:83-112. doi: 10.1016/j.addr.2020.12.014.
8. Schoenmaker L et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability.
Int J Pharm. 2021 (May 15);601: 120586. doi: 10.1016/j.ijpharm.2021.120586.
9. Xu S, Yang K, Li R, Zhang L. mRNA vaccine era-mechanisms, drug platform and clinical
prospection. Int J Mol Sci. 2020 (Sep 9);21(18):6582. doi: 10.3390/ijms21186582.
10. Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC. mRNA vaccines manufacturing:
challenges and bottlenecks. Vaccine. 2021 (Apr 15);39(16):2190-2200. doi: 10.1016/j.
vaccine.2021.03.038.
11. Maruggi G, Zhang C, Li J, Ulmer JB, Yu D. mRNA as a transformative technology for vaccine
development to control infectious diseases. Mol Ther. 2019 (Apr 10);27(4):757-772. doi:
10.1016/j.ymthe.2019.01.020.
12. Nicholas A. C. Jackson, Kent E. Kester, Danilo Casimiro, Sanjay Gurunathan and Frank
DeRosa; “The promise of mRNA vaccines: a biotech and industrial perspective”, Published:
04 February 2020. https://www.nature.com/articles/s41541-020-0159-8
13. BioNTech - https://biontech.de/
www.americanpharmaceuticalreview.com | | 35
» DRUG DELIVERY »

Introduction to mRNA- Introduction

LNPs, Their Manufacture

The emergence of mRNA-LNP based products into the market was
accelerated by need but was enabled by significant underpinning
research. Although currently most attention has focused on vaccine

and Future Perspectives

applications for infectious diseases and oncology, applications in
non-viral gene therapy such as in-vivo CAR-T therapy are emerging1,2
which will provide new therapeutic approaches. As a technology, it is
important not only that the products have clinical efficacy but are also
able to be manufactured consistently with such platforms also able
to adapt to the rapid rate of change seen with the technology. This
review will briefly introduce mRNA-LNP technology, considerations
to manufacture and future perspectives, considering first the RNA
payload followed by the lipid nanoparticle vector.
Dr. Philip M E Probert1*
Head of Technical
RNA Background
Dr. Juliana M Haggerty1
Two major flavors of RNA are in development as therapies - non-
Head of LNP Centre of Excellence
replicating mRNA and, self-amplifying mRNA (saRNA). Non-replicating
mRNA as used in marketed COVID-19 vaccines (Comirnaty and
Dr. John M Liddell1
Chief Technologist Spikevax) encode the antigen of interest and contain 5' and 3'
untranslated regions (UTRs). saRNAs encode not only the antigen
Mr. Graham Worrall1 but also the viral replication machinery that enables intracellular RNA
amplification and subsequent protein expression. saRNA applications
Chief Technologist
are not as far advanced as mRNA and to date products are still at the
Centre for Process Innovation, UK.
1 clinical trial stage.
Corresponding author
*
Whichever mRNA or saRNA the manufacturing method is the same
using a DNA dependant polymerase (typically T7 polymerase),
a linearized DNA plasmid template together with a supply of
nucleotides. mRNA contains four nucleotides – cytosine, adenine,
guanine and uridine. An important finding for mRNA therapies was
made in 2008 that the in-vivo effectiveness was increased by using

36 | | May/June 2022
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mRNA Therapeutics,
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» DRUG DELIVERY »
the uridine analogue, 1-methyl uridine.3 This is easily achieved in
the process of making mRNA from a DNA template by including
the modified nucleotide rather than uridine. The architecture of
RNA sequences to achieve effective expression has been the major
development challenge for this type of product. Additionally, the
formulation methods, lipid nanoparticles (LNPs) necessary to achieve
in vivo delivery and expression mean that mRNA and its delivery
approach are difficult to separate.
Summary of Different Systems
The required components of a successful mRNA therapeutic are shown
in Table 1 and described diagrammatically in Figure 1.
Table 1. mRNA/saRNA components and function.
Structure Approach
Co-capping with cap analogues or post
transcription enzymic capping. Authentic cap-1
5’ cap
structure essential to avoid activation of the innate
immune system and clearance of the mRNA.
Serves two key functions: to stabilize mRNA and
5’ UTR to facilitate scanning by small ribosome subunit to
localize the start codon.
Self-amplifying mRNA has two open reading frames.
The first frame, like conventional mRNA, codes for
Replicase
the antigen of interest. The second frame codes for
non-structural
an RNA-dependent RNA polymerase (and its helper
proteins
proteins) which replicates the mRNA construct in
the cell.
Sequence encoding for the protein to be made from
Transgene
the mRNA.
Role in gene expression by influencing the
3’ UTR localization, stability, export, and translation
efficiency of an mRNA.
Contains binding sites for poly(A) binding proteins
(PABPs), affecting the export, stability, decay,
and translation of an mRNA. PABPs bound to the
poly(A) tail may also interact with proteins, such
Poly A tail
as translation initiation factors, that are bound to
the 5' cap of the mRNA. This interaction causes
circularization of the transcript, which subsequently
promotes translation initiation.
Figure 1. A - mRNA architecture schematic.
B – saRNA architecture schematic.
38 | | May/June 2022
Manufacturing Considerations
for RNA
The way that RNA is produced, through an enzyme catalyzed reaction
from a DNA template compared to molecules like antibodies expressed
by cell culture processes, are that the impurities present at the end
of an in-vitro transcription (IVT) reaction are all defined and present
at known amounts. This facilitates the rational design of purification
process for RNA post IVT, with Figure 2 demonstrating an example
workflow for RNA synthesis and purification. DNA plasmid template
is linearized, purified and used as template for IVT following which
the mRNA is purified. RNAs are large molecules being 1-5kb (ca 2MD)
for mRNA and at least 12kb (ca 6MD) for saRNA. As such they are in
the same size range as the smaller viral vectors. Hence manufacturing
needs to be designed for particles rather than molecules in solution.
mRNA saRNA
An advantage of the large size of RNA molecules means that use
can be made of the size difference between RNA and IVT reaction
√ √
components (unreacted nucleotides, transcription enzymes, digested
DNA template components) so that significant purification can
√ √
be achieved by cross flow filtration. Subsequent chromatographic
purification is then required for polishing of impurities and product
related impurities (dsRNA specifically).
√
Some specific manufacturing considerations for mRNA include the
extreme sensitivity of the molecule to adventitious RNase activity
√ √
which needs to be controlled in manufacture. Failure to do this
results in rapid loss of RNA in processing and batch failures. Extreme
√ √
precautions are required to ensure that RNase activity is eliminated
through effective clean-in-place regimes, inclusion of RNase inhibitors
in the process and use of high-grade reagents which need to be
√ √ confirmed to be RNase activity free.
Another manufacturing consideration is the specialized nature of the
supply chain for RNA where many key reagents (including capping
reagents, IVT enzymes, encapsulation lipids) are only available from
single suppliers which means careful management of raw material
inventories to avoid manufacturing timelines being governed by
material supply.
Figure 2. Schematic of the RNA manufacturing process.
Post TFF2, RNA enters formulation.

The Need for Drug
Delivery Systems - LNPs
The molecular characteristics of nucleic acids provide significant
barriers to clinical development and commercialization, with
challenges around stability (as they are susceptible to degradation
in the body) and successful delivery into target cells (saRNA and
mRNA for example are large anionic molecules which will not cross
the cell barrier). Such challenges can be addressed when the nucleic
acid payload is encapsulated in a delivery vehicle such as polymeric
nanoparticles, liposomes or lipid nanoparticles, the latter being
the most clinically advanced drug carrier and a critical element of
the nucleic acid vaccine candidates as well as for other nucleic acid
therapeutics currently in clinical evaluation. LNPs provide a stable
structure in which the nucleic acid can reside, as well as facilitating
entry into target cells to give the desired therapeutic benefit and have
the potential to revolutionize the treatment of many diseases.
The development of robust and scalable processes for LNP-based
vaccines and therapeutics has recently received a great deal of attention
resulting in processes that have now been proven at the clinical scale.
CPI gained experience developing and handling lipid-based delivery
systems beginning with the more established liposomal systems to
encapsulate doxorubicin hydrochloride4 before progressing to those
multi component delivery vehicles containing cationic or ionizable
lipids. The use of ionizable lipids (those that have a positive or negative
charge depending on the pH of the medium in which they reside) to
complex with nucleic acid has been developed during the last twenty
years with successful candidates completing Phase III trials. This
Figure 3. Lipids used in the mRNA-LNP COVID-19 vaccines BNT162b2 (Comirnaty) and mRNA-12736
« DRUG DELIVERY »
approach was used by Alnylam to produce Patisiran, a prescription
medicine that treats the polyneuropathy caused by a hereditary illness
called ATTR amyloidosis, the first LNP delivery system to be approved
by the FDA in 2018.5
More recently work to improve the characteristics of ionizable lipids
has continued focusing on improved biodegradability, faster clearance
and RNA escape.6 New ionizable lipids e.g., ALC-0315 and SM-102 have
been developed and are now used in the BioNTech (BNT162b2) and
Moderna (mRNA-1273) vaccines respectively (Figure 3).
Manufacturing Considerations
for LNPs
The use of passive mixing systems to manufacture LNPs is one of the
most popular mixing methodologies used to date and includes such
devices as T-piece mixers, microfluidic channels with internal baffled
designs and membrane technologies. These micro-mixing techniques
provide the potential for enhanced process control and predictability
that has removed barriers to clinical development. CPI has experience
in using both T-piece and baffled micro-mixers to produce LNPs.
Successful encapsulation of RNA is achieved by bringing together
two liquid feed streams, one aqueous containing the RNA and the
second, organic, containing the dissolved lipids. Typically, there are
four lipids dissolved together in solvent each having a specific role
in successful LNP formation. Arguably it is the ionizable lipid that has
the most important role to play as it interacts directly with the nucleic
acid to initiate encapsulation, contributes towards stabilizing the RNA
against nuclease attack and also controls RNA release once inside the
www.americanpharmaceuticalreview.com | | 39
» DRUG DELIVERY »

Figure 4. Schematic of the LNP manufacturing process.

cell. The other lipids add structural benefits and act to make the LNP
outer surface more hydrophilic to avoid detection by the body’s own
immune system. Once formed it is important to maintain a stable
particle throughout the remainder of the process by maintaining a
constant particle size, particle size distribution and encapsulation
efficiency (amount of RNA trapped inside the LNP). With LNPs, and
indeed all nanotherapeutic delivery systems, precise control of the
product Critical Quality Attributes (CQAs), such as particle size, surface
properties and microstructure, is essential during manufacture to
deliver the required biological performance. These CQAs are a
combination of the raw material properties of the composition and
the processing conditions and controlling this at a scale suitable for
clinical development and commercial supply is an industry challenge.
Immediately following encapsulation (see Figure 4), the LNPs are
diluted with buffer solution to reduce the solvent concentration,
high levels of which can destabilize the LNPs, before entering the
formulation phase of the process in which the LNPs are washed and
filtered prior to being loaded into vials for the clinic. In addition, the
LNPs must be formulated to remain stable during transportation
and storage as has been highlighted by the requirements for cold
chain storage for some if not all of the current mRNA COVID vaccines,
the aim being to have an RNA-LNP that is stable at room or fridge
temperatures for many months. This challenge has yet to be fully
addressed and is providing an interesting area of research that once
understood will further enhance the applicability of biological LNP
therapeutic formulations.
The amount of product produced depends on the flow rates within
the device (flow rate ratio and total flow rate) and its period of activity
(how long it is run for) rather than its physical characteristics (i.e., its
volume, as in batch processes) and by scaling-out rather than scaling-
up, there are fewer variables to impact product quality, which will
allow production of large volumes required for clinical development.
Future Perspectives
mRNA-LNPs have entered the market quickly and catalyzed by interest
and investment, the technology is advancing rapidly. There are various
areas where improvements are being targeted. Within the scope of
this review, and of particular importance to be addressed are Cost of
Goods (COGs) and vector targeting.
On COGs, analysis and our own experience shows that the majority
of costs associated with mRNA-LNP manufacture are associated with
the raw materials, and in particular template DNA, enzymes and RNA
cap.7,8 Whilst these costs have dropped in many cases with expansion
of capacity in the supply chain, as new patented products come to
market and demand increases, it would be expected that this will
continue to be a challenge. This is particularly given since therapeutic
applications are likely to require higher doses than vaccines, as well
as more frequent applications. To overcome this, saRNA is favored
by a number of organizations as their platform of choice given the
potential to give lower doses whilst still achieving comparable and
longer action than for mRNA.9 Circular RNA is a potential alternative,
not self-amplifying but more stable and giving more persistent
and thus cumulative expression than mRNA.10 Approaches for
manufacture of mRNA we have seen broadly apply to saRNA, the
same which would be expected to be true for other RNA forms, albeit
with need for adaptation of analytical methods and modification of
manufacturing conditions for a given product format. Regardless
of form of RNA, conventional batch based IVT is a wasteful process,
involving the discard of high value raw materials after each batch. The
reuse of these materials, through continuous based processing has
the potential to significantly reduce COGs.8 The intensification of the
IVT step, if combined with continuous purification and encapsulation,
would further reduce COGs, whilst also improving the sustainability of
manufacture and reducing the size of plant needed.

40 | | May/June 2022

On vector targeting, current approved vaccines are delivered by direct
injection, overcoming issues of tissue targeting that are required for
therapeutic applications. Whilst some groups have explored direct
injection to non-muscle tissue, for example direct heart injection,11
or as an aerosol to target the lungs,12 the ability to introduce a
mRNA systemically and it specifically target a given tissue would
significantly improve utility of the technology. Currently used LNP
formulations accumulate in the liver, which has significant filtration
capacity and large surface area. Given the number of diseases that
result from a deficiency or failure of liver function, this should enable
application of mRNA-LNPs for treatment of various hepatic disorders.13
For extrahepatic delivery, use of different lipids and ratios has been
shown to change tropism of the LNP from the liver to other tissue/cell
types through passive targeting.14 Active targeting of mRNA such as
through use of nanoparticles coated with antibodies is an alternative
approach.15 Increasing complexity of nanoparticle, or alternative lipid
packaging systems will complicate manufacturing including necessary
raw materials, though is undoubtedly something able to be overcome
given the need and potential benefit of such novel therapies.
Conclusions
mRNA-LNP based vaccines have been an effective weapon against
SARS-CoV-2, with potential to be similarly effective against other
infectious and non-infectious disease. The technology and associated
manufacturing approaches have and continue to develop rapidly
which will support its wider application for prevention and treatment
of disease.
Author Biographies
Dr. Philip M. E. Probert is Head of Technical at CPI,
overseeing delivery of projects encompassing the range of
biologic products from mRNA, viral vectors, recombinant
proteins and mAbs, expressed in cell-based or cell-free
systems. He has expertise in high throughput process development,
including mRNA design and synthesis and scale up.
Dr. John M. Liddell is Chief Technologist at CPI, a UK-
based technology innovation centre and part of the UK
Catapult network. Broad biopharmaceutical experience
encompasses , including novel protein formats, antibodies,
viral vectors, vaccines and mRNA technology. He provides support to
biopharmaceutical development to increase access to new therapies
through the application of innovative technologies.
Dr. Juliana M. Haggerty is Head of Centre of Excellence
– Lipid Nanoparticles at CPI, leading the establishment
and delivery of a distributed UK capability to advance LNP
capability and expertise to support delivery of nucleic acid
therapies and vaccine. Expert in public-private collaboration, innovation
funding, digitalisation and biopharma process development.
« DRUG DELIVERY »
Mr. Graham Worrall is Chief Technologist at CPI with
25 years practical experience in the colloid, coatings and
formulation areas understanding particle interactions
relating to product stability. Has expertise in metal/metal
oxide nanoparticle synthesis, emulsion systems, micro-mixing technology
and liposome and lipid nanoparticle assembly.
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9. Bloom K, van den Berg F, Arbuthnot P. Self-amplifying RNA vaccines for infectious diseases.
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11. Anttila V, Saraste A, Knuuti J, et al. Synthetic mRNA Encoding VEGF-A in Patients
Undergoing Coronary Artery Bypass Grafting: Design of a Phase 2a Clinical Trial. Mol Ther
Methods Clin Dev. 2020;18:464-472. doi:10.1016/j.omtm.2020.05.030
12. Zhang H, Leal J, Soto MR, Smyth HDC, Ghosh D. Aerosolizable Lipid Nanoparticles
for Pulmonary Delivery of mRNA through Design of Experiments. Pharmaceutics.
2020;12(11):E1042. doi:10.3390/pharmaceutics12111042
13. Witzigmann D, Kulkarni JA, Leung J, Chen S, Cullis PR, van der Meel R. Lipid nanoparticle
technology for therapeutic gene regulation in the liver. Adv Drug Deliv Rev. 2020;159:344-
363. doi:10.1016/j.addr.2020.06.026
14. Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ. Selective ORgan Targeting
(SORT) nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing. Nat
Nanotechnol. 2020;15(4):313-320. doi:10.1038/s41565-020-0669-6
15. Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev
Genet. 2022;23(5):265-280. doi:10.1038/s41576-021-00439-4
16. Dahlman JE, Kauffman KJ, Xing Y, et al. Barcoded nanoparticles for high throughput in
vivo discovery of targeted therapeutics. Proc Natl Acad Sci U S A. 2017;114(8):2060-2065.
doi:10.1073/pnas.1620874114
www.americanpharmaceuticalreview.com | | 41
» BIOPHARMACEUTICALS »

A Next-Generation
Biotechnology has changed the face of pharmaceutical discovery,
research, and development by replacing small molecule drugs with
macromolecules produced in cultured cells and microorganisms.

Workforce for Next-

Through an equally monumental paradigm shift, Cell- and Gene-
Based Therapies (CGTs) represent the next generation of biotherapies
in which, rather than serving as expression systems, the cells and

Generation Therapies
genetic constructs themselves become the therapy.
CGTs both involve the transfer of a novel gene or genes into patients.
Gene therapy does this directly, by inserting new genes in vivo. Cell-

Preparing for Cell and

based treatments harvest a patient’s cells (typically immune system
cells), genetically modify them ex vivo, then re-introduce them into
the patient.

Gene Therapies
Emily Moran
Vice President, Viral Vector Manufacturing
The Center for Breakthrough Medicines (CBM)
Although the U.S. Food and Drug Administration has only approved 23
cell and gene therapies, more than 1200 such treatments are currently
undergoing clinical evaluation (about 10% in phase 3), with thousands
more at discovery and preclinical stages. This unprecedented surge in
interest in CGTs has created a significant gap between the needs of the
industry and the skilled resources available.
The manufacture of CGTs, moreover, tends to occur near patient
centers at several (or many) smaller facilities instead of at mega-plants.
Production tends to be decentralized, with many smaller teams and
operations, thus adding to the already formidable technical and
logistical complexity.
To succeed in this marketplace, therefore, developers must attract,
cultivate, train, and retain talented, motivated individuals to form
high-functioning production teams.

Where Do We Start?
Most individuals enter the biomanufacturing workforce directly
from high school or via undergraduate, graduate, and post-graduate
academic laboratories.

Most academic training environments focus on bioprocesses involving

mammalian or microbial cell cultures as the central upstream unit
operation, at “typical” scales of 5-100 liters for development and

42 | | May/June 2022
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100-plus liters for clinical supply and post-approval production. The
principal downstream unit ops are chromatography and filtration.
Since production scales for CGTs are typically much lower than
for therapeutic proteins, and cells or gene delivery systems are
the product, trainees entering emerging biotherapy markets
require a different skill set compared with someone applying to a
generic “biotechnology company.” Training programs must include
preparation in the unit operations involved in CGT production, e.g., cell
expansion and harvesting, transfection, viral vectors, cold chaining,
cell analysis, process analytics, Quality by Design, etc. It must also go
beyond basic technical expertise to include the core aspects of current
Good Manufacturing Practices (cGMPs), in particular aspects related
to documentation, contamination control, data integrity, automation,
and operating protocols.
Essential Skills
As academic skills catch up with current industry needs, degree and
certification programs need to stress not just the acquisition of specific
scientific skills, but a greater appreciation for regulatory compliance
and “good practices” on which trust in our medical system depends.
These include:
Good Laboratory Practices (GLPs) FDA defines GLP as “a quality
system concerned with the organizational process and the conditions
under which non-clinical health and environmental safety studies are
planned, performed, monitored, recorded, archived and reported.” The
objective of GLPs is to ensure the uniformity, consistency, reliability,
reproducibility, quality, and integrity of laboratory results.
Good Manufacturing Practices (GMPs) GMPs are wide-ranging
guidelines for manufacturing human and veterinary drugs. The
objectives of GMPs include risk minimization, product consistency,
process control, and ultimately, Quality by Design. GMPs cover all
aspects of production from ingredients, production facility, equipment,
and personnel training—anything that may affect the quality of the
finished product.
Good documentation practices Documentation of laboratory
or production results, although not an official FDA designation or
initiative, is defined as “measures that collectively and individually
ensure [that] documentation, whether paper or electronic, is secure,
attributable, legible, traceable, permanent, contemporaneously
recorded, original and accurate. FDA has published extensively on
data compliance particularly, with respect to electronic data, through
CFR Part 11.
Automation Most bio-industry workers first encounter laboratory
and process automation on the job, requiring rapid education with a
steep learning curve. Automation has long been viewed by companies
as a necessity rather than a luxury, but the same has not been true for
academic laboratories. If we are to industrialize CGT-related processes
to reduce cost of goods and make these therapies available to more
44 | | May/June 2022
patients, this must change. As more companies migrate to a “Biopharma
4.0” environment, process automation and machine learning will
become a critical skill, required in facility design and operation.
Data capture and management Commercial laboratories, including
service groups at biotech companies, now routinely employ Electronic
Laboratory Notebooks (ELNs), Laboratory Information Management
Systems (LIMSs), and data capture/entry and process monitoring
via portable, cloud-connected devices. As with automation, these
are viewed as luxuries in academic labs. At commercial scale, the
incorporation of automation into an overarching Enterprise Resource
Planning system will further improve and simplify the supply chain
and preserve a favorable bottom line.
Ongoing training Academic programs tend to be transactional and
heavily geared towards acquiring specific credentials: a PhD or B.S.
degree, or specific skill sets (e.g., “HPLC”). The problem with this model
is, that it is dead-ended by design. Skills rapidly become outdated
as laboratory and manufacturing technologies evolve and may even
become obsolete in the face of paradigm-shifting events such as
CGTs. The key to retaining employees, then, is to make them more
valuable within your organization by encouraging (or even requiring)
continuing education. Ongoing training imparts essential skills and
also tends to improve worker loyalty.
Excellent resources exist for initiating and promoting continuing
education through academic-industry partnerships, for example the
National Institute for Innovation in Manufacturing Biopharmaceuticals,
and the National Institute for Bioprocessing Research & Training.
Conclusion
From the production floors to analytical labs and everything in
between, CGTs are changing the face of biotech. Companies hoping
to staff CGT programs face shortages related to reduced enrollment
across higher academia, plus the inevitable lag in the acquisition of
appropriate hands-on skills. To assure their graduates can compete
in this fast-changing environment, biotech training programs
must prepare its graduates for both operational and regulatory
competence—through training experiences that more closely
represent their eventual work environments.
Author Biography
Ms. Moran is an experienced leader in cell and gene therapy and biologics
manufacturing – with a focus on commercial readiness, industrialization,
and manufacturing stabilization. Ms. Moran is experienced in Viral
Vector Manufacturing, aseptic processing, upstream and downstream
technology, supply chain and demand planning, quality auditing, and
facility and organization while employing lean manufacturing standards.
Prior to CBM, Ms. Moran was the Head of Viral Vector Manufacturing for
Lonza in Houston, Texas, prior to Lonza, Emily worked in multiple areas of
operations at Sanofi Pasteur.
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» MICROBIOLOGY »

Optimizing a Introduction

Viral Testing Strategy

Dr. Tim Sandle, PhD
Head of GxP Compliance and Quality Risk Management
Bio Products Laboratory Limited
Viruses are found in almost every ecosystem on Earth and are the most
numerous types of biological entity. As such, viruses pose a challenge to
many aspects of biopharmaceutical manufacturing. These challenges
not only relate to viruses per se or viruses that pose a particular problem
to different cell lines or production processes, but also from the varied
nature of viruses themselves – the genetic material (molecules of DNA
or RNA that encode the structure of the proteins by which the virus
acts); the nature of the protein coat (the capsid, which surrounds and
protects the genetic material) and whether or not the virus possess
an outside lipid envelope.1 The shapes of these virus particles range
from simple helical and icosahedral forms to more complex structures.
These geometrically sophisticated architectures are influencing factors
where nanofiltration is used as a viral inactivation step. Given the risk
presented from both pathogenic viruses in starting materials and from
adventitious agents arising during bioprocessing, the testing strategy
becomes an essential part of the viral contamination control strategy.
In a previous issue of American Pharmaceutical Review, a strategy
was outlined for viral reduction and virus clearance within a facility.2
This companion article considers the testing component of the
overall viral strategy.

Virus Contamination
With the exception of Adeno-Associated Viruses (AAV) vectors
as platforms for gene delivery for the treatment of many human
diseases, specific viruses are unwanted in bioprocessing. Viral
contamination is a potential safety threat common to all animal and
human-derived biologics and it follows that ensuring virological
safety is challenging. Despite advances in processing technology, the
use of cell culture to produce recombinant proteins continues to be
susceptible to contamination with viruses. In particular, viral infection

46 | | May/June 2022
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» MICROBIOLOGY »
of mammalian cell culture presents a continued risk.3 At risk products
and manufacturing stages include:
• Products produced from in vitro culture of cells lines of animal or
human origin;
• Products produced from in vivo culture of cell lines;
• Products produced from organs or tissues of human origin;
• Products produced from blood or other human fluids.
Complete viral safety, as defined by absolute freedom from extraneous
viral agents, is not easy to achieve and some would argue that it is an
impossible task due to the difficulties in testing for the full range of
viruses and residual virion activity. What is important is excluding or
clearing viral pathogens of concern (by viral inactivation methods and
viral removal methods).4 When contamination with pathogenic viruses
occurs, such contaminations can potentially cost millions of dollars,
especially when a facility needs to be decontaminated and a recovery
process developed. Most importantly, enforced shutdown can lead to
patients not receiving therapies.
In terms of viruses of concern, the contaminated cell type,
contaminating virus and suspected source of contamination varies.
Perhaps the most common are: herpesvirus, human adenovirus type
1, parainfluenza virus type 3 and reovirus type 3. In addition:
• Minute virus of mice (MVM), of which there are different
infective species. An example is Rodent protoparvovirus. These
viruses are common infectious agents of laboratory mice,
easily spread between populations. In processing, the primary
risk is to mammalian cell culture, especially Chinese hamster
ovary cell-based production of biologic drugs. Also, at risk are
NS0 cells. These are a model cell line derived from the non-
secreting murine myeloma used in biomedical research and
commercially in the production of therapeutic proteins.
• Vaccinia virus is a large, complex, enveloped virus belonging
to the poxvirus family. This virus has a linear, double-stranded
DNA genome approximately 190 kbp in length, which
encodes approximately 250 genes. The most common control
mechanisms are effective HVAC systems.
• Rhabdovirus, which is one of the emergent novel viruses of
note. This virus is from a family of viruses responsible for rabies
and vesicular stomatitis of cattle and horses.
The virus has been associated with contamination of
some cell culture: Spodoptera frugiperda Sf21 cells
(IPLB-Sf21-AE), used in insect cell culture for recombinant
protein production.
Viruses can be enclosed in an envelope made of proteins (capsid),
carbohydrates, and lipids (enveloped) or alternatively they are
nonenveloped. An example of an enveloped virus is herpes; an example
of a non-enveloped virus is parvovirus. Generally non-enveloped viruses
are the most difficult to remove in viral clearance studies.
48 | | May/June 2022
Testing
With the detection of viruses, while there are some indicators
such as a change in cell culture parameters as a leading indicator
of a contamination, only a robust testing strategy can determine
presence and provide an estimation as to the titer and all major viral
contamination events can be virus-specific Polymerase Chain Reaction
(PCR) assay, provided a suitable method is selected and recovery can
be proven through validation. Viruses are composed of DNA (such as
herpes viruses) or RNA (such as hepatitis viruses) encapsulated by a
protein coat, and there are PCR approaches for both. PCR approaches
are superior to traditional infectivity assays and electron microscopy.
PCR is a laboratory method used for making a very large number of
copies of short sections of DNA from a very small sample of genetic
material.5 This process is called “amplifying” the DNA and it enables
specific genes of interest to be detected or measured. With viral
RNA, the single stranded nucleic acid molecule needs to be made
into DNA before it can be amplified. This is achieved by the enzyme
called Reverse Transcriptase (RT) and an antisense (reverse) primer.
PCR assays can be developed relatively quickly, which is advantageous
should a novel pathogen emerge. Furthermore, primers can be
combined to allow for the identification of multiple viruses in a single
test. This is normally up to a maximum of four, in terms of reliability for
beyond this test sensitivity decreases. PCR methods are also affected
by mutations, which also affect sensitivity, and which can lead to false
negative results.
An alternative to PCR is Next Generation Sequencing (NGS). In terms
of the advantages and disadvantages of the test approaches, real-time
PCR is arguably easier to use and more tolerant of variable DNA quality.
However, it has a more limited multiplex capability (that is, the ability
to screen for multiple targets). NGS, in contrast, allows simultaneous
analysis of various genomic loci and it can reveal the exact sequence
changes.6 An example is shotgun metagenomics and such technology
is used to determine the order of nucleotides in entire genomes or
targeted regions of DNA or RNA.7 These methods have opened up
our understanding of virus discovery, identification, sequencing, and
manipulation. However, NGS methods are generally more expensive
to run than PCR methods and not always as practicable. All assays
should include appropriate controls to ensure adequate sensitivity
and specificity.
Testing Strategy
The approach to viral testing needs to be planned out and this
should connect with process understanding, preventative measures,
and virus clearance steps.8 These should be brought together within
a contamination control strategy. The testing strategy should be
considered from the outset of new product development. Failure

to account for later stage regulatory requirements9 in the early
development phases is a common source of project delays and failures.
Once established, testing should involve starting materials and cell
cultures, including master and working cell banks. In addition, a risk-
based framework should be used to establish the use of analytical
tools used for materials, upstream and throughout the process.10 This
is to:
• Ensure the manufacturing processes can handle the product
and stay free from viral contamination. This is especially when
used upstream and throughout the process.
• Consider how real-time testing (as with qPCR methods) can
shorten the time required for batch release.
Risk assessment is best served through the application of HACCP
(Hazard Analysis and Critical Control Points). Here it is necessary to
determine Critical Process Parameters (CPP) and the specific Critical
Control Points (CCPs) in the manufacturing process. An effective HACCP
will begin with prevention, then consider where contamination could
occur as it tracks the process downstream. Examples of prevention
include selecting cell lines and other raw materials, including media
components, based on the likely absence of undesirable viruses which
may be pathogenic (and confirming this through testing).
« MICROBIOLOGY »
The risk approach can be enhanced by knowing the viral titer
upstream and how the downstream process can deal with that viral
load before lot disposition. Here it is good practice to demonstrate
the capability of the manufacturing process to remove or inactivate
potential contaminants. This will involve a small-scale simulation
and with using four to six viruses with a sufficient starting titer to
demonstrate a four-log reduction or greater. Alternatively, other
models utilizing bacteriophages or nanoparticles can be used. These
carry the advantage that higher reduction values can be measured.
The risk-based approach should aid in the identification of points of
viral contamination including raw materials, cell culture processes,
bioreactor contamination and downstream processing. Key risk
areas include:
• Incoming materials and contaminated excipients, including
animal-derived additives such as bovine serum albumin.
Many incidents of viral contamination stem from using poorly
characterized materials.
• Contamination of cell lines.
• Purification and formulation reagents.
• Presence of impurities leading to viral stability in
the process.
• Failure of controls within a viral secure area.
www.americanpharmaceuticalreview.com | | 49
» MICROBIOLOGY »
• Accidental contamination of a production system. Adventitious
contamination can arise from a variety of sources such as
reagents, the use of contaminated biological reagents such as
animal serum components, or from human operators.
• Incomplete inactivation of live viruses used in
biopharmaceutical production.
• With blood and plasma products an additional complication
arises with the source material: human plasma. Thus, an
important additional factor is the profile of the donor and the
tests carried out to confirm the donated plasma is free from
pathogenic viruses.
Minimum test points would comprise of unprocessed bulk, purified
bulk, and final products. With more sophisticated bioprocessing,
additional stages should be considered through the use of suitable
risk framework. Inputs into the assessment will include:
• The nature of the cell lines used to produce the
desired products.
• The results and extent of virus tests performed during the
qualification of the cell lines.
• The cultivation method.
• Raw material sources.
• Results of viral clearance studies.
• Trend data from batch manufacturing.
To enhance the accuracy of the data, the sample taken should be
representative of the manufacturing stage. Thought must also be
given to the sample size given that a sample of a few milliliters may or
may not contain infectious particles.
Increased viral risk can occur when:
• Changes in critical process parameters that alter the safety
profile take place;
• Failures of virus detection systems to detect low levels of
viruses. Weaknesses with current molecular methods include
limited assay sensitivity (enhanced by the low volume of
sample volumes assayed); limitations with detection methods;
unavailability of permissive cell lines to detect viral variants, and
so on;
• Data errors, for example, with the extrapolation of viral
inactivation data;
• New and emerging viral risks.
With manufacturing, it is not sufficient merely to note the risks.
These risks need to be identified through a formal risk management
strategy. This involves a combination of risk reduction steps and
selecting process stage points for testing. The term ‘combination’ is
relevant not only for fusing together viral clearance and testing, but
also because no single approach of viral clearance sufficient; it is only
through a combination of controls and viral treatment steps that
a suitable level of assurance is reached. Additional contamination
control practices like the adoption of single-use technologies can
also be factored into the control strategy. Hence, adopting one
50 | | May/June 2022
approach is rarely sufficient. Many manufacturers are moving
towards a combination of membrane chromatography, filtration, and
solvent detergent; or they are putting into place a combination of
UV inactivation and filtration. While testing is an important element
it must be considered alongside multiple controls (including
prevention, detection, and viral clearance) throughout the process.
The biggest risks are the viral load moving downstream since
clearance becomes more challenging. Here a well-defined testing
strategy can support this, and it is good practice to obtain as much
information that can be gathered from in-process testing.
Test Validation
Assessing assay reliability is important for understanding how well
controls are actually working, for release testing, and to demonstrate
robustness to regulatory authorities. While there is still work to be done
to harmonize divergent global practices for method development,
qualification, and sample analysis.
One of the variables is with different test methods and test kits that
form part of the same method, from different manufacturers. There
are variations with scientific interpretation and in terms of reaction
volumes or DNA quantities required. In addition, some variants of PCR,
like digital PCR, require considerably longer method development time
due to the need for assessment of various conditions not necessarily
required for traditional qPCR (which is something that needs to be
balanced with the additional advantages of digital PCR).
Much of the method development is aimed toward optimizing the
detection of a specified sequence. To start method validation it is
important to gather information regarding the type of cell or gene
therapy product (target DNA) to be tested, the host species and strain
to be treated with the test articles, and the standard DNA to be used
for determining the amount of target DNA within each sample.
Common problems with method validation include:11
• Issues of dilution.
• The accuracy of the standard DNA concentration is essential for
successful qPCR analysis – good practice to develop methods in
quadruplicate.
• Challenge with amplification efficiency.
• The importance of ensuring the lower limit of quantification
(such as with most qPCR assays, which require ≤50 copies of
vector test article per 1 μg of gDNA).
As well as specific methodological issues, test error needs to be
avoided such as contamination from adventitious viruses. The
separation of workstations and control of contamination through
thorough disinfection can aid the avoidance of contaminants.

Viral Testing: In-House
or Outsourced?
The location of viral testing capabilities is very much an independent
decision for a particular manufacturer. In house gives direct control
and enables the demands of process variability to be met. Moreover,
having employees working according to specific requirements
saves time and often ensures compliance with local and regulatory
compliance. Outsourcing, on the other hand, can help to achieve
better quality of testing where the contract facility employs analysts
who are experts in different areas of software testing methodologies.
Selecting between these two options requires an understanding of the
range of viruses that pose a risk to the process, which are likely to be
present, and hence need to be detected and from this the capabilities
to conduct the testing.
Summary
This article has considered viral risk to biopharmaceutical
manufacturing and the use of risk assessment to determine where
control and testing are required. In particular, risk assessments should
focus on selecting and testing source material for the absence of known
detectable viruses and assessing the capacity of the manufacturing
process to inactivate or remove viruses. The strategy must also extend
to testing the product at appropriate time points and process stages
for detectable viruses. While various test method are available, qPCR is
probably the optimal technology for determining viral clearance and
virus removal in a close approximation to real-time, enabling faster
responses and actions to reduce the risk to a specific batch or to the
entire manufacturing facility.
« MICROBIOLOGY »
References
1. Fermin G. & Tennant P. Chapter 1—Introduction: A Short History of Virology. In: Tennant
P., Fermin G., Foster J.E.B.T.-V. (Eds.) Viruses: Molecular Biology, Host Interactions, and
Applications to Biotechnology. Academic Press; New York, NY, USA: 2018. pp. 1–16
2. Sandle, T. Current Methods and Approaches for Viral Clearance, American Pharmaceutical
Review, September / October 2015: https://www.americanpharmaceuticalreview.com/
Featured-Articles/179320-Current-Methods-and-Approaches-for-Viral-Clearance/
3. Merten OW. Virus Contaminations of Cell Cultures: A Biotechnological View. Cytotechnol.
39(2) 2002: 91–116. doi: 10.1023/A:1022969101804
4. Jones N. Identification and Remediation of a Cell Culture Virus Contamination. PDA J.
Pharm. Sci. Technol. 65(6) 2011: 615. doi: 10.5731/pdajpst.2011.00822
5. Gadsby N.J. Comparison of the Luminex Respiratory Virus Panel fast assay with in-house
real-time PCR for respiratory viral infection diagnosis. J. Clin. Microbiol. 2010;48(6):2213–
2216
6. Baker K.S. Metagenomic study of the viruses of African straw-coloured fruit bats: detection
of a chiropteran poxvirus and isolation of a novel adenovirus. Virology. 2013;441(2):95–
106
7. Forbes, J. D., Knox, N. C., Peterson, C. L. & Reimer, A. R. Highlighting clinical metagenomics
for enhanced diagnostic decision-making: a step towards wider implementation. Comput.
Struct. Biotechnol. J. 16, 108–120. https://doi.org/10.1016/j.csbj.2018.02.006 (2018).
8. Metsky, H.C., Welch, N.L., Pillai, P.P. et al. Designing sensitive viral diagnostics with machine
learning. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01213-5
9. ICH 5A: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of
Human or Animal Origin. The International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use: Geneva, Switzerland,
October 1997
10. Shimoni Y, et al. A Risk-Based Approach to Supplier and Raw Materials Management.
BioProcess Int. 13(10) 2015: 10–15
11. Feng, W., Newbigging, A., Le, C. et al. Molecular Diagnosis of COVID-19: Challenges and
Research Needs, Analytical Chemistry 2020 92 (15), 10196-10209. DOI: 10.1021/acs.
analchem.0c02060
www.americanpharmaceuticalreview.com | | 51
» BIOPHARMACEUTICAL »

Assuring Quality of A Roadmap to Quality: Performing

CMC Activities with an NDA In Mind
Oligonucleotide APIs Oligonucleotide therapeutics development continues to accelerate
with progress in oligonucleotide chemistry and adoption of delivery

and DPs technologies. This article discusses necessary steps to execute and
assure quality of oligonucleotide Active Pharmaceutical Ingredients
(APIs) and Drug Products (DPs). It will also discuss compliance and
expectations from regulatory agencies.
The basis of this work begins with “good science” as well as a grounded
understanding of the drug development process.

Judy Carmody, PhD

Founder and Principal Consultant, Carmody Quality Solutions, LLC
Affiliations: Member, Society of Quality Assurance (SQA), New England Regional Chapter
Society of Quality Assurance (NERCSQA), Association of GXP Excellence (AGXPE),
and Women in Bio
Responsibilities of Sponsor
and Vendors
A company has a platform, a therapeutic, or several candidates.
They have conducted research, published papers, applied for and
obtained grants, and may have other sources of funding. So what
now? How do they efficiently bridge the gap between research
and regulated drug development to accelerate the time to market
without sacrificing quality?
Most start-up or mid-size life sciences organizations are faced with this
challenge. And also, many of these organizations outsource most, if not
all, of the drug development activities. As such, the first step towards
accelerating time to market and building quality into the product
is knowing that as the Sponsor, you must be the expert about your
products. This allows you to collaborate with vendors appropriately.
Sponsors must work to build a collaborative approach from the
beginning in order to ensure clear communication of responsibilities.
(See Figure 1).
Sponsors need to share important information with vendors to
inform drug and process development. And while information such

52 | | May/June 2022
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» BIOPHARMACEUTICAL »
Figure 2.
as journal articles, other company or therapeutic drug product labels,
API or DP specifications, and stability data can inform the process, it
must not be the sole basis for how to manufacture and/or test/release
a therapeutic. This also holds true when sharing information with
regulatory agencies.
Starting with a strong knowledge base around regulatory requirements
is vital to lay the foundation for a successful application review.
And while there are accelerated options for clinical development
and regulatory review, it is critically important to know there are no
meaningful changes to CMC/Quality requirements with any of these
options. Meaning, if any accelerated options are granted, there is less
time to produce the same amount of CMC/Quality work for submission.
The operational challenges involved with developing oligonucleotide
therapeutics can include having smaller and fewer batches that offer
limited batch history and stability data; managing novel chemistries,
conjugates, and delivery systems; dealing with longer and more
complex development timelines; and understanding regulatory
expectations. Once we recognize these challenges, we can begin to
proactively devise the plans necessary to accelerate drug development
while building quality into the product.
Strategic Development Plans With the
NDA In Mind
Typically, CMC plan development happens with a back-ended
approach; however, to accelerate drug development and ensure
54 | | May/June 2022
that the drug sold to the public is safe and meets appropriate quality
standards, consider front-loading your CMC plan. This approach
offers the opportunity to make informed decisions sooner related to
candidate selection and design. It also can help you select candidates
to promote through the development process faster based on criteria
associated with accelerated development such as ease of manufacture,
available regulatory guidance, and similar approved products or
special designations.
This type of approach can also be applied to developing toxicology
studies, so they align with process improvements and changes in
the formulation. Such types of changes typically require additional
toxicology studies and/or changes to bioanalytical methods resulting
in revalidation or bridging studies.
Operational and
Regulatory Considerations
Three areas to consider when developing an operational plan to
accelerate your development and ensure quality integration are (see
Figure 2):
1. Manufacturing
Strive to keep the process as simple as possible, perform
characterization early in development, and get control of amidites and
other key raw materials.
 « BIOPHARMACEUTICAL »

Figure 3.

Figure 4.

2. Quality Control 3. Stability

Work on developing specifications in compliance with ICH from the
beginning, and identify impurities and degradation products as
early as possible. Build a comprehensive control strategy from raw
materials, intermediate products, and final API, DP, and finished drug
product. Perform all experiments with appropriate controls, so as to
make available for filing as supportive data.
Be strategic in designing stability studies to allow for the filing of data
in a regulatory submission. Conduct forced degradation studies early
to inform and adjust method’s capabilities.
When considering regulatory plans, remember that oligonucleotides
are considered “big small molecules” and are expected to meet the ICH
guidelines for New Chemical Entities (small molecules).1

www.americanpharmaceuticalreview.com | | 55
» BIOPHARMACEUTICAL »
When characterizing your oligonucleotides, the expectations for
testing are clear (see Figure 3).
That said, collaborate closely with your vendor or internal stakeholders
to build an understanding of and an agreement on (see Figure 4):
• Where raw materials (for APIs) and excipients (for DPs) will
be sourced, what testing will be performed, and what the
associated specifications will be.
• What in-process controls will be used and the
associated specifications.
• What specifications and tests will be required for the API
and DP.
• What will be the stability requirements/design for the raw
materials, any process intermediates, and API/DP.
Capturing these details in a documented plan helps solidify and focus
efforts, yielding efficiencies that can accelerate development and
assure quality of the product.
Specifications: How Are They Set and
Who’s Responsible?
Four elements comprise specifications: product detail information, test
attributes, testing method, and acceptance criteria. In many instances,
the Sponsor delegates establishment of specifications to the CMO.
This may be fine for raw materials, like commonly used amidites, that
most CMOs have familiarity with, but not for specialty raw materials or
the product itself.
Since the Sponsor has the most knowledge of the product, it is critical
they drive these conversations and work collaboratively with the CMO,
internal stakeholders, and other parties (preclinical/tox CROs). Given
these are based on multiple sources (data from safety/tox studies,
process capability, drug stability and capability/limitations of analytical
methods), regulatory agencies rarely define specification limits
except for patient safety (e.g., residual solvents, heavy metals, sterility,
particulate matter, or endotoxin levels of parenteral dosage forms).
Setting specifications needs to be a well thought out exercise based on
data, rather than conjecture—“throwing darts at a dart board.”
As all parties and functions have contributions to make; setting
specifications takes a village.
• Pharmacology ensures that specifications allow the drug
to maintain its activity. This is particularly important during
optimization of drug product formulation and label claim.
• Toxicology makes sure the drug batch tested in safety studies
has the widest array and levels of all potential impurities while
maintaining integrity of the safety study. This key factor will
allow justification of the lower limits of drug purity, as it will
directly provide the highest exposures of key process related
56 | | May/June 2022
impurities. When toxicology studies are managed well, the
clinical drug quality can be incrementally improved to afford
an additional margin of safety while allowing flexibility in
manufacturing.
• Process Development/Manufacturing makes certain
the proposed quality specifications can be consistently
met, particularly when scaling up or moving to contract
manufacturing facilities and commercial manufacturing.
• Analytical Development/Quality Control confirms the methods
used have the capability to test to the proposed specifications
and will continue to operate in the manufacturing Quality
Control department of a commercial facility.
• Quality Assurance/Regulatory ensures that proposed Quality
specifications will meet expectations of all relevant regulatory
agencies.
To reduce churn and maintain access to institutional knowledge, the
Sponsor captures the supporting rationale and data for specifications
in a Justification of Specification (JOS) document. The JOS is revised
and updated as development progresses and more data informs
specification adjustments. When contemplating the development of
specifications, start wide and narrow as you obtain data. Remember to
justify limits on safety arguments, not process capability. Use preclinical
and clinical exposures to establish “threshold of safety” to “qualify”
key known/specified process related impurities. Managed well, these
“threshold limits” should be higher than what the manufacturing
process can effectively deliver, even upon scale up.
The justification of limits can then be argued on “process control”
grounds rather than patient safety. Such an approach will pay off
during late-stage development through product launch when the
process needs to be scaled up and potentially moved to multiple
manufacturing sites.
Changes to specifications may be made if managed carefully and
documented/justified properly. Tightening specification limits may be
done at any time, assuming the Sponsor is confident that the process
and methods can deliver to tighter limits, and there is data to support
them. Conversely, widening specification limits is possible provided
sufficient data exists to assure the safety of the new limit. Setting
specifications for impurities requires careful consideration to meet the
spirit of the reporting requirements as outlined in ICH Q3.2
Considerations for Release Versus
Stability Specifications
Release specifications involve criteria that must be met at the time of
release of a specific batch of drug substance or drug product. Stability
specifications involve criteria that must be met over the entire shelf-
life of the DP. Each require scientific justification using results from
accelerated and long-term stability studies.

Figure 5.
If the DP attributes do not change over shelf-life, release specifications
are the same as the stability. However, if DP attributes do change over
shelf-life, then release specifications are different (more restrictive)
than stability specifications. Differences, such as potency or impurities,
must account for the degradation. Shelf-life specifications must
guarantee Safety, Identity, Strength, Purity, and Quality (SISPQ) at the
end of shelf-life. Using formal change controls for modifications to
specifications and acceptance criteria will ensure adequate scientific
justification for changes, alignment with all regulatory filings, as well
as CMO specifications.
Analytical Test Methods Validation
Versus Verification vs. Qualification
Both generic/compendial and custom/oligonucleotide specific
analytical test methods will be necessary to characterize the drug,
release batches, and test stability samples. Generic/Compendial
test methods (see Figure 5) only require verification. Custom/
Oligonucleotide specific test methods require validation. Verification
and validation can be time-consuming activities, if not approached
correctly. To ensure methods are scientifically sound and appropriate,
and provide reliable data with integrity, approach method
development with validation in mind. Start early with documenting
the method’s intended use (purpose).
« BIOPHARMACEUTICAL »
Once the method’s intended use is defined, use the table in ICH Q23 to
classify the method into one of four categories shown (Identification,
Testing for Impurities—Quantitative, Testing for Impurities—Limit, or
Assay). Based on the classification, determine the required validation
characteristics for each method type. Demonstrate that the method
performs as intended through implementing controlled qualification
experiments, which use authentically prepared standards and markers
or crude samples. Develop appropriate system suitability criteria to
assure method performance. Use this data to inform decisions for
specification limits.
The timing for validation of analytical methods has historically been
a confusing topic, but if one takes a commonsense approach, it can
be simple. What CMC activity takes the longest to conduct, is critical
for an IND or NDA submission and requires analytical testing? The
answer—stability. Like pregnancy, it takes the time it needs, and no
amount of added money or resources will change that.
To conduct stability studies, analytical methods that have been shown
to be scientifically sound and appropriate and stability-indicating
are required. To meet these requirements, methods need to be
qualified and used to analyze samples that have been part of a forced
degradation study. Regulations state that these activities don’t need
to occur until Phase 3; however, if building quality into your product
while accelerating development is your goal, these activities must
happen much sooner.
www.americanpharmaceuticalreview.com | | 57
» BIOPHARMACEUTICAL »
Typical method validation timing that usually meets regulatory
expectations include qualifying non-safety methods for early clinical
development, as well as validating:
• All safety methods before any administration to humans and
all critical methods (e.g., potency and purity/impurities) before
initial use in routine testing (batch release).
• Methods when transferring to another laboratory.
• Whenever conditions or method parameters for which the
method has been validated change, and the change is outside
the original scope of the method.
• All regulatory methods before production of material for your
pivotal efficacy trials (usually Clinical Phase 3).
• Methods earlier at the request of a regulatory agency.
Stability
As previously indicated, stability studies are a necessary component
of any submission and also can have long timelines depending on
the study. If conducted early, they can accelerate development and
inform drug design, formulation, manufacturing, and clinical study
design decisions.
Three types of stability studies provide different information, with
storage stability studies having the longest timeframes:
• Storage stability studies establish acceptable
storage timeframes.
• Drug Product—establishes shelf-life or expiration. This
is the period during which a drug product is expected
to remain within the approved shelf-life specification,
provided that it is stored under the conditions defined
on the container label.
• Drug substance—establishes the retest period.
This is the period during which the drug substance
is expected to remain within its specification and,
therefore, can be used in the manufacture of a given
DP, provided that the drug substance has been stored
under the defined conditions.
• In-use stability studies establish the acceptable timeframe
between when a drug is prepared and administered; they
inform components of the pharmacy manual or DP handling
instructions used in the pharmacy.
• Stress (Forced Degradation) stability studies establish a method’s
stability indicating capability and the drug’s degradation
pathways under various conditions.
58 | | May/June 2022
Other stability studies that may be performed include freeze/thaw
studies that establish impact of multiple freeze/thaw cycles on the
drug substance or product, and multi-use studies that provide insight
into impact of multi-use containers (e.g., multiple septa pierces).
Assuring Quality and Compliance of
Your Oligonucleotide APIs and DPs
As the development of oligonucleotide therapeutics continues to
accelerate, it is vital to do good science and keep quality in mind
throughout the process. Given the pace of change in this area,
keeping up with evolving regulatory expectations is paramount, as is
understanding the development process or bringing on the people or
vendors who do—and listening to them. Should you have challenges
in complying with ICH guidelines for an oligonucleotide-based
product, it is incumbent on the Sponsor to engage with regulators and
gain alignment.
Plan all activities with a view to the long term. Evaluate, select, and
qualify the right vendors and consultants. You must also ensure
you have the right internal people to effectively manage external
relationships and drive programs.
Remember that making quality a priority is a vital element of the final
product. It begins with a company culture where people feel safe and
empowered to share concerns and voice them when appropriate.
References
1. ICH Q11 Development and manufacture of drug substances (chemical entities and
biotechnological/biological entities).
2. ICH Q3A Impurities in New Drug Substances and ICH Q3B Impurities in New Drug Products.
3. ICH Q2 Validation of Analytical Procedures: Text and Methodology
Author Biography
Judy Carmody, PhD is the founder and Principal Consultant of Carmody
Quality Solutions, LLC, a quality solutions provider to life science startups
and global Fortune 500 organizations who are passionate about keeping
patients safe and delivering quality products. Dr. Carmody has 25+ years
of expertise driving vision in quality and operations. She is the former
founder and president of Avatar Pharmaceutical Services, an FDA-
registered contract research organization and manufacturer which was
acquired by Vertex Pharmaceuticals in 2010.
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FACILITY TOUR

Growing to Serve: Eurofins’ Portage,

Michigan, Facility Expands to Offer
New Capabilities and Services
As the need for fast, efficient, and high-quality bio/pharmaceutical
testing services continues to grow, industry service providers are Background
expanding and upgrading their capabilities to best serve their current
The Portage facility was originally constructed in 1990 by AvTech
and future partners.
Laboratories as a 20,000 square foot laboratory to support the
Eurofins BioPharma Product Testing offers a network of more than 40 company’s microbiology, clinical trials, and GMP finished product
laboratories purposely designed to cover the entire drug development testing. Eurofins purchased the facility in 2005.

process with global coverage, uniform quality assurance systems, and
best-in-class testing services.
Eurofins BioPharma Product Testing’s Portage, MI, facility is one of the
sites in the company’s network that provides GMP testing services
to the bio/pharmaceutical industry and has seen incredible growth
recently in both the size of its facilities and with the addition of new
services and capabilities.
Angela Smith, a 17-year veteran scientist and leader with the Eurofins’
Lancaster, PA, site and new site director for the Portage facility, adds,
“This was actually the first site that Eurofins purchased within the
BioPharma Product Testing group in the United States. Shortly after it
was purchased, Eurofins added an additional building to expand the
capabilities here in Portage.”
The additional building doubles the capacity, giving Eurofins room for
future growth.

60 | | May/June 2022
 « FACILITY TOUR »

Current Services
Eurofins has been putting that space to good use as the site focuses
on small molecule finished product testing for chemistry and
microbiology services. This includes method development, method
transfer/validation, release, and stability storage for early to late phase
programs. New expansions in small molecule product testing included
adding additional instrument capacity and new instrumentation,
including IC and LC/MS in 2022. Within the microbiology business, the
site has been performing non-sterile product testing such as microbial
limits testing (MLT) and antimicrobial effectiveness testing (AET) for
many years. The addition of raw materials testing, along with sterile
product microbiology services complements the current Portage
offering while providing greater options for their clients.
Smith relates how the Portage site works directly with other sites in
the Eurofins BPT network, specifically the Lancaster, PA, facility, “What
we’re adding right now to our facility here in Portage is raw materials
capabilities, and we are working directly with the Lancaster site to
identify some of the most common raw materials. We’re making sure
that we have the instrumentation and the space design to meet those
needs for our clients.”
Smith continues, “Recently, we’ve been working with the Lancaster
facility to harmonize raw materials testing across both sites to serve as
additional capacity to support our clients deadlines.”
Eurofins’ Lancaster site has been offering raw material testing for
decades and offers an experienced, industry leading team of more
than 200 subject matter experts. These experts were intimately
involved in helping the Portage site establish their services, designing
and setting up the lab, and selecting instrumentation, along with a for microbial identification purposes on site along with endotoxin
myriad of other details. testing capabilities. The team at the Portage site also installed new
sterility isolators to mirror technology that the Lancaster team
Nathan Whitford, who had been the site director at Portage since has recently validated, which will allow for sterility testing to be
2012 and is currently moving on to become the President at Eurofins’ performed there. These capabilities at the Portage location will allow
Columbia, Missouri facility, added more detail to the collaboration for a more complete service offering of microbiology testing for the
seen in the Eurofins network, “We’ve been planning this and meeting company’s clients.
with the Lancaster raw materials leadership team for over a year now.
They’ve been integral to the process. It’s been awesome to collaborate Whitford explains, “The goal is to really round out the services that we
with them and go through the planning stages and really look to see can offer from a microbiology testing perspective. We’ve had great
expertise in what would be non-sterile product testing, and we do
what are those highly requested tests, and monographs, which will
a great job with those MLT and AET services. Our previous gap was
provide a strong service offering to our clients. The leadership team in
microbiology testing services required for sterile products. Onboarding
Lancaster also gave us advice and details to look for from a laboratory
isolator sterility and endotoxin fills that void, and gene sequencing for
setup instrumentation perspective. Training is also important and
identification purposes rounds out the overall capability. By adding
fortunately we have the flexibility of sending analysts back and forth
a new microbiology lab, similar to our raw materials lab, within our
between the labs for training as needed. The ability to be integrated
network we can improve business continuity, not only for ourselves
and harmonized with the rest of the organization is really a value-add
but for our clients.”
for our clients.”

Expanding Microbiology Services A Closer Look at

Raw Materials Testing
One of the main reasons for the building expansion at the Portage
site was to add to, and enhance, the company’s microbiology The supply, and accurate and timely testing, of raw materials has always
services. At the site, Eurofins added genetic sequencing capabilities been an important part of bio/pharmaceutical product development.

www.americanpharmaceuticalreview.com | | 61
» FACILITY TOUR »

The COVID-19 pandemic has brought this topic into sharper focus
as the need for unprecedented quantities of new vaccines and
therapeutics with short delivery times has expanded. Raw materials
testing requires fast turnaround time due to the demand it has for drug
product manufacturing. Eurofins BioPharma Product Testing offers a
multi-site approach for their clients to help deliver this critical testing
in the turnaround time required while expanding on its bandwidth for
more and more samples.
Smith explains, “Raw materials have always been a busy focus for
the Lancaster site, and the operation has continued to grow. We’ve
seen substantial growth as newer and faster technologies have come
in place for raw materials testing, and it’s one of the reasons we’re
focusing on that area here in Portage.”
Smith adds that since the pandemic began, they are observing that
their clients are often sourcing a higher volume of raw materials due to embraced this multi-site qualification process, which improves their
supply chain challenges to ensure that they have an adequate supply service through supply and demand difficulties as well as business
during this time period, which has driven increased testing demand. continuity improvements that have been shown to be critical
throughout the pandemic.
Whitford adds more detail regarding the seamless collaboration
The Benefits of a Lean Lab between Lancaster and Portage, “From 2012 there was a clear focus
to harmonize SOPs, LIM systems, and things of that nature to have one
Lean concepts have been applied to bio/pharmaceutical testing
product and ability to serve our clients at multiple locations. That was
and production for a while now. But the need to implement Lean
a key. Certainly, as we bring in different services and have new people
has never been more important than in the current industry climate
joining the organization, we’re constantly looking to see how we can
of getting treatments out to market as quickly, and efficiently as
do things in the most efficient manner. There are best practices that
possible. Eurofins recognizes that Lean is tremendously important for
are shared from either site back and forth. That’s what makes for a
its customers on many different levels. Designing laboratories with
continually improving environment from that perspective.”
Lean concepts in mind allows for improved flow of samples with faster
turnaround times. However, the concepts go well beyond speed by Whitford continues, “Continuous strategizing among staff and sites to
driving more organized laboratories that analysts have a greater desire further enhance the customer service experience and create a more
to work in while improving quality, the look of the lab, and a safer efficient and innovative workplace for employees is what drives us.
laboratory environment for all. Having been a laboratory leader in Lancaster for 15 years previously,
there is great trust in our colleagues in Lancaster to advise on helping
“We see an improved flow for samples, which results in faster
us make significant improvements at our growing Portage site, not
turnaround time, as well as reduced errors. In addition, we utilize
only from a facilities perspective, or a Lean organization perspective,
Lean concepts to organize and manage reagent and standard supply
but from an SOP perspective.”
to ensure adequate inventory and minimize disruptions to work flow,
allowing analyst to focus on the work,” says Smith.

Long-Term Goals
Seamless Global Services Looking forward, Eurofins and the team at the Portage site anticipate
future growth. The company is currently in the process of evaluating
When engaging the services of a contract research and testing
another laboratory expansion. By continuing to design Lean
organization, pharmaceutical companies are keen to receive the same
laboratories, Eurofins Biopharma Product Testing can add more
level of service wherever they may be in the organization’s network.
capabilities to ensure that they keep up with current and future client’s
Eurofins has seen a greater need and demand for business continuity demand for high throughput testing, with fast turnaround time and
and continued support from laboratories without having any uncompromised quality.
disruption of services to the overall supply chain. Having shared
instruments, platform methods, LIMS, and harmonized SOPs between
their sites offers greater flexibility for clients and improved service. The
company can offer the same, validated methodology at multiple sites
and (with permission from clients) can test their product, excipient, or
raw material at either site to allow for the best TAT available without
compromise to the quality of their product. Many clients have

62 | | May/June 2022

Combining the Best of Both
Worlds with Semi-Targeted
Metabolomics
Bashar Amer
Metabolomics Application Development
Thermo Fisher Scientific
As a member of the
vertical marketing
metabolomics team at
Thermo Fisher Scientific,
Bashar Amer aims to develop robust
and sophisticated metabolomics
applications by employing state-of-
the-art technologies and best practices
to meet market needs.
Before joining the Omics team at
Thermo Fisher Scientific in San
Jose, California, Bashar developed
and implemented targeted and
untargeted LC-MS metabolomics
workflows to support microbial- and
plant-based metabolomics studies
at the Lawrence Berkeley National
Lab in Emeryville, California. Bashar
received a Ph.D. degree in GC-MS and
LC-MS nutritional metabolomics from
Aarhus University in Denmark.
VENDOR VIEWPOINT
Introduction
Metabolomics, using Liquid Chromatography-Mass Spectrometry (LC-MS), is one of the most powerful
ways to deliver molecular profiles from patient samples. The clear annotation of metabolites enables
scientists to accurately characterize biomarkers for disease diagnosis, prognosis and treatment, while
quantification of metabolites enables validation of biomarker discoveries and provides benchmarks for
normal metabolite levels (e.g., normal and abnormal sugar concentrations in the blood).
The study of metabolites usually falls under one of two main analytical chemistry strategies:
targeted or untargeted analysis. Targeted analysis provides accurate quantitation and annotation
of a predefined group of metabolites, while untargeted analysis gives an overview of changes in
metabolite relative concentrations across known and unknown metabolites in a sample. Both
approaches have their own strengths and drawbacks when using LC-MS, so researchers are often
faced with a choice: a low accuracy overview of total molecular changes, or a detailed yet blinkered
snapshot of a select group of metabolites.
However, a new semi-targeted workflow combining both untargeted and targeted workflows has
recently emerged as a middle ground, addressing the limitations of these approaches. The primary focus
of this semi-targeted approach is the confident annotation, and optionally the accurate quantitation, of
a targeted set of metabolites. In conjunction, the secondary focus is to find new molecular connections
in a system by performing untargeted analysis on a single injection. Here, we discuss how semi-targeted
metabolomics could offer the best of both worlds, and how this technique can be implemented by
laboratories to overcome barriers across multiple fields for their analytical needs.
Targeted and Untargeted Metabolomics
To make meaningful biological interpretations of metabolomic data, researchers need two main
insights: specific identification and quantification of metabolite changes (targeted analysis), and an
overview of general changes to the metabolome (untargeted analysis).
Targeted metabolomics is used in hypothesis-driven research to annotate and quantify a biologically
relevant subset of known metabolites, defined at the start of the study before data acquisition. The
workflow requires ultimate sensitivity and specificity, and typically uses triple quadrupole mass
spectrometers where only the metabolites of interest are detected. For example, researchers can
identify a select group of metabolites associated with type II diabetes to be analyzed in a sample from
a suspected diabetes patient. These metabolites can then be chemically annotated and quantified,
making the approach highly accurate at detecting these specific metabolite changes. However,
targeted analysis such as this has a major drawback: a limited coverage of the metabolome. As such,
researchers might miss metabolite changes outside of their pre-defined subset — information that can
be of biological significance to the question at hand.
63
American Pharmaceutical Review | May/June 2022
 VENDOR VIEWPOINT
In comparison, untargeted discovery metabolomics offers a wider
overview of metabolites and their relative levels. Here, researchers
will acquire full scan MS1 data to capture all ions in the sample.
The advantage of this is that analysts can maximize the number of
metabolites detected, providing the opportunity to find unexpected
changes not part of the original study goal. But, of course, the
general, untargeted overview comes with several limitations. For
one, the varying physiochemical properties of the metabolome
require specific detection criteria for different metabolite groups. This
means researchers must compromise on a combination of analytical
parameters (e.g., stationary phase for separation and ionization mode
for MS-based detection), which may improve the detection of some
substances, but reduce the detection of others. Moreover, untargeted
analysis can suffer from signal bias and mass drift introduced by
complex sample matrix effects, which complicates metabolite
identification and reduces overall sensitivity. Therefore, it is critical
to use High-Resolution Accurate-Mass (HRAM) detection, such as
that provided by Orbitrap mass analyzers — when feasible — for
untargeted metabolomics studies.
What is Semi-Targeted Metabolomics?
The recently developed semi-targeted metabolomics technique
is a promising alternative, offering researchers a way to strike the
Figure 1. Illustration of the semi-targeted metabolomics workflow.
64
American Pharmaceutical Review | May/June 2022
balance between untargeted and targeted approaches in one single
experiment. Semi-targeted workflows begin in much the same way
as targeted approaches: researchers annotate and quantify a pre-
selected group of metabolites in a sample. However, the data can then
be reanalyzed (or retro-mined) to look for global metabolic changes
that were not part of the original focus. Semi-targeted analysis can,
therefore, identify other biologically meaningful metabolite changes
that the scientist may not have been aware of in their signaling
pathway of interest.
One of the biggest strengths of semi-targeted metabolomics is the
ability to perform targeted and untargeted analysis in a single sample
injection. Traditionally, in metabolomics experiments a sample is
injected (analyzed) twice; once for untargeted metabolomics analysis
and a second time for targeted analysis. In comparison, the single
injection workflow is particularly advantageous for laboratories
that have limited access to samples, time and resources, and offers
a powerful and efficient way to gain more knowledge from valuable
biological samples.
Exemplifying the potential of semi-targeted workflows, a recent study
demonstrated how the technique can benefit both hypothesis-based
targeted verification and discovery untargeted data acquisition. Here,
semi-targeted workflows were utilized for cancer metabolomics
using Ultra-High-Performance Liquid Chromatography coupled with

High-Resolution Tandem MS (UHPLC-HRMS/MS). In the workflow,
a targeted quantification of 110 cancer-related metabolites was
analyzed simultaneously with untargeted profiling of 4651 features.
In the targeted workflow, a total of 78 metabolites were confirmed
in Parallel Reaction Monitoring (PRM) and data-independent All-Ion
Fragmentation (AIF) methods, of which 88 were confidently identified
and precisely quantified. Meanwhile, in the untargeted workflow,
all 4651 metabolites analyzed had high reliability and validity. By
merging targeted and untargeted approaches into one injection and
workflow, the semi-targeted workflows used in this study provided
the authors with a flexible, accurate and confident method for large-
scale metabolomics analysis.
The Semi-Targeted Analysis Workflow
Figure 1 illustrates key aspects of the semi-targeted metabolomics
workflow: utilizing high-resolution accurate mass spectrometry on
Orbitrap technology; sophisticated data processing and analysis
application solutions for targeted confident identification and
quantification of metabolites; and untargeted differential analysis
and unknown annotation for biomarker discovery.
Overcoming Barriers to Adopting
Semi-Targeted Analysis
Despite the clear advantages that semi-targeted analysis offers for
metabolomics, its adoption has been relatively slow. Why is this, and
how are vendors overcoming these barriers to enable its wider use
in laboratories?
First, to perform accurate and confident annotation with semi-
targeted analysis, scientists must have access to pure and diverse
chemical standards for use in the MS workflow development. This is to
confirm the identity of metabolites by matching multiple properties
such as separation retention time, accurate molecular mass and MS
fragmentation patterns. While many of these authentic metabolite
standards are commercially available, there are plenty that are not.
The main barrier, therefore, is the need for custom chemical synthesis,
when true unknown compounds are identified, which significantly
increases the cost. The untargeted part of semi-targeted workflows
can help the community by providing insights toward important and
relevant metabolites to be synthesized, which could then feed the
targeted part of future semi-targeted studies.
Second, high-quality data must be obtained to ensure a confident
interpretation of the results. In short, analyte data measured with
LC-MS must have mass accuracy, high resolution, and biological
and statistical robustness to ensure confident annotation of
unknown metabolites and accurate differential analysis. While
older LC-MS systems have often struggled to meet these demands
for semi-targeted metabolomics, the latest commercially available
65
American Pharmaceutical Review | May/June 2022
VENDOR VIEWPOINT
Orbitrap-based MS technologies and approaches are designed to
do exactly that — providing researchers with the tools to gain high-
resolution MS data. Moreover, they are also ideal for performing
large-scale experiments with high throughput and robust analysis,
helping laboratories to analyze large numbers of samples with high
consistency and replicability.
As discussed earlier, one of the benefits of semi-targeted analysis is
enabling scientists to dive deeper into the sample, while providing
an overview of known metabolites. One way in which vendors are
helping scientists to achieve a deeper coverage of the metabolome
is by creating intelligence-driven data acquisition strategies, which
maximize the number of relevant compounds interrogated by MS/MS.
These strategies offer several benefits to users, including integrating
independent experiments into automated workflows. This thereby
increases the efficiency and ease-of-use of LC-MS-based semi-
targeted analysis, resulting in deeper coverage of the metabolome
with higher confidence in the annotation.
Importantly, data processing is another key component of semi-
targeted analysis, and retro-mining data requires powerful data
analysis capabilities. As such, laboratories need sophisticated and
comprehensive software solutions that any scientist can learn to use
without a major training burden. These solutions should be able to
enable fast data processing and analysis with accurate quantification
of metabolites. Moreover, data solutions should facilitate differential
analysis and confident metabolite annotation for accurate discovery
analysis. Luckily, powerful software solutions are becoming more
readily available to overcome these challenges. Thermo Scientific™
TraceFinder™ and Compound Discoverer™ applications, for
example, have demonstrated an extended capability for metabolite
quantification and biomarker discovery, respectively (Figure 1).
Such systems unify data analytics and retro-mining processes along
with spectral libraries and databases for compound identification/
annotation, statistical analysis and biological interpretation.
Can Semi-Targeted Workflows Increase Access
to Metabolomics?
Semi-targeted workflows offer laboratories a new way to approach
metabolomics. By enabling simultaneous acquisition of hypothesis-
led and discovery-led datasets, semi-targeted workflows allow
scientists to gain more knowledge about biological systems in a
single experiment. Weighing up the pros and cons of untargeted
and targeted metabolomics has often held back the tremendous
potential of metabolomics in life sciences research. Now, semi-
targeted analysis provides the best of both worlds to unlock this
potential for scientists worldwide.
 VENDOR VIEWPOINT

Method Changes for Bacterial

Endotoxins Testing (BET)
Steps to Follow for a Straightforward Process
Hayden Skalski Quality Control departments in the pharmaceutical industry often seek new, innova-
tive products to simplify their testing. They want compliant and reliable instruments
SUEZ Water Technologies that are easier for analysts to use and reduce their footprint in the lab. Often, a com-
pany wants to change or upgrade to new technology, but they may be reluctant due
to the changes that will have to occur.

The Bacterial Endotoxins Test (BET) is a critical release test that every QC lab must
perform prior to releasing product, but most methods and instrumentation used to-
day are outdated and cumbersome. When labs want to change methods or upgrade
to new BET technology, there are a few steps they will need to assess and complete,
a process that is straightforward with the Sievers Eclipse. Each pharmaceutical com-
pany is different in terms of its internal procedures, so individual guidelines will also
need to be incorporated into the change. The good news is that in terms of LAL test-
ing, the process of changing should be similar throughout the industry. Here are four
easy steps to follow when a QC lab wants to change LAL testing methods:

1. Establish a validation plan: First, the lab should define what samples and
product types they wish to test using the new method or product (e.g., water,
raw materials, in process, and/or final drug product). Once products are estab-
lished, the lab should outline revalidation requirements for each type. For in-
stance, if a lab wishes to revalidate using the Sievers Eclipse from the 96-well
plate method for a drug product, they should screen that product at several
dilutions up to the Maximum Valid Dilution (MVD) to ensure recovery is accept-
able. Once the outline is established and sample types are known, those spe-
cific samples can be requested and obtained for revalidation testing.
Hayden Skalski is the Life Sciences
Product Application Specialist for
the Sievers Instruments product line,
specializing in bacterial endotoxins
testing (BET). Hayden has over 8 years of experience
in the pharmaceutical industry and Quality Control
Microbiology and has presented on numerous topics
surrounding endotoxin testing.
Previously, Hayden held roles at Charles River
Laboratories, Regeneron and Novartis, validating
and executing method development protocols for
endotoxin testing, providing customer support,
troubleshooting and supporting high-volume product
testing. Hayden has a B.S. from the University at Albany
(SUNY) in Biology.

66
American Pharmaceutical Review | May/June 2022
 VENDOR VIEWPOINT

2. Perform method suitability: When it is time to perform meth-
od suitability for products, it is important to confirm endotoxin
limits and MVDs to ensure the correct dilutions are being made.
Once those are known, the inhibition/enhancement screen-
ing can begin to determine the best non-interfering dilution
to achieve optimum spike recovery, ideally between 75-125%.
These results should be documented and reviewed.
3. Execute formal validation: Once the dilution is determined
for a given product, the lab should execute testing on three
discrete lots of each product using that ideal dilution – then
document, review, and sign results.
Once these four steps have been completed, the QC lab can begin
routine testing for products that have been revalidated with the new
method/technique. With a well-established plan to revalidate prod-
ucts and samples, the process is not one to be intimidated by. Of
course, changes to internal procedures will differ company to com-
pany, but in general, changing LAL testing methods is a straightfor-
ward process.

4. Document internal procedures: When a product has gone

through the validation phase with three lots complete, the
lab should create validation reports with the new method/
technique. Once reports are finished and signed, a change
control can be implemented per internal Standard Operating
Procedures (SOPs) to successfully change product testing to
the new method. After the change control is complete, the
lab can internally update its LAL testing SOP and procedure
with the new method for its products.

67
American Pharmaceutical Review | May/June 2022
» INTERVIEW »
AN INTERVIEW WITH...
Tony Saavedra
Sievers DataShare Elite Software
Tony Saavedra is the Life Sciences Associate Product
Manager for Sievers Analytical Instruments, focusing on
total organic carbon (TOC) software and instrumentation.
Tony began his tenure with the Sievers product line as part
of GE Analytical Instruments in 2011 in the field service
organization and later held responsibility for leading the
North America Technical Services team where he oversaw
technical support, factory service, and refurbishment process
operations. Prior to his positions with Sievers products, Tony
served for 10 years in the US Navy where he supervised
flight inspections and performed quality assurance
inspections and maintenance of complex electronic radar
and communication suites. Tony holds a BS in Electronic
Engineering Technology from ECPI University and an MBA
from Colorado State University.
68 | | May/June 2022
What is Sievers DataShare
Elite Software?
DataShare Elite is an intuitive software program that provides
a convenient way to manage and store Sievers TOC instrument
data in a single repository from multiple sites and instruments.
This enables pharmaceutical manufacturers to make faster,
more informed, and actionable decisions for their processes,
whether monitoring at one location or at various sites across
the world. DataShare Elite is available to accompany current
Sievers M9, DataPro2, M500, and CheckPoint product lines
as well as legacy 900, DataPro900, and 500 RL products.
The software facilitates audit reviews throughout the data
lifecycle and improves data management.
How does it help TOC customers
and their current software needs?
DataShare Elite software eliminates the reliance on removable
media devices, such as USBs, to transfer data and strengthens
the chain of custody of data and records. The software allows
users to import the complete history of an instrument (newer
Sievers instruments, as well as, legacy instruments) including
calibrations, verifications, and validations along with data, meta
data and audit trails. Customizable reports can be exported to
any network location in one of six different file types to help
facilitate the integration into other data repositories, such as
LIMS. A main driver in developing DataShare Elite software
was to make it simpler to access and manage TOC data across
different sites or multiple instruments – at the same time
ensuring data integrity is at the forefront and that the software
is extremely easy to use.
What are some unique components
that separate DataShare Elite
from other software
management programs?
DataShare Elite software is very intuitive, very easy to use, and
is compatible with Active Directory (AD). This means users can
log into the software with the same credentials as their work
computer. The software also provides easier management to
review audit trails and allows the import of instrument audit
 « INTERVIEW »

trails and software audit trails, as well as maintains its own audit trail to show full chain of custody
for all data, metadata, calibrations, verifications, and validation protocols.

Another unique aspect of the software it its ability to import data from legacy Sievers TOC
instruments (such as the Sievers 900 with associated DataPro900 software, and the Sievers 500 RL
Analyzers), facilitating audit reviews throughout the data lifecycle. There is no longer a need to keep
an antiquated computer with an outdated operating system just to store historical data.

What are some current data management

challenges and how does DataShare Elite
mitigate those challenges?
Data Integrity is of paramount importance in today’s life sciences manufacturing environment.
While data integrity guidance is not a new concept, there is increased scrutiny of data, including
how data are managed, transferred, secured, and protected.

DataShare Elite Software was designed to meet and exceed Data Integrity rules, regulations,
and guidance issued by both government agencies and professional user groups. The software
eliminates the reliance on weaknesses such as printing or using USBs in the chain of custody of data
and records. Data may easily and securely be passed from a remote instrument, such as the M500
Analyzer, to a secure server without exposing those records to unsecure USB media devices, open
file types that could be edited without traceability, accidental loss or deletion, or any other human
interaction that could call into question the completeness, consistency, or accuracy of those data
and records.

How does DataShare Elite meet/maintain today’s data

integrity requirements?
The DataGuard option allows control of data through highly customizable roles with permissions
outlined by the user’s organization ensuring 21 CRF Part 11 compliance while maintaining the
principles of ALCOA+. This enables users to achieve a high degree of data integrity across numerous
instruments and locations. Users can also electronically sign data for online instruments and other
Sievers standalone units. This becomes part of the electronic record to support batch record review.

What configurations are available for DataShare Elite,

and how easy is it to get started?
DataShare Elite software is available for both enterprise and standalone installations and can be
configured in Client-Server, Client, or standalone configurations. The software is compatible with
Active Directory (AD) to enable users to log into the software with the same credentials as their
work computer. The software easily integrates with current and legacy Sievers TOC instruments, and
Sievers support is available to ensure quick and successful implementation.

www.americanpharmaceuticalreview.com | | 69
 EDITOR’S
» TOP|TECH »
Four-Side Seal Pouch Machine for
Packaging Flat Medical Products
Designed for the packaging of flat medical
products, the Shawpak 4SS can be particular-
ly useful to manufacture pre-made pouches
for manual packaging lines.
The Shawpak 4SS is designed to:
• Pack flat products to a maximum
thickness of 5mm in packs up to
600mm x 340mm
• Produce up to 4000 packs per hour
• Make 3-side sealed pouches with the
remaining side open, allowing medical
device customers to produce pre-made
packs that are manually filled and sealed
• Incorporate the latest technologies and
innovative functionalities
• Ensure high efficiency and
minimal maintenance
The Shawpak 4SS delivers multi-faceted ben-
efits despite a compact construction that
minimizes the machine’s footprint, sparing
valuable floor space. For ease of operation, its
ergonomic design utilizes modular assemblies
for improved accessibility and quick change-
overs using minimal, low-cost tooling.
BTS International
www.bts-intl.com
Mass Photometer Improves
AAV Workflows
The SamuxMP mass photometer overcomes
the drawbacks of the current gold standard
analytical methods by enabling users to rap-
idly and cost-effectively determine empty/
full capsid ratios for AAVs of any serotype at
the bench using very low sample volumes
and concentrations, and with minimal sam-
ple preparation. Scientists involved in the
development of gene therapies and AAV-
based therapeutics will now be able to en-
hance productivity and speed up workflows
through more frequent analyses in-house,
making R&D and process development sig-
nificantly more efficient.
Refeyn
www.refeyn.com
70 | | May/June 2022
Automated Sample Storage
System Family
The Verso® Q50 and Verso Q75 Automated
Sample Storage Systems offer compact, walk-
away sample storage and access to decrease
manual labor and increase overall labora-
tory efficiency. The high-density Verso Q50
and Verso Q75 Automated Sample Storage
Systems are designed to be conveniently lo-
cated next to the benchtop. In this manner,
users gain rapid and convenient access to
samples rather than traveling to locate them
in a different room or offsite location. The
new models offer up to 4 times more capac-
ity than the Verso Q20 yet are also only 0.8m
/ 2.7ft deep. The compact footprint facilitates
direct installation, even in crowded labs, and
sample collections as large as 152,000 type-
dependent tubes in ANSI/SLAS compliant
racks or up to 1150 ANSI/SLAS compliant mi-
croplates may be accommodated.
Hamilton Storage
www.hamiltoncompany.com
Micro Pump Offers Precise,
Gentle Bio/Pharmaceutical
and Chemical Dispensing
The i-FILL® Micro pump provides extremely
precise, repeatable dispensing of 100mcl to
15ml with single stroke capacity and larger
volumes are possible with multiple strokes.
The micro pump delivers repeatable liquid fill-
ing accuracy +/-.5% of the intended volume.
In comparison, conventional pump accuracy
is usually within 1-2%. To avoid wasting any
high-value product, which can take months
to formulate and be worth thousands of dol-
lars per ml, the device is designed to pump
dry and does not drip between dispensing
batches. This eliminates product loss at the
end of a batch run. Because liquids in the
pump are not squeezed by rollers, there is no
opportunity for cultures, delicate specimens,
or sensitive chemicals to be harmed when
flowing through the tubing. The dispense
cycle does not compress tubing so there is no
shearing. The design maintains the integrity
of tubing throughout the process, and pre-
vents tiny fragments of tube from spalling, or
breaking off, preserving product quality.
Intellitech
www.intellitech-inc.com
 EDITOR’S
» TOP|TECH »
Low Dead Volume Reservoir Trays
Reservoir trays are designed for simple integra-
tion with any robotic liquid handling system.
When pipetting higher value reagent - it is an
advantage to reduce the waste or dead volume
in your reservoir tray. Incorporating a low plate
profile and a novel 'pyramid bottom' design –
these reservoir trays provide easy access to all
liquid contents as well as ensuring a low re-
agent dead volume for 96- and 384-well filling
applications. The novel pyramid bottom design
advantageously provides facility for both low-
volume and bulk transfer. Manufactured in a
cleanroom production environment, reservoir
trays offer options for a single liquid or a par-
titioned space for several liquids. With a wide
choice of working configurations, as well as liq-
uid volumes, an optimized reservoir tray is now
available to suit liquid handling applications
using 8 or 12-channel pipettes right through to
96- and 384-tip automated pipetting systems.
Porvair Science
www.porvair-sciences.com
Cryo Label for Frozen Medications
The Freeze-Lock is a cryo label that perma-
nently withstands the sub-zero storage and
transport temperatures needed for many cell
and gene therapy medications or clinical tri-
als – including those for COVID-19 mRNA
vaccines. The new solution comprises two
interlocking label layers that ensure reliable
adhesive strength and product information
readability. First, the bottom label layer with a
specialty microfine surface texture is applied
to the empty, non-refrigerated container at
room temperature. Next, the container is filled
with the active ingredient and immediately
cooled down for storage at subzero tempera-
tures utilizing dry ice (-78 °C/ -108.4 °F) or liq-
uid nitrogen (-196 °C/ -320.8 °F). To mark the
frozen container, it is removed from cold stor-
age. The Freeze-Lock’s top label layer is dis-
pensed and firmly pressed against the bottom
label (should a layer of ice have formed, this
ice does not need to be removed). The deep
freeze adhesive of the top label combines
with the bottom label‘s texture and freezes in
a matter of seconds. The result is a permanent
bond between the label construction and the
container, which is then returned to storage
and transported in frozen condition.
Schreiner MediPharm
www.schreiner-medipharm.com
www.americanpharmaceuticalreview.com
Small-Batch Blister
Packaging Machine
The EZ Blister+ meets increased demand
for 21 CFR Part 11 compliant micro-batches
throughout the blister production lifecycle,
from R&D right through to commercial pro-
duction. The machine provides a 21 CFR Part
11-ready system for producing small batches
with electronic signatures and documenta-
tion. An intelligent evolution of the EZ Blister,
Sepha’s flagship low volume blister packag-
ing solution. It provides the same proven
blister packaging performance, but with
added software capabilities that enable the
creation of electronic signatures, batch re-
ports, audit trails and other electronic docu-
mentation. The Structured Query Language
(SQL) database system at the core of the EZ
Blister+ powers a host of applications on
the machine, from recipe creation and man-
agement to reporting, as well as facilitat-
ing integration with factory networks and
Management Execution Systems (MES).
Sepha
www.sepha.com
All-in-One Metal Detection,
Checkweighing and Vision Unit
The All-in-One Inspection Unit combines two
prime quality assurance functionalities – X-ray
foreign objects detection and precision check-
weighing – with an enhanced vision system
capable of verifying label placement, barcode
legitimacy, film detection and validation of
expiration. The All-in-One, which also includes
an advanced reject system that sorts rejected
products by pre-set categories, is controlled
by a single intuitive HMI. The “Weigh and Ray,”
the process features the company’s high-tech
Electro-Magnetic Force Restoration (EMFR)
weigh cells, a differentiating engineering de-
sign that guarantees precise weighing results.
From there, the unit’s seamless X-Ray scanner
can identify foreign objects made of metal
and other materials, and has filters available to
track for certain other abnormalities. Next, the
machine’s vision system can flag errors such as
an incorrect film, label placement or missing
labels, or other aesthetic-driven quality con-
trol issues.
WIPOTEC-OCS, Inc.
www.wipotec-ocs.com/us/
| | 71
» MANUFACTURING »

A Discourse on Pharmaceutical
cGMP FDA Form 483 Trends
Why are We Re-Living the Same Issues
Over the Last 23 Years?
Steven J. Lynn, MS
Executive Vice President, Pharmaceuticals
Regulatory Compliance Associates Inc.

from RCA’s partner, Redica Systems, and was mined for the
Introduction top 483 observations between 2018 to 2021.

The intent of this article is to stimulate more conversation and catalyze
more improvement across our industry relating to the repeat problems
we have seen for literally decades.
During research for the article the author discovered the top 10 FDA
Form 483 observations have mostly remained unchanged over the last
23 years. However, they likely have been the same going back further
in time, but FDA 483 trend presentations could only be found going
back to 2000.
The main questions to consider when reading this article are:
1. Why are the top 10 FDA Form 483 issues mostly the same the
past twenty-three years? What is/are the cause(s)?
2. How can we, as an industry (regulators and regulated), solve
these systemic repeat observations?
a. Information was compiled by utilizing Redica’s Annual
Trend Report with the following search criteria: Year
range = 2018 to 2022 + Human Drugs/GMP + Form
483 + All Firms Globally
2. Research, data compilation and analysis on 483 trend
presentations given by the FDA going back to 2000.
a. Information was compiled based on internet searches
for prior FDA presentations on FDA Form 483
observational trends (aka Top 10 observations within
the specific year).
3. A highly informal, anonymous survey of 30 industry
professionals. The individuals selected were known to the
author and had to fit the following criteria:

a. They were ex-FDA employees who worked in the

Methods pharmaceutical CGMP program within the Center

The data and thoughts detailed in this article were derived from the
following four areas:
1. Data utilized and presented during the Parenteral Drug
Association’s 2022 Annual meeting. The data was obtained
for Drug Evaluation and Research (CDER) and/or the
Office of Regulatory Affairs (ORA - FDA’s inspectorate).
They had to have worked for more than two
pharmaceutical companies (after FDA) and have over
20 years of professional experience, OR

72 | | May/June 2022

b. They are current industry quality and/or operational
professionals who have worked for more than two
pharmaceutical companies and have over 20 years of
professional experience.
4. The author’s tacit knowledge gained from working as an
executive at the FDA and an executive at both generic and
brand pharmaceutical firms, as well as a consultant to dozens
of firms.
Discussion
What and Why
Please do not get caught up in the numbers. They are important, but
the important take away from this article is our collective industry
continues to be cited by the FDA for the same observations year over
year. The same 10 observations pop up again and again going back to
at least 1999, over 23 years. In those 23 years we’ve seen a rapid increase
in knowledge; better equipment; systems; processes; therapies; and
technologies to name a few. It’s definitely been a volatile, uncertain,
complex and ambiguous (VUCA) world we’ve been living in and will
continue to be. With all the rapid advancements in processes, products
and services how are we still seeing the same CGMP observations in
our facilities year over year? What can we do as an industry to change
these repeat observations?
Background
FDA’s 2006 Guidance for Industry, Quality Systems Approach to
Pharmaceutical CGMP Regulations" details a six-system inspection
model. The six systems (1. The Quality System; 2. Production System;
3. Packaging and Labeling System; 4. Laboratory Controls System;
5. Facilities and Equipment System; and 6. Materials System) are
all interconnected. In fact, FDA states in the guidance: “the quality
system provides the foundation for the manufacturing systems that
are linked and function within it. The quality system model described
in this guidance does not consider the five manufacturing systems as
discrete entities, but instead integrates them into appropriate sections
of the model.” The data presented below will be divided into the six
systems for ease of presentation.
« MANUFACTURING »
Data Analysis
During the 2022 PDA Annual Meeting the author gave a presentation
entitled “Analyzing Global FDA 483 Observational Trends 2018-2021.
In the presentation top observations within each of the six systems
were detailed. Table 1 below details the total 483 observations by
category/system from 2018-2021. The data was derived from a search
of Redica Systems data.
Table 1.
The data shows the Production System accounting for the largest
percentage of total observations (27.4%) during the reporting period.
However, the Quality System and Laboratory System are close runners
up at 26.2% and 19.4%, respectively.
Table 2 depicts the top observational issues (i.e., what were the top
observations within the different systems) across all systems for the
same time period.
The table lists the System (i.e., one of the six systems) then the
secondary category of each observation and then the tertiary category
(if available). For example, the top issue lies in the quality system,
with the secondary category of in adequate deviations and tertiary
breakdown to inadequate investigations.
Each of the Six Systems are further detailed in Tables 3-9.
These graphs only depict the past four years. During the research for
the article internet searches for FDA presentations on the Top 10 483’s
per annum were compiled and analyzed. The data showed the same
top 483 observations remain mostly unchanged over the past 23 years.
For example, in 1999 an FDA official from the San Juan District
Office gave a presentation entitled “Current 483 Issues”.3 Within
the presentation the official noted the top 483 observations were
laboratory controls, investigations, stability issues, calibration issues,
environmental controls, records, procedural deficiencies, equipment
and NDA Field Alert Reporting. Fast forward close to 23 years later and
the observations have largely remained the same, as depicted in the
PDA presentation.
Another FDA officer gave a presentation in 2015 detailing, among
other things, the top observations from 2004 to 2014 (Table 9) mostly
remain the same.
www.americanpharmaceuticalreview.com | | 73
» MANUFACTURING »

Table 2.

Table 3.

Table 10 below displays the top common observations over the
past 23 years. The rankings (i.e., 1 to 10) are not important year over
year because, while the top 10 observations may change from one
ranking to another, these common observations routinely remain in
the top 10.
Potential Causes and Call to Action
In April 2014 Harvard Business Review (HBR) published an article
entitled Creating a Culture of Quality. In the article the authors
detailed the results of their study which included 60 multinational
corporations, an extensive review of research and a survey of more
than 850 employees. The study garnered a great deal of attention in
our industry upon publishing. Subject matter experts prominently
displayed some of the key study quotes such as “A company with
a highly developed culture of quality spends, on average, $350
million less annually fixing mistakes than a company with a poorly
developed one.”.
In addition to this captivating statistic the HBR authors outlined four (4)
essentials that drive quality in organizations: 1. Leadership Emphasis –
Managers are told that quality is a leadership priority and walk the talk.;
2. Message Credibility – Messaging is delivered by respected sources,
are appealing to associates and easy to understand. 3. Peer Involvement
– peers routinely raise quality as a team topic of discussion and hold
each other accountable (i.e., a speak up and accountable culture); and

74 | | May/June 2022
 « MANUFACTURING »

Table 4.

Table 5.

Table 6.

www.americanpharmaceuticalreview.com | | 75
» MANUFACTURING »

Table 7.

Table 8.

4. Employee Ownership – Associates understand how quality fits with

the job and are empowered to make quality decisions (ex: anybody can
raise a deviation) and are comfortable (non-punitive) raising issues. Do
you think you are following these at your organization? What about
the industry as a whole?

Informal Survey
To try and make sense of this problem, a very informal and anonymous
survey was sent to thirty different colleagues. The colleagues were
known to the author and were a mix of ex-FDA’ers and industry. They
also had to have worked at no less than two different pharmaceutical/
biotech companies (not including FDA) and have no less than 20 years
Table 9.
of experience in quality, compliance and/or inspections or audits,

The survey posed the same two questions noted at the beginning of
this article, specifically:

76 | | May/June 2022
 « MANUFACTURING »

Table 10.
CFR Citation System Breakdown
21CFR§211.22(d) Quality System Quality Unit Inadequate - The responsibilities and procedures applicable to the quality control unit are not (in writing) (fully followed).

Deviations – Investigation - There is a failure to thoroughly review (any unexplained discrepancy) (the failure of a batch or any of its components to meet
21CFR§211.192 Quality System
any of its specifications) whether or not the batch has been already distributed.

There are no written procedures for production and process controls designed to assure that the drug products have the identity, strength, quality, and
21CFR§211.100(a) Quality System
purity they purport or are represented to possess.

General Laboratory Requirements - Laboratory controls do not include the establishment of scientifically sound and appropriate (specification)
21CFR§211.160(b) Laboratory Controls (standards)(sampling plans)(test procedures) designed to assure that (components)(drug product containers)(closures)(in-process materials)(labeling)
(drug products) conform to appropriate standards of identity, strength, quality and purity.

Sterile Products – Microbial Controls - Procedures designed to prevent microbial contamination purporting to be sterile are not (established)(written)
21CFR§211.113(b) Production
(followed).

Facilities and Maintenance – Equipment - Written procedures are not (established)(followed) for the cleaning and maintenance of equipment, including utensils, used
21CFR§211.67(b)
Equipment in the manufacturer, processing, packaging or holding of a drug product.

Testing and release of drug products for distribution do not include appropriate laboratory determination of satisfactory conformance to the (final
21CFR§211.165(a) Laboratory Controls
specifications)(identity and strength of each active ingredient) prior to release.

Control procedures are not established which (monitor the output) (validate the performance) of those manufacturing processes that may be
21CFR§211.110(a) Production
responsible for causing variability in the characteristics of in-process materials and the drug product.

21CFR§211.166(a) Laboratory Controls Stability Program - There is no written testing program designed to assess the stability characteristics of drug products.

Why are the top 10 FDA Form 483 issues mostly the same for the past twenty-two plus years? What is the cause(s)?
High Level Category Details
An executive told me to never add more quality to your product if your customer will not notice it. The executive equated it to not being much different
than throwing money in the trash.

A CEO said their time is spent on 2 things and only 2 things. The next quarterly financial results and the yearly financial results.

C-Suite / non-quality and operational
management
If a site has a bad inspection the discussion isn't about the details of how to get back into compliance, it’s how much it will cost to get back into
compliance and the lost future revenue from the extra investment.
When companies get in compliance trouble, they often focus intently on remediation, but those efforts (and funding) quickly dissipate once the
immediate compliance risks are resolved. Thus, allowing companies to fall back into poor practice before getting another round of observations or
worse. Rinse, wash, repeat.

People have come to believe that pharma is a great way to make a lot of money, including consulting firms, engineering firms and the business whiz
kids. Stir in Wall Street and the obsession with better results and you’ve got a mess.

There is a hesitation at the leadership level to admit to certain problems because that would mean that we then have to fix them.

It’s not rocket science. Processes and basic pharmaceutical manufacturing processes have not greatly evolved over the last 30 years - we have greater
automation and controls / trending; however, fundamentals are unchanged.

Cut Cost$, Cut Cost$, Cut Cost$, which supports the revenue drivers, and also explains why quality and maintenance are declining.
Operations Outsourcing nearly every physical activity set, including drug development, is the root cause. Ones resulting in poor supply chains with no
accountability.

Many small firms are still paper-based, and do not have the necessary money and technology to analyze process and analytical data in such a way that
allows for proactive responses to trends.

Why are the top 10 FDA Form 483 issues mostly the same for the past twenty-two plus years? What is the cause(s)?
High Level Category Details
Inconsistent auditing and ability/skill level of auditors means, not all top 10 issues get 100% attention all the time and thus industry has not fully focused
on these – firms tend to cherry pick what to focus or worry about.

Our industry seems unable to worry about no more than 3 key areas/issues at a time because of being overwhelmed by additional site and corporate
requirements.

The quality organization in many companies is a talent development center for the rest of the business. Quality is not looked at as a destination for top
talent. There are many, many skilled quality professionals, but we need to continue to improve the image and provide opportunities for professionals to
advance in the field.
The Quality Unit
Incomplete root cause investigations, resulting in CAPAs that do not address the true, underlying root causes. Firms are not willing and able to admit
when the true root cause underlying their repeat issues is related to culture or leadership deficiencies, they are bound to land on a procedure revision or
retraining to address a problem. These seem like legitimate fixes because it can never hurt to make a procedure more clear or specific, or to re-educate
an employee on a task they perform daily. But the next time those operators are called upon to handle an issue, they are still operating in the same
culture with the same leadership pressures as before, and their decision-making process is still lacking a full understanding of the safety, quality, and
compliance implications of the situation. This not only leads to repeat issues, but to a constant level of issues – the whack-a-mole problem.

In many firms, leadership is happy to stick with the status quo until someone tells them it’s no longer acceptable. In my case, anything that ‘passes
inspection’ is considered good enough…

The regulations have not changed, for drugs at least, in a very long time. Therefore, the FDA observations are always based on the same regulations -
Regulators
and evolving beyond them tends to cause a fire storm of denial in the industry.

www.americanpharmaceuticalreview.com | | 77
» MANUFACTURING »

How can we, as an industry (regulators and regulated), solve these systemic repeat observations?
High Level Category Details
Build strong local leadership (not just quality and operational execs), which has a good understanding of quality culture and risk management

Implement robust risk management and compliance programs to proactively seek out changes to regulations, guidances, and standards. Companies
should be active in reviewing and commenting on proposed changes to ensure the voice of industry is heard by the Authorities

Ensure executive management support of quality goals. This does not mean that quality has the sole responsibility, but that exec management must
ensure that all areas of the business understand the need for quality.
C-Suite / non-quality and operational
management
Keep Wall Street and the greed out of it AND plan long term .... more than one year.

Companies must align their priorities and resources to match the agency’s trigger points. The “hope for best” or “maybe we’ll get lucky” approach to
inspections clearly does not work.

Perhaps forcing companies to suspend manufacturing until meaningful corrective action can be implemented will be the wake-up call that companies
need.

A well designed and maintained facility; a well designed and developed process with well understood key quality indicators and signals/trending when
Operations
normal process parameters for process/product start to drift or shift out of control.

Good alignment of local site culture versus Global company culture / standards

Establishing mechanisms to measure and improve the quality culture of a company will have a significant benefit long-term
The Quality Unit
Implementing, reviewing, and reacting to quality metrics is important to ensure companies continue to improve and do not fall back into areas of poor
compliance. Even when a compliance issue has been remediated, a robust metrics program can be used by senior management to ensure the progress
is maintained.

Fostering a proactive quality culture versus a reactive one is the only way that lasting quality can be achieved.

Industry-wide benchmarking should be accepted where companies can share experiences and work, as an industry, to improve. We all have patients as
Industry Collaboration
our number one goal, so we should be open to benchmarking and best-practice sharing.

I know the FDA has to enforce the CFR, but maybe if observations were written in more plain language, firms would interpret the concern more broadly
vs. more literally. They’re confusing and open to multiple interpretations, internal disagreements and variation.
Regulators If FDA had better inspection methods to determine the strength of the quality culture at a firm, and were able to come right out and say when they
think there is a cultural issue, maybe then firms wouldn’t be so literal and narrow-minded in responses and corrective actions, leading to actual
improvement and fewer repeat observations.

1. Why are the top 10 FDA Form 483 issues mostly the same for
the past twenty-three years? What is/are the cause(s)?
2. How can we, as an industry (regulators and regulated), solve
these systemic repeat observations?
The results were telling and seem to point to a deeper systemic issue,
both at the line level of the firm, within the quality units, laboratories,
operations and most importantly in the C-Suite. A sampling of the
results are listed in the table below. You be the judge.
Initiative alone. The success of the Initiative has been predicated on active
participation and input from experts in industry, academia, government,
and consumer groups…. The journey has just begun. There is still much
to learn and innovations to incorporate into all our processes. We, in the
agency, will continue to emphasize the importance of the Initiative and
look forward to many more improvements in our regulatory processes for
ensuring product quality.” This initiative started 20 years ago. The Top 10
483 observations have not changed in this same timeframe. Have we
stagnated, improved or fell behind? This is something for the regulator
and regulated to ponder and address – together.

Conclusion
I do not profess to have the answer(s) to this problem. During my 25+
References
year career I’ve seen a lot of very good CGMPs and a lot of not-so-good 1.
CGMPs. To be honest, I do not think one person, one firm, one trade 2.
organization, or one consultancy has the solution. It could be argued
3.
that the solution lies in our collective knowledge and experience.
4.
I’ll leave you with one additional parting thought: In May 2007, FDA/
CDER Center Director, at the time, Dr. Janet Woodcock posted an update
and message on the Agency’s Pharmaceutical Quality for the 21st 5.
Century,6 which kicked off in 2002. In Dr. Woodcock’s opening message,
she astutely pointed out “As you know, FDA cannot meet the goals of the 6.
Guidance for Industry (fda.gov) QS Guidance
FDA QS Guidance, pg. 7
Current 483 Issues Diana Amador, Director, Science Branch San Juan District Office 1999
Microbiological Inspections, Regulatory Investigator Update, Sharon Thoma, ORA National
Expert Pharmaceutical Inspections, 2014 9th Annual Global Conference on Pharmaceutical
Microbiology
Srinivasan, Ashwin and Kurey, Bryan, Creating a Culture of Quality, Harvard Business
Review, April 2014
Pharmaceutical Quality for the 21st Century A Risk-Based Approach Progress Report | FDA

78 | | May/June 2022
 EQUIPMENT
FOCUS
Fully Automated Sample
Preparation System for
Clinical Flow Cytometry
The CellMek SPS is a fully automated sample
preparation system (SPS) that offers on-de-
mand processing for a multitude of sample
types to help laboratories to expand capabili-
ties. The system incorporates features that en-
able lean workflows and bolsters lab efficiency
by streamlining and automating many out-
dated and manual preparation methods. The
innovative new system provides a continu-
ous output of samples ready to analyze with
full traceability. The system offers integrated
cell wash modules that enable an end-to-end
sample preparation workflow. Samples can be
washed, stained, and lysed without the need
for manual intervention. A refrigerated liquid
antibody carousel can hold up to 53 vials, in-
cluding the option to run third-party reagents.
The flexible system allows users to expand its
capacity with the use of preformulated reagent
cocktails in the new DURACartridge format for
the company’s DURA Innovation dry reagents.
Beckman Coulter Life Sciences
www.beckman.com/cellmek-sps
Transfection Kits
Increase Titers in Cell and
Gene Therapy Manufacturing
TransIT-VirusGEN® SELECT Transfection Kits are
designed for large-scale viral vector produc-
tion for cell and gene therapy applications.
The kits support pre-clinical and process de-
velopment activities and consist of the TransIT-
VirusGEN Transfection Reagent, complex
formation solution and enhancer. The full kits
are designed to improve the delivery of pack-
aging and transfer plasmid DNA constructs to
suspension and adherent HEK 293 cell types
for adeno-associated virus (AAV) or lentivirus
(LV) production. The kits are identical in for-
mulation to the fully synthetic and animal-
origin-free, TransIT-VirusGEN GMP Transfection
Reagent and Kits. All kits have quality release
testing to simplify the process of qualifying
ancillary materials used in the manufacture of
viral vectors.
Mirus Bio
www.mirusbio.com/SELECT
www.americanpharmaceuticalreview.com
Mobile Platform for
Cell and Gene Therapy
The bioGO® cGMP Mobile Cleanroom solves
complex logistical challenges facing the CGT
(cell and gene therapy) industry. The flexible
bioprocessing platform was designed in re-
sponse to critical constraints in the develop-
ment, manufacturing and delivery of new
and emerging curative therapies. The bioGO
mobile platform supports the scale-out pro-
cess by using standard, proven cGMP facility
designs that are pre-commissioned and pre-
qualified, significantly shortening the time to
process readiness. Process spaces can also be
reconfigured or interconnected to add bioGO
units, for a larger facility.
Germfree Laboratories Inc.
www.germfree.com
Syringe Filling &
Assembly System
Employing a versatile yet precise setup that
accurately fills syringes via ceramic piston,
peristaltic pump or direct draw from a res-
ervoir bag, the SimpliFil Syringe Filling &
Assembly System is suitable for small to me-
dium batches. SimpliFil is designed for ease
of use: its signature highlight is a walking
beam indexing configuration, which provides
intuitive operation and simplified, recipe-
based changeover. For heightened preci-
sion, TurboFil's unique TipFil™ technology al-
lows syringes to be filled through the tip – a
step-saving innovation eliminating the need
to insert plungers post-filling. Filling up to
36 syringes per minute, the SimpliFil system
smoothly handles syringes with tips outfit-
ted for press-on caps and luer caps, as well as
various safety caps recently incorporated for
oral syringes. For post-capping marking ap-
plications, TurboFil also offers an optional la-
beler that can wrap syringes with pre-printed
labels or, for medications requiring variable
marking, print individual labels per uploaded
input. An automatic syringe loading system
also is available, as is a volume and vision sys-
tem for fill and label matching verification.
TurboFil Packaging Machines LLC
www.TurboFil.com
| | 79
» MICROBIOLOGY »
Cleaning and Disinfection:
An Important Pillar of Contamination Control
Ratul Saha, PhD
Director, Contamination Control
Seres Therapeutics
Background Information:
Cleaning and disinfection of surfaces are not only the essential steps
for maintaining the cleanliness and control of the manufacturing
environment, but it is also one of the most important pillars of the
Contamination Control framework. The overall regulatory expectations
regarding this topic are well described:
US Code of Federal Regulation
Sec. 211.56 Sanitation.
a. Any building used in the manufacture, processing, packing,
or holding of a drug product shall be maintained in a
clean and sanitary condition, any such building shall be
free of infestation by rodents, birds, insects, and other
vermin (other than laboratory animals). Trash and organic
waste matter shall be held and disposed of in a timely and
sanitary manner.
b. There shall be written procedures assigning responsibility
for sanitation and describing in sufficient detail the
cleaning schedules, methods, equipment, and materials
to be used in cleaning the buildings and facilities; such
written procedures shall be followed.
c. There shall be written procedures for use of suitable
rodenticides, insecticides, fungicides, fumigating
agents, and cleaning and sanitizing agents. Such written
procedures shall be designed to prevent the contamination
of equipment, components, drug product containers,
closures, packaging, labeling materials, or drug products
and shall be followed. Rodenticides, insecticides, and
fungicides shall not be used unless registered and used in
accordance with the Federal Insecticide, Fungicide, and
Rodenticide Act (7 U.S.C. 135).
80 | | May/June 2022
d. Sanitation procedures shall apply to work performed
by contractors or temporary employees as well as work
performed by full-time employees during the ordinary
course of operations.
EudraLex Volume 4-Good Manufacturing Practice
(GMP) Guidelines
Part 1 Basic Requirements for Medicinal Products
Principle
Premises and equipment must be located, designed, constructed,
adapted and maintained to suit the operations to be carried out.
Their layout and design must aim to minimize the risk of errors and
permit effective cleaning and maintenance in order to avoid cross-
contamination, build-up of dust or dirt and, in general, any adverse
effect on the quality of products.
Revised Annex 1 Manufacture of Sterile
Medicinal Products
Disinfection
4.36 The disinfection of cleanrooms is particularly important. They
should be cleaned and disinfected thoroughly in accordance with
a written program. For disinfection to be effective, prior cleaning
to remove surface contamination should be performed. More than
one type of disinfecting agent should be employed to ensure that
where they have different modes of action, and their combined
usage is effective against all bacteria and fungi. Disinfection should
include the periodic use of a sporicidal agent. Monitoring should
be undertaken regularly in order to assess the effectiveness of the
disinfection program and to detect changes in types of microbial flora
(e.g., organisms resistant to the disinfection regime currently in use).
Cleaning programs should effectively remove disinfectant residues.

4.37 The disinfection process should be validated. Validation studies
should demonstrate the suitability and effectiveness of disinfectants
in the specific manner in which they are used and should support the
in-use expiry periods of prepared solutions.
4.38 Disinfectants and detergents used in Grade A zone and Grade
B areas should be sterile prior to use (disinfectants used in Grade C
and D may also be required to be sterile). Where the disinfectants and
detergents are made up by the sterile product manufacturer, they
should be monitored for microbial contamination. Dilutions should
be kept in previously cleaned containers and should only be stored
for defined periods. If the disinfectants and detergents are supplied
“ready-made” then results from certificates of analysis or conformance
can be accepted subject to successful completion of the appropriate
vendor qualification.
One of the more difficult tasks is the implementation of an effective
Cleaning and Disinfection Program. The problem is multifactorial
and therefore several aspects need to be considered to develop
and implement a compliant and effective Cleaning and Disinfection
Program.
a. What are we cleaning and disinfecting?
b. What are the sources of microbial contamination in a
cleanroom?
c. Why should we care?
d. What are the definitions?
e. What are the different types of chemical agents and their
modes of action?
f. What makes a chemical agent effective
against microorganisms?
g. Does your Standard Operating Procedure (SOP) have
the following?
h. What are some of the best practices of cleaning
and disinfection?
i. What is the importance of training and periodic
walkthrough?
j. Why the details matter?
A. What are We Cleaning and
Disinfecting?
This is one of the key questions we need to understand and answer.
Before answering the question, it is important to understand some
of the basic definitions of the terminologies associated. Cleaning is
the process of removing residues and soil from surfaces to the extent
that they are visually clean. Cleaning involves the use of a detergent.
Detergents are basically surfactants that work by reducing the surface
tension, which allows the removal of the soil. Cleaning is key before the
application of disinfectant (Sandle, 2012). Disinfectant is a substance,
or mixture of substances, that destroys or irreversibly inactivates
bacteria, fungi and viruses, but not necessarily bacterial spores, in the
« MICROBIOLOGY »
inanimate environment. (EPA, 2022). Disinfection describes a process
that eliminates many or all pathogenic microorganisms, except
bacterial spores, on inanimate objects (CDC, 2008).
One of the factors that affect efficacy of disinfection is the prior
cleaning of the object: - level of organic and inorganic load present.
Disinfectants are poor at penetrating soil and microorganisms
hidden within the soil cannot be easily reached by the disinfectants
without effective cleaning with a surfactant. Now as the purpose of
disinfection is to eliminate microorganisms it will be important to
define microorganisms.
An organism that can be seen only through a microscope.
Microorganisms include bacteria, protozoa, algae, and fungi (NIH).
What about viruses? According to the National Cancer Institute,
although viruses are not considered living organisms, they are
sometimes classified as microorganisms. Within viruses there are
enveloped and non-enveloped viruses.
When it comes to microorganisms five factors are key from a cleaning
and disinfection standpoint: a) they are invisible (due to the sizes) to
the naked eye; b) their number; c) their diversity; d) their persistence
on inanimate surfaces (varies both at species and at strain level) and
e) innate resistance of microorganisms (this also varies at species as
well as at strain level). When discussing microorganisms, it would
be prudent to talk about microbial structures such as bacterial
endospores and fungal spores. It is important to keep into perspective
that the behavior of the vegetative form of the microbial (bacteria and
fungi) cells are very different than the spores (bacterial endospores
and fungal spores) towards disinfectants.
To summarize, cleaning is required to remove soil to expose the
microorganisms so that the disinfectants could reach and eliminate
them. Nowadays most commercially available disinfectant solutions
have a detergent within the formulation and therefore the activity of
cleaning and disinfection could occur simultaneously, basically the
formulations act as a one-step cleaner.
Additional definitions related to cleaning and disinfection will be
discussed under the question around definitions.
B. What are the Sources of Microbial
Contamination in a Cleanroom?
Now that we understand what we are cleaning and disinfecting it would
be important to understand the sources of microbial contamination
within a cleanroom keeping into perspective that microorganisms
are ubiquitous. According to literature, there are five main sources of
microorganisms in a cleanroom environment (Sandle, 2011):
• Personnel
• Facility
• Utilities
• Materials
• Equipment
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» MICROBIOLOGY »

Out of the above five key sources, personnel and material-equipment Differences between Bacterial Endospores and Fungal Spores
transfer are the major contributors for surface contamination and
Bacterial Endospores Fungal Spores
therefore, personnel and material flow, gowning, cleanroom behavior
Bacterial endospores are dormant Fungal spores are reproductive structures present
and robust material/equipment transfer processes play a key role in structures present in prokaryotic bacteria in eukaryotic fungi
controlling the number and diversity of microorganisms in a cleanroom Fungal spores originate to the outside. Hence,
Endospore originates inside
they are exospores
environment. In addition to personnel, the material transfer process
Endospore has a thick structure with a
plays an important role in contamination control within the cleanroom Fungal spores are varied in size, shape, and color
spore coat
environment and therefore, it is key to understand the sources of
Dipocolinate is present in endospores Dipocolinate is absent in fungal spores
contaminants and their route of ingress. For example, wheels of carts,
In endospores, heat resistance is high Heat resistance is Low in fungal spores
cardboard boxes, wooden pallets, fibers, shoes etc. are important
Endospores are resistant to chemicals and In general, fungal spores are less resistant to
sources of microbial spores. radiation chemicals and radiation

Therefore, a sporicide is an agent that destroys bacterial and mold

C. Why Should We Care? spores. Because spores are more difficult to kill than vegetative cells, a
sporicide can also acts as a sterilizing agent. In this regard, a sterilant is
We should care about a robust and effective cleaning and disinfection
used to destroy or eliminate all forms of microbial life including spores.
program not because it is required by the law, but it is the right
thing to do from a product quality and patient safety standpoint. Whereas a sanitizer lowers the number of bacteria on surfaces to levels
Microorganisms could cause harm in several ways: that are considered safe by public health organizations. Sanitizers tend
to be faster and safer than disinfectants, but disinfectants usually have
• Mere presence of them in a product (parenterals)
broader kill and log reduction claims.
• Number and types (non-sterile products)
• Producing metabolites and toxins (both parenterals and non-
sterile products) E. What are the Different Types of
• Causing degradation or spoilage (non-sterile products) Chemical Agents and Their Modes
of Action?
D. What are the Definitions? As it is evident from the definitions above that an agent can be used
for different applications (sanitizer, disinfectant, sporicide, sterilant)
In addition to the definition of cleaning, disinfection and
depending on its claims therefore, indicating that there are different
microorganisms it is also important to understand other definitions types of chemical agents along with different mode of actions.
associated with cleaning and disinfection. In this respect, it is important
to understand spores and sporicidal agents, and more importantly the Broadly, there are two types of chemical agents: - a) non-oxidizing (e.g.,
difference between bacterial and fungal spores. Broadly there are two alcohols, aldehydes, amphoterics, phenolics, quaternary ammonium
types of spores: - a) bacterial spores (endospores) and b) fungal spores
(fruiting bodies)
a. Bacterial spores: Microorganisms respond to the lack
of nutrients and other environmental fluctuations by
undertaking different survival strategies, one of the
strategies is to form endospores. Bacterial endospores
are simplified forms of the bacteria, consisting of the
DNA genome, some small amount of cytoplasm, and
a specialized coating that confers resistance to heat,
radiation, and other harsh external conditions. Endospores
are virtually immortal, and can be re-activated, under
favorable growth conditions, after lying dormant for
hundreds or perhaps millions of years (Berman, 2012).
b. Fungal spores: Are microscopic biological particles that
allow fungi to be reproduced, serving a similar purpose
Figure 1. Mode of Actions of Chemical Agents
to that of seeds in the plant world. Fungal spores are used as Antimicrobial
adapted for airborne dispersal and might also withstand
unfavorable conditions.

82 | | May/June 2022

compounds) and b) oxidizing (e.g., peracetic acid, hydrogen peroxides,
sodium hypochlorite).
Based on this broad classification of whether an agent is non-oxidizing
or oxidizing there could be different mode of actions (Figure 1) leading
to growth inhibition of the microorganism or lethal action.
(https://www.pharmawareness.com/what-is-disinfectants-and-mode-
of-action-of-disinfection-solution/)
Possible mode of actions:
• Action on the external membrane of the cell
• Action on the cell wall
• Action on the cytoplasmic membrane
• Action on the cytoplasm and nucleus
• Action on spores
To learn in more detail please visit, Center for Disease Control and
Prevention (CDC) Guideline for Disinfection and Sterilization in
Healthcare Facilities (2008)
https://www.cdc.gov/infectioncontrol/guidelines/disinfection/
disinfection-methods/chemical.html
F. What Makes a Chemical Agent
Effective Against Microorganisms?
There are several factors that could affect the efficacy of disinfection:
• Number and location of microorganisms: All conditions
remaining constant the larger the number of microbes,
the more time a chemical agent would need to eliminate
them all. Therefore, controlling and reducing the number of
microorganisms potentially increases the efficacy of a chemical
agent. The location of microbes must also be considered.
Surfaces that have crevices, joints and angles are more difficult
than flat surface. In addition, surfaces that are in direct contact
with the chemical agent would get disinfected so it would be
important to avoid air pockets. Therefore, adequate coverage of
surfaces as well as strictly adhering to the vendors application
method would be key.
• Types of microorganisms: Microbes vary greatly in their
resistance to chemical agents. The variation could be due
to cellular composition, structure, and other structures
(e.g., spores).
• Concentration of disinfectants: It is important to pay
attention and adhere strictly to the concentration of the
chemical agent recommended by the vendor or supported by
disinfectant efficacy testing (Saha, 2019) to achieve expected
efficacy. In this regard, it is important to follow the procedure
and document the preparation of the in-use solutions where
« MICROBIOLOGY »
in-house dilution of concentrated chemicals might be required
before application for disinfection. Similarly, considering the
length of the disinfection time, which depends on the potency
of the disinfectant is also important during selection of
chemical agents for surface application.
• Physical and chemical factors: Temperature, pH, and hardness
of water plays an important role in the efficacy of the chemical
agent. According to the CDC, the activity of most disinfectants
increases as the temperature increases, but some exceptions
exist. Furthermore, too great an increase in temperature causes
the disinfectant to degrade and weakens its germicidal activity
and thus might produce a potential health hazard. An increase
in pH improves the antimicrobial activity of some disinfectants
(e.g., glutaraldehyde, quaternary ammonium compounds) but
decreases the antimicrobial activity of others (e.g., phenols,
hypochlorites, and iodine). The pH influences the antimicrobial
activity by altering the disinfectant molecule or the cell surface.
Water hardness (i.e., high concentration of divalent cations)
reduces the rate of kill of certain disinfectants because divalent
cations (e.g., magnesium, calcium) in the hard water interact
with the disinfectant to form insoluble precipitates. Taking
into considerations these aspects and challenging them in the
disinfectant efficacy testing is prudent to have an efficacious
chemical agent. Similarly, adhering to these factors during
application is equally important.
Relative humidity is the single most important factor influencing
the activity of gaseous disinfectants/sterilants, such as EtO, chlorine
dioxide, and formaldehyde.
• Soiling Matter: The type and amount of soil can have an impact
on the efficacy of the chemical agent. Organic soil could interact
with the chemical agent making it ineffective or less effective.
Microorganisms could also hide within soiling agents and
inaccessible to the chemical agent.
• Wet Contact Time: It is one of the critical parameters that
directly has an impact on the efficacy of a disinfectant. It is
also known as “dwell time” and “contact time”. The wet contact
time is determined during disinfectant efficacy testing using
coupons representative of different facility surfaces on which
the disinfectants will be applied. It is important to note that
disinfectant manufacturers register their disinfectants with
specific wet contact time based on studies performed on
limited surface types to meet relevant authority’s regulations,
but pharmaceutical manufacturers must then demonstrate
disinfectant efficacy on their broad cleanroom’s surfaces
(USP<1072>) to qualify their chemical agents. As mentioned
within the article (Azab, 2020) that the disinfectant efficacy
testing performed within a laboratory in general only takes into
account microbial kill, disinfectant formulation, disinfectant
concentration and cleanroom surface but in real world
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» MICROBIOLOGY »
setting wet contact would be impacted by temperature and
humidity in the cleanroom, air exchanges in the cleanroom,
application techniques, surface cleanliness and actual time for
the disinfectant to dry. Therefore, there are two approaches
to have an efficacious disinfection from a wet contact time
standpoint: - a) if the surface dries out before achieving the wet
contact time, then reapply the chemical agent and b) perform
the disinfectant efficacy testing with the actual wet contact time
(time the surface remains wet within a cleanroom with a single
application of the chemical agent) demonstrating efficacy of the
disinfectant.
The criticality of wet contact time highlights the importance of
documenting it adequately during a cleaning and disinfection
session to meet both quality and compliance requirement.
• Other Factors: In addition to the above obvious factors there
are other less obvious factors which are equally important and
have impact of the efficacy of the chemical agents. Such factors
are:
a. frequency of disinfection,
b. rotation of disinfection (for more information refer to
Hollands, 2021),
c. technique of cleaning and disinfection (for more
information refer to Sandle, 2016)
d. state of repair and storage of accessories used for the
purpose of cleaning and disinfection,
e. disinfectant residue removal,
f. hold times of disinfectants,
g. transfer process of cleaning and disinfection materials
in and out of the cleanroom,
h. training of cleaning crew
G. Does your SOP have the following?
Since we now understand what factors could affect the efficacy
of disinfection it is important to incorporate the elements within
the cleaning and disinfection SOP with sufficient details so that the
information is not open to interpretation and the steps could be
performed in a consistent and standardize manner each time every
time. Sometimes it might be prudent to have a bilingual SOP to make
it more effective and easier to follow.
• Preparation: The types of chemical agents to be used and their
concentration.
• Regimen: The frequency and rotation of disinfectants.
• Regimen: The frequency of cleaning and disinfection.
84 | | May/June 2022
• Materials: A list of suitable cleaning materials and
their expiration.
• Application Methods: Step by step cleaning methods.
• Wet Contact Times: Length of time surfaces must stay wet to
be effective.
• Residual Removal: Steps to ensure the chemical agent is
adequately removed.
• Transfer: Process for the transfer of chemical agents and
related materials such as mops, buckets, etc. in and out of the
clean room(s).
• Expiration: Hold times for chemical agents.
H. What are Some of the Best Practices
of Cleaning and Disinfection?
Do…. Do not….
Perform cleaning prior to or simultaneously Use mops/buckets or other tools in poor
with disinfection to ensure disinfection efficacy shape
Inspect buckets/mops and other tools for Continue to use a visibly soiled mop head
integrity prior to use and replace as required or cloth
Use a double bucket system, at a minimum. Use anything less than a double
Can also use a triple bucket system bucket system
Utilize uni-directional strokes (including
parallel overlapping strokes and/or the Use circular motions
serpentine method)
Leave a room until wet-contact time
Ensure water quality for preparation
is ensured
Clean from more clean to less clean, i.e., top
to bottom (ceilings, walls, floors), back to front Walk on wet surfaces until they are dry
(entrance to exit)
Clean “less clean” areas prior to cleaner
areas (i.e., do not clean the floor prior to the
Follow cleanroom behavior and SOPs
wall) and Clean during ongoing production
activities in the same area
I. What is the Importance of Training
and Periodic Walkthrough?
Cleaning and disinfection consists of:
• several variables
• manual in nature
• performed during odd hours
• often work performed by contractors
• language barrier
• potential of high turnover
Therefore, it is needless to say the importance of training (not only
read and understand of the SOP but to have On-the-Job Training)
and oversight for a critical task like cleaning and disinfection. It is
important to note that for any manual activity it is key to ensure
that it is performed in a standardized and consistent manner to
ensure its effectiveness.

EudraLex Volume 4 Guidelines on Good Manufacturing Practice Specific to
Advanced Therapy Medicinal Products states within section 3.2 Training
• Cleaning and maintenance personnel should receive training
relevant to the tasks performed, in particular on measures to
avoid risks to the product, to the environment, and health risks.
• Training can be provided in-house. The effectiveness of training
should be periodically assessed. Records of training should be
kept.
In addition to the training periodic walkthrough of the facility to
observe cleaning and disinfection activities is equally value added
from an oversight standpoint to implement a robust and effective
cleaning and disinfection program. A checklist such as below could be
used to record and learn about the strength and weakness of both the
SOP and the training program.
• Gowning performed appropriately for area being cleaned
• Wipe down of Cleaning Cart into clean space performed
appropriately, as applicable
• Cleaning agents and supplied used within expiration
• Order of room cleaning is aligned with the procedure
• Sequence of cleaning is aligned with the procedure
• Proper technique used when cleaning
• Following proper cleanroom behaviors
• Mops/buckets changed out appropriately between rooms or
cleanroom grades, as per procedure
• Required contact times for disinfectants met as defined in the
procedure
• Copy of effective SOP or Job Aid present during cleaning
• Logbook entries made for cleanings, as required
per procedure
In conclusion the answering the final question why do the details
matter? The details matter because
• The number of microorganisms matters
• The diversity of the microorganisms matters
• The sources and location of the microorganism matter
• The mode of action and the application of the
disinfectant matters
• The manual nature of the cleaning and disinfection matters
• All the other factors matter!
References
1. Azab, W.El. (2020) A refresher on disinfectant wet contact time, Cleanroom Technology.
https://www.cleanroomtechnology.com/news/article_page/A_refresher_on_
disinfectant_wet_contact_time/167596 accessed April 04.
« MICROBIOLOGY »
2. Berman, J.J. (2012) “Class Bacilli Plus Class Clostridia” Chapter 12. In Berman, J. Taxonomic
Guide to Infectious Diseases. Academic Press, pp.65-71.
3. Center for Disease Control and Prevention (CDC) (2008) Introduction, Methods, Definitions
of Terms. Guideline for Disinfection and Sterilization in Healthcare Facilities. https://www.
cdc.gov/infectioncontrol/guidelines/disinfection/introduction.html accessed April 04.
4. EudraLex Volume 4 (2013) Good Manufacturing Practice (GMP) Guidelines. Part 1 Basic
Requirements for Medicinal Products
5. EudraLex Volume 4 (2017) Guidelines on Good Manufacturing Practice Specific to Advanced
Therapy Medicinal Products, Section 3.2 Training
6. EudraLex Volume 4 (2020) Good Manufacturing Practice (GMP) Guidelines, Revised Annex 1
Manufacture of Sterile Medicinal Products, Disinfection. https://www.gmp-compliance.org/
files/guidemgr/2020_annex1ps_sterile_medicinal_products_en.pdf accessed April 04.
7. Hollands, W. (2021) Disinfectant rotation and the frequency of use of a sporicidal agent.
American Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/
Featured-Articles/581815-Disinfectant-Rotation-and-the-Frequency-of-Use-of-a-
Sporicidal-Agent/ accessed April 04.
8. Saha, R. (2019) Disinfectant Efficacy: How can we make it effective? American
Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/Featured-
Articles/364046-Disinfectant-Efficacy-How-Can-We-Make-It-Effective/ accessed April 04.
9. Sandle, T. (2011) A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA
Journal of Pharmaceutical Science and Technology, Vol. 65, No. 4, July–August 2011,
pp392-403.
10. Sandle, T. (2013) “Application of Disinfectants and Detergents in the Pharmaceutical
Sector”. In Sandle, T. The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms.
Grosvenor House Publishing: Surrey, UK, pp. 168-197.
11. Sandle, T. (2016) Pharmaceutical Facility Sanitization: Best Practices Considered. American
Pharmaceutical Review. https://www.americanpharmaceuticalreview.com/Featured-
Articles/184449-Pharmaceutical-Facility-Sanitization-Best-Practices-Considered/
accessed April 04.
12. United States Code of Federal Regulation Sec. 211.56 Sanitation.
13. United States Environmental Protection Agency (EPA) (2022) Pesticide Registration
Manual: Chapter 4-Additional Consideration for Antimicrobial Products. https://www.
epa.gov/pesticide-registration/pesticide-registration-manual-chapter-4-additional-
considerations accessed April 04.
14. United States Pharmacopoeia (USP) (2021) USP <1072> Disinfectants and Antiseptics.
Disclaimer: This article reflects the views and opinion of the author and
should not be construed to represent any company’s views or policies. My
communication represents my own best judgment.
www.americanpharmaceuticalreview.com | | 85
 Pharmaceutical
P.I.N.
Points
Patent Innovation News
The purpose of this column is to highlight
and summarize recent key patents in the
pharmaceutical arena issued by the US
Patent Office in April 2022.
Neelam Sharma, MS, Lavanya Kundurthy,
BE and Hemant N. Joshi, PhD, MBA*
Tara Innovations, LLC
www.tarainovations.com and www.tara-marketing.com
*hemantjoshi@tarainnovations.com
Pharmaceutical Compositions
for Combination Therapy;
M. Pruzanski and L. Adorini;
Intercept Pharma; USA; U.S. Patent
#11,311,557; April 26, 2022.
Elevated concentrations of circulating lipid compounds in
the blood, such as cholesterol and triglycerides, accompany
many conditions, some include Type II diabetes, Primary
Biliary Cirrhosis (PBC), and various chronic hepatitis states
(Hepatitis B and C). The treatments of these conditions
need an improved therapy. The present invention relates to
a pharmaceutical composition comprising a combination
of an FXR (Farnesoid X Receptor) agonist and at least one
lipid lowering agent (e.g., PPAR-alpha agonist, PPAR-delta
agonist, PPAR-alpha and delta dual agonist, and/or a statin).
Also, disclosed are the use of combinations for the treatment
of the FXR-mediated condition, such as Primary Biliary
Cirrhosis (PBC), Primary Sclerosing Cholangitis (PSC), portal
hypertension, bile acid diarrhea, NAFLD (nonalcoholic fatty
liver disease), NASH (non-alcohol-induced steatohepatitis),
other chronic liver diseases with elevated lipid and liver
enzyme levels.
86 | | May/June 2022
Method for Enhancing Wound
Healing by Administrating Adenine;
H.M. Chen, C.Y. Cheng, C.F. Huang, J.T.
Lin; Energenesis Biomedical Co., TW;
U.S. Patent #11,311,546; April 22,2022.
Adenine is one of the four nitrogenous bases found in DNA
and RNA, and also, an important component of Adenosine
Triphosphate (ATP), which is a molecule that is responsible for
delivering energy to cells. The inventors have incorporated
adenine as is or as a salt in a formulation to enhance the healing
of wounds in the following tissues – skin, mouth, gingiva and
corneal epithelium. The composition activates Adenosine-
5’-Monophosphate Protein Kinase (AMPK) in the patients. It
suppresses fibroblast proliferation and prevents scar formation
during wound healing. The rate of healing wounds, such as
chronic wounds, including diabetic ulcers, pressure ulcers, and a
burn may be enhanced by adenine and/or the pharmaceutically
acceptable salt.
Compositions and Methods for
Targeting Cancers;
R. Bindra, P. Glazer, P. Sulkowski, and B.
Shuch; Yale University, USA; U.S. Patent
#11,311,538; April 26, 2022.
Genomic instability is a hallmark of cancer cells. Cells possess a
complex network of surveillance and repair pathways to confront
DNA damage directly. The deregulation of DNA repair pathways
is associated with the initiation and progression of cancer.
Present patent discloses compositions and methods for targeting
cancers. The present invention relates to the unexpected
discovery of novel compounds that sensitize a tumor cell to
anticancer therapies and that comprise succinate, fumarate,
and/or derivatives or analogues. The present invention also
includes methods for treating or preventing cancer in a subject,
wherein the cancer cells have a Fumarate Hydratase (FH) and/or
Succinate Dehydrogenase (SDH) mutation. The method includes
administering to the subject a therapeutically effective amount
of at least one compound selected from the group consisting of
a DNA repair inhibitor, a DNA strand break repair inhibitor, and a
Homologous Recombination (HR) repair inhibitor.
Lymph Directing Prodrugs;
C. Porter, J. Simpson, N. Trevaskis, T.
Quach, S. Han, and L. Hu; Monash
University, AU; U.S. Patent
#11,311,512; April 26, 2022.
The lymphatic system consists of a specialized network of
vessels, nodes and lymphoid tissues that are distributed
throughout the body in close proximity to the vascular
system. Targeted drug delivery to and through the lymphatic
system has been suggested as a means to improve both
pharmacokinetic and pharmacodynamic profiles. It has the
potential to enhance oral bioavailability through avoidance
of first pass metabolism, to alter systemic drug disposition,
and to enhance efficacy against lymph or lymphocyte
mediated pathologies. The present invention relates to
compounds in the form of prodrugs that promote transport
of a pharmaceutical agent to the lymphatic system.
Efficient Tobramycin Compound
Conjugate Compositions;
M.V. Shah, V. Subramanian, and
I. Subramanian; Somerset Therapeutics,
USA; U.S. Patent #11,298,368;
April 12, 2022.
Tobramycin is an aminoglycoside that has been used in
ophthalmological and other settings as an antibiotic.
Tobramycin, is unique among major aminoglycoside antibi-
otics. Tobramycin products show limited concentration, as
well as low corneal permeability. Despite significant investi-
gation and a multitude of formulation approaches, no tobra-
mycin or tobramycin derivative product address the limita-
tions associated with on-market tobramycin products. The
present patent discloses compositions comprising tobramy-
cin or a tobramycin derivative that exhibit improvements
in permeation of corneal cells, retention in corneal cells, or
both. Compositions can comprise tobramycin or tobramycin
derivative(s) complexed to an agent (PCL-PVCc-PEG). Such
conjugate compositions facilitate improved permeation of
corneal cells, retention in corneal cells, or both. The inven-
tion also relates to a process of preparing such compositions.
Triamcinolone and Moxifloxacin
Compositions;
M.V. Shah; I. Subramanian; V. Subramanian,
and A. Trehan; Somerset Therapeutics, USA;
U.S. Patent #11,298,315; April 12, 2022.
The eye is a sensitive and complex system. Numerous known challenges
exist in formulating ophthalmological products. Inventive approaches
are required to develop new effective ophthalmological products that
exhibit desirable properties such as reduced inflammation risk, uniform
API distribution, and API stability. Present patent describes physically
and chemically stable pharmaceutical suspension compositions to be
used before or after ophthalmic procedures. Compositions comprise
therapeutically effective amounts of one or more moxifloxacin
compounds and one or more triamcinolone compounds in a
suspension. Composition also includes an effective amount of least one
non-ionic suspension agent or one ionic suspension agent or both. It
also includes an effective amount of a chelating agent. Compositions
do not contain HPMC, and any block copolymer of poly(ethylene oxide)
and poly(polypropylene oxide).
Composition for Inhibiting Growth
of SARS-CoV-2 and Method of
Preparing the Same;
D.H. Na, K.P. Kang, J.H. Jun, G. Go, H.J.
Na, S.J Kang, Y.H. Na, S. J. Yoon, and S.J.
Na; Medicare Pharmaceuticals, KR; U.S.
Patent #11,292,789; April 5, 2022.
The present disclosure relates to a composition for inhibiting the
growth of SARS-CoV-2, and more particularly including a component
derived from Phallus indusiatus. In the SARS-CoV-2 replication cycle,
Post-Translational Modification (PTM) occurs during viral assembly
and maturation and it refers to the covalent modification of a protein
after it is translated by the ribosome. PTM involving structural
changes in polypeptides includes proteolytic cleavage and disulfide
bond formation and phosphorylation, glycosylation and lipidation
(palmitoylation and myristoylation). In addition, proteins may be
modified by covalent conjugation of one or more small proteins or
peptides. Accordingly, the present disclosure sets a virus assembly
part as a clear treatment target to solve the above problems, and
proposes a substance capable of targeting glucosylation of the PTM
stage that is the last stage of the SARS-CoV-2 replication cycle.
www.americanpharmaceuticalreview.com | | 87
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