Volume 2 Issue 4

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Cell Therapy
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Is Liver Biopsy a Gold or an Old Standard

In NAFL and NASH

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Best Practices of IoT Implementation

For Smart Drug Research

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II INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY Winter 2019 Volume 2 Issue 4
 Contents

04 Foreword

RESEARCH / INNOVATION / DEVELOPMENT

06 Cell Therapy: Challenges and Perspectives

The achievements of cell-based therapeutics over the last

DIRECTORS: decades have bolstered efforts in recent years to bring more
Martin Wright of these products to market and across an ever more diverse
Mark A. Barker range of applications. These advanced therapeutics offer
promising potential to treat conditions which, to date, have
BUSINESS DEVELOPMENT:
defied traditional treatment modalities. Anna Gregson and
Mark Sen
Dean Houston at Mathys & Squire provide an overview of some
mark@ibijournal.com
of the key applications of cell therapies, as well as looking more
EDITORIAL: closely at the challenges facing the evolution of this field.
Virginia Toteva
virginia@pharmapubs.com 10 Modelling Cancer for Immuno-oncology Applications:
Past, Present & Future
DESIGN DIRECTOR:
Jana Sukenikova Cancer therapeutics have come a long way in a short time span,
www.fanahshapeless.com leading to significant paradigm shifts in drug discovery. The
emergence of checkpoint inhibitors that target T lymphocytes
FINANCE DEPARTMENT: has dramatically altered cancer treatment, availing a plethora
Martin Wright
of new opportunities that leverage the immune system either
martin@ipimedia.com
as a monotherapeutic, or increasingly within combinatorial
RESEARCH & CIRCULATION: treatments, in the clinical management of patients. Ivan
Ana de Jesus Gladwyn-Ng at Taconic Biosciences reveals how a review of
ana@pharmapubs.com the past, present, and future of humanised models to support
immuno-oncology demonstrates how these essential tools are
COVER IMAGE: helping researchers in their quest to advance cancer therapies.
iStockphoto ©
CLINICAL RESEARCH
PUBLISHED BY:
Pharma Publications 14 Congenital Birth Defects Following Use of Dydrogesterone
50 D, City Business Centre Versus Micronised Vaginal Progesterone as Luteal Phase
London, SE16 2XB Support: A Retrospective, Observational Study
Tel: +44 (0)20 7237 2036
Fax: +44 (0)01 480 247 5316 Incidence of congenital anomalies is reported to be higher
Email: info@ibijournal.com in children born to couples with infertility compared to their
www.biopharmaceuticalmedia.com fertile counterparts. Parental (chromosomal, genetic) and
environmental factors are major contributory factors. In
All rights reserved. No part of this publication may be this study, Dr. Baidyanath Chakravarty et al. compare the
reproduced, duplicated, stored in any retrieval system or incidence of congenital anomalies in babies born to infertile
transmitted in any form by any means without prior written women with in-utero exposure to either dydrogesterone or
permission of the Publishers. micronised vaginal progesterone (MVP) following assisted
reproductive technology (ART) and non-ART treatment, and
The next issue of IBI will be published in Spring 2020. in infertile women conceiving normally and not receiving any
ISSN No.International Biopharmaceutical Industry ISSN drug for luteal phase support (LPS).
1755-4578.

The opinions and views expressed by the authors in
this magazine are not necessarily those of the Editor or
the Publisher. Please note that although care is taken in
preparation of this publication, the Editor and the Publisher
are not responsible for opinions, views and inaccuracies in the
articles. Great care is taken with regards to artwork supplied,
the Publisher cannot be held responsible for any loss or
damage incurred. This publication is protected by copyright.
2019 PHARMA PUBLICATIONS / Volume 2 Issue 4 – Winter 2019
20 Is Liver Biopsy a Gold or an Old Standard in NAFL and NASH?
When defining the term gold standard, the most appropriate
definition is “a benchmark test that is the best available under
reasonable conditions”. Rafal Ziecina at Worldwide Clinical
Trials demonstrates how when considering this definition, it
becomes clear that non-invasive imaging is replacing liver
biopsy as the gold standard for evaluation of fibrosis in
non-alcoholic fatty liver disease (NAFLD).
MANUFACTURING / TECHNOLOGY PLATFORMS

24 Split Butterfly Valve Technology and Eradicating the

Risk of Contamination in Biologics Manufacturing

As new biological therapies continue to be developed, the

market has seen an increased need for innovation. Demand

www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 1

 Contents

has grown for improved operational efficiency and achieving explains that it is essential that pharmaceuticals are protected
greater quality and cost reductions in manufacturing throughout the supply chain end-to-end, as temperature
processes. Christian Dunne at ChargePoint Technology excursions during transportation can even cause them to
shows how one of the key challenges facing biopharma become toxic.
manufacturers is eliminating the risk of contamination, which
requires effective containment strategies and monitoring of
critical manufacturing processes.

REGULATORY / QUALITY COMPLIANCE

28 Meeting the Standards Required for Effective

Biorepository Management

Biorepositories are a key asset for many organisations

including those in the biotechnology, pharmaceutical and
medical research areas. As they may contain human tissue and
other material, possibly together with personally identifying
information (PII), security is key and they can be subject to
stringent regulations. Simon Wood at Autoscribe Informatics
explores how the use of an appropriately configured
laboratory information management system (LIMS) can
contribute significantly to the efficient management of any
biobank facility.

30 Is the Absence of Data Integrity Software Affecting

the Assurance of Gel Clot Assays In Bacterial Endotoxin
Testing?

Data integrity remains an important topic in pharmaceutical

and biopharmaceutical manufacturing science. The purpose
of data integrity is to ensure that the accurate process of
production and quality of the products are shown through the
documentation that is associated with them. It is essential that
facilities report what is occurring in their labs’ processes to
ensure that good laboratory ethics are continuously practised
throughout the production of their products. Although
this is nothing new to the community, LaToya Mayfield at
FUJIFILM Wako Chemicals U.S.A. Corporation states that
every manufacturer around the world must ensure that
the data related to their products has not been affected or
compromised.

34 Best Practices of IoT Implementation for Smart Drug

Research

Drug discovery takes years to complete. Testing a single

compound can cost more than £1.5 billion. Experiments on
animals often fail to predict human behaviours and responses,
because traditional animal anatomy often does not accurately
mimic the human body. For these reasons, there is a vast need
for other ways to emulate human diseases in vitro in order
to accelerate the research and development of new drugs.
Anindya Mookerjea, founder of S-Cube Technologies, explains
why, to get the best of drug research using organ-on-a-chip
(OOC) methods, connecting it to an IoT sensor would be the
most effective.

40 Managing Global Supply Chains to Mitigate Risks in

Pharma Logistics

As temperature-sensitive pharmaceutical products are

increasingly being shipped globally to more remote regions,
there is an even greater demand for the effective and efficient
management of global supply chains. Whether shipping
finished products, transporting clinical trials materials or
delivering sample drugs, Adam Tetz at Peli BioThermal

2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY Winter 2019 Volume 2 Issue 4

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 Foreword
Following the approval, in recent years, of
the first immune checkpoint inhibitor, there
has been an explosion in the development
of immuno-modulating pharmacological
modalities for the treatment of various
cancers. From the discovery phase to late-stage
clinical testing and regulatory approval, challenges in the
development of immuno-oncology (IO) drugs are multi-fold
and complex. In the preclinical setting, the multiplicity
of potential drug targets around immune checkpoints,
the growing list of immuno-modulatory molecular and
cellular forces in the tumour microenvironment – with
additional opportunities for IO drug targets, the emergence
of exploratory biomarkers, and the unleashed potential of
modality combinations all have necessitated the development
of quantitative, mechanistically-oriented systems models
which incorporate key biology and patho-physiology
aspects of immuno-oncology and the pharmacokinetics of
IO-modulating agents. In the clinical setting, the qualification
of surrogate biomarkers predictive of IO treatment efficacy
or outcome, and the corresponding optimization of IO trial
design have become major challenges.
In this issue of IBI Ivan Gladwyn-Ng at Taconic Bio-
sciences reveals how a review of the past, present, and
future of humanised models to support immuno-oncology
IBI – Editorial Advisory Board
• Ashok K. Ghone, PhD, VP, Global Services MakroCare, USA
• Bakhyt Sarymsakova – Head of Department of International
Cooperation, National Research Center of MCH, Astana,
Kazakhstan
• Catherine Lund, Vice Chairman, OnQ Consulting
• Cellia K. Habita, President & CEO, Arianne Corporation
• Chris Tait, Life Science Account Manager, CHUBB Insurance
Company of Europe
• Deborah A. Komlos, Senior Medical & Regulatory Writer,
Clarivate Analytics
• Elizabeth Moench, President and CEO of Bioclinica – Patient
Recruitment & Retention
• Francis Crawley, Executive Director of the Good Clinical Practice
Alliance – Europe (GCPA) and a World Health Organization (WHO)
Expert in ethics
• Hermann Schulz, MD, Founder, PresseKontext
• Jeffrey W. Sherman, Chief Medical Officer and Senior Vice
President, IDM Pharma.
Ad Index IBI Winter
Page 7 Abzena Ltd
Page 31 ARTES Biotechnology GmbH
Page 3 BioGenes GmbH
IBC Expres2ion Biotechnologies Aps
Page 25 GenXpro GmbH
4 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
demonstrates these essential tools are helping researchers
in their quest to advance cancer therapies.
Within the Clinical Research Section, Dr. Baidyanath
Chakravarty, a renowned IVF Phisician and his team,
compares the incidence of congenital anomalies in babies
born to infertile women with in-utero exposure to either
dydrogesterone or micronised vaginal progesterone (MVP)
following assisted reproductive technology (ART) and
non-ART treatment.
Within the Regulatory Section, Simon Wood at Autoscribe
Informatics explores how the use of an appropriately
configured laboratory information management system (LIMS)
can contribute significantly to the efficient management of
any biobank facility, and Anindya Mookerjea, founder of
S-Cube Technologies, explains why, to get the best of drug
research using organ-on-a-chip (OOC) methods, connecting
it to an IoT sensor would be the most effective.
The IBI Team wishes all our partners, a wonderful start to
the year 2020, my team and I look forward to bringing more
exciting articles and features in the next issue of IBI.
Virginia Toteva,
Editorial Manager
• Jim James DeSantihas, Chief Executive Officer, PharmaVigilant
• Lorna. M. Graham, BSc Hons, MSc, Director, Project Management,
Worldwide Clinical Trials
• Mark Goldberg, Chief Operating Officer, PAREXEL International
Corporation
• Maha Al-Farhan, Chair of the GCC Chapter of the ACRP
• Rick Turner, Senior Scientific Director, Quintiles Cardiac Safety
Services & Affiliate Clinical Associate Professor, University of
Florida College of Pharmacy
• Robert Reekie, Snr. Executive Vice President Operations, Europe,
Asia-Pacific at PharmaNet Development Group
• Stanley Tam, General Manager, Eurofins MEDINET (Singapore,
Shanghai)
• Stefan Astrom, Founder and CEO of Astrom Research
International HB
• Steve Heath, Head of EMEA – Medidata Solutions, Inc
• T S Jaishankar, Managing Director, QUEST Life Sciences
Page 5 Nemera
Page 35 Pharma Publications
IFC PlasmidFactory GmbH
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Page 21 SONOTEC GmbH
Winter 2019 Volume 2 Issue 4
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www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 5

 Research / Innovation / Development
Cell Therapy: Challenges and Perspectives
The achievements of cell-based therapeutics over the
last decades have bolstered efforts in recent years to
bring more of these products to market and across an
ever more diverse range of applications. These advanced
therapeutics offer promising potential to treat conditions
which, to date, have defied traditional treatment
modalities. Interest and investment in this sector are at
an all-time high and whilst many are hopeful of a boom
in the number of approved therapies in the coming years,
the industry still faces significant challenges, particularly
with regard to the manufacture and regulation of these
cell-based products. In this article, we will provide an
overview of some of the key applications of cell therapies
as well as look more closely at the challenges facing the
evolution of this field.
To date, the applications of cell therapies have largely fallen
into two broad categories; tissue regeneration and immuno-
modulation. With regard to the former, cell therapy has been
viewed as one of the most promising techniques for the
repair of damaged tissue, with applications in cardiovascular
disease, neurodegenerative disease (for example, Parkinson’s
and Alzheimer’s), musculoskeletal injury or degeneration and
endocrine dysfunction (for example, type I diabetes).
Cell therapies have proven particularly effective in the repair
of articular cartilage, for which the intrinsic capacity for repair
is low. The most established of these therapies have employed
the patient’s own cells, i.e. autologous cells. In brief, harvested
chondrocytes are expanded ex vivo, seeded into a collagen
matrix and then re-implanted into cartilage defects in joints.
Such products have been available for around a decade now
(ChondroCelect, developed by TiGenix was first approved in the
EU in 2009) and have shown considerable efficacy, although
use of these advanced options is still low when compared to
traditional treatment modalities (for example, joint replacement
and analgesics). Whilst cartilage repair applications have tended
to employ the terminally differentiated chondrocyte, bone repair
applications have made use of the regenerative capacity of
stem and progenitor cells. Bone marrow-derived mesenchymal
stem cells (MSCs) have been proven in a range of orthopaedic
applications over recent decades, including in the treatment
of infants with osteogenesis imperfecta and in the repair of
non-union fractures. Unfortunately, obtaining sufficient yields
of pure MSC populations from bone marrow has proven difficult
and there has been a switch in recent years to utilise MSCs derived
from other sources, such as adipose tissue.
Autologous cell therapies like those discussed above all
depend on obtaining sufficient cell numbers from the donor
patient and the ability to expand functional cells ex vivo. Off-the-
shelf cell therapies, which clinicians can employ for a range of
patients, as and when needed, without concerns over yield or
6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
PEER REVIEWED
expansion protocols, are likely to represent the future of cell
therapy. UK-based biotech, ReNeuron, is one such company
forging ahead with allogeneic cell therapies. Interestingly,
ReNeuron’s neural stem cell line for the treatment of the disabling
effects of stroke were cryopreserved prior to utilisation in the
PISCES I (Phase I) clinical trial. Cryopreservation is just one of a
number of advancements which will be necessary to bring off-the-
shelf cell products to reality.
Whilst the regenerative applications of cell therapies have, at
the very least, been researched for some time now, the immuno-
modulatory applications of cell therapy, in particular, chimeric
antigen receptor (CAR) T cells, is a more recent development.
Indeed, it was only in the early 90s when first-generation
CAR T cells (which contained an antibody/T cell receptor
fusion molecule) were developed and around the same time
researchers were investigating adoptive transfer of patient-
derived virus-specific T cells. Since these early days, significant
leaps forward have been made. In 2017, Novartis’ Kymriah
(tisagenlecleucel) became the first CAR T cell therapy to be
approved by the FDA, with Kite Pharma’s Yescarta (axicabtagene
ciloleucel) following shortly thereafter. Data from the UK’s Cell and
Gene Therapy Catapult clinical trials database indicates that there
were around 22 clinical trials investigating the safety and efficacy
of CAR T cells in the UK alone in 2018. The success of CAR T cells
to date has largely been shown for haematological malignancies
(indeed, Kymriah and Yescarta are approved for the treatment
of acute lymphoblastic leukaemia and large B-cell lymphoma
respectively). In contrast, despite extensive research, CAR T cell
therapy for solid tumours hasn’t had the same impact, not least
because of the challenges of targeting solid tumours including
identifying a suitable target antigen and homing the cells to
the hostile, tumour microenvironment. Nonetheless, strides are
being made by combining CAR T cell therapy with other biologic
agents, specifically checkpoint inhibitors such as pembrolizumab
and nivolumab which target programmed cell death protein 1
(PD-1) a key regulatory protein found on T cells. The University of
Pennsylvania, for example, is recruiting for a Phase I clinical trial
assessing the safety of a CAR T cell/pembrolizumab combination
therapy for the treatment of glioblastoma. This follows preliminary
evidence from the Memorial Sloan Kettering Cancer Center that
showed both safety and efficacy of a mesothelin targeting CAR
T cell and pembrolizumab combination therapy in patients with
malignant pleural disease. Thus, the use of CAR T cells for the
treatment of solid tumours appears to be progressing.
Immuno-modulatory cell therapies other than CAR T cells are
also being investigated in the clinics. By way of example, Fate
Therapeutics is currently assessing the safety of its off-the-shelf
Natural Killer (NK) cell therapy. Unlike traditional CAR T cells,
Fate’s NK cell therapies are derived from an induced pluripotent
stem cell (iPSC) line allowing the production of large numbers of
well-defined cells without relying on a patient’s own immune cells
(which are often depleted in many cancers). Preclinical studies
showed the efficacy of these cells in the treatment of checkpoint
Winter 2019 Volume 2 Issue 4
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 Research / Innovation / Development
inhibitor resistance tumours. At present, Fate has a pipeline of at
least five different NK cell therapies.
As well as the immuno-oncology applications, cell therapies
are also being trialled for immuno-regulatory applications such
as in the treatment of autoimmune disease and graft versus host
disease. These trials have largely involved the use of autologous,
expanded, regulatory T cells (Treg cells) which, through a range
of mechanisms, are able to suppress a variety of immune cells.
Treg cells used in studies to date have been isolated from both
umbilical-cord blood and peripheral blood. A variety of Phase I
studies have been completed or are in the process of assessing the
safety of Treg cells for the treatment of type I diabetes. Although
in the early stages of development, data to date is showing that
Treg cells are well tolerated in patients and the ex vivo expansion
methods are capable of generating sufficient numbers of stable
and functional Treg cells. Future Phase II/III trials will of course be
needed to reveal the true potential of these cells.
Global investment in cell-based therapies increased to US$7.6
billion in 2018, a 64% increase from the previous year. In spite
of this, the sector still faces a number of significant challenges
before these advanced therapeutics become widely used.
Research and development in this sector is undeniably
booming, though difficulties in expanding, manufacturing and
transporting cell products may be hampering the commercial
viability and ultimate availability of these products. Achieving
the quantity of cells needed with current production methods,
especially if uptake of these therapies becomes more widespread,
is one of the major hurdles facing the industry. By way of example,
the recommended dose of ChondroCelect is 1 million cells/cm2
of cartilage defect. CAR T cell therapy Yescarta is dosed at a
8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
staggering 2 million cells per kg (around 140 million cells for an
average adult male). The issue is magnified somewhat by the focus
of today’s research on the cell product per se; emerging biotech
companies with innovative cell therapies should, at an early
stage, consider the processes that will be necessary to achieve
the desired cell numbers for later Phase II/III trials and beyond.
These challenges also bring opportunities however, and there
are now a number of innovative companies seeking to develop
solutions for the industry, to simplify, accelerate and improve cell
therapy manufacturing and supply.
Automation of the manufacturing processes is currently
of significant interest to the community. At present, the
manufacturing processes employed in the generation of
cell therapies largely resemble those utilised in other
biopharmaceutical areas (for example therapeutic antibodies).
Unlike therapeutic antibodies production, however, cell
therapies (especially those relying on patient or donor cells)
vary significantly from batch to batch, requiring complex and
adaptive processes to generate consistent products within the
regulatory confines. Through the implementation and training
of a variety of mechanisms, e.g. sensors, robotics and image
acquisition as well as processing software, researchers believe
variability and reliability of current manufacturing processes
can be improved.
Whilst improvements in the manufacturing processes will
hopefully lead to a reduction in the costs associated with the
production of cell therapies, it should be noted that, unlike
traditional therapeutic modalities, cell therapies are often a
one-off treatment option for patients. Biotech companies must
bear this in mind when attempting to recoup their research
and development costs and, as such, costs are always likely to
Winter 2019 Volume 2 Issue 4
 Research / Innovation / Development

be higher than traditional biologics. As it stands, the high costs

associated with these therapies is proving challenging for
healthcare providers to justify.

The cost of these therapies is at least in part due to the

convoluted path from bench to bedside. Cell therapies are
considered differently to the conventional biopharmaceutical
agents and have to undergo even more rigorous regulatory and
quality assessments. This of course ensures public safety, but has
also put the brakes on the number of cell therapies actually being
approved (despite the ample number of trials). As is so often the
case, the regulatory frameworks in place have not been able to
keep up with the unprecedented scientific advances in this field.
What’s more, the absence of harmonisation across jurisdictions
has placed undue burden on the smaller players in this field. The
lengthy timescales involved in obtaining regulatory approval
(even after showing clinical efficacy) are exemplified by Holclar,
an autologous cell therapy (comprising human corneal epithelial
cells and limbal stem cells) for the repair of damaged cornea,
which despite having shown clinical efficacy as early as 1997,
only obtained regulatory approval in 2015.

Regulation is of course paramount to ensure the safety of

patients receiving advanced therapeutics (including cell and gene
therapies) which have long been shrouded in safety concerns.
These concerns are not without basis. Indeed, safety has been a
major sticking point for stem cell therapies. The primary concern
regarding stem cell therapies is unwanted differentiation, as has
been shown in the cardiovascular setting, where calcifications
have been identified in the myocardium of patients treated with
MSCs following infarction (MSCs, of course, give rise to cells of
bone and cartilage as well as muscle). Tumorigenesis has also been
a concern for stem cell therapies, although this appears to have
been unwarranted based on current data. In the immuno-oncology
field, CAR T cells have also been associated with safety concerns
Anna Gregson
including the development of cytokine release syndrome in
Anna is a partner in Mathys & Squire’s
patients receiving CAR T cell therapies, the engagement of target biotech team. She has over 10 years’
antigens on non-pathogenic tissues and host immune response to experience working with a diverse client
the specific recombinant proteins found in these cells. Pleasingly, base; from university technology transfer
the industry is seeking solutions to these problems and research organisations to international corporations. Anna’s
is ongoing to improve the safety profile of these therapies. In the expertise covers a wide range of biotechnology and life
CAR T cell space, the incorporation of suicide or elimination genes sciences subject matter, with a particular emphasis on the
into delivered cells is being investigated as a means to selectively cell and gene therapy space, including CAR T cells, iPSCs,
deplete these cells in the body when necessary. The approved cell neural regenerative medicine, cell culture technologies,
therapies are largely still in their infancy and data from future and viral/non-viral gene therapy vectors.
Phase IV clinical trials will be indispensable in assessing the
long-term safety of these therapies. Email: algregson@mathys-squire.com

The number of cell therapies actually approved for clinical
use remains small. This highlights that, despite the significant
scientific advances and investment, the sector is largely still at the
research and development stage. Having said that, the industry
appears to have reached a critical mass and with the number of
clinical trials in this field growing steadily, we can only assume
that we will be seeing more and more of these therapies in the
clinics. The industry seems to have clicked and more emphasis
is now being placed on the challenges of efficiently, yet safely,
manufacturing these products. Improvements in this key area
could pave the way for wider implementation and access to these
therapies. A multidisciplinary approach will be essential in the
coming years to increase the number of approved therapies whilst
still ensuring affordability and, importantly, patient safety.
Dean Houston
Dean is a trainee patent attorney in Mathys
& Squire’s biotech team. He has a strong
background in the cell and gene therapy
sector, having completed his PhD at The
Roslin Institute in Edinburgh. Dean has experience in the
global prosecution of patent families across a diverse
range of technologies, including therapeutic toxins,
antisense oligonucleotides, vaccines and methods of
neuronal stem cell culture.
Email: dahouston@mathys-squire.com

www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 9

 Research / Innovation / Development
Modelling Cancer for Immuno-oncology Applications:
Past, Present & Future
Cancer therapeutics have come a long way in a short
timespan, leading to significant paradigm shifts in drug
discovery. The emergence of checkpoint inhibitors that
target T lymphocytes have dramatically altered cancer
treatment, availing a plethora of new opportunities
that leverage the immune system either as a
monotherapeutic, or increasingly within combinatorial
treatments in the clinical management of patients. This
successful targeting of T cells in oncotherapies has led
researchers to explore the possibilities of harnessing
other immune cell types that also contribute to cancer
biology in immunotherapies. With the advancement
of such treatment modalities, there has been a
corresponding advancement in the animal models that
serve as preclinical tools, most notably mouse models
in which a human immune system is engrafted into
an immunodeficient host, enabling the more faithful
recapitulation of targets within the immune system. A
review of the past, present, and future of humanised
models to support immuno-oncology demonstrates how
these essential tools are helping researchers in their
quest to advance cancer therapies.
The Past: T Cells Take Centre Stage
There is no denying the substantial contribution of immune
checkpoint inhibitors (ICIs), such as anti-PD-1, anti-PD-L1, and
anti-CTLA-4, to the oncology field, as evidenced by the awarding
of the 2018 Nobel Prize in Physiology or Medicine to James
P. Allison and Tasuku Honjo – two immunologists whose work
led to pioneering discoveries on the manipulation of immune
checkpoints for cancer treatment. ICIs work by blocking immune
checkpoint proteins from binding to their partner proteins, which
prevent an “off-signal” from being sent to immune cells. The
approved ICIs mentioned above prevent cancerous cells from
evading attack from immune T lymphocytes, thereby improving
the latter’s ability to mount an immune response against the
tumour cells.
Whilst the breakthrough in immuno-oncology is built upon a
wealth of preclinical findings from in vitro and in vivo research
– the latter comprising syngeneic and xenograft rodent models
– the recent advancement in immunotherapies is increasingly
facilitated by humanised mouse models. In greater detail,
immuno-deficient mice are engrafted with either human
hematopoietic stem cells (HSCs) or peripheral blood mononuclear
cells (PBMCs) – or specific immune cells such as natural killer
(NK) cells – in order to reconstitute the human immune system
(or specific immune sub-compartments). The resulting models
are known as humanised immune-system (HIS) rodent models.
These HIS models are then implanted with human tumour cells
derived from immortalised cell line- or patient-derived xenografts
(CDX and PDX, respectively). In one early study, HIS mice were
implanted with human leukocyte antigen (HLA) partially matched
10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
tumours of various histological types from either PDXs or from
a triple-negative breast cancer CDX. After treatment with the
anti-PD1 pembrolizumab (Keytruda®), growth of the PDX and CDX
tumours was significantly inhibited in the humanised immune
system mice.1
The Present: A Broader Perspective
With the increasing successes of preclinical investigations
and clinical trials since the 2000s that led to the approval of
blockbuster ICIs such as pembrolizumab, nivolumab (Opdivo®)
and ipilimumab (Yervoy®) in the early 2010s, many subsequent
preclinical studies are investigating the use of ICIs in combination
with other therapeutic agents. The efficacy of pembrolizumab
was explored in combination with ONCOS-102, an oncolytic
adenovirus which the researchers hypothesised could serve
as an immunosensitiser when used with an ICI. In HIS mice
engrafted with human A2058 melanoma cells, treatment with
pembrolizumab alone showed no therapeutic effect, whereas
treatment with ONCOS-102 led to a significant reduction in tumour
volume, and combinational co-treatment of pembrolizumab and
ONCOS-102 reduced tumour volume to a greater degree.2
While checkpoint inhibitor therapy has become the treatment
of choice for an increasing number of oncology indications – both
as monotherapy and in combination – with greatly improved
treatment outcomes, especially for a subset of cancer patients that
were previously refractory to other treatment modalities, marked
differences exist between responders and non-responders.
Additionally, these T cell-based immunotherapies are associated
with risks such as cytokine release syndrome and neurotoxicity,
and they are not always feasible for patients who are already
immuno-depressed after receiving a first-line cancer treatment.
These limitations have led oncology researchers to venture far
beyond the boundaries of T cells and ICIs.
A natural evolution of the prior immunotherapies described
above has been to enhance the efficacy of T cell-based
therapies, which is being accomplished in several ways. Current
immuno-oncology research is focusing on a wider range of
therapeutic approaches and cell types. One such approach is
chimeric antigen receptor (CAR)-T cell therapy, which involves
genetically modifying a patient’s T cells to render them capable
of recognising and killing cells that express the target protein.
The first two CAR-T cell-based therapies were approved by the
US Food & Drug Administration (FDA) for haematologic cancers
(Kymriah® from Novartis and Yescarta® from Gilead Sciences) and
reached the market in 2017, contingent on physicians completing
adverse effects management training due to the known risks
of cytokine release syndrome and neurotoxicity.3 In late 2019,
Gilead released data that reported a three-year survival rate of
47% in patients with refractory large B cell lymphoma treated
with its Yescarta® CAR-T cell therapy. Concurrently, Johnson &
Johnson’s investigational CAR-T therapy (JNJ-4528) received the
Breakthrough Therapy Designation from the FDA after a clinical
Phase Ib trial in which patients with relapsed or refractory
Winter 2019 Volume 2 Issue 4
 Research / Innovation / Development
multiple myeloma showed a 100% response rate at a median
six months follow-up post-treatment.
Haematologic cancers have been the initial focus of adoptive
cell transfer of T lymphocytes, which has proven efficacious in
some tumour types. In a preclinical study of prostate cancer,
adoptive transfer of naïve T lymphocytes (including CD4 T
cells) from female mice were transplanted into irradiated male
syngeneic mice that were subsequently implanted with TRAMP-C2
prostate tumour cells. The female lymphocytes slowed the growth
of the implanted cancer cells, with no worsening of graft-versus-
host disease (GvHD) despite implanting cells across genders.4
Although CAR-T therapy has been approved for haematological
cancers such as Non-Hodgkin’s lymphoma and acute
lymphoblastic leukaemia, ongoing preclinical work is addressing
its toxicity and efficacy, as well as the challenge of CAR-T cells
migrating into solid tumours. In NOG mice expressing human
IL-2 cytokine, CAR-T cell therapy was effective in targeting
both xenografted uveal and cutaneous melanoma cells.5 These
findings were consistent with the results of clinical trials, even
in patients who had proven resistant to adoptive cell transfer of
autologous tumour-infiltrating T lymphocytes (TILs). Nevertheless,
the potential efficacy of CAR-T therapy must be balanced by the
presence of similar risks as found in other T cell therapies, as well
as a risk of cerebral oedema. Continued preclinical work aims to
improve upon CAR-T therapy to overcome these drawbacks.
Though these approaches have demonstrated good efficacy
in some applications, T cell-based immunotherapy risks have
prompted investigators to explore other immune cell types to
develop newer therapeutic modalities. NK cells are one option
www.biopharmaceuticalmedia.com
gaining traction because they can be mobilised to target tumour
cells by using various germ line-encoded cell surface receptors,
in order to circumvent the risks and limitations of T cell therapies.
Whilst early efforts to engraft NK cells into immune-deficient
models have been limited by poor cell uptake and a short survival
window for the recipient mice, research at the Central Institute
for Experimental Animals (CIEA) demonstrated improved NK
cell engraftment and survival is feasible using a mouse model
that transgenically expresses human interleukin 15 (IL-15). In
comparison to the conventional NOG mouse, the engraftment of
human PBMCs in this hIL-15 NOG mouse led to significantly higher
levels of human NK cell reconstitution, with a longer duration of
survival without indications of GvHD, all of which resulted in a
feasible study window for assessing NK cell therapeutics.6
Recognising the role of cytokines in immune response, further
characterisation of IL-15 and IL-2 is underway to determine their
ability to stimulate NK cell anti-tumour immunogenicity, in order
to overcome limitations in NK cells’ ability to zero in on tumours.7
Concurrently, clinical trials are investigating the subcutaneous
administration of IL-2 in low doses in conjunction with NK cell
therapy, based on preclinical studies demonstrating that low
doses of IL-2, following chemotherapy-induced cytopenia,
promoted anti-tumour response.
One of the known challenges of modelling the interactions
between the immune system and tumour cells is that the
development of certain human immune cell lineages (including
myeloid cells) is restricted in most HIS mouse models. In order
to enable a more comprehensive reconstitution of the human
immune system and improve the levels of human myeloid cells
following HSC engraftment in mice, researchers are investigating
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 11
 Research / Innovation / Development
the efficient development of neutrophils, granulocytes, and
macrophages in HIS mice. In turn, preclinical mouse models that
express the human cytokines IL-3 and granulocyte-macrophage
colony-stimulating factor (CM-CSF) are becoming more widely
used in immuno-oncology, as these cytokines are critical for
myeloid development and differentiation. These models,
including the NOG-EXL and huNOG-EXL, support expanded
myeloid lineage development and improved T cell engraftment,
making them especially suitable for studying ICIs in combination
with other therapies. Using a breast tumour model with expanded
development of lymphocytic and myeloid lineages, researchers
investigated the effects of niraparib, a PARP-1/2 inhibitor,
on the efficacy of an anti-PD-1 therapeutic and discovered
the administration of these therapeutics together resulted in
synergistic anti-tumour activities in both BRCA-proficient and
BRCA-deficient tumours.8
Oncological Vaccines on the Rise
While ICI therapy has become a standard of care in oncology,
the relatively high percentage of non-responders continues to
encourage researchers to explore new alternative treatments,
such as increasing investigations into therapeutic cancer vaccines.
Such vaccines are designed to elicit an immune response
against tumour antigens, which are known to play a role in the
development, progression and metastasis of cancers. Oncological
vaccine research is yielding new insights and developments, in
both target and antigen selection and in vaccine technology.
Antigen selection can be especially problematic, as few antigens
meet all the criteria considered necessary for cancer vaccine
efficacy, including the need for the antigen to be expressed by
cancer cells only, present on all cancer cells, essential for cancer
cell survival, and highly immunogenic.9
The primary oncological vaccine targets are tumour-associated
antigens (TAAs) and tumour-specific antigens (TSAs), including
neoantigens. Many studies demonstrate that response to ICI
therapy is often correlated with a high number of predicted
neoantigens.9 Yet, most neoantigens are unique to the patient and
their number varies by tumour type, necessitating a personalised
approach that can prove cost-prohibitive. Nevertheless, preclinical
proof-of-concept studies in mice have tested the feasibility
and efficacy of employing vaccines targeting neoantigens.
12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Post-vaccination with synthetic long peptide neoantigens, three
mouse models experienced noteworthy anti-tumour response,9
while a mouse model of Lynch syndrome (associated with a high
risk of colorectal cancer and increased risk of other cancer types)
was vaccinated with four tumour neoantigens and subsequently
experienced significantly reduced tumour burden in the intestines
and improved survival as compared to non-vaccinated mice.10
Nucleic acid vaccines, such as DNA-based vaccines, represent
another avenue of promising immuno-oncology research. Most
DNA vaccines are plasmids that deliver genes encoding tumour
antigens, which in turn elicit an immune response against
tumour cells bearing those antigens. While DNA vaccines have
proven to be well tolerated without significant risk of major
adverse effects, they tend to demonstrate limited therapeutic
effects due to poor immunogenicity.11 A number of strategies
are under investigation to improve DNA vaccine efficacy,
including improving immunogenicity through selection of the
optimal antigens for insertion into the DNA, or combining a
DNA vaccine with other therapies in order to modulate tumour
immunosuppression or improve immune cell volume and
activity.12
A third approach to oncological vaccines is to adapt a patient’s
dendritic cells to express tumour-associated antigens, thereby
encouraging T  cells to attack cancer cells. Currently, efficacy
limitations have hampered the success of this approach in clinical
trials, but several strategies may enhance efficacy, including the
identification of subsets of dendritic cells that express high levels
of targeted antigens and the improvement of vaccine delivery to
lymph nodes.13
The Future: Modelling Pharmacokinetics and
Pharmacodynamics
Building on efforts to improve immunotherapy efficacy, future
immuno-oncology research is likely to focus on enhancing the
pharmacokinetic (PK) and pharmacodynamic (PD) profile of
immuno-oncology therapeutics. A better understanding of the
absorption, distribution, metabolism and excretion (ADME) of an
immunotherapeutic, as well as its mechanistic action (including
response magnitude and duration), is critical to improving the
viability of these therapeutics, yet much remains unknown.
Winter 2019 Volume 2 Issue 4
 Research / Innovation / Development
A critical foundational tool for this work is preclinical
models capable of modelling human pharmacokinetics and
pharmacodynamics more faithfully, which remains a challenging
endeavour. Syngeneic mouse models have been used to study
the toxicity of immunotherapies, since such models represent the
interactions between murine host cells and a competitive immune
system.14 Hence, ongoing development of preclinical models is
targeted towards improving their ability to accurately assess
appropriate dosing levels that balance the efficacy and safety of
immunotherapies, making this an essential area of emphasis in
the evolution of model development.
One of the most significant obstacles to gaining a better
understanding of the PK and PD profiles of an immunotherapy is
the potential for the human microbiome to impact drug activity,
metabolism and toxicity. Enright et al reviewed the various ways
in which human microbiota may increase a drug’s activity, render
it inactive, or lead to the accumulation of toxic metabolites.15 In
fact, preclinical and clinical studies have found that patients’
marked differences in response to ICI therapy may be a function of
differences in their gut microbiome compositions. When patients
with non-small cell lung, kidney or bladder cancer received
antibiotics before or shortly after treatment with an anti-PD-1
therapeutic, they experienced shorter progression-free and
overall survival than those who had not received antibiotics.16
When germ-free mice then received faecal microbiota
transplantation (FMT) from patients, anti-tumour activity was
evident in the mice that received FMT from responder patients
and anti-PD-1, a response that was correlated with one bacterial
strain.16 Using melanoma models, researchers at the University of
Texas MD Anderson Cancer Center and the University of Chicago
found similar correlations between the microbiome composition
and response to anti-PD-1 or anti-PD-L1.
The field of immuno-oncology has experienced both dramatic
breakthroughs and steady improvements, all contributing to
significant advances in how cancer is treated. Preclinical models
have served and will continue to serve as effective tools for the
study of immunotherapy efficacy and safety, across an expanding
array of immune cell types and therapeutic approaches. As
immuno-oncology investigators continually push the boundaries
of their research, seeking the most efficacious treatments with
the most desirable safety, PK and PD profiles, these models will
evolve to stay in step with investigative trends.
REFERENCES
1. Wang M., Yao L.C. et al. Humanized mice in studying efficacy and
mechanisms of PD-1-targeted cancer immunotherapy. FASEB J. 2018
Mar;32(3):1537-1549. doi: 10.1096/fj.201700740R. Epub 2018 Jan 3.
2. Kuryk L., Møller A.W., Jaderberg M. Combination of immunogenic
oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab
exhibits synergistic antitumor effect in humanized A2058
melanoma huNOG mouse model. Oncoimmunology. 2018 Oct
29;8(2):e1532763. doi: 10.1080/2162402X.2018.1532763.
eCollection 2019.
3. June, C.H., O’Connor, R.S. et al. CAR T cell immunotherapy for human
cancer. Science. 2018 March 23; 359, 1361–1365. doi: 10.1126/
science.aar6711.
4. Jenq R., Curran, M.A. et al. Repertoire enhancement with adoptively
transferred female lymphocytes controls the growth of pre-implanted
murine prostate cancer. PLoS One. 2012;7(4):e35222. doi: 10.1371/
journal.pone.0035222. Epub 2012 Apr 6.
5. Jespersen H., Lindberg M.F. et al. Clinical Responses to Adoptive
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T-Cell Transfer Can Be Modeled in an Autologous Immune-Humanized
Mouse Model. Nat. Commun. 2017, 8 (1), 707. doi: 10.1038/s41467-
017-00786-z.
6. Volden, P. New PBMC-humanized Mice Support Efficient NK-cell
Engraftment https://www.taconic.com/taconic-insights/oncology-
immuno-oncology/humanized-mice-nk-cell-engraftment.html
(accessed Mar 11, 2019).
7. Childs R., Carlsten M. Therapeutic approaches to enhance natural
killer cell cytotoxicity against cancer: the force awakens. Nat Rev Drug
Discov 14, 487–498 (2015) doi:10.1038/nrd4506.
8. Wang Z., Sun K. et al. Niraparib activates interferon signaling and
potentiates anti-PD-1 antibody efficacy in tumor models. Sci Rep.
2019 Feb 12;9(1):1853. doi: 10.1038/s41598-019-38534-6.
9. Hollingsworth R.E., Jansen K. Turning the corner on therapeutic
cancer vaccines. npj Vaccines 4, 7 (2019) doi:10.1038/s41541-019-
0103-y.
10. Gelincik O., Ibrahim H. et al. Frameshift neoantigen vaccination
prevent Lynch syndrome mouse model intestinal cancer. Proceedings
of the AACR Annual Meeting 2019; 2019 Mar 29-Apr 3;: AACR; Cancer
Res 2019;79(13 Suppl):Abstract nr 2723. 
11. Yang B., Jeang J. et al. DNA vaccine for cancer immunotherapy.
Hum Vaccin Immunother. 2014 Nov; 10(11): 3153–3164.
doi: 10.4161/21645515.2014.980686.
12. Lopes A., Vandermeulen G., Préat V. Cancer DNA vaccines: current
preclinical and clinical developments and future perspectives. J Exp
Clin Cancer Res. 2019; 38: 146.10.1186/s13046-019-1154-7. doi:
10.1186/s13046-019-1154-7.
13. Riley R.S., June C.H., Langer R.  et al.  Delivery technologies for
cancer immunotherapy. Nat Rev Drug Discov 18, 175–196 (2019)
doi:10.1038/s41573-018-0006-z.
14. Ochoa de Olza M., Oliva M. et al. Early-drug development in the era
of immuno-oncology: are we ready to face the challenges? Annals
of Oncology, Volume 29, Issue 8, August 2018, 1727–1740, https://
doi.org/10.1093/annonc/mdy225.
15. Enright E., Gahan C.G.M. et al. The impact of the gut microbiota on
drug metabolism and clinical outcome. Yale J. Biol. Med. (2016).
16. Routy B. et al. Gut microbiome influences efficacy of PD-1-based
immunotherapy against epithelial tumors. Science (80-. ). (2018).
doi:10.1126/science.aan3706.
Ivan Gladwyn-Ng
Ivan Gladwyn-Ng, PhD, is a Field Applications Scientist at
Taconic Biosciences. He earned his PhD at the Australian
Regenerative Medicine Institute at Monash University
in Melbourne, Australia, where he studied cortical
development, epilepsy and autism in children using
transgenic mouse models. He completed postdoctoral
fellowships at the GIGA-Neuroscience Institute and the
Institut Pasteur.
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 13
 Clinical Research
Congenital Birth Defects Following Use of Dydrogesterone
Versus Micronised Vaginal Progesterone as Luteal Phase
Support: A Retrospective, Observational Study
Objectives: To compare the incidence of congenital
anomalies in babies born to infertile women with
in-utero exposure to either dydrogesterone or
micronised vaginal progesterone (MVP) following
assisted reproductive technology (ART) and non-ART
treatment, and in infertile women conceiving normally
and not receiving any drug for luteal phase support
(LPS).
Methods: This retrospective observational study was
carried out at a tertiary level infertility centre, in
Kolkata, India. Women (n=6549) in the age group of
21–45 years with a history of primary or secondary
infertility were screened from January 2002 to June
2017, who had undergone ART or non-ART procedures,
and had received either MVP or dydrogesterone as
LPS. Infertile women with natural conception and not
exposed to drugs for LPS served as control/s.
Results: 6298 women met all criteria and were
sub-stratified into women with natural conception
not receiving LPS (n=2003; Group A), women who
received MVP (n=2103; Group B) and those who
received dydrogesterone (n=2192; Group C) as LPS. All
demographic parameters were comparable amongst
the three groups. The overall pregnancy rate was
32.01%, 29.15%, and 30.72% for Groups A, B, and C,
respectively. Intrauterine foetal outcomes and neonatal
characteristics were also comparable amongst the three
groups. A total of 1851 children were born to 6298
women; 613 in Group A, 589 in Group B and 649 in
Group C. Congenital (Group A: 2.45%, Group B: 2.89%,
Group C: 3.08%), functional (Group A: 3.92%, Group B:
4.58%, Group C: 4.16%) and chromosomal (Group A:
0.49%, Group B: 0.68%, Group C: 0.31%) anomalies
were comparable in all three groups.
Conclusions: Dydrogesterone did not result in increased
incidence of congenital malformations compared with
MVP as LPS, and had similar incidence to those observed
in infertile women who conceived naturally and did not
receive LPS.
Introduction
Incidence of congenital anomalies is reported to be higher
in children born to couples with infertility compared to their
fertile counterpart/s1–3. Parental (chromosomal, genetic) and
environmental factors (such as endocrine, infective-viral e.g.
rubella, body mass index and smoking, etc.) are major contributory
factors.
Infertility treatments may have additional impact for higher
incidence of congenital anomalies through various technologies
involved, or from hormonal and non-hormonal drugs
14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
PEER REVIEWED
administered 3,4,5 during the treatment procedure/s. Available
data is, however, not conclusive on the actual risk involved from
infertility treatments2,6–12. In fact, a longitudinal study of the Danish
national birth cohort showed that congenital malformations in
children born to couples with infertility were higher, irrespective
of treatment1; and that the congenital malformations were
partly due to the underlying infertility or its determinants.
Hence, based on current evidence, there are no clear factors
to distinguish congenital anomalies arising from the effect of
parental sub-fertility or from those occurring as a consequence
of infertility treatment3.
Infertility treatments consist of two areas: procedural impact
and use of drugs. A study by Pinborg et al. in 2013 indicates that
overall risk of procedural impact is not increasing over time and
there is a tendency for decline in the prevalence of congenital
anomalies in the younger ART generation3. Procedural impact in
ART or non-ART treatment drugs used in infertility management
and their possible role on enhancing incidence of foetal congenital
anomalies has not been specifically ascertained and highlighted
in the majority of reviews published so far10. However, two recent
reports indicate that amongst various drugs used in infertility
management, progesterone may have an increased adverse impact
on foetal congenital disease or deformities11,12. But a possible
mechanism and large-scale evidence-based study has not been
reported through either of the publications.
Progesterone is a natural gestational support which has to
be supplemented compulsorily as a luteal phase support (LPS)
in pregnancies following assisted reproductive technologies
(ART) 13,14 and possibly in other non-ART pregnancies achieved with
or without treatment in women with infertility. The usefulness
of progesterone14,15, especially in pregnancies following ART
treatment and also in threatened and unexplained miscarriage,
is well established16–18. The vaginal route is the preferred
mode of administration for progesterone worldwide, and the
pharmacological preparation commonly used is micronised vaginal
progesterone (MVP).
Dydrogesterone, an orally active progestogen with enhanced
bioavailability, is similar to endogenous progesterone in its
molecular structure and has a high affinity for progesterone
receptors19. Dydrogesterone is approved in several countries
for the treatment of progesterone deficiencies and has been
used as a hormone replacement therapy since the 1960s. More
recently, the non-inferiority of oral dydrogesterone to MVP
(capsule or gel) for LPS in fresh-cycle IVF was demonstrated
in the Phase III, randomised LOTUS I and LOTUS II studies20,21.
Several other smaller studies including those by our team, and
a meta-analysis have also shown that dydrogesterone is at least
as effective as MPS for LPS22–28, with a favourable benefit-risk
profile comparable with MVP20,22,26. Based on the LOTUS I and
II studies, dydrogesterone has now been approved in several
countries including India for use in LPS as part of ART treatment21.
There are, however, conflicting reports on risk of congenital
Winter 2019 Volume 2 Issue 4

anomalies with dydrogesterone use, but reports on this topic are
not univocal20,21,29,30,31.
This discrepancy motivated us to study through retrospective
analysis of our own data to evaluate the occurrence of congenital
birth defects in children born to Indian women with infertility
receiving dydrogesterone or MVP as LPS following ART and/
or non-ART treatment (ovulation induction [OI], intrauterine
insemination [IUI]) and also with babies born to infertile women
conceiving naturally and not receiving any LPS.
Materials and Methods
Study Design
This retrospective observational study was conducted at the
Institute of Reproductive Medicine, Salt Lake City, Kolkata, India,
a tertiary level infertility centre. Medical records at the institute
were screened to identify and follow-up babies born between
January 2002 and June 2017 to women with infertility who
received dydrogesterone or MVP as LPS following ART or non-ART
procedures; additionally, women who conceived naturally during
this period without LPS were included for comparison.
The study protocol was approved by the Research Ethics Board
of the institute prior to the commencement of the study. All women
Fig 1. Study design. OI: Ovulation induction, IUI: Intrauterine insemination, IVF: In-vitro fertilisation, LPS: Luteal phase support, IUGR: Intrauterine growth restriction, IUFD:
Intrauterine foetal death, APGAR: Appearance, pulse, grimace, activity, respiration; NICU: Neonatal intensive care unit, RDS: Respiratory distress syndrome
www.biopharmaceuticalmedia.com
Clinical Research
consulting at the institute signed consent forms allowing the use
of their health information except personal identification for the
purpose of research or publication. Simultaneously, on admission
for delivery, a consent form was received allowing the use of the
newborn’s medical information for research purpose. As this was
a retrospective study, data was obtained from the medical files
of mothers and newborns. The trial is registered at http://www.
ISRCTN.org (Trial ID: ISRCTN55659103).
Patient Selection
Women in the age group of 21–45 years with a history of primary
and/or secondary infertility, e.g. fallopian tube obstruction,
polycystic ovary syndrome, endometriosis and/or having
partners with male factor infertility were included. Women with
adenomyosis, hyperprolactinemia, hypothyroidism, congenital
uterine anomalies, uterine synechiae, or baseline follicle
stimulating hormone >12 international units, and those who were
donor oocyte recipients or gestational surrogates were excluded
from the study.
All women were carefully scrutinised for the presence of the
following risk factors for congenital malformation: history of
congenital/chromosomal anomaly in the previous pregnancies;
recurrent pregnancy loss; family history of birth defects; presence
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 15
 Clinical Research
of medical disorders; concomitant use of other drugs; smoking;
alcohol consumption during pregnancy and occupational exposure
to endocrine disruptors/ radiation.
Exogenous Progesterone Supplementation for LPS
Women undergoing ART or non-ART procedure/s received either
MVP (Susten®, Sun Pharmaceutical Industries Ltd, India) 200 mg
thrice daily, or oral dydrogesterone (Duphaston, Abbott India Ltd)
10 mg thrice daily as LPS for up to 12 weeks of gestation (Fig. 1).
Table 1: Demographics and baseline characteristics of three groups with or without
treatment of luteal phase support with micronised vaginal progesterone and/or
dydrogesterone
LPS was initiated on day 16 and continued for 10 days in women
undergoing OI, from day after insemination in women undergoing
IUI, and from day of embryo transfer (ET) in women undergoing
in-vitro fertilisation (IVF) or IVF/intra-cytoplasmic sperm injection
(ICSI). All medicines were procured by patients.
Assessments
Serum β-human chorionic gonadotropin (β-hCG) level was
estimated 13 days after OI / IUI / ET to confirm pregnancy. Clinical
pregnancy was defined as the presence of a viable foetus in an
ultrasound scan performed seven weeks after ET. The presence of
at least one viable foetus at 12 weeks of gestation was classified
as an ongoing pregnancy. Following successful conception, all
women were followed up regularly for antenatal check-up at
our institute. Pregnant women who could not come for regular
follow-up were advised to visit at least thrice for antenatal
check-up and at least once at six weeks post-conception. Foetal
viability scan and detailed anatomy scans were performed at 6–7
weeks of gestation. Ultrasonography was performed at 12 weeks
of gestation (completion of first trimester) and between 20 and 22
weeks of gestation to assess for congenital anomalies.
The definition of congenital malformation, deformations and
chromosomal abnormalities as stated in Chapter XVII, ICD-10
World Health Organization, International Statistical Classification
of Diseases and Related Health Problems was used for the study
purpose. According to the World Health Organization, congenital
anomalies are also known as birth defects, congenital disorders
or congenital malformations. Congenital anomalies were defined
as structural or functional anomalies, including metabolic
disorders, which are present at the time of birth. A Samsung
SonoAce R7 (Samsung Medison; Seoul, South Korea) was used for
the assessments, and the same trained sonologist carried out all
assessments at every visit.
The women were routinely examined by the obstetrician and
the babies were thoroughly examined by a paediatrician during
their postnatal visit. Electronic records till their postnatal check-up
were maintained for all women. Demographic characteristics,
pregnancy rate, miscarriage rate, congenital anomalies and foetal
outcome were recorded. Information related to growth, learning
16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Table 2: Causes of infertility categorized by type of treatment procedure in women
receiving MVP or dydrogesterone
abilities and psychologic functioning was recorded by trained
child psychologist, paediatrician and trained nurses (S1 File).The
functional abnormalities in children were closely monitored. The
follow-up reports were collected using various methods such as
analysis of birth records, regular health check-up of children by
the paediatricians and paediatric-psychologist during the annual
baby get-together, and parents’ feedback.
Statistical Analysis
The proportion of congenital malformations between the study
groups and treatment groups were compared by Yate’s corrected
chi square test. Anomaly risk was quantified by univariate odds
ratio (OR) with 95% confidence interval (CI). Unpaired t tests were
used to compare the mean centiles between each group. Age and
birth weight between different groups were compared by student
t test. Data were expressed as mean ± SD and analysed using SPSS
20.0 statistical software (SPSS Inc., Chicago, Illinois, USA). A value
of P<0·05 was considered significant. Statistical power of the study
was obtained to be >95%, with a type I error of 5% based on null
hypothesis and by G*Power software, available at http://www.
gpower.hhu.de .
Results
Six thousand two hundred and ninety eight records were included
after screening of 6549 history files of women reporting for
infertility treatment between 2002 and 2017. The patients were
stratified into three groups based on type of treatment received.
Group A included women with natural conception and without
any LPS (n=2003); women who underwent ART or non-ART
treatment and received MVP as LPS (Group B; n=2103) and
women who underwent ART and/or non-ART treatment with oral
dydrogesterone as LPS formed Group C (n=2192). All demographic
parameters were found to be comparable between the three
groups (Table 1). The overall pregnancy (Group A: 32%; Group B:
29.15%; Group C: 30.7%) and the miscarriage rate/s (Group A:
10.61%; Group B: 14.19%; Group C: 13.52%) were comparable.
No major pregnancy complications were observed in any of the
intervened or control group/s.
Causes of Infertility
Tubal factor, PCOS, endometriosis, and male factor in >800
women each were the reported causes for infertility; the cause
was unexplained in the remaining women (Table 2). Since
both Groups B and C were comprised of patients treated with
a variety of regimens – oral clomiphene citrate, injectable
gonadotropins, timed intercourse, IUI, IVF and IVF/ICSI and
represent a heterogenous treatment regimen/s, chi square test
was applied to evaluate the uniformity of patient distribution
in each treatment arm.
Winter 2019 Volume 2 Issue 4

Table 3: Fetal and neonatal outcomes of three groups with or withouat treatment of
luteal phase support with micronized vaginal progesterone and/or dydrogesterone
Table 4. Congenital anomalies and functional and chromosomal abnormalities observed
in children in three groups with or without treatment of luteal phase support with
micronised vaginal progesterone and/or dydrogesterone
Table 5. Congenital anomalies and functional and abnormalities observed in children
born to women in three groups, by tretment procedure
Foetal and Neonatal Outcomes
Intrauterine foetal outcomes such as gestational age,
intrauterine growth restriction (IUGR) and intrauterine foetal
death (IUFD), neonatal characteristics such as number of live
births, body weight, and Appearance Pulse Grimace (reflex)
Activity Respiration (APGAR) score; neonatal intensive care
unit (NICU) admission; respiratory distress syndrome (RDS);
neonatal jaundice; hypoglycemia; and hypocalcemia did not
vary significantly between the three groups (Table 3).
A total of 1851 children were born to 6298 women. The
babies were sub-stratified into (n=613; Group A), (n=589;
www.biopharmaceuticalmedia.com
Clinical Research
Group B), and (n=649; Group C). The overall occurrence
of congenital anomalies, functional and chromosomal
abnormalities observed in these children (aged 0–5 years)
were comparable between the three groups (Table 4).
Congenital anomalies were observed in 2.45% of patients in
Group A, 2.89% of patients in Group B, and 3.08% of patients
in Group C (OR [95% CI]: A vs B: 1.18 [0.58–2.39]; A vs C:
1.26 [0.64–2.49]; B vs C: 0.93 [0.48–1.8]). Similar comparable
findings were documented in both functional (OR [95% CI] A
vs B: 1.17 [0.67–2.06]; A vs C: 1.06 [0.61–1.86]; and B vs C:
1.11 [0.64–1.91]) and chromosomal abnormalities (OR [95%
CI] A vs B: 1.31 [0.61–6.23]; A vs C: 0.63 [0.11–3.77]; and B vs
C: 1.03 [0.52–1.85]). No obvious differences were observed
in congenital anomalies or functional abnormalities by type
of treatment procedure in the groups treated with MVP or
dydrogesterone (Table 5).
Discussion
Progesterone plays an important role in the establishment
and maintenance of pregnancy33–35. It is well documented
that stimulation of ovaries as part of infertility treatment
procedures can cause luteal phase deficiency and a negative
effect on implantation and maintenance of gestation36–38.
Hence, LPS during these procedures is considered a standard
practice to support implantation and improve pregnancy
rates 39,40.
The luteal phase, which from an embryologic point of view,
ranges between the period of ‘dividing totipotent’ cells of
cleaving embryo to the period of ‘differentiating pluripotent’
cells of expanding blastocyst, is the phase of embryonic
development and organogenesis. While damage to the
totipotent cells is more amenable to repair mechanisms 41–43,
scope of repair slowly diminishes as the cleaving embryo
progresses towards pluripotency, due to loss of unique
transcriptome and/or modification in epigenetic and chromatin
features, leading to a defective blastocyst44. This may be
a possible explanation of teratogenic impact of the drug
(progesterone) administered during the phase of embryonic
organogenesis, i.e. during the luteal phase of an ovulatory
cycle.
However, in addition to several epidemiological studies
which have shown no teratogenic effect of progesterone45,
the ASRM committee in 2008 also suggested that there is
no evidence which has been documented so far to indicate
the maternal exposure to progesterone during pregnancy to
increased risk for birth defects39. Dydrogesterone has been
in use in pregnancy from the 1960s, with a total cumulative
exposure of 26 million patient years up to 201846. While the
extensive use theoretically rules out any substantial foetal risk,
an overview of birth defects reported with dydrogesterone
from 1977 to 2005, and a systematic review of all studies
conducted until 2010, showed minimal risk of birth defects
with dydrogesterone use.32,47
However, a cause-effect relationship may be presumed
when a similar type of defect or disorder is detected in
the offspring delivered following use of a specific drug or
specific procedure used as part of infertility treatment. One
retrospective study identified a positive association between
dydrogesterone and congenital heart defects30. However,
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17
 Clinical Research
counter reports have provided evidences to suggest that this
study has several methodological constraints21,46. A recent
patient database study has also suggested increased risk of
teratogenic effect/s following exposure to dydrogesterone in
pregnant women31.
In the Phase III LOTUS I study, the incidence of congenital,
familial and genetic disorders in the foetus or infants was
limited and similar amongst dydrogesterone (1.0%) and MVP
(1.2%) and in the group of women with no luteal support.
A substratification of the LOTUS I trial in a subset of 216
Russian patients also demonstrated similar percentages of
congenital anomalies in the two groups as those observed
in the overall population; more importantly, no health issues
were reported in the infants at six-month follow-up48. In
the LOTUS II study, the incidence of congenital, familial and
genetic disorders was 6.3% with dydrogesterone and 5.0%
with MVP21. Consistent with findings in the LOTUS I and LOTUS
II studies, we observed no notable differences in the incidence
of congenital malformations and also no distinct pattern of
defects in children born to mothers following dydrogesterone
or MVP supplementation. Furthermore, the outcome for both
the groups was comparable with that of spontaneously
conceived cases, along with functional and chromosomal
abnormalities in children of each of the three groups.
Our findings are also in agreement with previous studies of
dydrogesterone in other indications. El-Zibdeh and co-workers
in 2005 reported no significant differences with respect to
pregnancy complications or congenital abnormalities among
women with a history of recurrent, unexplained spontaneous
abortion treated with oral dydrogesterone, intramuscular
human chorionic gonadotrophin or no additional treatment
until 12 weeks of gestation17. Furthermore, Pandian et
al., in 2009 stated absence of congenital anomalies after
dydrogesterone supplementation for threatened miscarriage16.
In a subsequent study in 200918, the authors again found no
statistically significant differences in congenital abnormalities
associated with dydrogesterone support in threatened
miscarriage cases compared with women receiving no
treatment. Despite our comparable findings, it is important
to consider that infants born from singleton pregnancies
after ICSI are at an increased risk of developing congenital
malformations when compared with similar naturally
conceived children49.
This study is the first to our knowledge where congenital
abnormalities were assessed in a real-life observational
18 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
setting in children born to women with infertility receiving
dydrogesterone following ART or non-ART procedures. The
limitations of the study are its retrospective nature, and it
being a single-centre study in women with the same ethnicity.
In conclusion, the study results demonstrate that
dydrogesterone as LPS is associated with a similar rate
of congenital anomalies. The proportion of children with
congenital anomalies was similar to those observed with MVP
as LPS and in women with spontaneous pregnancy achieved
in infertile women and receiving no luteal support. Our
findings also add to the existing evidence in support of oral
dydrogesterone as an effective and well-tolerated drug for LPS
in women with infertility.
Acknowledgements
The authors thank Sushanta Chakraborty and Sharmista Kundu
Nag for assisting in collection of data.
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30. Zaqout M, Aslem E, Abuqamar M, Abughazza O, Panzer J, De Wolf D. The
impact of oral intake of dydrogesterone on fetal heart development
during early pregnancy. Pediatr Cardiol. 2015;36(7):1483-1488.
31. Koren G, Gilboa D, Rotem R, Levy A, Daniel S, Shalev V. Fetal outcome
following dydrogesterone exposure in pregnancy. Arch Dis Child.
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32. Queisser-Luft A. Dydrogesterone use during pregnancy: overview of
birth defects reported since 1977. Early Hum Dev. 2009;85(6):375-377.
33. Csapo AI, Pulkkinen M. Indispensability of the human corpus luteum
in the maintenance of early pregnancy. Luteectomy evidence. Obstet
Gynecol Surv. 1978;33:69–81.
34. Couzinet B, Le Strat N, Ulmann A, Baulieu EE, Schaison G. Termination of
early pregnancy by the progesterone antagonist RU 486 (Mifepristone).
N Engl J Med. 1986; 315:1565–1570.
35. Silvestre L, Dubois C, Renault M, Rezvani Y, Baulieu EE, Ulmann A.
Voluntary interruption of pregnancy with mifepristone (RU 486) and a
prostaglandin analogue. A large-scale French experience. N Engl J Med.
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36. Macklon NS, Fauser BC. Impact of ovarian hyperstimulation on the luteal
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phase. J Reprod Fertil Suppl. 2000;55:101–108.
37. Beckers NG, Macklon NS, Eijkemans MJ, Ludwig M, Felberbaum RE,
Diedrich K, Bustion S, Loumaye E, Fauser BC. Nonsupplemented
luteal phase characteristics after the administration of recombinant
human chorionic gonadotropin, recombinant luteinizing hormone, or
gonadotropin releasing hormone (GnRH) agonist to induce final oocyte
maturation in in vitro fertilization patients after ovarian stimulation
with recombinant follicle-stimulating hormone and GnRH antagonist
cotreatment. J Clin Endocrinol Metab 2003;88:4186–4192.
38. Kolibianakis EM, Bourgain C, Platteau P, Albano C, Van Steirteghem AC,
Devroey P. Abnormal endometrial development occurs during the luteal
phase of nonsupplemented donor cycles treated with recombinant
follicle-stimulating hormone and gonadotropin-releasing hormone
antagonists. Fertil Steril 2003;80:464–466.
39. Practice Committee of the American Society for Reproductive Medicine.
Progesterone supplementation during the luteal phase and in early
pregnancy in the treatment of infertility: an educational bulletin. Fertil
Steril. 2008;89:789–792.i. van der Linden M, Buckingham K, Farquhar C,
Kremer JA, Metwally M. Luteal phase support for assisted reproduction
cycles. Cochrane Database Syst Rev. 2011(10):CD009154.
40. Hansis C, Grifo JA, Krey LC. Candidate lineage marker genes in human
preimplantation embryos. Reprod Biomed Online. 2004;8(5):577-583.
41. Jedrusik A, Parfitt DE, Guo G, Skamagki M, Grabarek JB, Johnson MH, et
al. Role of Cdx2 and cell polarity in cell allocation and specification of
trophectoderm and inner cell mass in the mouse embryo. Genes Dev.
2008;22(19):2692-2706.
42. Sun JH, Zhang Y, Yin BY, Li JX, Liu GS, Xu W, et al. Differential expression
of Axin1, Cdc25c and Cdkn2d mRNA in 2-cell stage mouse blastomeres.
Zygote. 2012;20(3):305-310.
43. Niakan KK, Han J, Pedersen RA, Simon C, Pera RA. Human pre-implantation
embryo development. Development. 2012;139(5):829-841.
44. Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P. Progesterone
for the luteal support of assisted reproductive technologies: clinical
options. Hum Reprod. 2000;15 Suppl 1:129-148.
45. Kaur KK, Allahbadia G, Singh M. Luteal phase support using oral
dydrogesterone-a prospective treatment for future replacing
micronized vaginal progesterone. Open Acc J Repro & Sexual
Disord. 2014;1(4). OAJRSD.MS.ID.000119. DOI: 10.32474/
OAJRSD.2018.01.000119
46. Carp H. A systematic review of dydrogesterone for the treatment of
threatened miscarriage. Gynecol Endocrinol. 2012;28(12):983-90.
47. Sukhikh GT, Baranov II, Melnichenko GA, Bashmakova NV, Blockeel C,
Griesinger G et al. Lotus I: A Phase III randomized controlled trial of
oral dydrogesterone versus micronized vaginal progesterone for luteal
support in in vitro fertilization, with focus on the Russian subpopulation.
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(in Russian)
48. Lacamara C, Ortega C, Villa S, Pommer R, Schwarze JE. Are children
born from singleton pregnancies conceived by ICSI at increased risk
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2017;21(3):251-259.
Shovandeb Kalapahar, MS, DNB,1
Tushar K. Das, PhD,1
Sunita Sharma, MD, FNB,1
Sourav RoyChoudhury, PhD,1
Ratna Chattopadhyay, MBBS, PhD,1
Koel Chaudhury, PhD,2
Pratip Chakraborty, PhD,1
Baidyanath Chakravarty, MO, FRCOG, DSc.1
1. Institute of Reproductive Medicine, Salt Lake, Kolkata, West
Bengal, India
2. School of Medical Science and Technology, Indian Institute
of Technology, Kharagpur, West Bengal, India
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 19
 Clinical Research
Is Liver Biopsy a Gold or an Old Standard
in NAFL and NASH?
When defining the term gold standard, the most
appropriate definition is “a benchmark test that is the
best available under reasonable conditions.” 1 When
considering this definition, it becomes clear that
non-invasive imaging is replacing liver biopsy as the
gold standard for evaluation of fibrosis in non-alcoholic
fatty liver disease (NAFLD).
Over the last 40 years, NAFLD has evolved from an
unrecognised entity to a heterogeneous collection of overlapping
liver diseases with a common phenotype of hepatic steatosis.
Although NAFLD is quite common, affecting approximately
25% of the world’s adult population, it is increasingly clear
that subjects with non-alcoholic steatohepatitis (NASH), and
especially those with significant fibrosis, are at the greatest
risks for excess mortality and adverse clinical outcomes, as well
as impairment of patient reported outcomes and significant
economic burden.
Patients with NASH do not present with any obvious clinical
signs until they are near liver failure. Despite the growing
recognition of this important burden, there are significant
challenges to accurately and non-invasively diagnose the
progressive form of NAFLD. Although liver biopsy (LB) is still
considered the current “gold” standard for diagnosing NASH and
staging fibrosis, it is an invasive procedure with some variability
in assessment of the key features of NASH.
LBs require significant expertise both to perform and to
interpret the results. Two clinicians are involved in obtaining
and interpreting LB which represents a huge clinical process,
especially in the settings of clinical trials. Given the significant
number of patients with NAFLD, the limited number of
hepatologists represents a serious bottleneck, often leading
to delays in biopsy reads and confirmation of results. The
accuracy of LB to assess fibrosis has also been questioned due
to sampling errors and intra- and inter-observer variability that
may lead to over- or under-staging, with even highly skilled and
trained pathologists showing inter-observer concordance rates
of less than 80%.2 The size of the biopsy specimen, which varies
from 10 to 30 mm in length and from 1.2 to 2 mm in diameter,
represents 1/50,000 of the total mass of the liver and is therefore
subject to a significant risk of sampling error.3
Although LBs remain the gold standard for confirmation
of liver fibrosis staging, they are costly, painful and pose risk
of complications such as bleeding, damage to other organs,
and potentially, although rare, death. Multiple LBs present a
significant challenge in recruitment and retention of subjects in
clinical trials due to above states risks and subject discomfort.
Ideally, less invasive tests that are more widely available, less
costly, and accurate can be confirmed and widely accepted by
clinicians and regulatory bodies as the new gold standard. There
are a number of such non-invasive tests such as radiographical
20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
modalities, serum markers, and non-invasive predictive
algorithms undergoing investigation for identifying those with
increased risk of NAFLD and NASH and for confirmation of or
staging of NASH fibrosis. The ideal test to discriminate advanced
liver fibrosis due to NASH would be non-invasive, widely
available, affordable, accurate, and reproducible.
Imaging Modalities
In the context of NAFLD, the first diagnostic challenge is to
accurately show the presence of fat in the liver. Fat is thought to
have its own chemical signature, which can be detected directly
by magnetic resonance spectroscopy (MRS). MRS quantifies the
proton density fat fraction (PDFF), a standardised measure of liver
tissue.4 However, MRS has several limitations, including: limited
availability, need for expertise in protocol prescription, data
collection, and the requirement for spectral analysis. Furthermore,
MRS is not available on routine scanners. Therefore, now, magnetic
resonance imaging (MRI) based methods have been developed
using MRI-PDFF to quantify liver fat without needing spectroscopy
coils, using routinely available clinical MRI scanners.4,5
In contrast, fibrosis has no molecular signature that can
be detected by current imaging techniques, so all imaging
tests for fibrosis attempt to detect fibrosis indirectly using
proposed biomarkers, which include: stiffness, diffusion,
perfusion, metabolites, and image texture. Testing for the
presence of advanced fibrosis is a primary concern when
evaluating a patient with suspected NASH. It is known that
fibrosis is the only independent predictor of associated
morbidity and mortality. Confirmed presence of advanced
fibrosis alters clinical management and consideration for
treatment, potentially in the context of clinical trials. The
leading biomarker of fibrosis is liver “stiffness” (or “elasticity”)
and its family of related parameters.4
The most accurate non-invasive methods to assess the
stiffness of the liver and to classify the patient into advanced
versus non-advanced fibrosis include transient elastography
(TE), magnetic resonance elastography (MRE), and emerging
techniques such as shear wave elastography (SWE) and acoustic
radial force imaging (ARFI).
TE has been clinically useful as well, and has the means to
replace LB as the gold standard. TE has high accuracy when
identifying patients with F3-F4 fibrosis who are at greater risk
for worse clinical outcomes.6 TE has been shown to have an
area under the curve (AUROC) of 0.83 for advanced fibrosis
when compared to blood tests. The most remarkable advantage
of TE is that the procedure is non-invasive, without any of the
complications associated with liver biopsy. In addition, its cost
is one-fourth of that of liver biopsy, and it can be done in five
minutes in the outpatient setting without any associated pain.
MRE has the AUROC of 0.98 in identifying patients with F3–F4
disease, so at the very least is non-inferior to liver biopsy.7 Given
Winter 2019 Volume 2 Issue 4
NON-INVASIVE FLOW SENSORS
SONOFLOW CO.55 V2.0

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SONOTEC GmbH
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www.sonotec.eu INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21
 Clinical Research
its accuracy, MRE may also offer a good non-invasive tool to
monitor changes in liver fibrosis. In a placebo-controlled trial of
sitagliptin in NAFLD, MRE was shown to have robust correlation
coefficient between baseline and 24 w   eeks.8 Longitudinal studies
of contemporaneous MRE and liver biopsy are underway, and
their results are eagerly awaited.
Controlled attenuation parameter (CAP) is a novel ultrasound
technique that measures steatosis simultaneously with liver
stiffness during vibration-controlled transient elastography.
Overall, CAP is a relatively simple and inexpensive method for
steatosis assessment that is reasonably accurate for the diagnosis
of steatosis. When combined with other clinical assessments, it is
likely to help clinicians diagnose or exclude steatosis.
Non-invasive Biomarkers in NASH
In addition to the non-invasive tests based on the imaging
modalities, there is an attempt to define non-invasive biomarkers
using predictive models or serum biomarkers. These non-invasive
markers include those that are based on alanine aminotransferase
(ALT) levels, those that include components of metabolic
syndrome, measuring circulating keratin18 fragment levels,
as well as tests based on soluble markers such as FibroMeter,
microRNA (miRNA) panels, and lipidomic panels, etc. The NASH
test combines demographic characteristics (age, sex, and BMI),
serum parameters (aminotransferases and lipids), and alpha-2
macroglobulin, apolipoprotein A1, and haptoglobin.
Predictive Models for Advanced Fibrosis
Serological markers of fibrosis evaluate alterations in hepatic
function as well as collagen turnover. The severity and progression
of liver fibrosis plays a key role for predicting outcomes and for
making therapeutic decisions in NASH patients.
22 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
AST/ALT ratio: The aspartate aminotransferase (AST)/alanine
aminotransferase (ALT) ratio helps distinguish alcoholic hepatitis
from NAFLD and NASH. Using these non-invasive tests to
diagnose for NASH, current studies have found that the frequency
of NASH in individuals with normal ALT (<35 U/L) was 11%
whereas the frequency was 29% in those with elevated ALT (
35 U/L); and if the ALT was two times the upper limit of normal
(>70 U/L), predicting NASH was found to have a 50% sensitivity
and 61% specificity for NASH.9  However, another study found
that individuals with NAFLD can have normal ALT levels as the
disease progresses.10
Fibrosis-4 index: This scoring system combines age, AST, ALT,
and platelet count. It has a negative predictive value of more than
90% for advanced liver fibrosis, according to experts. Results can
be subject to rapid AST and ALT changes, though.
BARD score: Calculated from body mass index, the ALT/AST
ratio, and the presence of diabetes, this score, reported on a 0-4
scale, is routinely used to predict liver fibrosis in NAFLD patients.
Scores less than 2 have a strong negative predictive value for
advanced liver fibrosis associated with NAFLD.
Enhanced liver fibrosis (ELF) test: Though not yet approved by
the Food and Drug Administration and not sensitive to early-stage
fibrosis, this panel is an algorithm comprised of three fibrosis
markers – amino-terminal propeptide of type III procollagen,
hyaluronic acid, and tissue inhibitor of metalloproteinase. It
detects advanced fibrosis with high accuracy in both adult and
paediatric patients.
Tailored Approach
Significant progress has been made regarding the non-invasive
Winter 2019 Volume 2 Issue 4

assessment of liver disease in patients with NAFLD. Regarding
detection and grading of steatosis, MRI-PDFF is the most
accurate method but appears better suited for assessment
and follow-up of selected patients in clinical trials, whereas
conventional ultrasound, and if no steatosis is shown, CAP,
as a point of care technique, could be used as triage in large
unselected populations. As for the identification of advanced
fibrosis, MRE, TE, as well as FIB-4 are the most accurate and
validated methods. FIB-4 is best suited as a first-line tool in a
primary healthcare setting to confidently exclude advanced
fibrosis, whereas TE and MRE are more suited for referral centres.
It is postulated that combinations of NITs in sequential algorithms
can accurately detect advanced fibrosis while eliminating the
risks associated with biopsy and reducing costs by minimising
unnecessary testing.11
Regarding NASH, no highly sensitive and specific blood
tests are available to differentiate NASH from simple
steatosis. Neither imaging modality can reliably discriminate
NASH from simple steatosis, although MR-based modalities
are showing promise. Finally, there is increasing evidence
that serum markers and liver stiffness, measured using TE,
accurately identify the subgroup of patients with NAFLD at a
higher risk to reach the outcome of liver-related complications
and death/liver transplantation, especially when analysed
together. Screening data from Phase 2 ATLAS study evaluating
combinations of investigational cilofexor, firsocostat and
selonsertib in advanced fibrosis due to NASH has been recently
presented. This analysis demonstrates that the use of currently
available NITs can accurately identify patients with advanced
fibrosis due to NASH and potentially reduce the need for LB.
When used in combination, the ELF test and FibroScan® (TE)
accurately identified advanced fibrosis in >805 of patients
(presented at The International Liver Congress 2019, Vienna).
Looking Ahead
Adopting new biomarkers in clinical trials requires
substantial efforts and investment to validate the reliability
of these biomarkers as surrogate endpoints. In an effort to
address this challenge, developers are currently integrating
exploratory markers as secondary endpoints in Phase II and
Phase III studies. The field has also seen the formation of two
multi-stakeholder consortia, LITMUS and NIMBLE, aimed at
accelerating validation of non-invasive markers by sharing
resources and patient samples.
Histological diagnosis of NASH is still required in clinical
trials, however non-invasive modalities can be used more
frequently to follow at-risk patients over time, as well as be
instituted for screening evaluations in the absence of the
morbidity that unfortunately comes with LB. MRE has emerging
data to support its non-inferiority to LB in terms of accuracy in
fibrosis staging and, combined with the dramatic risk profile
differences, should be soon considered a superior test.
REFERENCES
1. Claassen JAHR. The gold standard: not a golden standard.
BMJ 2005;330:1121
2. Bedossa P, Bioulac-Sage P, Callard P et al. Intraobserver
and interobserver variations in liver biopsy interpretation in
patients with chronic hepatitis C. Hepatology 1994;20:15-20
3. Bedossa P, Dargère D, Paradis V. Sampling variability of liver
www.biopharmaceuticalmedia.com
Clinical Research
fibrosis in chronic hepatitis C. Hepatology 2003;38:1449-1457
4. Dulai PS, Sirlin CB, Loomba R. MRI and MRE for noninvasive
quantitative assessment of hepatic steatosis and fibrosis in
NAFLD and NASH: clinical trials to clinical practice. J Hepatol
2016;65:1006–1016.
5. Park CC, Nguyen P, Hernandez C, Bettencourt R, Ramirez K,
Fortney L et al. Magnetic resonance elastography vs transient
elastography in detection of fibrosis and noninvasive
measurement of steatosis in patients with biopsy-proven
nonalcoholic fatty liver disease. Gastroenterology 2017;152:598–
607.e2
6. Boursier J, Vergniol J, Guillet A, Hiriart JB, Lannes A, Le Bail B
et al. Diagnostic accuracy and prognostic significance of blood
fibrosis tests and liver stiffness measurement by FibroScan in
non-alcoholic fatty liver disease. J Hepatol 2016;65:570–578.
7. Loomba R, Wolfson T, Ang B et al. Magnetic resonance
elastography predicts advanced fibrosis in patients with
nonalcoholic fatty liver disease: a prospective study. Hepatology
2014;60:1920-1928.
8. Cui J, Philo L, Nguyen P, Hofflich H, Hernandez C, Bettencourt R
et al. Sitagliptin vs. placebo for non-alcoholic fatty liver disease:
a randomized controlled trial. J Hepatol. 2016;65(2):369–76.
9. Verma S, Jensen D, Hart J, Mohanty SR. Predictive value of ALT
levels for non-alcoholic steatohepatitis (NASH) and advanced
fibrosis in non-alcoholic fatty liver disease (NAFLD). Liver Int
2013;33:1398–1405.
10. Rinella ME, Loomba R, Caldwell SH, Kowdley K, Charlton M, Tetri
B, Harrison SA. Controversies in the Diagnosis and Management
of NAFLD and NASH. Gastroenterol Hepatol 2014;10:219–227.
11. Anstee QM, Lawitz EJ, Alkhouri N et al. Noninvasive tests
accurately identify advanced fibrosis due to NASH: Baseline
data from the STELLAR trials. Hepatology 2019;70(5):1521-1530
Rafal Ziecina
Rafal Ziecina MD, PhD is Executive Director
of Scientific Solutions at Worldwide Clinical
Trials. Dr Ziecina is a board-certified
pharmaceutical physician specialising in
design and execution of cardiovascular and metabolic
trials. He provides consultancy services around filing
strategies, regulatory & safety strategies, regulatory
agency interactions as well as protocol and drug
development plans writing.
Email: rafal.ziecina@worldwide.com
Scott Beasley
Mr. Beasley has more than 23 years clinical
research experience and program leadership
within the CRO and pharmaceutical industry.
Mr. Beasley has successfully led a significant
portfolio of several hepatology phase II and phase III
programs in NAFLD/NASH as well as other metabolic
disorders. Over the most recent years, Mr. Beasley has led
the development of a NASH initiative training program
to address the unique challenges associated with clinical
trials in NASH/NAFLD. He has provided team expertise in
managing complex NASH programs including development
of operational strategies to proactively identify and
mitigate risks to limit any potential impact. Mr. Beasley
currently serves as Executive Director, Project Management
and Franchise Area Lead for the NASH/NAFLD and Liver
disease program at Worldwide Clinical Trials.
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 23
 Manufacturing/Technology Platforms
Split Butterfly Valve Technology and Eradicating the Risk
of Contamination in Biologics Manufacturing
As new biological therapies continue to be developed,
the market has seen an increased need for innovation.
Demand has grown for improved operational efficiency
and achieving greater quality and cost reductions in
manufacturing processes. One of the key challenges
facing biopharma manufacturers is eliminating the risk
of contamination, which requires effective containment
strategies and monitoring of critical manufacturing
processes.
Biopharmaceutical Market Landscape
The biopharma sector has witnessed huge growth over recent
years. Total annual revenue has increased from $4.4 billion in
1990 to around $275 billion at the end of 2019, an increase of
6,250%, and biopharmaceuticals now make up more than 25%
of the total pharmaceutical market1. New technologies that can
ensure vigorous and flexible containment strategies while also
reducing a product’s time to market offer arguably the greatest
benefits to manufacturers as the industry continues to evolve.
Containment Strategies
Manufacturing environments are open to many sources of
potential contamination, which if not correctly controlled, pose
possible hazards during the manufacture of biopharmaceutical
products. Patient safety could ultimately be put at risk should
microorganisms, particles or endotoxins enter a manufacturing
environment.
Potential sources of contamination include the equipment,
materials used and the people within the manufacturing
environment. Multiple technologies have been developed in
response to the need to ensure the safe and sterile transfer of
biologics, biosimilars and ingredients during aseptic processing.
Restricted access barrier systems (RABS) and isolator technologies
have become widely used in recent years. Isolators provide an
airtight barrier around the aseptic processing line and can be
employed in cleanroom environments to minimise the risk from
contaminants. RABS provide a barrier between workers and
processing lines, while offering operators the opportunity to
interact with products as necessary.
Despite this, both systems have disadvantages. Manufacturers
using isolator technology may face difficulties when transferring
materials in and out of the chamber, which can delay the shut-down
and start-up process. The closed solution provided by RABS
technology provides lower integrity chambers, and the technology
relies on manual cleaning processes such as steaming in place
(SIP) or sterilisation between uses which can be time-consuming
and create delays.
SBV Technology as a Solution
SBVs are made up of an active half and passive half and enable the
transfer of an active pharmaceutical ingredient (API) or product
from a process vessel, container, isolator or RABS to another
24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
PEER REVIEWED
without jeopardising sterility. When in use, the active half of the
SBV is attached to the receiving container, while the passive half
is attached to the flexible bag or discharging API or drug product
container. When the two halves of the disc are brought together,
a single plate is created, which allows the product to flow on
the internal surface of each half. Thus, when the two halves are
detached, the external faces remain clean and can be safely
exposed to the process environment.
When the valve of the SBV is sealed, an opening is created
between the discs, which means decontaminating gas can be
flushed through and decontamination can take place in a closed
environment. Validation occurs using chemical indicators, which
confirm full coverage of the enclosure has been attained. This is
followed by biological indicators, to ensure a 99.9999% reduction
(or 6-log reduction) in bacterial spores has been achieved.
Case Study
Sterile API Addition to a Mixing Vessel
The CDMO is a full-service pharmaceutical company that leverages
blow-fill-seal technology. Its capabilities extend well beyond
manufacturing, with an in-house development team specialising in
all aspects of bringing a product to market – from lab scale batches,
regulatory filings, scale-up, manufacturing and distribution.
Challenge
The CDMO was looking to solve the issue of charging sterile API
into a mixing tank. This is a common problem with all aseptically
prepared pharmaceutical products.
Critical to the process was maintaining sterile conditions whilst
docking a container to the vessel and then transferring solid API to
form a liquid suspension. With a fully dissolved liquid, the product
could be sterile filtered to ensure sterility as it was passed to the
filler. Although in this case, the product being passed to the filler
was a suspension and so this option was not possible.
This required the whole process to be performed under aseptic
conditions and as such would normally mean one of the following
upgrades would be required.
1. Upgrade the whole room from a grade C cleanroom to grade A.
2. Upgrade the room to a grade B environment and additionally
introduce an over-pressurised grade-A area around the point
of fill.
3. Implement a laminar flow system around the point of fill, plus
additional control due to the lack of a barrier.
4. Upgrade the room to a grade B environment and additionally
introduce a RABS system at the point of fill or full vessel.
5. Maintain the grade C cleanroom but introduce isolator
technology around the point of fill or full vessel.
Traditionally RABs and isolator technology would have been
favoured here due to the benefits they bring in terms of improved
sterility assurance, employing the fundamental techniques of
Winter 2019 Volume 2 Issue 4
www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 25
 Manufacturing/Technology Platforms
separation and decontamination. That said, when considering some
of the negative constraints which come with these technologies,
such as high initial capital investment, space, ergonomics and
ongoing cost and energy consumption, the company decided
to look elsewhere to find a solution that was more suited to this
critical task.
Solution
An aseptic bio-valve product was selected as an ideal solution
to this problem, providing a sealed powder transfer in a small
footprint mounted to the inlet port of the vessel. The valve can
be pre-steam sterilised along with the vessel, unlike traditional
SBVs or other conventional connections (see illustration 1a/b). On
final connection, it also removed any room contamination from the
mating faces of the transfer in a controlled and validated manner
(see illustration 2a/b).
Dock SIP Cap illustration 1a
SIP through Cap – Pre-sterilising Active & Vessel illustration 1b
The AseptiSafe Bio valve works by creating a sealed
chamber between the transfer container (passive section) and
vessel (active section). When the two halves dock together, the
sealed chamber is then bio decontaminated with vaporised
hydrogen peroxide (VHP).
Illustration 2a
This removes any biological contamination to a validated
6-log reduction and leaves the space and mating faces clean
and ready to fully dock together. Once fully mated, the disc
26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
can be opened, which allows the product to be transferred
from transfer container to vessel, free from the risk of
contamination. Performing this transfer still within the grade
C space provided enormous cost and production benefits,
although the process needed to be fully validated to ensure
the initial perceived benefits could be proven.
Illustration 2b
Validation
The first step in microbiologically validating the process
was to generate a validated decontamination cycle for the
hydrogen peroxide gassing phase. This typically consists of
four distinct phases, which the generator will run through to
ensure a validated gassing cycle is performed each time. All
the four phases are set on time.
1. Dehumidification – Where the chamber being gassed
reduces the humidity within the chamber to provide ideal
condition for biological kill.
2. Conditioning – Where VHP is introduced into the chamber
to build up to levels to achieve good decontamination.
3. Decontamination – VHP concentration is maintained in
order to deactivate any microbiological activity within
the chamber.
4. Aeration – Where on completion of the biological
decontamination, the VHP is removed from the system
so that no harmful levels of residue are left. Normally
the acceptance level is 1ppm, although in this instance
0.4ppm was used as the acceptance level. The client used
a lower residue limit to ensure they had a really robust
system and no chance of contamination of their product
due to gas residue.
The full decontamination cycle can be achieved in as little
as four minutes, although 20 minutes is more typical. For this
application the process was only being performed once a
day and, to ensure a robust cycle was produced, additional
time was added to each of the critical phases, ensuring that
decontamination was confirmed, and gas was aerated from the
system. This resulted in a 41-minute full cycle.
Initial cycles utilised chemical indicators (CIs) to determine
H202 distribution. When satisfactory CI results were achieved,
biological indicators (BIs) were introduced to the process
to confirm the process was successfully achieved. Upon
completion of each cycle, all BIs and CIs were collected.
The CI strips were then checked for colour change to ensure
uniform vapour distribution. The BIs were transferred to a
suitable growth media, in this case Spordex culture media, and
incubated at 55°C to 60°C for seven days. They were observed
daily for any microbial growth.
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 Manufacturing/Technology Platforms
Acceptance criteria for the cycle included:
A) All CI strips used in the cycle must have changed colour.
B) The positive control BI must demonstrate growth.
C) At least one BI from each location must not demonstrate
growth.
Once the cycle was developed it was then executed in
triplicate to form the performance qualification (PQ) for this
element of the process.
In order to fully validate the system, the process was
challenged with multiple media runs prior to validation.
These successful media challenges were then carried
forward with three media runs at PQ. The sterile hold
was demonstrated at greater than 10 days with product
transferred to the vessel and with the bio valve held in the
closed interlocked position. The sterile hold period was
demonstrated for the passive section (product in transfer
container) for 48 hours, which was more than adequate as
typically this would be at most half this time.
Conclusion
The installation is now operational and in full production.
The original benefits seen at the outset of the project, such
as low capital equipment cost, smaller footprint and ease of
installation, have now been matched by improved sterility
assurance, ease of use for operators, and low maintenance.
The system is straightforward to use, easy to install / validate
and has certainly improved the CDMO’s process.
One learning from this project was at the dispensing stage.
At the time of validation, the system installed was a fully rigid
reusable solution where pre-sterilised API was supplied to the
client in bags. These bags were opened and then subdivided
and dispensed within an aseptic isolator to the pre-autoclaved
transfer container and bio valve passive section. It would have
been beneficial to sterilise the product, container and transfer
connection in one step (gamma irradiation), although due to
the constraints associated with gamma sterilising stainless
steel and elastomeric assemblies as one item, this was not
possible.
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As an alternative to this process, the CDMO could
choose a non-sterile API which is easier to handle, dispense
this into pre-sterilised single use bags with the integral
passive half of the valve which can be docked and product
transferred. The whole package could then be sent away for
gamma sterilisation, instead of having multiple individual
sterilisation and aseptic assembly steps, again making
the process more streamlined, easier to handle and more
cost-effective.
The benefits seen by adopting this method appear to be
growing and, according to a report by ResearchAndMarkets,
the global market for single use technology is estimated to
be worth $7 billion by 2024.
Final Thought
As the requirements of biological products continue to evolve,
it will become increasingly important for technologies in the
sector to be agile in order to adapt to new demands.
REFERENCES
1. https://www.pharmamanufacturing.com/articles/2018/
biopharma-market-an-inside-look/
Christian Dunne
Christian Dunne is the Global Head of
Sterile Solutions at ChargePoint Technology
for the AseptiSafe®range of products for
sterile containment. He works on the
advancement of ChargePoint Technology's split butterfly
valve technology, designed to handle highly potent/sterile
powders and small-scale components, where product and
operator protection are paramount. While working on
many aseptic applications, Christian integrated several
different bio-decontamination systems and has an in-depth
understanding of their performance and application.
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27
 Regulatory/Quality Compliance
Meeting the Standards Required for Effective
Biorepository Management
Biorepositories are a key asset for many organisations
including those in the biotechnology, pharmaceutical and
medical research areas. As they may contain human tissue
and other material, possibly together with personally
identifying information (PII), security is key and they can
be subject to stringent regulations.  With every aspect
of specimen handling from collection through storage,
processing, distribution, and eventual disposal to be
considered, as well as the need to manage all the associated
data and information, the use of an appropriately configured
laboratory information management system (LIMS) can
contribute significantly to the efficient management of
any biobank facility. Guidance for managing biobanks is
available from two highly respected sources. The ISBER
Best Practices: Recommendations for Repositories Fourth
Edition1, published by ISBER (International Society for
Biological and Environmental Repositories) presents a
set of recommendations for the  most effective practices
for managing biological and environmental specimen
collections and repositories. These are either evidence-
based or consensus-based practices for collection,
long-term storage, retrieval and distribution of specimens. 
In addition, the recently published ISO 20387:2018(en)2
Biotechnology – Biobanking – General requirements
for biobanking specifies general requirements for the
competent, impartial and consistent operation of biobanks
including quality control requirements for ensuring the
quality of biological material and data collections.
Creating a Robust Biobank Management System using LIMS
By providing the functionality to control, manage, organise and
document information within a dedicated database, a LIMS can
meet many biobank management needs. Specifically, it can
become a powerful component of an overall biobank quality
management system. ISO 20387:2018(en) fully supports the
use of computer software and hardware for data storage and
tracking. However, a well-designed LIMS will also address
issues around data security and access that help prevent loss
or corruption of data. For example, commercially available LIMS
provide functionality such as automatic data capture, username
and password management, audit trails and database backup
facilities that help ensure data integrity and prevent data loss.
In addition, the data is held in a single place within an industry
standard database for as long as required, with easy access
using industry standard tools. From within the LIMS application,
users have a user-friendly interface to search for, and easily
filter, relevant data. However, the data they can access can be
controlled to ensure they see only what they are authorised to
see. Beyond just specimen management data, LIMS can have an
important role to play in overall laboratory and organisational
management, for example in monitoring equipment and
instrument maintenance and calibration, managing staff training
and competencies and monitoring corrective and preventative
actions (CAPA).
28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Specimen Handling
Biobanks can contain a wide range of different specimen types with
different storage requirements. The handling of these specimens,
which may be defined by regulatory requirements, is of critical
importance. LIMS provides a complete specimen management
solution from sample collection and registration to sample
disposition and eventual disposal through the allocation of unique
IDs and tracking of actions performed on the specimens. The exact
physical location of each specimen can be defined in terms of the
storage hierarchy, for example facility, room, freezer, shelf, and
rack. Container types, for example 96-well plates, can be specified
as required and can include unique sample position information
(Figure 1). All changes in location are recorded, giving a complete
record of where a specimen has been. Aliquoting and sub-sampling
can be managed with full history and chain-of-custody reporting,
allowing each of these aliquots and sub-samples to be tracked and
their relationship to the parent sample maintained. Randomised
location auditing built into the LIMS provides evidence that
specimens are where they should be.
Figure 1. Sample storage management
Handling Other Specimen-related Data
In addition to data related to the type and location of the
specimens, there is a significant amount of other specimen data
and information to be managed. This can include patient consent
data and potentially other patient-related data or specimen-
specific information. The LIMS must be capable of recording
this information (Figure 2). The biobank can define acceptance
criteria for biological material and associated data, including
biosafety, biosecurity and intellectual property rights. Collection
procedures must be specified and documented. When samples are
transferred out of the biobank, the minimum/maximum shipping
temperatures and other key data should be recorded as required.
Any processing or testing procedures required for any sample
must be specified and documented. These various sample-related
stages, such as collection, accession, acquisition, identification,
preservation, long-term storage, quality control, transport,
disposal, etc., must be described in a workflow (Figure 3).
The issue with this is that all biobanks may work in different ways
and record different information (indeed an individual biobank
may have very different processes for different specimen types).
Therefore, a LIMS must allow for these different working practices
while maintaining control in terms of data integrity and security.
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
Figure 2. Sample registration
Figure 3. Biobank manager workflow
This is much easier to achieve in a truly configurable LIMS that can
be set up to reflect the exact workflows and needs of the individual
biobank. All procedures need to be specific to the biological
material and associated data and should be fully documented. The
LIMS can provide access to the specific documentation or standard
operating procedures (SOPs) as required.
Biobank Facilities and Competency
Clearly, it is essential that equipment such as refrigerators
and freezers within a biobank are kept fully operational and
calibrated. In order to meet best practice requirements, the use
of a LIMS allows the management of scheduled and unscheduled
maintenance and calibration actions. Equipment can be flagged
as out of service either automatically, for example if maintenance
or calibration is past its due date, or manually in response to
an unexpected event. Any equipment that is out of service
can be prevented from being used. A permanent record of all
actions can be maintained for regulatory and audit purposes.
It is also necessary to check environmental conditions within
areas of the biobank or indeed within specific equipment such
as freezers, which could influence the quality of the biological
material. A regime of spot checks may be set up to regularly
monitor individual sampling points within locations throughout
the biobank (Figure 4). These results can be recorded within the
LIMS to ensure a permanent record of effective quality assurance.
Failures and trends may lead to the raising of corrective actions
that can also be tracked and managed through the LIMS. In fact,
all non-conformances can be logged, tracked and corrected to
provide management of QA preventive and corrective actions.
It is also essential to ensure that personnel that carry out any
action within a biobank have the appropriate level of training and
expertise for the specific activity. A LIMS must allow the status of
competency and training of biobank personnel to be recorded,
with automated checks when specific actions are undertaken that
prevent untrained or uncertified personnel carrying out the action.
www.biopharmaceuticalmedia.com
Figure 4. Environmental monitoring
Reporting
Given the amount of biobank data that can be managed by a
LIMS, the ability to report on that data is essential. The LIMS must
provide extensive reporting capabilities across all the available
data, for example specimen reporting, management reporting and
monitoring of key performance indicators. Typical reports may
include the number of specimens accessioned over a period of
time, specimens dispatched but not returned within the required
time, or specimens due for disposal. Reports and searching
functions can also be used to identify specimens that meet specific
selection criteria for inclusion in projects or studies.
Harnessing the Power of LIMS
The ability of a LIMS to manage the varied nature and amount of
data associated with a biobank means that it can make a powerful
contribution to biobank best practice. However, the very fact that
each biobank may be different and the data so diverse requires a
flexible solution. A LIMS that is specifically designed for biobanks
and biorepositories is clearly attractive, but it must also retain
the flexibility to adjust the configuration to meet the exact needs
of the particular organisation, rather than impose a particular
workflow that is not appropriate. The ability to configure a LIMS
without needing to change the underlying code offers significant
benefits. It delivers a specific solution that does not compromise
the underlying software and allows future changes to the system,
for example in response to changing business needs or regulatory
requirements, again without modification of the underlying
software. This future-proofs the biobank solution and provides
long-term protection for the investment made in the system.
REFERENCES
1. https://www.isber.org/page/BPR
2. https://www.iso.org/standard/67888.html
Simon Wood
Simon Wood PhD, Product Manager at Auto-
scribe Informatics, has  30 years’ experience
in the commercial LIMS environment. He is an
acknowledged expert in the field of scientific
and laboratory informatics.  Autoscribe is a global supplier of
LIMS to both the laboratory and the wider business markets,
with distributors in every continent offering localised
technical support. Visit www.autoscribeinformatics.com for
more information.
Email: simon.wood@autoscribe.co.uk
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 29
 Regulatory/Quality Compliance
Is the Absence of Data Integrity Software
Affecting the Assurance of Gel Clot Assays
in Bacterial Endotoxin Testing?
Data integrity remains an important topic in
pharmaceutical and biopharmaceutical manufacturing
science. The purpose of data integrity is to ensure that
the accurate process of production and quality of the
products are shown through the documentation that is
associated with it. It is essential that facilities report what
is occurring in their labs’ processes to ensure that good
laboratory ethics are continuously practised throughout
the production of their products. Although this is nothing
new to the community, every manufacturer around the
world must ensure that the data related to their products
has not been affected or compromised.
Data Integrity
Regulators have been cracking down on manufacturers to
confirm that they have an adequate representation of their
data. Quality assurance departments must enforce and have
complete control over the data that is produced. This ensures
that information is not manipulated during any stage of
testing.
In FDA’s Data Integrity and Compliance with Drug cGMP
Guidance for Industry it states that “data integrity is critical
throughout the CGMP data life cycle, including in the creation,
modification, processing, maintenance, archival, retrieval,
transmission, and disposition of data after the record’s retention
period ends. System design and controls should enable easy
detection of errors, omissions, and aberrant results throughout
the data’s life cycle.” The lack of information of what accurately
occurred versus what is presented to regulatory agencies
presents an issue of incompleteness which could be hard to
determine when there is a product failure. Having the full
30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
understanding helps to reduce the chance of recalls and ensure
that any potential failure in testing cannot be simply tested over
without a trace. Data integrity forces companies to identify the
cause of a potential failure rather than exchange information
as if it never occurred. Regulators are strict about data integrity
due to:
1. Alteration of raw data and manipulation of values
2. Changing or eliminating points without justification
3. Backdating results
4. Changing lot information or product information on the
paperwork
5. Allowing technicians the ability to edit prior to approval
6. Security of the data is left unattended
When these data integrity issues occur, facilities must become
more stringent to ensure that the data generated have the
integrity needed to be accepted by regulators. The system that is
encouraged by regulators to be used for the integrity guidelines
of facilities is to confirm that their data is Attributable, Legible,
Contemporaneous, Original (true copy) and Accurate – the ALCOA
Principle.
The Effect of Data Integrity on Bacterial Endotoxin Testing
Endotoxin testing has been around to test pyrogens for many
years. Bacterial endotoxin testing uses limulus amebocyte
lysate (LAL) to detect pyrogens (specifically endotoxin) in
pharmaceuticals, biologics and medical devices. Endotoxin is a
toxin released from the lipid A portion of a lipopolysaccharide
by a living or lysis gram negative bacteria. This release causes an
adverse effect when it encounters the immune system of a living
organism. The detection of this pyrogen is important to ensure
the safety of end product testing of drugs prior to public release
for human and veterinary use.
Winter 2019 Volume 2 Issue 4
www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31
 Regulatory/Quality Compliance
Endotoxin testing can be performed by three different
methods; gel clot, turbidimetric and chromogenic. The ability to
test endotoxin by the change in turbidity or chromophore release,
the turbidimetric and chromogenic methods can be tested by a
microplate reader. This can only happen when the instrument
works in conjunction with software to ensure that data integrity of
the testing was performed. In contrast, the gel clot assay cannot be
performed on the microplate reader; it is the only method that is
unable to prove that the integrity of the data was held throughout
the testing process with software.
Is the Gel Clot Method Enough Without Data Integrity Software?
The gel clot method is an acceptable end product test to release
products. It is also known as the “traditional method” because
it is the first method for LAL testing. The gel clot method can
determine the presence of endotoxin both qualitatively and
semi-quantitatively. The method is relatively simple: the sample
is incubated at 37± 1°C for 60 ± 2 minutes using a water bath or
heating block. After the incubation, each sample tube is removed
and slowly inverted at 180° to determine if there is a firm gel
formed at the bottom of the tube. If the integrity of the gel within
the tube is intact with no deformation, the result is positive for
endotoxin. If the gel does not retain its integrity and collapses,
the result is negative for endotoxin. The result of the gel clot
method concludes the decision for that sample.
Although the testing of this method is based on a subjective
interpretation of the user, many can question if the user is
trained properly to be able to have an accurate interpretation
of the results. Performing such a subjective test can potentially
differ from person to person, due to the judgement of a gel after
inverting the test tube to 180°. Some users of this method have
a second person to confirm the results as well. This could still
pose an issue as well. The gel clot method itself is risky because
it may be difficult to determine if the testing was carried out
properly. Other common concerns associated with gel clot lack of
data integrity are shown below, which leave room for the ALCOA
principle to not be fulfilled:
1. Ability to manipulate data
2. Providing a minute amount of information
3. Unavailability of a second person to confirm the testing results
4. Misinterpretation of gel
5. Lack of audit trail
6. Lack of unique identifier of each user
Data Integrity with Tube Reader
There is instrumentation that is capable of gel clot evaluation.
32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Like the heat block and water bath, this incubator can evenly
distribute the temperature of 37 ± 1ºC for heat stability
and vibration control to avoid inaccurate determination of
results. Unlike the traditional water bath and heat block, the
tube reader has user-friendly software to provide a visual
trend of what is occurring with the sample and the direction
the test is going in real time. This benchtop instrumentation
is specific for endotoxin data processing. It is based on the
protocols complying with three pharmacopoeias (USP, EP, JP)
for bacterial endotoxin testing capable of a successful output
of very detailed reports.
The software has audit trail capabilities to help to ease audits
by regulators. The robustness of the reader’s data processing
functions allows statistical processing of the mean of the
activation times and the standard deviations of the samples.
The software permits the user to define the sample types, such
as standard sample, negative control, positive control, etc. The
system collects the data and ensures that the integrity of the
actions abide to the 21 CFR Part 11 compliant regulation by
providing:
• Audit trails
• Electronic signatures
• Raw data identification
• Archiving
• Backing up data (user management and system)
• Showing revisions when information is edited
• Unique user identification and level of access
Confirmation of Data Integrity for the Tube Reader
As stated, FDA 21 CFR Part 11 is a feature of most tubers. This
allows the tube reader to be able to have the proper electronic
data and signatures from all parties involved in the testing of
the product. There is a function that will show warnings if the
user is not abiding by the guidelines set. In this case, no error in
data entry can occur, further ensuring that the facility performing
the test has the needed data integrity for their gel-clot assay.
Workflows are created to ensure the protocol has been reviewed
prior to performing the testing.
Another advantage of performing the gel clot method on
the tube reader is being able to see what is occurring as the gel
is forming. On the tube reader, an indication that the sample’s
gel is not forming, in the process of forming, or that gelation
has occurred, is the colour of the light adjacent to each well. A
time course plot gives the user the ability to be provided with
a graph of the behaviour representation of the results in order
to determine the outcome of the results quantitatively. This
can solidify the user’s testing ability and provide that needed
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
additional confirmation of quantitative accuracy of the gel. As
a result, an extensive report can be printed to use as backup
information. This will show more evidence of the test added to
the qualitative positive or negative results for confirmation of
the gel clot method.
Transitioning Gel Clot Method from the Water Bath/Heating
Block to a Tube Reader
Validation of products is always tedious and time-consuming,
however, it is a necessary aspect of ensuring that the product
testing is accurate and reliable. It also confirms the suitability
under actual conditions of use. Since this is the case, most
facilities object to going through the process of revalidating a
product unless enforced by a regulator or bringing a new product
online. The gel clot transition from traditional instrumentation to
a tube reader has become easier because the two are very similar.
Testing performed on the tube reader should resemble testing
with the traditional gel clot instruments. This similarity should
eliminate the stress of having to change protocol.
Transitioning from Gel Clot Method to Kinetic Methods using
the Tube Reader
This instrumentation and software are great for clients who
are using the gel clot method but would like to start measuring
endotoxin by the chromogenic or turbidimetric method. This
technology gives the user time to gradually convert from the gel
clot method to the kinetic application over time, while still being
able to carry out the protocol they have already established using
www.biopharmaceuticalmedia.com
the same instrumentation for both. This technology allows for
testing to be done by every method. It also gives the customer
flexibility when introducing a new product to be tested for
endotoxin testing.
Most samples require dilution prior to testing due to the
samples’ ability to interfere with the endotoxin or the LAL.
In response, interference can be eliminated by diluting with
bacterial endotoxin testing (BET) water or by a buffer. Gel
clot assays restrict the user from making larger dilutions due
to the maximum valid dilution that can be set from the lysate
sensitivity that the user uses to test the sample. On the contrary,
the kinetic method has a higher sensitivity than the gel clot assay
and allows for a greater maximum valid dilution for samples.
The kinetic method allows for a wider range of standards rather
than a set two-fold bracket of concentration to use as a standard
for comparison. Kinetic assays are typically faster and more
accurate results than the gel clot assay. This will permit the user
to gradually change from one method to another.
Should there be More Emphasis on Data Integrity Software for
Gel Clot Assays?
The answer to this question is that it depends on how necessary
it is to have supported documentation, security and backup
quantitative results on the gel clot analysis to confirm what
was proven qualitatively. Data integrity will continue to be an
important topic in this industry. Whether the information is all
recorded by handwritten documentation or by software that can
ensure the data is kept secure and free of manipulation, ensuring
that the intact data integrity should be the number one goal.
If the paper system is ever questionable or human subjection
causes a difference in results for a gel clot assay, it is imperative
that the proper measures are in place to ensure the data is not
at risk. There is much success in the tube reader and software
approach for the completion, confirmation and accuracy of the
data integrity to support the qualitative results of the gel clot
assay.
REFERENCES
1. U.S. Food and Drug Administration. Data Integrity and
Compliance With Drug CGMP Questions and Answers Guidance.
Available at: https://www.fda.gov/regulatory-information/
search-fda-guidance-documents/data-integrity-and-compliance-
drug-cgmp-questions-and-answers-guidance-industry. Last
accessed October 2019.
LaToya Mayfield
LaToya Mayfield is a technical specialist
for the LAL Division at FUJIFILM Wako
Chemicals U.S.A. Corporation. She has a
background in quality control and quality
assurance. She is knowledgeable in cGMP, ISO and CAP
regulations. LaToya performs customer trainings to assist in
achieving successful product validations that comply with
regulations. She is the founder and president of GiSTEM,
Inc., a non-profit organisation designed to encourage girls
to pursue and thrive in STEM fields.
Email: latoya.mayfield@fujifilm.com
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33
 Regulatory/Quality Compliance

Best Practices of IoT Implementation

for Smart Drug Research
Drug discovery takes years to complete. Testing a single
compound can cost more than £1.5 billion. Experiments
on animals often fail to predict human behaviours and
responses because traditional animal anatomy often does
not accurately mimic the human body. For these reasons,
there is a vast need for other ways to emulate human
diseases in vitro in order to accelerate the research and
development of new drugs.
IoT technology can evolve the sensors’ integrated OOCs. To
achieve this, the first step is to integrate sensors to monitor
physical and biochemical parameters associated with the
functionality of OOC models.
What is IoT in the Drug Industry
Artificial intelligence (AI) in pharmaceutical companies has the
ability to emulate human learnings in the analysis of complicated
drug data without direct human input. AI technology gains
information and processes it to give a well-defined output to
the end-user through machine learning algorithms.

The Internet of Things (IoT) in pharmaceutical companies is an

application of the IoT for drug- and health-related purposes, data
collection and analysis for research, and monitoring.

IoT in pharmaceutical companies can be interpreted as

connecting the drug discovery devices to the network to allow
the scientists and researchers who may be located in remote
locations and can still do the exchange of data and information
and be able to take intelligent decisions for better drug research.

Is There An Alternative?
In this context, organs-on-a-chip (OOC) has emerged as a new
tool for drug discovery. Organs-on-a-chip is an in vitro  tissue
culture platform w  hich provides better evaluation of the effects
of various chemicals on human tissue.

Organ-on-a-chip
An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic
cell culture chip that simulates the activities, mechanics and
physiological response of entire organs and organ systems, a
type of artificial organ. OOC use microfluidics to reproduce the
way a tissue or part of an organ works. Organ-on-a-chip can Drug Life Cycle
partially simulate organ function including lung, intestine, kidney,
skin, bone marrow, blood-brain barrier, etc.

Although the field of OOCs has been looed at over some

time, there is still a need to digitise the OOCs that will
provide real-time monitoring and capturing the continuous
information about the metabolic activity of the tissue from
remote locations.

Benefits of Integrating IoT Sensors in OOCs

34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY Winter 2019 Volume 2 Issue 4

 Volume 9 Issue 1 INSIGHT /
Volume 9 Issue 1 - Spring - 2017

International Pharmaceutical Industry

Supporting the industry through communication
Peer Reviewed
KNOWLEDGE /
FORESIGHT
IPI – International Pharmaceutical Industry

MALDI Mass Spectrometry in Drug Discovery

Gaining A Deeper Understanding

SUPER PUBLICATIONS
Three Ways to Mitigate the Risk of
Late-Stage Failure in CNS Drug Development

Data
The Foundation of Clinical Trials

Temperature Management
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Keep Your Cool

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FOR SUPER PHARMACEUTICALS
IPI
Peer Reviewed, IPI looks into the best
practice in outsourcing management
for the Pharmaceutical and Bio
Pharmaceutical industry.
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JCS
Peer Reviewed, JCS provides you
with the best practice guidelines for
conducting global Clinical Trials. JCS
is the specialist journal providing you
with relevant articles which will help
you to navigate emerging markets.
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Volume 4 Issue 1
Volume 4 - Issue 1

Supporting the Development of Veterinary Drugs, Veterinary Devices & Animal Feed PEER REVIEWED

Applying Game Theory to One Health

Modelling Veterinary Healthcare Delivery
International Animal Health Journal - Supporting the Development of Veterinary Drugs, Veterinary Devices & Animal Feed

Mastitis due to Mycoplasma bovis

Insights

Pet Obesity
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Leadership Skills of
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Official Supporting
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IAHJ IBI
Peer Reviewed, IAHJ looks into the Peer reviewed, IBI provides the biopharmaceutical industry with
entire outsourcing management of the practical advice on managing bioprocessing and technology,
Veterinary Drug, Veterinary Devices & upstream and downstream processing, manufacturing,
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www.animalhealthmedia.com delivery, analytical testing and more.
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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35
 Regulatory/Quality Compliance
Working Principle of IoT in the Drug
Industry
Let us first understand the working
principle for IoT: Collect – Connect –
Process – Present
1) Collect: Drug discovery devices
with enabled sensors collect live
data from the live environment.
2) Connect: The sensor sends
collected data to a cloud infra-
structure using various mediums of
communications, including mobile
phones or satellite networks,
Bluetooth, WI-FI, WAN, etc.
3) Process: Once data is collected,
and it gets to the cloud, the IoT
software processes data into valuable information.
4) Present: The information needs are then presented to be
available drug researchers.
Challenges of IoT
Data Collection
• Unstructured data
• Security and privacy issues
Device Connection
• Internet outages
• Power cuts
• Security issues
• Lack of device performance
• Industrial IoT integration – data loss, ineffective
connectivity and desynchronisation
Data Processing
• Analysis of unstructured data
• Inaccurate analysis due to flaws in the data
Displaying the Information
• Unpredictable situations
• Incorrect information
The IoT technology is expanding into the entire pharma-
ceutical and drug space. However, due to the challenges
mentioned above and the lack of resources to overcome those
challenges, companies may struggle to integrate sensors to
OOCs. Research estimates that almost three-quarters of these
projects fail due to these reasons. A recent forecast suggested
that £192 billion in pharmaceutical sales could be at risk by
2024.
This article aims to discuss the best practices of IoT
implementation to empower the world of drug discovery. This
is done by integrating the IoT implementation framework in
the drug development stages, for seamless integration of IoT
sensors to OOCs.
Best Practices to Address These Challenges and
Successfully Implement IoT Solutions
The below figure illustrates the integration of IoT life cycle
stages into the drug life cycle phases.
36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
• Phase 1: Pre-clinical Research
Stage 1: Inception
Stage 2: Planning
Stage 3: Design
Stage 4: Prototyping and verification
• Phase 2: Clinical Approval and Launch
Stage 5: Validation and implementation
• Phase 3: Commercials
Stage 6: Maintenance of IoT framework
Phase 1 – Pre-clinical Research of the Drug
The following can be achieved with the correct implementation
of IoT technology (Stage 1 to Stage 4)
• Improved drug discovery through improved information
• Unmet need met with real-world data
Stage 1 – Inception – IoT Implementation
This stage includes understanding users’ needs of the drug and
the drug discovery devices required to identify and assess the
scope and value of the IoT implementation. The vast and diverse
data collected from large sets of real-world cases will increase the
genuineness of user research. However, the data must be correct
and complete and of high quality. If the data is not high quality,
then data mining must be performed:
Data mining: Most of the time, medical research users lack
critical real-world information. It mostly uses leftovers, controlled
environments and volunteers for medical examination. Data
mining ensures availability of meaningful and valuable data.
By generating more practical and reliable data, IoT yields better
solutions and discovery of issues that were previously unknown.
That’s why improving data quality for IoT applications should be
one of the most important activity to get the best ROI through
IoT implementation. The below are the suggested best practices:
• Gather data from large sets of real-world cases.
• Prepare data for mining.
• Apply the mining algorithms to data.
• Display mined data to the end users (patients/HCPs).
At the end of this stage, an intended use and indication of
use document (covering all the points) having a clear and exact
understanding of why IoT is needed in the drug discovery device
space should be produced. The below are the suggested best
practices:
• Document clearly what is the purpose of the drug discovery
device.
• Document why IoT should be implemented for this particular
drug discovery device.
• Document what clinical solution IoT will bring for this drug.
• Document the short-term and long-term usage of using IoT
technology.
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
• Document the end users – usage environment, age group,
gender.
• Document the frequency of the drug usage.
• Document the user interaction methods with the drug
discovery device.
• Document the risks of the category of drug and how IoT
technology control the risk.
• Document the frequency of the drug usage.
• Document the interoperability needs with other medical
devices.
Review of Stage 1:
• Does the drug manufacturing company have complete clarity
of why IoT implementation is needed for their drug devices
and exact understanding of the IoT system requirements?
• Is the risk management framework (identification-assessment-
evaluation-control) understood?
Stage 2 – Planning – IoT Implementation
Once the user needs are well understood, the next step is to plan
for the IoT implementation, considering multiple aspects of the
devices. Find below the suggested best practices:
• Design planning of the IoT implementation: Successful IoT
solutions require a solid strategy and workflow, combined
with risks and human factors considerations. At this stage
a plan document should be produced as per the given
guidelines:
• Plan for development kit – IoT hardware device, IoT
software device, drug discovery device.
• Plan for the infrastructural needs.
• Plan for the risk management – technical and clinical
risks.
• Plan for human factors – formative and summative
activities.
• Plan for end-to-end security and privacy aspects for the
end users in the IoT ecosystem.
• Plan for cold and hot path analytics.
• Plan for standards to be followed enabling companies
to accelerate time to market and maximise the market
size.
• Plan for post-market clinical follow-up.
• Plan to communicate with regulatory body to comply
with regulations.
• Resource planning: Some of the experts should be planned
early for the IoT team.
• Computer Engineer:  a computer engineer specialising
in embedded systems.
• Software Engineer: t  o design and implement computer
programs, perform unit and integration testing, perform
code reviews.
• Electronic Engineer:  to design circuit boards to be used
in the IoT systems.
• Mechanical Engineer:  to design the hardware IoT
components.
• Mechatronic Engineer: specialising in sensor use.
• Automation Engineer: automation of production
processes.
• IT Expert:  to implement and maintain the internet
www.biopharmaceuticalmedia.com
network.
• Manufacturing & Production Engineer:  someone with
knowledge of the production processes, needs, and
limitations.
• Telecom specialist:  to deal with the variety of protocols
and gateways for IoT data transfer.
• Security & Vulnerability Engineer: someone with
end-to-end security and vulnerability knowledge and
formulate the security strategy to protect the entire IoT
ecosystem.
• Planning for maintenance and upgrades of IoT infra-
structure.
Review of Stage 2:
• Has the drug manufacturing company done sufficient
planning to accomplish all the work?
• Has the risk management plan (identification-assessment-
evaluation-control) been implemented?
Stage 3 – Design – IoT Implementation
By now, the user needs are well understood and the plan is in
place to build a strong and reliable IoT system. At this stage, you
have to start designing the IoT framework that comprises IoT
hardware and IoT software.
To have an effective design, below are the suggested best
practices:
• Choosing the IoT platform
• Choosing the IoT hardware
• Choosing the IoT software
• IoT security solutions
Choosing the IoT Platform
The parameters for choosing the IoT platform could be on the
basis of the below suggested best practices:
• Security
• Performance
• Storage and retention
• Scalability
• Usability
• Interoperability
• Adaptability with the legacy architecture
• Message protocols
• Disaster recovery
• Maintenance & decommission support
Choosing the IoT Hardware
The below are the suggested best practices to choose the IoT
hardware:
Device to be connected: Choose the drug discovery device as per
the intended use.
Data acquisition: This is the component that contains the sensors
that acquire real-world signals such as temperature, pressure,
density, motion, light, vibration, etc. Decide the type and number
of sensors you will need on your application. IoT sensors are the
most critical IoT hardware. These devices consist of a variety of
modules such as energy modules, RF modules, power management
modules, and sensing modules.
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37
 Regulatory/Quality Compliance
Data processing: This is the main unit that processes the data
and performs operations such as local analytics, and stores data
locally.
Data display: It enables communications with the third-party
system locally or on the cloud.
For example, wearable electronic devices are the devices that
can be worn on the body to get real-time information.
Choosing the IoT Software
The below are the suggested best practices to choose the IoT
software:
The Internet of Things (IoT) is an entirely new platform for
developers, but one thing which remains consistent in this new
world is the programming language. Developers utilise the same
languages for their projects, while also integrating some specific
changes for IoT. What languages are best for IoT? Selecting
a language for IoT projects could be as difficult as selecting an
IoT hardware platform. IoT software comprises a wide range of
software and programming languages. C/C++, Python and Java
are the most popular I  oT programming languages.
• C/C++ is great for writing hardware-specific code and it works
really well in Linux operating system (OS), which is the most
popular IoT  OS. C/C++ is made to handle the hardware and
complex processing at the same time, making it ideal for
running on embedded systems. This language was written for
the hardware systems, which makes them so easy to use. C is
considered the most useful for I  oT  devices because it doesn't  but also ensure interoperability between them.
require  a lot of processing power.
• Java:  While C and C++ are hardware-specific, the code in
JAVA is more portable. It is more like a write once and read
anywhere language. Programming   with  Java  makes   IoT  compromising the patient’s sensitive data from malicious attacks.
devices more efficient in exchanging information and making
proper use of the information when and where it is needed.
So, the device becomes more integrated.
• Python: Python is a good choice for data analysis in IoT
systems. The language is simple and can be easily deployed.
Its large community helps in providing help and libraries as
and when required, which makes it an ideal language for IoT
systems, especially for data-intensive applications.
• JavaScript: JavaScript works best in a wide range of
environments, and is best in gateways and the cloud. It is
very efficient when it comes to sensors because of having an
event-driven modality option.
• PHP: PHP is a good option to develop apps using the GPS data
from IoT devices.
Low-level programming languages: B#, a language built from
the ground up for very low power devices. It is similar to C#, but
fitted with real-time control functions. Assembler is probably
among the low-level languages, capable of running on just about
everything. The downside is there’s no hand-holding at all, so
if your code doesn’t work, or if a new processor doesn’t accept
Assembler code, then you are in a really difficult situation. Weave
(Google) could become popular if it receives more support from
developers. Apple offers its open source language, Swift, currently
marketed at iOS and Mac OS developers. To interact with the
iPhones and iPads with your home hub, Swift is a good choice.
IoT Security Solutions
IoT security is the technology area for protecting the connected
drug devices and networks in the IoT system.
38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Challenges
Lack of awareness: Design is one of the most sensitive phases in
building the IoT system. The majority of vulnerabilities discovered
in software are due to lack of awareness towards security aspects
during the design phase. Statistically, more than 80% of all
complex and expensive software refactoring is due to this issue.
Low in priority: Security is often considered as an “afterthought”
and as a result, the cost of fixing security issues in live software
is much higher than the cost of implementing the secured design
techniques at the design stage (before even beginning to code).
Obsolete technology: Often, the connected legacy drug devices
may not be inherently designed for modern IoT systems; hence
the modern threats cause vulnerabilities.
Weak password: Hardcoded or default passwords can lead to
security breaches.
Security patches: Mostly IoT devices (drug device sensors) are
placed on a machine and left until end of life. They hardly ever
receive security updates or patches and hence often cannot handle
advanced encryption or other modern security measures.
Lack of industry-accepted standards: While many IoT security
standard frameworks exist, there is no single agreed-upon
framework which makes it difficult not only to secure systems,
Effect
Apart from the financial and reputational losses, the worst could be
Injecting security solution on the basis of the suggested best
practices:
• Incorporating security features at the design phase
• Strong encryption protocols adapted for secured
communication between devices
• Strong passwords policy in the IoT system
• Updated digital certificates and continuous software updates
for connected drug devices and IoT systems
• API security for data integrity to ensure secured data
transfers from devices to IoT backend systems
• Unique device identifier (UDI) providing unique identifier
for each device
• Hardware security
• Protecting IoT network by ensuring security gateways,
port security, firewalls
• Inventory management of IoT devices
• Awareness and training – keep staff up to date with new
systems, architectures and programming languages so
they are ready for new security challenges. 
Review of Stage 3:
• Has the drug manufacturing company done the effective
design to meet all the user requirements?
• Has the risk management framework (identification-
assessment-evaluation-control) been implemented?
Stage 4 – Prototyping & Verification – IoT Implementation
Now that IoT hardware and software are chosen, a secured IoT
system prototype for the drug discovery device can be built. It
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
is the IoT hardware and devices enhanced with smart sensors
and embedded systems using many off-the-shelf components
like sensors, circuit boards, and microcontrollers.
Verification of the prototype shall act as proof that the concept
will work the way it was envisioned.
Verification of the prototype on the basis of the below
suggested best practices:
• Code reviews
• Unit testing – verifies that the code works
• Integration testing – verified that the coded items are
integrated with no interface challenges
• System testing – testing the drug device in the prototype
of the IoT system
• Formative testing – early end use feedback. This can lead
to some design changes
• Cold path analytics – testing the batch processed data
• Security testing – data integrity testing
Review of Stage 4:
• Has the drug manufacturing company built the IoT System
prototype for the drug discovery devices, and done
sufficient verification?
• Has the risk management framework (identification-
assessment-evaluation-control) been verified?
Phase 2 – Clinical Approval & Launch
The following can be achieved with the correct implementation
of IoT technology (Stage 5)
• Ensure effectiveness of drug
• Meet post-market expectations
Stage 5 – Validation & IoT Implementation
Once the prototype is verified, the IoT system implementation
for the drug discovery device can be accomplished. Before it
gets implemented in the patients’ and HCPs’ world, a thorough
validation must be performed.
• Validate end-user requirements
• Validate risk and usability factors of patients and HCPs –
summative testing with the reference of intended use of
the drug device in the real IoT system
• Perform a risk-benefit analysis
• Compatibility testing of drug device in IoT ecosystem
• Clinical evaluations of device in IoT system
• Summative testing using real users in the real IoT system
• Performance testing
• Validate the hot path analytics
• Interoperability testing
• Security testing.
Review of Stage 5:
• Has the drug manufacturing company implemented the
IoT system prototype for the drug discovery devices in
the real-world environment, and has it done sufficient
validation in terms of summative testing and clinical
evaluation?
• Has the risk management framework (identification-
assessment-evaluation-control) been validated?
www.biopharmaceuticalmedia.com
Phase 3 – Commercial Phase of the Drug
The following can be achieved with the correct implementation
of IoT technology (Stage 6)
• Monitor outcomes against competitive products
• Monitor safety.
Stage 6 – Maintenance of IoT Framework
• Performance monitoring of the IoT system and measure
of its ROI
• Post-market clinical follow-ups (PMCF) - the data
derived from the post-market clinical follow-up study
is used to provide clinical evidence to support
the  post-market surveillance of the IoT system for the
drug discovery device
• Corrective action (CA) – rectify a task, process, product
that has caused an issue
• Preventive action (PA) – change implemented to address
a weakness in the IoT system that is not yet responsible
for causing an issue.
Review of Stage 6:
• Is the PMCF process effective?
• Is the CAPA process in place?
Conclusion
To get the best of drug research using organ-on-a-chip
(OOC) methods, connecting it to an IoT sensor would be the
most effective. This article is an attempt to suggest the best
practices of IoT implementation in the drug research method,
based on my rich experience in digital therapeutics and
converging digital technologies with pharmaceutical products.
REFERENCES
1. https://en.wikipedia.org/wiki/Internet_of_things
2. https://en.wikipedia.org/wiki/Digital_health
3. https://en.wikipedia.org/wiki/Drug_development
4. https://en.wikipedia.org/wiki/Organ-on-a-chip
Anindya Mookerjea
Anindya, Founder & CEO at S-Cube Techno-
logies, is an Entrepreneur, Thought Leader,
Business Visionary, Strategist & Founder
of S-Cube Technologies, an IT Digital
Solutions Company using Internet of Things (iOT), Artificial
Intelligence (AI)/ Machine Learning (ML) & Robotic Process
Automation (RPA) with potential contribution towards
bringing digital technology in Healthcare, Drug and Life
Sciences industries. His core specialisations include
Advanced Digital Technologies, Connected Devices &
Cloud-based App Development.
Email: anindya@scubetech.co.uk
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39
 Regulatory/Quality Compliance
Managing Global Supply Chains
As temperature-sensitive pharmaceutical products are
increasingly being shipped globally to more remote
regions there is an even greater demand for the effective
and efficient management of global supply chains.
Increasingly there is a rising requirement for innovative
solutions being placed on the temperature-controlled
packaging industry, coupled with a rising requirement to track
and trace pharma payloads throughout transit.
Whether shipping finished products, transporting clinical
trials materials or delivering sample drugs, temperature
excursions can mean the difference between success and
failure, profit and loss.
It is essential therefore, that pharmaceuticals are protected
throughout the supply chain end-to-end as temperature
excursions during transportation can even cause them to
become toxic.
There is increasing recognition within the supply chain of
the vital role smart packaging plays and in response to the
stringent regulatory requirements, the packaging industry is
taking a proactive approach.
An impetus for the latest generation of high-performing
packaging products comes as the pharma industry continues
to grow at a significant rate, with an estimated global worth of
$400 billion by 2020 according to the World Health Organization.
Maintaining end-to-end pharma supply chain integrity is
critical to mitigate risks within the pharma-logistics cold chain
and better ensure the safe and secure transportation of health-
giving and life-saving pharmaceutical products.
The market for packaging to transport temperature-
sensitive materials for the global life sciences industry, such
as pharmaceuticals, blood, tissue and organs, is currently valued
at $2 billion and expected to grow to approximately $5 billion
by 2026.
The global life sciences industry faces several complex
challenges – protecting the integrity of their temperature-
sensitive high-value payloads is an important challenge. But
the industry also must concern itself with mitigating costs,
managing and tracking the assets within a complex cold chain
closed-loop logistics system, meeting stringent global regulatory
standards and navigating complicated global shipping lanes and
unforeseen challenges.
Various pharmaceutical compounds, utilised within the
sector, are developed under certain temperature control
conditions or designed to be stored at specific temperatures
to maintain their stability.
40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
Therefore, it is vital, when shipping pharmaceutical products
between locations, they remain at their storage condition
temperatures to maintain their effectiveness at the point of use.
Within the industry there are standard temperature ranges such
as deep-frozen (below -50° Celsius), frozen (-50° C to -20° C),
refrigerated (4° C to 8° C) and room-temperature (15° C to 25° C).
With the pharmaceutical companies developing ever more
complex and temperature-sensitive drugs, there is a greater
demand in the cold chain industry to meet the growing market
demand for supply as well as improved packaging performance
and efficiency. To help mitigate supply chain risks, the industry
is seeing a greater demand for higher performing packaging
products.
Coupled with the rise in regulatory requirements, there is an
even greater emphasis on supplying the global market with more
advanced, smart packaging that does more than act as a container
for precious, high-value payloads being transported to emerging
markets via complex shipping lanes.
The expertise of experienced engineers is a vital component
within the packaging industry, with providers deploying
increasingly high performing packaging systems that need to
mitigate supply chain risks and minimise temperature excursions.
Any spikes or deviations in temperature, beyond the range
specific pharmaceutical products are required to be stored and
shipped at, could have a devastatingly detrimental effect on the
payload, damaging the container’s contents and impacting on
the efficacy of the products being transported.
This could have financial repercussions equating to losses of
hundreds of thousands of pounds; however, more importantly, it
could have catastrophic consequences for the end user/patients
reliant on the drugs being delivered remaining intact.
More global clinical trials are requiring stricter temperature
regulations, which command compliant cold chain conditions
and increasingly innovative packaging solutions.
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
Temperature restrictions when transporting these pharma
payloads present their own challenges, coupled with the fact
more are being shipped to emerging markets where there are
also extreme temperature ranges to contend with.
An impetus for the latest generation of high-performing
packaging products are these advancements in drug
developments, which includes the increase in more fragile and
temperature-sensitive pharmaceutical products.
A driver for a substantial proportion of this projected growth
in the life sciences sector includes the rise in temperature-
controlled biosimilars and biologics, which are biologically-based
pharmaceuticals as opposed to chemical-based. It is predicted
more than 50 per cent of approved new drugs are going to be
biologics or biosimilars in the next few years.
The evolution of this latest drug development presents its
own supply chain challenges when it comes to safe storage and
transportation of these temperature- and time-sensitive pharma
products.
With the rapid rise of biologics and biosimilars within the
pharma development sector, the need for temperature control
during transportation is ever increasing. Any temperature excursion,
from minor to major, within the supply chain can have costly
consequences for pharmaceutical companies. Particularly serious
would be a temperature excursion which impacts patient health.
More complex distribution lanes, with emerging markets,
geographies and increasing regulatory compliance conditions are
some of the challenges when transporting these temperature-
sensitive biologics/biosimilars.
Innovation and new technologies are proving pivotal to the
emergence and evolution of smart temperature-controlled
packaging protecting pharmaceutical payloads worldwide, and
successful management of supply chains is essential to provide
pharma payload protection.
Packaging companies continue to incorporate innovative
design features and are utilising more advanced technologies,
to produce and manage pioneering products and ever more
sophisticated systems, which help eliminate excursions in
temperature in cold chain.
www.biopharmaceuticalmedia.com
Blockchain is another technology finding its place in
supporting pharmaceutical manufacturing’s cold chain logistics
processes. Blockchain is having a real impact on pharmaceutical
shipments, from prevention of theft and counterfeiting of
pharmaceuticals, to tracking root cause of a dangerous event
that sickens a patient, to government tracking of source of origin
to properly assess duties and taxes for imports.
The industry is seeing an increase in the introduction of
information-centric capabilities to assist with the safe shipping
of pharmaceuticals around the globe. Packaging companies are
increasingly utilising advanced asset management software
systems, which are in place specifically to ensure pharmaceuticals
are shipped to the right location, at the right time and critically,
that they arrive in the right condition.
Companies deploying pharmaceutical shipments worldwide
benefit from the introduction of new technological advancements
including web-based asset management software solutions,
designed to track individual shipments globally. Integrating these
cloud-based systems offers a range of capabilities benefiting the
industry, including options to set up automatic maintenance, next
shipments alerts and produce customisable reports.
The industry is also seeing a growing trend to deploy
reusable systems coupled with asset management SaaS
(software as a service) and reaping the associated benefits.
These systems can automatically collect and analyse data
from company data logger outputs. Currently operating in
the market is a range of SaaS products providing collection
and analysis of brand-agnostic sensor data, as it’s linked to a
variety of smart packaging options allowing packaging vendors
to track a diversity of data including vibration, light, humidity,
temperature and more.
These software platforms capture and monitor information
throughout the course of the shipments trip. The data retrieved
and shared can help pharmaceutical companies make more
informed choices on the most appropriate packaging systems
to deploy, depending on the specific shipping lanes and routes
their payload will navigate.
Utilising asset management software within the supply chain
process help life science industry clients reduce payload risk,
distribution costs and their environmental impact, ensuring
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41
 Regulatory/Quality Compliance
temperature-sensitive, critical and high-value payloads reach
their destination safely.
Integrating the cloud-based system supports and enhances
engineering expertise that is incorporated into the development
and design of the increasingly sophisticated systems utilised by
the life science industries.
Increasingly passive and active bulk systems are incorporating
data loggers to track the temperature throughout the course of
the trip throughout the supply chain.
Issues can arise because often a parcel shipment will need to
be opened to access the temperature logger stored inside.
Alternatively, data logger devices can be attached to a
specialised container to ship a pallet of products providing an
isolated monitoring option to pick up data, which can be saved to
the cloud via Bluetooth or radio-frequency identification (RFID).
The latest development within the packaging industry
transporting pharma shipments globally is the move toward GPS
tracking, which would need to be managed via Bluetooth, RFID
or manually scanned barcodes whereby pharma companies can
track packages, and their shipment progress, online.
GPS is the latest development in response to ensuring
the protection of high-value pharma payloads. It is predicted
advancements in GPS tracking options via a SaaS system will
be part of the industry in the near future. There are benefits to
pharma companies, including knowing where their shipment is
throughout its transportation trip.
If payloads are lost or get delayed en route, the pharma
company take steps to intervene and recharge or replace coolants
so the package or the bulk system gets delivered before expected
temperature duration is exhausted, and this would help mitigate
a temperature excursion caused by a delay.
What’s exciting about leveraging all of this smart technology
is the promise of the capabilities to have data logger sensors and
SaaS platforms communicate directly with each other.
The latest developments will be something that interests
the pharmaceutical companies; the availability of information
through a SaaS platform will be a market differentiator for the
companies that produce this type of smart packaging option.
Currently the information being captured is primarily via
barcode, which requires human intervention to manually collect
and then input the information into a SaaS system.
It’s predicted there will be a move to a system whereby
the relevant information will be captured and then stored in
a centralised database. That information could be updated
frequently at check-in points or instantly via Bluetooth.
Additionally, forensically these smart packaging systems
would offer insight to find out at what point the systems
failed and where that happened with a combination of GPS
and temperature data. This would allow pharma companies to
discover when and where a failure occurred and then diagnose if
it’s a problem with the shipment, with logistics, or with the local
customs office or similar.
42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY
On the horizon is the integration of GPS and temperature
logging to a SaaS system in an effortless way that doesn’t involve
human interaction to leverage the movement towards an Internet
of Things (IoT), which allows all devices to be assigned an IP
address, allowing each device to interact as a unique entity on
the internet.
Leveraging a movement to IoT will help packaging
manufacturers with making future packaging truly smart, by even
providing information on the shipment’s status, temperature,
current location and more back to its owner in a central office.
It also allows for mid-transit interventions if needed or
examining efficiencies of scale identified, such as using different
shipping lanes or different ways to palletise or bulk-ship smaller
products.
Specialised software systems can also aid reverse logistics
within the industry and can provide real-time tracking and
trading through the entire end-to-end distribution cycle.
These optimisation tools can help ensure payload efficacy
and efficient life cycling of reusable packaging inventory assets,
providing a high return on investment. Often easy-to-learn
and use, these superior software systems help the global life
sciences industry manage its demanding and expanding cold
chain logistics supply chain operations while critically meeting
the stringent requirements of a highly regulated industry.
All of these innovations based on clever uses of smart
technology build upon the robust performance of advanced
thermal shippers. Traditionally packaging vendors relied on
more basic thermal control methods providing passive packaging
products, where the key forms of technologies deployed were
ones incorporating insulating material for the outer box of
polystyrene or foams, providing protection and insulation.
To maintain the temperature within the packaging,
combinations of chilled and frozen water were utilised.
Because of the requirement to carry a lot of water to control
temperature and often bulky insulation, these systems proved
heavy and their performance against highly variable external
temperature challenges was not reliable.
Winter 2019 Volume 2 Issue 4
 Regulatory/Quality Compliance
Although useful for an anticipated and unchanging external
environment, these antiquated systems are being replaced more
regularly with technologically advanced packaging systems.
These advanced temperature-controlled packaging systems were
developed to meet the demand and regulatory requirements
for pharmaceutical companies and their cold chain supply chain
service providers.
Most recently the technological advancements introduced
to the market have seen the advent of better insulation options
by incorporating vacuum insulated panels (VIPs), reducing the
thickness of the insulation required and improving performance.
The traditional water-based systems are rapidly being
replaced with ones using phase change materials (PCMs) where
the melting point of the coolants deployed is designed to the
ideal temperature required, while holding that temperature for as
long as a week without any additional, external thermal energy.
These latest advancements mean temperature-controlled
shipping systems using PCMs are far more reliable, providing
thermal stability within the payload space at the desired
temperature while using less weight and space.
The payload efficiency of these newer systems can be more
than twice that of the traditional water-based and foam-insulated
shippers, proving more cost-effective when it comes to logistics
services. Although these more sophisticated shippers can be
more expensive, the trade-off is you are less likely to experience
temperature excursions en route, which would lead to damaged
products providing a costly consequence in the long run.
In line with the increase in more complex shipping lanes and
limited infrastructure in place in some developing destinations,
the need for smarter, more secure, robust packaging has become
more prevalent.
The more sophisticated a shipper, the more the unit cost can
be, so increasingly there is a requirement for reusable systems
to provide better return on investment and which are more
www.biopharmaceuticalmedia.com
cost-effective in the long run. Additionally, reusable systems
provide considerable environmental benefits.
Provided the infrastructure is in place to recapture and
reuse higher performing systems, that contain higher-value
components, reuse makes economic and environmental sense.
The rise in reuse has trigged a corresponding increase in the
global network of service centres being established to facilitate
the reconditioning and repurposing of these smarter packaging
options.
It has also sparked the rise in SaaS systems within the
packaging industry with cloud capabilities that better enable
collaboration in the sometimes complex packaging supply chain
and the ability to centralise data focuses the management of
packaging services intelligently.
Ultimately the aim always is to continue to reduce the supply
chain costs and improve performance and reliability.
Adam Tetz
Adam Tetz is the Director of Worldwide
Marketing at Peli BioThermal and has more
than 25 years of marketing experience. He
is responsible for telling the story of Peli
BioThermal to our worldwide audiences, including brand
identity, product launch and communication strategy.
Prior to Peli BioThermal, Tetz held positions in product
management and marketing communication across a
variety of industries, including medical software, financial
software, information services and professional consulting
services. H
  e holds an MBA in marketing from the University
of Saint Thomas, a BA in advertising from the University
of Minnesota and is a veteran of the United States Coast
Guard.
Email: adam.tetz@pelican.com
INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 43
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www.biopharmaceuticalmedia.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 44
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45 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY Winter 2019 Volume 2 Issue 4