Insights on Successful
Gene Therapy Manufacturing
and Commercialization
Insights from industry experts on gene therapy
manufacturing, including the latest in manufacturing
methods, analytical analysis, regulatory considerations
and reimbursement strategies.
Topics to be discussed include:
• Planning for clinical and commercial manufacturing,
including upstream and downstream bioprocess
development, scale up and current best practices.
• Advancements in gene therapy analytics and
incorporating in-process analytics.
• Ensuring scalability and efficient timelines in
manufacturing while maintaining reasonable cost.
• Reimbursement strategies for effective
commercialization planning.
• Current regulatory considerations and outlook for
the future.
• Current state of the industry and areas for
improvement.
Foreword
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Gene Therapy An Industry Coming of Age
In 2018, Cell Culture Dish worked with a talented
group of authors, all experts in the field of gene
therapy, to create an Ebook that was meant to
serve as a guide for identifying key
considerations in gene therapy manufacturing
and commercialization. The publication was
very well received and earlier this year, we
decided that it was time to publish a new guide
based on the advancements that we had seen
over the past two years. The process of writing
this updated publication provided wonderful
insight as we compared the state of the industry
in 2018 to today. What we found is that
significant strides have been made across
manufacturing and commercialization, however
there is still much to be done to reach gene
therapy’s full potential.
In our previous Ebook, we referred to gene
therapy as the future of medicine and while I
still believe that term is accurate, as we have
plenty more to do, the truth is that the future
is here. We see this in the six gene therapy
products approved in the US and in the
hundreds of gene therapy products in the
clinical pipeline. The enthusiasm for what
this technology can do hasn’t waned; in fact
we have compelling data on how these
approved therapies are helping patients.
In a recent presentation, Alliance for
Regenerative Medicine Gene Sector Overview
July 2020, the patient impact of recently
approved products was shared. The results are
quite impressive. Zolgensma®, approved by the
FDA in May 2019, is a gene therapy treatment
for pediatric patients less than two years of
age with spinal muscular atrophy (SMA). The
Alliance for Regenerative Medicine (ARM)
shared data that 93% of SMA Type 1 patients
treated were alive without permanent
ventilation at 24 months post-treatment.
Luxturna®, a gene therapy treatment for certain
inherited retinal diseases had 93% of patients
who showed an improvement of at least one
light level from baseline. In a recent press
release, Kite shared data on its CAR T therapy
for Mantle Cell Lymphoma, Tecartus™. In a
single-arm, open-label study, 87% of patients
responded to a single infusion of Tecartus,
including 62% of patients achieving a complete
response. These are just a few examples of the
remarkable outcomes that these therapies can
provide to patients.
© 2020 The Cell Culture Dish, Inc.
However, these results did not come easy. The
tremendous potential of gene therapy is the
result of decades of research, implementation
of select best practices from the field of
biologics development, and impressive clinical
results that collectively have led to significant
industry investment and a pathway for
regulatory approval. Gene therapies represent
a new medical paradigm and that impacts
regulatory approval, reimbursement methods,
and manufacturing strategies. While there is
much to be learned from following the example
of almost 40 years of recombinant antibody
manufacturing and commercialization, there
are many differences that need to be addressed.
What makes gene therapies so different?
• They offer the possibility, in many
instances, of an actual cure instead of
chronic treatment.
• Frequently the treatment is a one-time
only treatment.
• Gene therapies can be expensive to
produce and administer which can be a
challenge in terms of reimbursement.
• Gene-modified cell therapies are highly
customized, patient-specific therapies,
which present unique challenges from a
logistics and distribution perspective.
These differences represent what makes gene
therapies so compelling, but also represent the
challenges that the industry faces. In this new
2020 Ebook, Insights on Successful Gene
Therapy Manufacturing and Commercialization,
our aim was to share insights and lessons
learned on how to manufacture and
commercialize gene therapy products. We were
lucky to bring together a talented group of
authors from several different companies, who
all saw the importance of coming together to
create a piece that is educational and identifies
the key challenges, new developments, recent
Gene therapies represent
a new medical paradigm
that impacts regulatory
approval, reimbursement
methods, and
manufacturing strategies.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page i
Foreword
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
© 2020 The Cell Culture Dish, Inc.
successes, and areas for improvement. Our goal
was to address some key areas important to the
successful commercialization of gene therapies.
This included discussion about gene therapy
manufacturing, the latest in manufacturing
methods, analytical analysis, key regulatory
considerations and reimbursement strategies.
The key areas we identified include:
• Planning for clinical and commercial
manufacturing, including upstream and
downstream bioprocess development,
scale up and current best practices.
• Advancements in gene therapy analytics
and incorporating in-process analytics.
• Ensuring scalability and efficient timelines
in manufacturing while maintaining
reasonable cost.
• Reimbursement strategies for effective
commercialization planning.
• Regulatory considerations and outlook for
the future.
• Current state of the industry and areas
for improvement.
The industry faces some formidable
challenges ahead, but there are enabling
technologies being developed, excellent
resources and experts available, and
importantly, a regulatory and manufacturing
pathway that has been paved for other
companies to follow. In this Ebook, with the
help of experts sharing their perspectives on
various topics, we examine the current state
of the industry and how we can continue to
advance these life-changing medicines for
the benefit of patients. We sincerely hope
this serves as a valuable resource for those
wishing to learn more about gene therapy, its
manufacture, and commercialization.
Brandy Sargent
Editor-in-chief
Cell Culture Dish & Downstream Column
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page ii
Author Bios
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Jane F. Barlow, MD, MBA, MPH
Tony Hitchcock
Chief Clinical Officer
Real Endpoints
Technical Director
Cobra Biologics
Senior Advisor
MIT Center for Biomedical Innovation
Dr. Jane Barlow is an independent
board director and strategic healthcare
executive consultant with over 25
years of leadership experience. She is Chief Clinical Officer of
Real Endpoints, a market access company, serves as Senior
Advisor to the MIT Center for Biomedical Innovation project
on Financing of Curative Therapies and is board director of
Momenta Pharmaceuticals.
Dr. Barlow previously held senior leadership positions at
CVS Health, Medco Health Solutions and IBM. Her diverse
background also includes positions with the U.S. military,
federal government, industry and private practice.
She attended medical school at Creighton University and
completed her residency training in occupational medicine
at Johns Hopkins University. She holds masters’ degrees in
business administration and public health and is a Certified
Physician Executive with a certificate in Health Information
Technology from the American College of Physician
Executives. Dr. Barlow is board-certified in occupational
medicine and is a fellow of both the American College of
Occupational and Environmental Medicine and the American
College of Preventive Medicine.
Tracy L. TreDenick-Fricke
Head of Quality & Regulatory
BioTechLogic, Inc.
Tracy TreDenick-Fricke is one of the
founding partners of BioTechLogic,
formed in 2004. She is the Head of
Regulatory and Quality and has over
20 years experience in Pharmaceutical
Quality, Manufacturing and Regulatory. Most recently she has
supported the development of 7 Breakthrough therapy
products including 4 gene and 3 cell therapies, including one
that is approved. This responsibility included development of
combination product CMC strategies, preparation of CMC
sections for IND and BLA submissions, as well as development
of strategies related to vendor qualification, risk management
and cross-contamination controls. This experience enabled a
clear understand of the expectations for the development
and commercialization of Breakthrough therapy products.
Prior to joining BioTechLogic, Ms. TreDenick directed the
validation and pre-approval readiness programs for
Biopharmaceutical products within Pfizer (formerly
Pharmacia Corporation), including the process validation,
registration and commercialization of Somavert®, a protein
product. Ms. TreDenick received her B.A. in Biology/Pre-Med
from Indiana Wesleyan University. She also completed
graduate study courses in Business Management at Keller
Graduate School of Management.
Clive Glover, PhD
Director, Gene Therapy Strategy
Pall Corporation
Dr. Clive Glover is Director of Gene
Therapy Strategy at Pall Corporation
part of Danaher. Clive has wide
ranging
experience
developing
manufacturing tools for the cell and
gene therapy space including stem cells, T cell therapies and
viral vector production He has held business development,
marketing and innovation positions at STEMCELL
Technologies, GE Healthcare Lifesciences (now Cytiva) and
Pall Corporation. He obtained his PhD in Genetics from the
University of British Columbia.
© 2020 The Cell Culture Dish, Inc.
Tony Hitchcock has over 35 years of
experience
in
the
large-scale
manufacture of biopharmaceuticals.
As a founding staff member of Cobra,
Mr. Hitchcock has been responsible for
the development of much of Cobra’s manufacturing
technologies in the field of DNA and virus production. He has
held a number of senior roles, managing both manufacturing
and development functions within the company, working on
over 40 programmes for the development of novel
therapeutic products including protein, recombinant virus,
bacteriophage and plasmid DNA products.
His current role is based in the commercial group and
supports external collaboration and outreach activities.
Debbie King
Scientific Technical Writer
Debbie King is a freelance technical
writer and a frequent contributor to
The Cell Culture Dish, Inc. specializing
in editorial content in the cell culture
and gene therapy space. Her writing
style is technical, yet approachable
and engaging. She currently works with many
biotechnology and pharma clients to provide her writing
expertise. Her extensive cell culture experience from her
previous positions at STEMCELL Technologies Inc in QC
and product development has been an asset to her as a
technical writer. She is proud to have been an integral
member of the process development team to commercialize
the mTeSR/TeSR hPSC media.
Mani Krishnan
Vice President & General Manager
CE & BioPharma Business Unit
SCIEX
Mani Krishnan is the Vice President &
General Manager of the CE &
BioPharma Business Unit at SCIEX.
For over 25 years, Mr. Krishnan has
worked with biopharmaceutical developers, helping to
innovate technologies used in the manufacture, purification
and analysis of proteins, viral vectors and pDNA. His team’s
current focus is on developing rapid at-line or near-line
analytical methods to enable our customers to optimize
viral-vector upstream and downstream purification
processes, and demonstrate that the process is in control,
which ultimately contributes to accelerating the successful
release of these novel and effective therapies.
Prior to joining SCIEX, Mr. Krishnan was Vice President,
Biotech Technical Services and Scientific Affairs at Pall
Corporation. He also spent more than 20 years at Millipore in
both technical and leadership roles. Mr. Krishnan holds a
Bachelor of Technology in Chemical Engineering from the
Indian Institute of Technology, Madras, India and a Master of
Science in Chemical Engineering, from the University of
Calgary in Calgary, AB, Canada.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page iii
Author Bios
Foreword
Author Bios
Introduction
Regulatory
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Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Rachel Legmann PhD
Mark Schofield PhD
Director, Technical Consultancy
- Gene Therapy and Viral Vectors
Pall Corporation
Senior R&D Manager
Pall Corporation
Dr. Rachel Legmann is partnering with
sales, marketing and Pall’s technology
teams to build company prominence as
a preferred partner for the development
and manufacturing of virus based therapeutics with a focus on
Gene Therapy. In addition to supporting global customers and
building high level networks, Dr. Legmann is supporting various
internal cross-functional activities including due diligence and
new product development.
Dr. Legmann has more than 20 years of experience in the
field of scalable biologics and gene therapy manufacturing
of therapeutic products, viral vector and proteins for gene
therapy and biologics. She completed her Ph.D. in Food
Engineering and Biotechnology at the Technion-Israel
Institute of Technology, Israel. Dr. Legmann joined Pall in
2014 and currently serves as a process development services
senior lab manager in charge of upstream and downstream
process development, scale-up, and manufacturing support
activities for the gene therapy and biologics markets. Prior
to joining Pall, Dr. Legmann held several scientific and
leadership roles at Microbiology & Molecular Genetics
department at Harvard Medical School, SBH Sciences,
Seahorse Biosciences, and Goodwin Biotechnology
Dr. Steve Pettit, PhD
Scientific Technical Writer
Dr. Steve Pettit is a scientific technical
writer and frequent contributor to the
Cell Culture Dish. Steve specializes in
editorial
content
on
viral
manufacturing
and
upstream
monoclonal antibody bioprocessing.
He received his Ph.D in Biochemistry/Virology from the
University of Alabama at Birmingham and continued to
work for 15 years in virology at the University of North
Carolina at Chapel Hill. Dr. Pettit also served for 10 years as
the Director of the cell culture program at InVitria.
Brandy Sargent
Editor in-chief
Cell Culture Dish & Downstream
Column
Brandy Sargent is the Editor in-chief
and frequent author of The Cell
Culture Dish and The Downstream
Column, She has worked in the
biotechnology industry for over twenty years, first in
corporate communications and public relations, then in
technical sales and marketing, and most recently as a writer
and publisher. She strives to introduce topics that are
interesting, thought provoking, and possible starting points
for discussion by the biomanufacturing community. She has
been fascinated by the different applications of
biotechnology since she first started working in the industry
and continues to be fascinated as the industry evolves.
© 2020 The Cell Culture Dish, Inc.
Dr. Mark Schofield (Senior R&D
Manager) holds a Ph.D. from the
University of Dundee. He has 15 years’
experience in R&D and new product
development for biopharma related
industries. Currently leads the chromatography applications
team at Pall developing continuous processing solutions.
Edita Botonjic-Sehic, PhD
Director, Global Analytics,
Biotechnology, Analytics & Controls
Pall Corporation
Dr. Edita Botonjic-Sehic, a graduate of
Assumption College with BA degrees
in chemistry and mathematics and
the University of Rhode Island with a
PhD in the field of Analytical Chemistry. Dr. Botonjic-Sehic
joined Pall Corporation in 2016 and is currently Director,
Global Analytics, Biotechnology. In her current position, she
is responsible for conceptualization and implementation of
process analytical technology (PAT) in continuous
bioprocessing. With a PhD program concentrating in
spectroscopy and chemometrics and multiple years in PAT,
Dr. Botonjic-Sehic brings a highly refined experience to the
table. This experience includes over 16 years of research in
spectroscopy and chemometrics for PAT and the
development and utilization of advanced analytical
techniques in pharmaceutical, biotechnolgoy and homeland
security arenas.
Don Startt
Director of Upstream Process
Development
REGENXBIO
Don Startt leads the upstream
process development group for
REGENXBIO. His team is responsible
for the development, optimization,
and scale-up of AAV gene therapy manufacturing processes.
Prior to his current position, he has held roles at REGENXBIO,
GSK and Human Genome Sciences in various CMC-related
positions including Manufacturing Sciences, Project
Management, Quality, Operations and Facility Design
working on biotechnology-based processes and products at
all lifecycle stages.
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Introduction
Foreword
Author Bios
Introduction
Regulatory
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Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Insights on Successful Gene Therapy
Manufacturing and Commercialization
By: Brandy Sargent, Jane Barlow, Clive Glover, Tony Hitchcock
Industry Outlook
The first approved gene therapy study was
conducted by the National Institutes of Health
(NIH) in 1989 and provided evidence for the
first time that human cells could be genetically
modified and returned to the patient without
harm. Since then, the industry has grown
significantly with 22 gene and gene-modified
cell therapies approved by regulatory bodies
from various countries as of August 2019.1 As
of October 2020, six gene and gene-modified
cell therapies have been approved for use in
the United States, including the latest approval
in July 2020 for the gene therapy Tecartus™.
Clinical trials have continued to grow with 352
gene therapy and 452 gene-modified and
cell-based immunotherapies in clinical trials
globally at the end of last year.2 While the
majority of current trials are still in Phase I
or Phase II ~91% for gene therapy and ~97%
for
gene-modified
and
cell-based
immunotherapies, there are nearly 50 trials
globally that are in Phase III. This suggests
there could very well be dozens of gene
therapy product approvals in the coming
years. In fact, the FDA expects to see a
doubling of new gene therapy applications
each year and Scott Gottlieb, the former FDA
commissioner, predicted that by 2025, the US
would be approving between 10 and 20 gene
therapies each year.3
Supporting this clinical and commercial
progress has been substantial investment and
company growth. Gene and gene-modified cell
therapy had its second highest year of
investment with $7.6 billion raised in 2019 and
there are 496 gene therapy companies working
globally.2 However, this success has not been
without its challenges. As we cover in this
Ebook, gene therapies stand to revolutionize
the current healthcare paradigm from patient
treatment to drug supply logistics and the best
way to execute on the promise of these
therapies is still evolving.
How Does Gene Therapy Work?
Gene therapy is the use of a gene-modifying
technology to repair, replace or correct
damage in the body. For gene therapies to
work, genetic material must be introduced into
© 2020 The Cell Culture Dish, Inc.
cells to treat disease, which is most effectively
achieved using a vector delivery system.
Viruses make good vectors for delivering
genetic material because they have evolved to
do just that—deliver genes by infecting cells.
Viral vectors for gene therapy are modified to
ensure that they don’t cause infectious disease
in the patient. The most commonly used viral
vectors for gene therapy include retrovirus,
adenovirus, adeno-associated virus (AAV), and
lentivirus. While non-viral approaches for
delivering gene therapy are being explored,
viral vectors are still the most popular approach
with two-thirds of the clinical trials to date
delivered via viral vector.4
Clinical trials have
continued to grow with
352 gene therapy and 452
gene modified and cellbased immunotherapies in
clinical trials globally at
the end of last year.
Gene therapies can be delivered by injecting the
vector directly into the patient (in vivo) or the
vector can be delivered to specific cells harvested
from a patient’s blood or tissue collection (ex
vivo). The vector is introduced into the patient’s
cells using a method called transduction. With ex
vivo approaches, the modified cells are
subsequently expanded in cell culture before
they are injected back into the patient.
Non-viral approaches, offer some advantages
including delivery of larger genes, simplified
production, and reduced biosafety concerns.
However, they have been shown to be less
efficient at delivering the genetic material, and
in some instances, the therapeutic benefits
have been short term. Recent improvements in
non-viral methodologies are increasing interest
in this approach.
Indications
Perhaps the most talked about gene therapies
have been the gene-modified cell therapies for
cancer immunotherapy and with good reason.
The American Society of Clinical Oncology’s
(ASCO) Clinical Cancer Advances 2018 report
named adoptive immunotherapy with chimeric
Insights on Successful Gene Therapy Manufacturing and Commercialization
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antigen receptor T cells (CAR T) as the most
important clinical cancer advance of the year.
CAR T therapies are both gene therapies and
immunotherapies, often called gene-modified
cell therapies. In brief, CAR T takes immune
cells, called T cells, from a patient then
genetically engineers them to express chimeric
antigen receptors (CARs) that recognize the
patient's cancer cells. Cells are then infused
back to the patient (this process is called
adoptive cell transfer, or ACT). These engineered
cells circulate in the bloodstream, becoming
“living drugs” that target and kill the antigenexpressing cancer cells. With many early
successes in clinical trials using CAR T, there is
great hope that it can be used to treat a wide
variety of blood and solid tumor cancers.
Not surprisingly, cancer is by far the largest
category of indications being investigated with
65% of the gene therapy clinical trials in this
area. The second most popular category of
indications is inherited monogenetic disease
with 11.1%, followed by infectious diseases (7%)
and cardiovascular diseases (6.9%) rounding
out the top four indications.4
Gene Therapy From the Lab to
Commercialization
As discussed, gene therapies represent a
new paradigm both therapeutically and with
respect to how they are managed within the
larger scope of healthcare. This has wide
ranging impacts on several key areas
including: regulatory, manufacturing, patient
education, reimbursement, and logistics and
distribution. While the process can be
modeled on existing biologics, there is a
great deal that is different about gene
therapies that needs to be addressed.
With clinical success and increased investment
from the market, many gene therapy companies
are looking toward manufacturing and
commercialization of their lead therapies.
However, there are still several challenges that
must be considered when looking at how these
products will be manufactured safely, at
appropriate scale, and with reasonable cost.
Complicating this task is the fact that there is
no “one size fits all” approach as gene therapy
products are complex and can be manufactured
in a variety of ways, using a variety of vectors
and cell lines.
Gene Therapy Manufacturing
Gene therapy manufacturing is a critical part
of whether a gene therapy will be successfully
commercialized or not. It is important for
stakeholders to include process development
© 2020 The Cell Culture Dish, Inc.
and manufacturing programs as early as
possible in development to keep pace with
the rapid movement of gene-based products
through the clinical landscape. There is a key
balance to be struck between investing too
early in manufacturing technology before the
product has been fully characterized and
running the risk that the manufacturing
process doesn’t produce the correct product.
On the opposite end, investing too late means
attempting to scale up a process that may not
meet needs, which could become very
expensive and risky. For the purposes of this
publication we will focus on viral vector
manufacturing.
Manufacturing capacity for the industry is also
of great concern, with viral vectormanufacturing capacity estimated to be 1–2
orders of magnitude lower than what is needed
to support current and future commercial
supply requirements.5 The COVID-19 pandemic
has added to strained capacity as some of the
COVID-19 vaccine development programs also
use viral vectors. Work is being done to reduce
the capacity crunch in the short term by adding
new manufacturing facilities and additional
production capacity. However the most
durable capacity increase would come from
improving manufacturing practices to increase
process productivity. Advancements including
engineering cell lines for increased productivity,
improving plasmids and constructs and
boosting process recovery are all areas
currently being explored. For example, current
process recovery is quite low, less than 20%. If
you increase recovery to 50%, manufacturing
requirements can be cut by two and a half.
Gene therapy companies also need to
understand what regulatory pathways are
available to them based on whether their
product fills an unmet need or addresses a
serious, life-threatening condition. As such,
there are several expedited pathways that may
be available for gene therapy developers. If the
product is designated under an expedited
timeline then this will impact manufacturing
timelines and thus needs to be considered
during process design, scale-up, qualification,
and continuous verification.
Evaluating the Existing Process
Many pre-clinical processes for making viruses
are based on academic protocols where scale
and quality are not of the highest importance. It
is important to determine whether the current
process is scalable to clinical and commercial
manufacturing and whether quality demands
can be met. The Good Manufacturing Practices
(GMP) requirements for raw materials, cell
Insights on Successful Gene Therapy Manufacturing and Commercialization
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substrates and process consumables are much
more demanding and the potential long lead
times for obtaining those critical items can be
challenging. Therefore, evaluating existing
processes will help design process development
for clinical and commercial manufacturing. It
will also help to develop accurate time lines as
academic processes that need more changes
will take longer to move to a clinical/
commercially compatible process.
Scalability
Scale is one of the biggest considerations
when designing the manufacturing process.
This can be a challenge to achieve, particularly
if a product’s approval timeline is accelerated.
Determining scale means identifying how
much material is needed for clinical trials and
ultimately
commercial
manufacturing.
Projected manufacturing scale is calculated by
taking the number of patients to be treated
per year x dose / desired number of batches
per year. So for example, if you plan to treat
200 patients per year at a dose of 1014 viral
genomes (vg) per patient, then one needs to
understand how much a process and a facility
can make in a single batch.
Because product demand and the number of
patients treated will usually increase as a
product progresses through clinical trial
phases and on to commercialization, it is
important that the designed process is scalable
and that critical quality attributes are well
characterized. Therefore, investing in process
development and optimization must be
appropriately timed, particularly if the product
has been granted an accelerated approval
process by regulators.
It is also possible that a gene therapy will not
need to be scaled-up, for instance in rare
monogenic diseases with very low patient
populations. In these cases it may be possible
to meet all product demands without the need
to scale-up. This is why understanding the
scale of a disease indication is so critical in
manufacturing process design.
In-house or Outsourced Manufacturing
It is estimated that greater than 65% of cell and
gene therapy manufacturing is outsourced to
contract manufacturers and that gene therapy
developers may need to wait up to 18 to 24
months
before
accessing
a
Contract
Development and Manufacturing Organization’s
(CDMO) production capacity.6 This makes it
critical for developers to establish a
manufacturing plan early in the development
timeline by securing either in-house or
outsourced manufacturing resources.
© 2020 The Cell Culture Dish, Inc.
The patient population being treated is a key
consideration in deciding between in-house
manufacturing
versus
outsourced
manufacturing to a CDMO. In conventional
medicine, the ratio of the incidence to
prevalence of patients for a given disease is
expected to be high so that capital
expenditures, such as those required to build
a manufacturing facility, can be amortized
over a number of years. In contrast, for
genetic diseases, the ratio of incidence to
prevalence population can be quite low. This
means that the prevalence population gets
treated in the first few years after launch and
then a manufacturing facility is only required
to treat the incidence population which can
be very small for rare and ultra-rare diseases.
This means the capital associated with
building a manufacturing facility for a single
gene therapy has to amortized over just a
couple of years after product launch. This
can make the business case for building your
own facility quite high.
Beyond the expense and dedication of capital
to build manufacturing capacity, time and
resources required of gene therapy companies
and their management must be considered.
According to a recent report on the current and
forecasted skills demand for the cell and gene
therapy industry in the UK, there will be a 112%
increase in jobs from 2019 to 2024. There will be
a 126% increase in bioprocessing roles including
manufacturing, supply chain and logistics,
process development and total quality. This
increase in the need for skilled employees raises
concerns about recruitment or retention
required for industry growth. Companies
involved in the report also expressed concern
that academic courses are not producing
industry ready graduates and post graduates.7
To help meet this need, gene therapy training
centers have been established. For example,
BioCentriq™ is the Cell and Gene Therapy
Development and Manufacturing Center and
Center of Excellence at New Jersey Innovation
Institute (NJII). Biofactory Competence Center
(BCC) based in Fribourg, Switzerland is
another group that provides practical training
on gene therapy manufacturing.
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Companies must be able to establish the design
and scale requirements for the facility, often with
limited certainty as to the scale ultimately
required, the potential launch quantities, and
market penetration to guide planning.
Outsourcing can serve as an effective and
economical bridge until greater certainty is
gained as to product demand. However, for gene
therapy companies who either have difficulty
finding a CDMO partner or securing production
slots in the timeframe they need, building a small,
early phase GMP facility may make sense. There
are also unique partnership agreements that can
be established in lieu of full outsourcing, for
example, a monoplant or condo arrangement.
Ultimately gene therapy companies will have
to weigh several factors before deciding
whether to outsource manufacturing or
manufacture in-house and the appropriate
time to implement each option.
Manufacturing Processes
Viral vector systems are by far the most widely
used methods to delivery therapeutic gene
products because of their infectious nature and
ability to introduce specific genes into a cell. In
addition to creating a safe and efficacious
product, the industry must also look at methods
to increase capacity, reduce cost of goods,
improve therapeutic efficacy and employ
analytics that not only enable process
optimization, but also provide information
critical to regulatory approval. There is progress
on several fronts to improve upon existing
methods and some key areas of focus have
emerged. These will be covered extensively in
the manufacturing articles that follow.
Aseptic Manufacturing Controls
Aseptic processing is stated as “Handling sterile
materials in a controlled environment, in which
the air supply, facility, materials, equipment and
personnel are regulated to control microbial
and particulate contamination to acceptable
levels.”8 While aseptic controls are commonplace
in pharmaceutical manufacturing, aspects of
gene and gene modified cell therapies make
aseptic processes even more critical.
For viral vector production it is important to
understand the level of aseptic control that is
built into the process. Aseptic control is greater
in processes with closed manufacturing and
less in systems with manual, open processes. In
addition, it is important to consider whether
the vector bulk can be sterilized. For instance,
viral clearance is used to some degree in both
AAV and Lentivirus processes, which confers a
level of viral safety.9 The level to which bulk can
be cleared of contaminants and the methods
available for that dictate the area with the
most risk of contamination.
The level of risk is higher with gene-modified
cell therapies because there are typically more
open operations in these processes. These
cultures often include higher risk, animal
derived raw materials as well.8 Furthermore
commonly used viral clearance and sterile
filtration methods are not possible with these
therapies. Thus, a focus on prevention of
contamination is the best approach. This
means good aseptic techniques, transfer to
automated processes and removal of operator
handling as much as possible. In addition,
sterile filtration of raw materials to remove or
inactivate any virus or bacteria prior to use in
culture is important.
Regulatory Considerations
Currently there are six approved gene therapies
in the US and hundreds of products in the
pipeline. The Food and Drug Administration
(FDA), has responded to the challenge by
hiring more reviewers and rapidly developing
the much needed regulatory framework.
Image courtesy of Pall Corporation
© 2020 The Cell Culture Dish, Inc.
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Introduction
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Regulatory
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Manufacturing
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Reimbursement
However, in 2020 there were regulatory
disappointments as well. This included the
Food and Drug Administration (FDA) denying
approval of BioMarin’s hemophilia A gene
therapy. Another setback came when Audentes
Therapeutics reported a third death in its
halted gene therapy clinical trial for a rare
genetic neuromuscular disorder. The three
patients who died were all from the high-dose
AAV vector cohort. There has been concern
around the use of high dose AAV vectors in
treatments. Researchers are investigating
methods for engineering capsids that would
target specific tissues, thus enabling lower
dosing
requirements
(see
Upstream
Manufacturing article for more details). The
takeaway from these setbacks is that there is a
need for improvement in purification and
analytics and that the FDA will likely be
expecting more data as developers and
regulators expand their understanding of gene
therapy characteristics. A more comprehensive
regulatory review will be provided in the
regulatory article that follows.
Supply Chain Security
The traditional biologics supply chain has been
developed and refined over many years. It has
been vetted to ensure that every aspect of the
supply chain has safety measures and
redundancies to mitigate any risks. With gene
therapies and gene-modified cell therapies in
particular, several of these measures are not
possible. For instance, gene modified cell
therapies
are
personalized,
autologous
treatments. This requires not only delivering
the therapy to the patient, but also coordination
and collection of patient material to the
manufacturing location. Furthermore, these
activities must be conducted following very
tight timelines. For instance, Novartis has
stated that their target manufacturing
turnaround time from receipt of patient
material to return of product is only 22 days.
Gilead has stated that the median turnaround
time for Yescarta® is 17 days. Unlike traditional
biologics, these therapies have no redundancy.
They are personalized to the patient; there is
no back up and no margin for error.
Reimbursement Strategies
Gene therapies introduce new uncertainties for
payers. From the payer’s point of view the risk is
high for an expensive one-time therapy. This is
not surprising if you consider how traditional
reimbursement occurs, a payer pays a lower
cost for a therapy over a longer period of time.
In this model, the payer accrues value on their
investment over time. With the gene therapy
model, they pay a large cost upfront and the
value for this investment isn’t accrued until
much later. To complicate matters, if the
subscriber leaves a plan the payer has no
opportunity to reap the value of their investment.
Since gene therapies are a new type of medicine,
there is still uncertainty around performance
durability, the hope is that the treatment will be
a cure or will last for a very long time, but that
has yet to be proven. Smaller plans will have a
more difficult time trying to plan for these rare
conditions and define how many cases their
plan will have to budget for.
The good news is that the vast majority of payers
cover and reimburse gene therapies today. They
are working with gene therapy developers to
create new financing and reimbursement
solutions to ensure sustainability for all plan
members as payers transition payment models
to address the challenges of one-time high cost
gene
therapy
treatments.
For
specific
reimbursement solutions being used by payers
today, please see our article on reimbursement.
© 2020 The Cell Culture Dish, Inc.
Image courtesy of Cobra Biologics
These therapies are also complex to transport,
they require strict temperature controls,
product security and must be constantly
monitored for compliance. According to a
recent article by Cryoport on the cell and gene
therapy supply chain, they state that “A shipping
system should include the following elements:
validation and shock absorption through
advanced internal and external packaging that
exceeds ISTA standards; state-of-the-art dewar
technology and revalidation processes that
ensure cryogenic holding times are actively
monitored and confirmed for every shipment;
active shipment level tracking that is based on
redundant GPS, cellular and WiFi networks
constantly monitored around the globe; and
24/7/365 support personnel who can intervene
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Introduction
Foreword
Author Bios
in emergencies. No exceptions. These are the
standards that must be adopted globally to
ensure patient safety and Cell and Gene Therapy
(CGT) success.”10 Thus the supply chain logistics
must be carefully planned with a great deal of
expertise and no room for error.
Introduction
Provider and Patient Education
and Access
Regulatory
It is important that providers and patients have
access to the product. This access can be in the
form of logistics, for example with cell-based
gene therapies, making sure patients can get to
a site to collect their cells or tissue and provide
infusion of the treatment. It is important that
providers understand proper collection and
infusion methods. Cost is also a part of access,
so securing reimbursement coverage is
important. In addition, patients are increasingly
becoming advocates of their own treatment
and connection with thought leaders and
patient advocacy groups can be invaluable in
helping to educate the community on the
product, as well as working with payers to
ensure coverage. Having a commercial group
involved early in a company’s evolution can be
very beneficial. These professionals can make
commercial projections, identify and interact
with patient advocacy groups, and establish the
Upstream
Manufacturing
Downstream
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Analytics
Reimbursement
framework for a suitable Target Product Profile.
Initial and ongoing stakeholder education is a
critical part of any commercialization plan.
In Closing
It is clear that gene therapies hold a tremendous
amount of promise when it comes to treating
disease, but not unlike any emerging therapy
there are challenges that are still being
overcome. The good news is that recent
product approvals and clinical successes have
secured investment and resources to improve
upon current manufacturing processes and
build the supporting infrastructure required to
see the industry reach its full potential.
Suppliers have done a good job creating
innovative, fit for purpose products to solve
the unique challenges that gene therapy
products present and there is still more
innovation needed. Developers are effectively
leveraging lessons learned from monoclonal
antibody production to speed the development
of new tools to ensure continued success. In
looking back, gene therapies have come a long
way since that first clinical study in 1989 and
looking
forward
gene
therapies
have
tremendous potential to deliver on the
designation - the future of medicine.
References
1.
Ma, C., Wang, Z.-L., Xu, T., Z-Y, H. & Wei, Y.-Q. Approved
gene therapy drugs worldwide: from 1998 to 2009. Biotechnol
Adv 40, 1-13 (2020).
2.
Alliance for Regenerative Medicine. Advancing gene, cell,
& tissue-Based Therapies-ARM annual report & sector year in
review: 2019. Accessed Oct. 11, 2020: Retrieved from
https://www.alliancerm.org/sector-report/2019-annualreport/
3. US FDA (Jan. 15, 2019). Statement from FDA commissioner
Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of
the Center for Biologics Evaluation and Research on new
policies to advance development of safe and effective cell and
gene therapies Accessed Sept. 9, 2020: Retrieved from
https://www.fda.gov/news-events/pressannouncements/statement-fda-commissioner-scottgottlieb-md-and-peter-marks-md-phd-director-centerbiologics
4. Ginn, S. L., Amaya, A. K., Alexander, I. E., Edelstein, M. &
Abedi, M. R. Gene therapy clinical trials worldwide to 2017:
An update. J Gene Med 20, 3015 (2018).
5.
© 2020 The Cell Culture Dish, Inc.
van der Loo, J. C. & Wright, J. F. Progress and challenges
in viral vector manufacturing. Human molecular genetics 25,
R42-52 (2016).
6. Maingi, S. (Mar. 19, 2020). Visualizing the future of contract
development and manufacturing for cell and gene therapies.
Accessed Sep. 20, 2020: Retrieved from https://www.
pharmasalmanac.com/articles/visualizing-the-future-ofcontract-development-and-manufacturing-for-cell-andgene-therapies#:~:text=With%20traditional%20
biologics%2C%20approximately%2035,65%25%20
or%20more%20is%20outsourced.
7.
Catapult. UK cell and gene therapy skills demand report
2019. Accessed Oct. 14, 2020: Retrieved from https://
ct.catapult.org.uk/sites/default/files/publication/
Catapult_01_UK%20Skills%20Demand%20Report%20
2019_published_v2.pdf
8. Parenteral Drug Association (2011). Process simulation for
aseptically filled products PDA task force-2011. Accessed
Oct. 11, 2020: Retrieved from https://store.pda.org/
TableOfContents/TR2211_TOC.pdf
9. Barone, P. W. et al. Viral contamination in biologic
manufacture and implications for emerging therapies. Nat
Biotechnol 38, 563-572 (2020).
10. Wilson, P. (Nov. 21, 2019). Avoiding catastrophe: Addressing
the supply chain risks in cell and gene therapy development.
Accessed Oct. 11, 2020: Retrieved from https://www.
cryoport.com/biological-shipping-blog/supply-chainrisks-cell-gene-therapy
Insights on Successful Gene Therapy Manufacturing and Commercialization
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A Remarkable Year for Gene
Therapies
Maturing regulatory framework and increasing product and process
understanding advances the gene therapy sector.
By Tracy TreDenick-Fricke
Amid a chaotic year in many areas, 2020 has
been an eventful year for gene therapies—a lot
of progress coupled with some setbacks.
Notable events so far this year have included a
new gene therapy approval, finalization of FDA
guidances, FDA striving to manage an
increasing number of applications, a widely
expected approval not granted, and a trial
halted due to patient deaths.
Unexpectedly, BioMarin, the frontrunner in the
hemophilia A gene therapy race, was denied
approval. Its application for Roctavian included
several years of data from a phase 1/2 study and
interim results from an ongoing phase 3 study.
However, the FDA asked for two years of data
from the phase 3 trial to show "substantial
evidence of durable effect" on the bleeding rate
of trial participants.6
FDA Prepares for Increasing
Reviews and Approvals
While undoubtedly painful for BioMarin, this
rejection signals that the sector's knowledge
base and the understanding of an acceptable
gene therapy’s characteristics are advancing.
The bar is being raised, signaling gene therapies'
march toward more widespread adoption.
To date, one gene therapy approval has been
granted in 2020, but there are many products in
the development pipeline.1 In fact, the FDA hired
more than 50 reviewers2 to handle the over 900
new investigational gene or cell therapy drug
applications.3 To put this into perspective, the
FDA expects to see a doubling of new gene
therapy applications every year. In fact, Scott
Gottlieb, the former FDA commissioner, predicted
that by 2025, the US would be approving between
10 and 20 gene therapies each year.4
US Gene Therapy Regulatory
Framework Maturing
In terms of the gene therapy regulatory
framework, 2020 has brought a bounty of
riches—six guidances were finalized, and a new
draft guidance was introduced.
Reflecting the diversity and specificity of gene
therapies, some finalized guidances were quite
specific—notably addressing hemophilia and
retinal disorder. Other final guidance’s confirm
issues
like
long-term
follow-up
after
administration of human gene therapy products
and CMC for gene therapy INDs.5 The sector's
developing regulatory maturity will allow the
industry and regulators alike to progress quicker
and with increased confidence.
High-Profile Rejections Rattle the
Industry While Indicating Progress
There has been a reasonably widespread
perceived leniency, particularly for the review of
one-time
therapies
for
rare
diseases.
© 2020 The Cell Culture Dish, Inc.
Scott Gottlieb, the former
FDA commissioner,
predicted that by 2025, the
US would be approving
between 10 and 20 gene
therapies each year.
High-dose AAV Toxicity Called
into Question
While adeno-associated viral (AAV) vectors
have proven to be extremely effective genetic
material delivery vehicles, toxicity concerns
for high-dosage systemic administration
applications are increasing. Despite the
setbacks, the industry is committed to working
to understand the relationship of high-dose
systemic administration of AAV with deadly
toxicity.7 Ultimately, given the widespread use
of AAV vectors, this increased understanding
will benefit the entire gene therapy sector.
Analytical Methods Advancing
Despite Ongoing Challenges
Perhaps the most nerve-wracking aspect of
gene therapy development is that while
product and process understanding and
regulatory frameworks are maturing, there are
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Author Bios
Introduction
Regulatory
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Manufacturing
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Manufacturing
Analytics
Reimbursement
still plenty of unknowns that can vex analytical
development. This is not a state that is likely to
change; as both the industry and regulators
increase their knowledge and experience
levels, the bar will continue to rise. A few of the
common challenges associated with analytical
development are discussed below.
Method Validation
As for other biotherapeutics, gene therapy
process validation requirements are well defined,
and the process performance qualification (PPQ)
protocol includes predetermined acceptance
criteria. However, it is difficult to set acceptance
criteria due to limited manufacturing data and
the possibility of method variability.
In addition, many methods, such as titer, are
rushed through development and validation
because of the aggressive timelines associated
with a breakthrough therapy. Often, they are not
fully developed to prevent high levels of
variability. Further adding to validation
challenges is that as process and product
knowledge increase, methods are being further
optimized or redeveloped in tandem with PPQ.
However, these optimization efforts sometimes
result in failure of predefined acceptance criteria.
In other cases, due to the known variability of the
method, the acceptance criteria are set so wide
that it raises questions as to the correlation of
the actual specification with clinical experience.
Notable Gene Therapy Method
Validation Challenges
In Vitro Potency is the largest method
development challenge gene therapy developers
face. Per the FDA guidance, the established
method must reflect the product’s relevant
biological properties: the ability to transfer the
gene (infectivity) and the biological effect of the
expressed genetic sequence (potency).8 For
many gene therapies, the mechanism of action is
not fully understood; therefore, the downstream
impact of the expressed gene is not known. This
makes it incredibly difficult to generate a
consistent potency method.
Detection of host cell protein (HCP) impurities
can be a stumbling block largely because many
companies are not developing in-house
methods. Rather, they are using commercially
available kits that are eventually discontinued
due to kit depletion. As a result, the next
generation kit may result in major changes to
the detectability and/or sensitivity of the
results. Also, as they evolve, HCP kits’ sensitivity
can increase reflecting higher residual HCP
levels—resetting specifications and additional
method validation can result.
© 2020 The Cell Culture Dish, Inc.
Cell-based assays, such as in vitro potency or
infectivity, always include inherent variability
due to the nature of the cells. To reduce
variability, many companies are increasing the
n for the number of independent assays or
independent samples run under the same
operating conditions to achieve one reportable
result. A n=3 is becoming the standard to
decrease the variability.
The empty to full capsid ratio has been yet
another consistent challenge. Some companies
initially rely on two highly variable methods,
vector genome (vg) titer and capsid titer, to
calculate the empty to full capsid ratio. Due to
the inherent variability in each method, the
variability in the final reportable result is
compounded. For example, if the determined
vg titer is at the lower end of the specification
and the capsid titer is at the higher end, the
resulting ratio will demonstrate a lower full
capsid ratio. As a result of the variability in
historical data, many teams are developing new
methods such as analytical ultracentrifugation
(AUC) to determine the empty to full capsid
ratio later in development or even during PPQ.
Analyst to analyst variability due to operator
error must be sharply reduced to deliver needed
consistency. Avoiding manual manipulation
wherever possible is one important measure.
For example, electronic cell counters generating
consistent results should be used to determine
cell concentration rather than manual cell
counting. Other sources of analyst to analyst
variability include inconsistent or insufficient
training and too few analysts.
FDA & Gene Therapy Process
Validation
Process validation studies are typically not
required for early-stage manufacturing;9
therefore, most IND submissions will not
include them. Instead, the FDA asks gene
therapy developers to use early-stage
manufacturing experiences to evaluate the
need for process improvements and to support
process validation studies in the future. This
said, too many changes cannot be made to the
initial process, as initial processes generated
the material used in the Phase 1 trial to obtain
breakthrough therapy designation.
As challenging as it is, effective validation
requires a thorough understanding of the
product’s critical quality attributes (CQAs).
Additionally, developers need to determine the
critical process parameters (CPPs) that will
impact the CQAs—a risk-based approach must
be employed to identify the most meaningful
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Regulatory
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Author Bios
process control strategy. In addition to
gaining a thorough understanding of as many
process parameters as possible, a risk-based
approach must recognize processes for which
understanding and control are lacking.
Critical Quality Attributes (CQA)
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
To what degree must CQAs and mechanisms
of action be understood in order to project
likely clinical outcomes? What levels of
understanding do developers and regulators
need to gain confidence in the product?
These are not easy questions to answer.
Although gene therapy development has
advanced markedly in recent years, our
understanding of structural-function is still
evolving. In short, there are still gaps in our
knowledge as to why a gene therapy works or
does not work. Or more specifically, why one
batch has a higher titer and/or has more or
fewer empty, partial, or full capsids than the
next batch. Despite the observation of promising
clinical outcomes, the causes of process
variability are not always well understood.
titer, infectivity, HCP, residual DNA, aggregates,
etc. Like all product attributes, product
characterization must continue to become more
robust as product development advances.
Conclusion
The gene therapy sector suffered some setbacks
in 2020 including the FDA denying a widely
anticipated approval and three deaths in a trial
using high-dose AAV vectors. These incidents
were humbling reminders that the industry and
regulators alike have much work ahead.
However, with now six approved gene therapies
in the US and hundreds of products in the
pipeline, the gene therapy sector is making
tremendous strides. Additionally, the muchneeded regulatory framework is rapidly
developing. Ultimately, gene therapy developers
will continue to make progress in product
consistency and understanding so that the lifeenhancing and life-saving promises of gene
therapies can be realized.
To achieve a sufficient level of understanding
and process control, developers must develop
assays specific to their gene therapy
platforms including vector genome titer, capsid
References
1.
US FDA (July 24, 2020). Approved cellular and gene
therapy products. Accessed Oct. 9, 2020: Retrieved
from www.fda.gov/vaccines-blood-biologics/cellulargene-therapy-products/approved-cellular-and-genetherapy-products
6. Taylor, N. (Aug. 20, 2020). FDA rejection of BioMarin
gene therapy raises durability questions. Accessed Sep.
9, 2020: Retrieved from www.biopharma-reporter.com/
Article/2020/08/20/FDA-rejection-of-BioMarin-genetherapy-raises-durability-questions
2.
Weintraub, A. (Dec. 19, 2019). Pharma's gene and cell
therapy ambitions will kick into high gear in 2020—
despite some major hurdles. Accessed Oct 9, 2020:
Retrieved from www.fiercepharma.com/pharma/
gene-and-cell-therapy-r-d-will-kick-into-high-gear2020-despite-hurdles-experts-say
7.
3. US FDA (Jan. 20, 2020). FDA continues strong support
of innovation in development of gene therapy
products-News release. Accessed Sept. 9, 2020:
Retrieved from www.fda.gov/news-events/pressannouncements/fda-continues-strong-supportinnovation-development-gene-therapy-products
4. US FDA (Jan. 15, 2019). Statement from FDA
commissioner Scott Gottlieb, M.D. and Peter Marks,
M.D., Ph.D., Director of the Center for Biologics
Evaluation and Research on new policies to advance
development of safe and effective cell and gene
therapies Accessed Sept. 9, 2020: Retrieved from www.
fda.gov/news-events/press-announcements/statementfda-commissioner-scott-gottlieb-md-and-peter-marksmd-phd-director-center-biologics
5.
© 2020 The Cell Culture Dish, Inc.
raps.org (Jan. 29, 2020). FDA finalizes 6 Gene therapy
guidances, unveils a new draft. Accessed Sep. 9, 2020:
Retrieved from www.raps.org/news-and-articles/
news-articles/2020/1/fda-finalizes-6-gene-therapyguidances-unveils-a
Paulk, N. K. (Sep. 2020). Gene therapy: It’s time to talk
about high-dose AAV. Accessed Oct. 9, 2020: Retrieved
from www.genengnews.com/commentary/genetherapy-its-time-to-talk-about-high-dose-aav/
8. US FDA (Jan. 2011). Potency tests for cellular and gene
therapy products final guidance for industry: January
2011. Accessed Sep. 9, 2020: Retrieved from www.fda.
gov/regulatory-information/search-fda-guidancedocuments/potency-tests-cellular-and-gene-therapyproducts
9. Preti, R. (Oct. 2016). Process improvement and
accelerated CMC development workshop, Cell and
Gene Meeting on the Mesa (video recording). Accessed
Sep. 9, 2020: Retrieved from www.youtube.com/
watch?v=t05tD1UizHk
BioTechLogic, Inc. provides regulatory and manufacturing
consulting services with strategic and hands-on experience
to help clients bring their products to market quickly and
successfully by augmenting and optimizing their
organization's resources.
We are a team of professionals with expertise in Project
Management, Process Development, Manufacturing,
Process Validation, Analytical Testing/Quality Control,
Quality Assurance, Regulatory Submissions, Supply Chain
Management, and Combination Products.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 9
Upstream Manufacturing
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Author Bios
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Manufacturing
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Manufacturing
Analytics
Reimbursement
Upstream Manufacturing of Gene
Therapy Viral Vectors
By: Steve Pettit, Clive Glover, Tony Hitchcock, Rachel Legmann, Don Smartt
Overview
The majority of gene therapy applications in
development utilize viral vectors to carry the
therapeutic gene into the target cells. Cells
may be genetically modified either in vivo or ex
vivo. In ex vivo applications, cells are modified in
culture, which also allows for cell expansion
and analytical characterization prior to reinfusion of the treated cells. This process has
an advantage in that cells can be examined
pre-treatment and post-treatment for viability,
density, expression level, etc. The ex vivo
process can sometimes result in more efficient
transduction due to the greater control of the
conditions. Alternatively, cellular modification
by vectors can also be performed in vivodirectly in the subject. This approach avoids
the need for cellular implantation.
There are several possible viral vectors systems
available and the decision of which to use
depends on many factors such as tissue tropism,
desire for integrative or non-integrative
modification, in vivo or ex vivo process, prior
immune exposure, whether the target cell is
replicating or non-replicating, safety, and
others. Transduction at a high efficiency and
robust levels of transgene expression are
desired outcomes in viral vector applications.
A safety concern with viral vectors is generation
of wild type infectious virus from vector
components. For this reason, viral vectors are
typically manufactured from 2, 3 or even several
separate expressible units in order to
significantly reduce the possibility of forming
wild type virus via recombination. The units are
typically separate plasmids introduced to
producer cells either by transfection or through
“helper” transducing viruses.
The vector units can be further engineered for
safety by the placement of mutations or
deletions, which would disable the function of
the wild type virus should it be formed via a
recombination event. For example, newer HIV
lentiviral (LV) vectors delete genes for
virulence factors tat, vpr, vpu, nef and/or have
gag and pol on separate plasmids from rev and
env1 and/or contain other mutations such as
deletions in the 3’ LTR and some contain only a
small percentage of complete HIV genome.2,3
Multi-plasmid transfection can also be favored
© 2020 The Cell Culture Dish, Inc.
for convenience, or by necessity in order to
avoid toxicity of a vector component to
producer cells (Figure 1).
pTransgene Vector
pHelper Vectors
Transfection
Replication
Transcription
& Translation
293 Cell
AAV ssDNA
AAV Extraction Solution
AAV2 Particles
Figure 1. Schematic of the production of AAV vector via
transfection.
AAV vector components and therapeutic gene are transfected,
usually as separate expression cassettes from different
plasmids. Expression cassettes are expressed within the cell
resulting in viral proteins and a genomic ssDNA containing the
expression cassette for the therapeutic gene(s). In the AAV
system, viral particles containing the transgene assemble in
the cytoplasm. Particles are released to the media via cellular
lysis prior to further purification and characterization.
Major viral vectors in use
Retroviral (primarily gamma-retroviral) and
adenoviral vectors were among the first cell
modifying vectors and have generated a 30-year
history to date.4 More recently, vectors derived
from adeno-associated virus (AAV) and lentiviral
(LV) vectors have advanced in development
primarily due to enhanced safety and improved
target tissue expression profiles. Other vectors
such as herpes simplex virus and pox/vaccinia
vectors, are entering the field and show promise
in some applications such as oncolytic vaccines.
AAV is the most commonly used vector for in vivo
genetic modification, and different serotypes
(naturally occurring or recombinant) can be
used to target different tissues within the body.5
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AAV has been thought to be well tolerated, and
results in a lower inflammatory response6,7, and
generally thought to have a good safety profile.
Moreover, unlike other viral vectors such as
lentivirus that integrate into the host cell genome,
AAV primarily remains episomal4,8 and has long
term expression of the transgene.9
One possible limitation to the use of AAV is its
lower transgene packaging capacity relative to
other viruses at ~ 4.7 Kb (Table 1).6 This capacity
limitation is not a major problem with many
diseases being targeted and can sometimes be
circumvented by minimizing promoter and ITR
sequences or by the use of mini-genes of
expression targets. Recent vector engineering
efforts have demonstrated that AAV capacity
can be increased to ~ 10 Kb by the use of split
vectors to exploit the fact that AAV genomes
form
head-to–tail
concatemers
through
homologous recombination within cells.9,10 Thus,
by splitting the transgene between two vectors,
the capacity of AAV can be roughly doubledwith current tradeoffs of reduced transgene
expression and possible expression of unwanted
products from reverse concatemerization.9,10
However, this work is still in experimental form.
Another challenge is that AAV can have a
propensity to package any DNA in the vicinity,
which may include host cell DNA and plasmid
DNA.11,12 Other challenges can include lower
transduction in humans than animal models,
and the current methods of purification are
somewhat immature. Nevertheless, AAV vectors
have proven their potential and there is an
ongoing effort to improve these vectors further
through engineering.13
There have been some recent setbacks to the
AAV platform. In 2020, three patients died in
the high dose arm of the clinical trial of Audentes
Therapeutics’s AT132, an AAV8 vector that
delivers a copy of the myotubularin 1 gene.14,15 A
few other patients in the higher dose arm
receiving the higher dose also experienced
adverse events. This event indicates that a
clearer understanding is needed of the dosing
limitations of an AAV vector. Nevertheless,
despite these limitations and recent setbacks,
AAV vectors show great promise for treatment
of a wide variety of diseases, and there are a
several AAV based therapies that have been
approved worldwide.9,13 As a growing platform
for gene therapy, AAV based therapeutics have
a promising future.
Lentiviral vectors have gained much use
recently, particularly in ex vivo applications, and
are advantageous due to their ability to
transduce both dividing and non-dividing cells.
Current LV vector are primarily based on HIV-1,
a virus that have been intensively characterized
over the past years.
LV vectors have been key in the advancement of
chimeric antigen receptor (CAR) T cell
therapeutics which involve the transduction of
primarily CD19 T cells, and CAR T therapies
generated by retroviral vectors have been
approved world wide as of 2019.13 LV transduction
of slowly dividing CD34+ human stem cells has
been applied to several genetic diseases, including
ß-thalassemia16, X-linked adrenoleukodystrophy17,
and metachromatic leukodystrophy.18
Another advantage to using LV vectors is that
there are fewer challenges associated with
insertional oncogenesis compared to gamma
retroviral vectors (example MuLV), like those
observed in the early clinical progress of gene
therapies.1,19 LV vectors have advanced through 4
generations as of 2020 so that newer vectors
contain as little as 4.8% sequence similarity to
wild type HIV-120, which further advances their
safety profile (for review see3).
The table below summarizes the physical
properties of the four major viruses that are
used for gene therapy:
Parameter
Retrovirus
Lentivirus
AAV
Adenovirus
Coat
Enveloped
Enveloped
Non-Enveloped
Non-Enveloped
Packaging capacity (Kb)
8
8
~4.7(a)
7.5
Tropism/infection
Dividing cells
Broad
Broad excluding
hematopoietic stem cells
Broad
Inflammatory potential
Reduced
Reduced
Reduced
High
Host genome interaction
Integrating
Integrating
Integrating/ non-integrating
Non-integrating
Transgene expression
Long-lasting
Long-lasting
Potentially long-lasting
Transient or longlasting depending on
immunogenicity
Table 1. Overview of common viruses used for generating gene therapy viral vectors
a) Recent AAV engineering efforts have demonstrated that AAV capacity can be doubled via the use of split vectors.9,10
© 2020 The Cell Culture Dish, Inc.
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Overview of viral vector
manufacturing
The manufacture of viral vectors can be a
complex process that may require several
manufacturing phases or platforms. Initially, the
materials
needed
to
manufacture
the
therapeutic viral vector must be generated.
These materials differ between vector types
and design, but may include plasmids encoding
helper-virus functions and the therapeutic gene,
cell lines used to manufacture the vector, and
others (See Figure 2, Left, generation of
plasmids). In some cases, helper transducing
viruses may be substituted for plasmids, which
also need to be generated via cells. There are
established packaging cell lines for some vector
systems, such as gamma-retrovirus, while
others are in development. One advantage to
the use of stable lines for packaging is that they
can reduce or eliminate transfection and/or
transduction steps, simplify the production
process, and lower costs.
The next step in vector manufacturing involves
the generation of viral vector (Figure 2, Middle).
Often cells are transfected with plasmids to
generate the viral vector, which is harvested.
Harvested vector is then concentrated,
purified, titrated, characterized, and stored for
later ex vivo or in vivo use. In the case of AAV
and a few other vectors, viral particles may
accumulate in both in the cytoplasm and the
media. In that case, lysing the cells may
enhance the total yield of vector.
If ex vivo transduction is being used (Figure 2,
Right), target cells are collected and modified
by the viral vector in a clean room. Following
modification,
the
cells
are
harvested,
characterized, and formulated prior to
transplantation. In some processes, transduced
cells may be expanded in cell culture prior to
the infusion into patients.
Generation of viral vectors by
transfection or infection
majority of vectors are currently generated by
transient
production
systems
due
to
convenience, or because of the lack of a stable
producer cell line, or for other reasons.
Manufacturing viral vectors by transfection
offers advantages and disadvantages. It is
flexible and efficient, does not require timeconsuming development of stable cell lines, and
allows for successful vector generation should
any viral component produce cellular toxicity
upon expression.22 Producers that have a need
to get to market quickly may favor manufacture
by transfection because of its faster approach.
Transfection based methods do have
disadvantages due to the requirement for
costly GMP grade plasmids and the possible
requirement of additional purification steps to
remove DNA such as benzonase treatment.
Currently there are many producers that
produce plasmid, however, only a few offer
GMP plasmid at scales required for large
manufacturing. Moreover, transfection of
suspension cells at larger scale can prove tricky
to implement. Transfection can require time to
optimize at any scale, and inconsistences
between production batches is a concern in a
manufacturing setting.
Among
the
transfection
methods,
polyethylenimine (PEI) transfection has
advantages over calcium phosphate. It is
Key Manufacturing Platforms
Viral Vector
Manufacturing
Plasmid
Manufacturing
Expand
Fermentation
Isolate & Enrich
(E. coli)
Transfect
Plasmid
Recovery
Activate and
Tranduce
Clarification
Purification
In general, there are two modes of vector
production. The first is through a transient
production system. Transient production
systems involve either 1) transfection of one or
several plasmids encoding the helper virus
functions or 2) viruses to provide helper
function.21 The second major mode of vector
production uses a stable producer cell line.
Stable producer lines do not require
transfection or transduction to enable vector
packaging, however, they do require substantial
effort to generate. At the present time, the
© 2020 The Cell Culture Dish, Inc.
Cell
Processing
Expand
Concentration
Formulation
Harvest
Purification
Formulation
Sterilization
Figure 2. Example of 3 manufacturing platforms for the
generation of modified cells for ex vivo gene therapy via viral
vectors produced by transfection.
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generally less toxic to cells than other methods
and the use of PEI can eliminate the need for a
media exchange. PEI transfection is also less
dependent on pH, the presence of serum, and
is applicable for both adherent and suspension
cultures.23 Drawbacks, however, include the
large amount of costly plasmid required for
large suspension culture and the absence of an
analytical method to quantify PEI in the
purified vector preparation.24 Lipid-based
transfection reagents are effective but their
cost may make them impractical for use at
production scales.25 Non-chemical methods
such as electroporation show promise,
however, the need to concentrate cells can also
make its use impractical at larger scales.24 Thus,
transfection-based methods, while fast and
efficient, can present significant challenges
which include development time and concerns
about consistency, particularly at larger
manufacturing scales.
Use of baculoviral vectors for
therapy or to produce other
viral vectors
Low toxicity and the inability of baculoviruses
to replicate in mammalian cells make them
interesting candidates for therapeutic gene
delivery.26 Baculovirus-mediated gene delivery
into dividing and non-dividing mammalian
cells has demonstrated therapeutic efficacy in
both ex vivo and in vivo gene therapy studies.27-29
A suitable expression cassette and/or a
pseudotyped envelope such as VSV-G can be
used to transduce various cell types.30,31
Baculoviral vectors can mediate high-level
transient expression of transgenes in many
stem cells32 and can be modified to permit
stable transgene expression.33 The high-level
of transgene expression from baculoviral
vectors is well suited for cancer gene
therapy29,34 and the lack of pre-existing
antibodies to baculovirus in humans is an
advantage for in vivo therapy.
Baculoviral vectors have advantages and
disadvantages. Baculoviral vectors offer an
alternative to transfection for vector
generation and have been used in the
production of vectors such as AAV from insect
cells35 and LV vectors from suspension cells.36
Baculovirus
and
other
infection-based
systems for the generation of vector are
typically easier to work with at large scale.
There are potential drawbacks to the use of
baculoviral systems in that additional
manufacturing steps are required in order to
produce the baculoviral vector and remove it
© 2020 The Cell Culture Dish, Inc.
during downstream processing. Moreover,
some cell culture media formulations can
result in reduced transduction efficiency.27
Also, post-translational modification of
viruses produced in insect cells can sometimes
result in a reduction of potency. Overall, there
is much promise for the use of baculoviral
vectors and their use is growing in gene
therapy vector development.
Stable producer cell lines
Cellular toxicity from the expression of a
required vector component has complicated
the establishment of stable producer cell lines
for many vector systems. Toxicity from LV
components (pro, pol, rev) has thwarted the
development of stable LV producer cell lines. In
the past few years, methods have been
established for generating clinical-grade cell
lines that continuously produce LV vector
however many of these approaches currently
lack desired robustness.37,38
Some stable producer lines utilize inducible
promoters to minimize toxicity, and most
stable MuLV retroviral cell lines are inducible.21
One example of cellular toxicity produced from
a vector component is the pseudotyped
retroviral envelope VSV-G1, which is can be
substituted with an alternate envelope to
improve toxicity.37 Toxicity is one aspect.
Another is the prospect of making infectious
virus without the transgene packaged. That
effectively becomes an impurity that must be
cleared via the purification process.
While vectors produced by transient expression
of vector components continue to dominate
the field, there has been a notable increase in
the past couple of years in the use of stable
production lines so that they now comprise
about 30% of the products entering
manufacturing.39 While the productivity of
producer cells are currently no greater than
transfection based methods39, their use can
simplify manufacturing steps, lessen regulatory
oversight, and lower cost. Thus, there can be
significant advantages in the use of a stable
producer cell line for therapies with large
therapeutic demand or for those requiring
high titers of vector.
Choice of producer cell line
Many factors influence the choice of cell line for
vector generation. A key factor is production
titer. Increased production titers of therapeutic
vector can greatly reduce production scale and
lower costs. For vectors produced by
transfection, transfection efficiency is a major
factor. HEK293, HeLa, and HT1080 cells
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generally have good transfection efficiency and
can also produce high titers of vector.21,40 Among
the many HEK lines, HEK293T cells reportedly
produce increased titer because of the presence
of SV40 large T-antigen.1 More recently “Fast”
HEK293FT cells, which reportedly have shorter
doubling times and greater production have
been developed.41 Thus, many companies are
presenting commercial cell lines with reportedly
favorable properties, although there is always
room for future improvement, such as with cell
line engineering.
Note that the FDA suggests that HEK293T
cells should not be used because of present
technical and safety considerations with the
presence of the SV40 T-antigen42. In addition,
the FDA has undertaken recent studies looking
at the feasibility of the use of T-antigen deleted
HEK293T clones as a substitute for production
of LV and AAV vectors.43
According to current US Food and Drug
Administration (FDA) guidance for human
gene therapy investigational new drug
applications, if cells with a tumorigenic
phenotype, such as HEK293T cells, are used
for viral vector production, residual substrate
DNA must be limited to assure product safety.
In addition to controlling host cell DNA
content; the level of transforming sequences
in clinical products should be tightly controlled
to limit patient exposure. Clinical products
produced in HEK293T cells will require testing
for residual adenovirus E1 and SV40 large
T-antigen sequences.43,44
Scaling of adherent cells for
manufacturing vector
The majority of the cell lines used to generate
viral vectors are naturally adherent, with the
exception of some tumor cell or blood cell lines
which can grow in suspension.21 For adherent
cells, the surface area of the culture device
limits cell expansion. Hence, production scaling
is accomplished either by increasing the
number of identical culture systems units
(flasks, roller bottle, cubes, HYPERStacks®)
(scale out) or using successively larger devices
(scale-up).45,46 In a manufacturing setting
problems arise if production is scaled out since
the number of increasing culture units can
become unmanageable. Thus, scale-up into
even larger units, such as bioreactors, can
become an attractive option for increasing
production scale.
Vector manufacturing has used “flatware” unit
production systems of adherent based processes
for some small-scale needs or for smaller clinical
© 2020 The Cell Culture Dish, Inc.
samples. These have mostly involved multilayer
cell factories (CF) or HYPERFlasks®/Stacks (HY)
(Corning). Cell factories have been used for the
production of preclinical and clinical vector
batches of ß-retroviral, lentiviral47 or AAV
vectors.48 CFs can provide up to 2.5 m2 per unit,
which is an increase over a roller bottle with area
up to 0.17 m2.
Roller bottle expansion can be assisted by
automation, which is commonly used in the
vaccine industry; however, this requires
significant
utilization
of
specialized
manufacturing facilities that limits feasibility.
Cell factories are more difficult to scale than
roller bottles and limitations in gas exchange in
some conditions have been shown to decrease
viral titer for AAV production.49 Some cell
factories systems are available in semi-closed
loops, providing some advantage.50
HYPERFlasks and HYPERStacks have a
membrane for gas exchange which has been
shown to increase LV production.51 These are
more flexible than cell factories in media/
surface area volumes and they offer areas of up
to 1.8 m2 /unit. However, these also require the
addition of more units for a significant increase
in production scale. The Corning CellCube®
system can scale-up to 34 m2. However, the
CellCube system is only partly single-use.21
The use of bioreactors has advantages to
increase production scale in that they
uncomplicate increasing production scale by the
use of fewer, larger vessels. Other advantages
include easier monitoring and control of
processes, reduced record keeping, fewer unit
operations, lower contamination risks, reduced
facility space, and lower operation costs.21,52,53
The improved control of culture condition in
bioreactors may also result in improved
productivity. Furthermore, many bioreactor
systems can also be used in a perfusion mode
which can be attractive for some vector systems
such retroviral and lentiviral.21
Hollow fiber bioreactors, such as the Quantum®
(Terumo BCT) utilize hollow fibers to provide
an adherent surface for cells54, thus providing
increased production scale in a small
footprint.55 Fixed-bed bioreactor technology
has been widely accepted in the industry. For
example, the iCELLis® fixed-bed bioreactor
(Pall Biotech) is used in the production of
Zolgensma®, an AAV-based viral vector gene
therapy used in the treatment of spinal
muscular atrophy. The iCELLis bioreactor
family currently consists of two models. The
iCELLis Nano bioreactor is a benchtop unit
used for process development and clinical
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production of virus at small scale. When larger
scales are required, processes developed in the
iCELLis Nano bioreactor can be easily scaledup to the iCELLis 500+ bioreactor (Figure 3),
the manufacturing-scale model.
The fixed-bed of the iCELLis bioreactor
consists of polyethylene terephthalate (PET)
macrocarriers (13.9 cm2 each) fixed inside a
housing where media flows from the bottom
to the top. The height of the bed can be
adjusted as well as the compaction density
of carrier. This enables a wide range of
scalability options up to 500 m2 (Table 2). At
maximum scale the iCELLis offers area that is
roughly equivalent to 5900 roller bottles,
780 10-layer stacks, or 280 HYPERStacks
(Table 2). In addition, the iCELLis bioreactor
enables high cell density growth of 300,000
cells/cm2 and produces similar cell-specific
titers compared to adherent culture.56 The
iCELLis ™bioreactor is also provided on an
industrial automation platform, easing the
integration of this technology into existing
manufacturing IT infrastructure.
Pall Biotech recently reported iCELLis
performance and usability, cell distribution,
pH, dissolved oxygen (DO), and metabolite
profiles compared to flatware controls.57 The
tests demonstrated a well-controlled system
with homogeneous cell distribution. Pall
Biotech has also demonstrated seed train
usability through benchtop to industrial scale.
Thus, these studies indicate that the iCELLis
fixed bed systems provide confidence in
moving rapidly from initial vector development
to commercial scale production. These
systems are suitable for the production of cell
and gene therapy viral and vectors as well as
for viruses used in vaccines.
With the cell lines typically used for gene
therapy production, the iCELLis at the 500 m2
scale has roughly the capacity of a 500L-1000L
suspension cell, stirred tank, bioreactor. Thus,
the iCELLis fixed bed reactor is an attractive
Bed Size
Image courtesy of Pall Corporation
Figure 3. The iCELLis 500+ System
enabling technology for therapeutics that
require large or flexible production scale. This
is particularly enabling for producers seeking a
rapid time to market (producing a suspension
cell line can take a year or more time) or for
cell systems that are limited to adherent cells.
The cost benefits of the iCELLis bioreactor
over multi-stack culture systems has been
demonstrated in a modeling study that
estimates cost reduction in a 100L to 1000L
scale out scenario. The study modeled a
typical LV production campaign via a
transfection-based method. The results
estimated the iCELLis would generate a 50%
reduction in cost of goods, a 1.6x to 1.9x
reduction in cost of goods through
optimization (perfusion and plasmid use),
and a 3x increase in throughput with
perfusion. Furthermore, process complexity
was significantly reduced. Thus, the iCELLis
bioreactor produces numerous benefits over
traditional flatware, that include significant
cost savings and a simplified process.
Surface Area/m2
Equivalent Units
Bioreactor
Bed Diameter
(cm)
Bed Height
(cm)
Carrier Compaction
Low-High
850-cm
Roller Bottles
10-Layer Cell
Stacks
HYPERStack
36
iCELLis Nano
11
2
0.53 - 0.8
6.2-9.4
0.04-0.08
0.3-0.4
iCELLis Nano
11
4
1.06 - 1.6
12.4-18.8
0.2-0.5
0.6-0.9
iCELLis Nano
11
10
2.65 - 4.0
31.8-47.1
10-25
1.5-2.2
iCELLis 500+/100
86
2
66 - 100
776-1116
104-157
37-56
iCELLis 500+/200
86
4
133 - 200
1565-2353
209-314
74-111
iCELLis 500+/500
86
10
333 - 500
3918-5882
524-786
185-278
2
Table 2. Scalability options for the iCELLis Nano and 500+ bioreactors.
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Rocking bioreactors with adherent cell/
microcarrier or single cell suspension culture
are a potential option to produce smaller
quantities of vector for research or clinical
studies. Currently, their capacity is limited to
500L. Rocking bioreactors are disposable
and can also be used to provide cell seed for
larger bioreactors. Microcarriers can be
effective for some cell types; however, their
use requires much optimization to provide
consistency as far as microcarrier type and
surface, and of cell attachment protocol.
Moreover, the ability to transfect/transduce
cells growing on a microcarrier needs to be
understood early in development.
Stirred tank bioreactors for
manufacture of vector
While hollow fiber and fixed-bed bioreactor
systems offer significant increases in scale,
the scale is currently limited compared to
stirred tank bioreactors that can extend into
the thousands of liters. Virus production in
the vaccine industry has been successfully
scaled up to more than 2000 liters using
microcarriers in stirred tank bioreactors59
using adherent Vero or MRC5 cell lines. A
2000L bioreactor operating at 10 cm2/mL
microcarrier density can theoretically offer
2,000 m2 of surface area, if the complex
technical optimizations involving the use of
microcarriers can be perfectly optimized
and developed.
Stirred-tank bioreactors are by far the most
prevalent bioreactor used for the commercial
manufacturing of mAbs and recombinant
proteins. As a result, the technology is very
well characterized and both industry and
regulatory authorities are very familiar with
their operation. They are the most efficient
means of scaling up to large volumes. While
these bioreactors can be used to grow
adherent cells with the use of microcarriers,
they are best adapted to the growth of
suspension cells. HEK293 mammalian cells as
well as SF9 insect cells can be adapted to
suspension culture.
Stirred tank bioreactors are offered in a variety
of size and configuration options. For example,
the Allegro™ STR bioreactor (Pall Biotech) is a
single-use bioreactor that is available in 50,
200, 500, 1000 and 2000 L scales (Figure 4).
In addition, the Allegro STR bioreactors have a
compact footprint due to their cubical design,
allowing for an installed height of less than 3 m
even at the 2000 L scale. BrammerBio has
shown that insect cell culture can be
successfully carried out in the Allegro STR
bioreactors, allowing for cell density up to 7
million cells/mL.60 The wide range of models
offer flexibility of choice in production scale as
well as a convenient path though cell seed
generation through seed train.
One of the important considerations in
selecting bioreactors for up-scaling of
production is performance as well as ease of
use and compatibility through the sizes.
Studies have shown linear scalability from
Allegro STR 50L to 500L bioreactors for the
production of AAV from HEK293 suspension
cells and that there is no significant difference
between the two bioreactors as long as the
same process is performed (Figure 5).58
Moreover, cell doubling time was reduced in
the Allegro STR 50 bioreactor (26 hours)
compared to shake flasks (34 hours). This
would be expected from the controlled
conditions and improved monitoring.
Image courtesy of Pall Corporation
Figure 4. Allegro STR 50, 200, 500, 1000, and 2000 L bioreactors.
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Scaling of suspension cells for
manufacturing vector
A major advantage of single-cell suspension
culture over adherent cells is that they can
easily be scaled up from spinner, to laboratory
scale bioreactor, to production bioreactor
without burdensome cell detachment steps.
The use of animal free media is a desirable
feature for manufacturing due to the
decreased risk of adventitious agents and the
reduced purification burden. Several HEK293
cell lines used for vector production (293T,
293FT) are prone to adaptation to suspension
culture in chemically defined media61 and
suspension HEK293 cell lines pre-adapted to
commercial media are available on the market.
Bauler et al. recently described the creation of
a GMP compatible suspension HEK293T cell
line that doubled LV production compared to
flatware and a 10x increase in transducing
units compared to two commercial systems.62
Published reports have shown that high titers
of LV vector can be achieved using a
suspension adapted helper cell line in smallscale bioreactors.62,63
Current challenges and limitations
of suspension cell culture
It can take some time to adapt a custom cell line
to high growth, up to a year and more, which
can result in a delay of time to market for a
therapeutic. Moreover, there is no guarantee
that the adaptation effort will result in a
suspension cell line with equal or greater cellspecific productivity than the originating
adherent cells. In addition, there are currently
challenges associated with transfection of cells
at scales >200L which can present a limiting
factor to the adoption of suspension culture for
transfection
dependent
processes.24
Furthermore, extensive development time is
usually required to optimize the efficiency of
transfection at larger scale.
Another challenge is that many of the cell lines
currently used in vector production do not
necessarily achieve high single-cell density
growth. For example, HEK293 cells can be
prone to clumping at densities above 2 million
cells/mL in current media formulations.39 Thus,
it is important for the vector developer to
understand
the
full
advantages
and
disadvantages of the use of suspension culture
for their particular vector, cell, and market.
While the use of suspension cell lines currently
remains a smaller fraction of the market, there
has been a notable increase in their adoption in
the past couple of years.39
© 2020 The Cell Culture Dish, Inc.
Figure 5. Yield reproducibility in three STR 50 bioreactor runs
and linear yield scalability from STR 50 to STR500 bioreactor
scale was demonstrated. The Allegro STR bioreactor
performed similar to the other cylindrical bioreactor at the
same scale. Provided the stirred tank bioreactor demonstrates
good AAV yield, then one should take into consideration
additional criteria when selecting a bioreactor, such as ease
of use, height, secured supply and service.
Cost considerations
Vector based gene therapies can be expensive,
and up to $1-2 million/dose. It is to be expected
that production costs will decrease in the
future as new technologies advance in the
emerging vector production industry. Currently,
there are several factors that can influence the
cost of viral based gene therapy manufacturing.
These include production titer, the ability to
up-scale production processes, regulatory
burden, and other factors.
Production titer is a key factor as higher titer
results in a smaller, more cost-effective
production process. High titer production of
vector can lower reagent demand, labor, and
facility requirement, in addition to enabling a
more efficient downstream operation. One
possible route to increase titer is through
control of the cell environment and of
production conditions. In this regard, controlled
bioreactor vessels can present an advantage
over flasks, roller bottles, CFs, or other flatware
in productivity.
In addition, use of bioreactors can simplify
scale-up of a process as previously described.
From a capital expenditure, point of view,
scaling
up
multilayer
stacks
requires
considerable space. Since clean room space is
expensive, this can be a very significant cost. In
contrast, implementation of a bioreactor
system can be done in the same space required
for a modestly size multilayer stack system.
Application of a bioreactor system can
decrease labor and consumables costs 3-fold
and there is considerably less plastic waste
generated with a bioreactor system resulting
in reduced disposal costs. Most bioreactor
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systems come with some degree of automation,
which manifests itself as a 3-fold decrease in
labor, compared to more manual systems like
multilayer stacks. A greater than 50% reduction
in costs can be achieved by implementing a
bioreactor system compared to a multilayer
stack of similar scale.64 Scaling out a process
that uses adherent cells in multilayer CF
requires many connections and incubators, so
contamination risk increases along with capital
expenditure costs. Finally, disposable systems
like the iCELLis bioreactor, or other single use
systems can provide savings as cleaning,
sterilization as well as extensive validations are
reduced or eliminated. The use of bioreactors
also results in lower risk due to the reduction
of production units, especially compared to a
scale-out, multi-production unit configuration.
Other expenses include screening for producer
cells and vector batches for replicationcompetent viruses and other regulatory
requirements.65 Limitations of vector sterilization
may also influence costs given that large viruses,
such as HSV, may not be filter sterilizable, thus
requiring aseptic processing, and validation
that goes with that. As some gene therapy
products are relatively new for regulatory
authorities, it is highly likely that regulatory
guidance will increase as more products are
taken to market, which may add to future
costs.66
Process design and regulatory
burden as a cost determinant
Production design can have a large effect on
cost. While vector production by transfection
is easy, and flexible, and quick to market, it
increases in costs and presents technical
challenges as scale increases. The cost of GMP
plasmid can be one of the largest cost drivers
of upstream operations.
The use of helper viruses (for direct infection)
rather than plasmids may reduce transfection
needs and provide a less expensive path for
scaling up production. In this regard, the use of
a stable producer cell line to reduce or eliminate
transfections and transductions can be of
great benefit through reduced manufacturing
steps, which lower costs.
The use of a stable producer cell line over
transfection methods may also facilitate process
consistency, which is an attribute for GMP vector
production.65 Since all components of vector
production intended for therapy require GMP
manufacture, optimization or elimination of
costly GMP steps is an important consideration.67
© 2020 The Cell Culture Dish, Inc.
Meeting regulatory specifications is critical
requirement. Optimization of the overall process
design in light of regulatory burden for purity
and safety can greatly lower production costs.66
These include the presence of potentially toxic
or immunogenic impurities, contaminants such
as HCP proteins and DNA, plasmid DNA,
reagents
from
transfection,
potential
transduction inhibitors, induction agents,
antibiotics, and others.68 Other optimizations
include optimizing transfection to minimize
costly plasmid consumption, optimizing cell
culture medium feed to improve productivity,
and optimizing cell harvest from large bioreactors
so that they are pooled to enter downstream
processing in a single path.
While there is significant work in optimizing
the overall process there are significant longterm benefits. Overall, it has been estimated a
non-optimized production process, like those
used in academia, can increase costs 40% and
can reduce dose throughput 33%.39
Process development timeline
considerations
Required speed to market and overall costs are
two considerations of process development.
Adherent based bioreactor systems such as
the iCELLis bioreactor can have advantages
over suspension cell systems in terms of
process development timelines. There is less
required cell development and optimization
can move relatively fast and adaptation of an
adherent cell process from cell factories can be
relatively short. By contrast, adapting an
adherent cell system to suspension cell system
can be a very lengthy process (8-18 months)which can delay the time of product to market.
Overall, it has been
estimated a non-optimized
production process, like
those used in academia, can
increase costs 40% and can
reduce dose throughput 33%
Transfection based approaches often enter
production because they are inherited from
academic development of the vector.
Transfection based processes generally are
quick and flexible and can move at a faster
pace into market. However, they are expensive,
and may induce technical and cost difficulties
at larger scale required for some viral vectors.
Many producers are currently entering market
using transfection and adherent cells because
it is fast and provides a fast route to market.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 18
Upstream Manufacturing
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
For products that require high yield, lower
costs, and have a longer allowed time for
introduction
to
market,
undergoing
development of suspension cells or a stable
producer line can have long term benefit,
even though it can take months or years to
develop either. Indeed, some vector producers
choose to enter market using a transfection/
adherent cell process while concurrently
developing stable producer cells, suspension
cells, or both for future use.
Process development must be done smartly
with defined goals in mind. As such it can
take considerable time to develop a process
that meets strict regulatory standards. There
are many parts to an upstream process such
as media optimization, cell line optimization,
creation of cell banks and use of (stable) cell
lines. All of these require considerable
consideration and time to be successful in
the market.
Analytical considerations
The final vector product intended for gene
therapy and vaccine applications must be well
characterized and of proper quantity, purity
and potency for both producers and regulatory
authorities. This can be achieved only if
adequate characterization methods are in
place and the viral production process is well
established, understood and reproducible.
Control and product characterization are not
only important for the final product, but also
within the different steps of the production
process. In depth knowledge of both up- and
downstream processing is crucial since the
upstream production greatly affects the
downstream process. Sometimes even slight
changes in the upstream process can result in
a less efficient downstream process.
There has been continued advancement of
analytics for vector production. The wellestablished framework for traditional biologics
(i.e.
recombinant
proteins,
enzyme
replacement
therapies,
monoclonal
antibodies) is increasingly being applied to
the field of gene therapies. As such, even
more investment in real-time, high-throughput
technologies to characterize both process
and product intermediates is needed for
vector manufacturing.
Impact of upstream on
downstream purification
Many vectors are secreted into the media. In this
case, virus can be collected from the media,
which will substantially reduce downstream
© 2020 The Cell Culture Dish, Inc.
burden. With some vectors, a portion of the virus
remains in the cells and so cell lysis with either
detergent or physical means may be critical to
improve yield. There are a wide variety of
processes that can be used for purification,
including
clarification
by
filtration
or
centrifugation; chromatography steps for
affinity, ion exchange, sizing, multi-modal and
other resins; concentration steps with TFF or
ultracentrifugation; and enzymatic treatments to
reduce nucleic acids. When the cells must be
lysed to release the vector products, multiple
steps may be needed to optimize the purification.
The choice of upstream production process
can affect downstream purification burden.
For example, shear forces on adherent cells on
microcarrier systems or other systems with
fluid motion can increase cell debris. Another
example is that a fixed-bed bioreactor can
offer some benefit since some impurities can
be trapped into the bed. For downstream
production engineers, the greatest concerns
are the productivity, quality and consistency of
material that arrives from upstream operations.
There has been increased interest and work in
the area on how vector design and upstream
process can impact final vector product
infectivity and quality - and lessen purification
burden. Two aspects of quality of a vector
preparation are the levels of 1) full particles to
empty particles and 2) the level of infective
capsids to non-infective particles. These
essentially determine the potency of the product
and potentially the levels of side effects, such as
loss of potency or immune (CTL) response.69
Theoretically, a perfect vector preparation would
have a total particle (vp) to infectious particle
(iu) ratio of 1.70 It has been noted that highly
purified preparations of wild type AAV2, for
example can contain nearly this ratio.70 However,
identically purified rAAV vector preps can have
an infectivity of less than one out of 100
particles.70 Thus, there is room for improvement
in vector design, cell lines, or methods for the
production of recombinant vectors.
It has been noted that several factors can
influence these quality parameters, such as
culture methods (shake flasks vs fed batch flask
vs bioreactor)71, production via transfection vs
transduction71,72, and others. It has also been
noted that the relative distribution of AAV
particles in cell lysate and culture media can be
dependent on the particular vector.73 Thus, there
is much to learn and improve for the production
of vector with high specific productivity.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 19
Upstream Manufacturing
Room for Improvement
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
There are several areas of improvements for
viral vectors for gene therapy. One of the
current challenges in the market is to
understand the product and how the
production process can impact the product.
There is generally good knowledge of both
the individual virus and production processes.
However, there is not much current experience
with the interplay of the two in producing a
viral product. This is an emerging industry and
there is much to learn as it moves forward.
As mentioned earlier, transfection can be both
problematic at larger scale and expensive to
produces doses required for some clinical
applications. More advancement in the use of
stable producer lines is needed. The development
of stable producer cell lines that are also adapted
to suspension may further increase production
by enabling the use of large tank bioreactors.
These, in turn, can be used in closed systems
with less risk of contamination and the use of
serum-free, animal-free media will lessen
purification burden and improve regulatory
safety profiles.
The cost of vector and vector-derived therapies
continue to present a challenge towards
advancement. Methods need to be developed
which result in lower costs similar to what has
occurred in the monoclonal antibody industry.
Current methods for lysing cells (example
AAV) are also crude and there is room for
improvement. Alkylphenol ethoxylate (APE)
surfactants such as nonylphenol ethoxylates
(NPEs) and octylphenol ethoxylates (OPEs),
have restrictions on use in the EU and will be
severely restricted in Jan. 2021.74 This restriction
will include Triton X-100, which is has been one
of the most widely used lysing agents.
Despite the ongoing need for improvements in
therapeutic vector production, there is much
optimism for the future of viral based gene
therapy. In the future, vector based gene therapy
may not only offer gene replacement, but could
advance to offer gene editing functions as well.
Conclusion and recent shifts in
upstream processing
One of the most notable shifts in the past 2-3
years has been the approval of numerous vector
based therapies by regulatory authorities. This
serves as validation of this new, emerging
industry and it is a significant move forward. In
addition, there is more experience and
recognition of the production scales needed for
some vectors, such as AAV, to go to market.
© 2020 The Cell Culture Dish, Inc.
Over the past 5 years, production has
gradually shifted towards suspension cells, a
trend which appears to have accelerated in
the past 12 months.75 Just a few years ago,
nearly 100% of products were produced via
transfection of adherent cells. At the present
time, adherent cells are used in roughly 70%
of products in development39, including two
FDA approved gene therapies. The shift to
suspension cells requires developmental
effort that is ongoing and although there are
currently no approved products that use
baculovirus vectors13, there has been a notable
increase in their development as well.
While there is a perception that cost of goods
would be lower for a suspension process, this
may not be true currently. On average these cell
lines currently produce less virus than their ad­
herent counterparts, which in turn would increase
the cost per dose both in terms of cell line and
plasmid raw material requirements.75
There has also been a notable increase in the
use of stable producer cells, particularly for the
production of LV vectors.39 LV vectors to date
have been primarily utilized for ex vivo CAR T
therapies, which do not require large titers of
virus and is well suited for production via
transfection. The use of stable cell lines and
the advancement of LV vectors could
potentially assist the expansion of LV therapies
into in vivo applications.
There has been more recognition by the industry
that the molecular biology of the cell, the
expression system, and the vector matter for
production yield- or for clinical therapeutic
properties. It is entirely possible that we are at
the early stages of the re-engineering of systems
so that they are designed for improved outcome.
On the production side there is more work being
performed on suspension cells, which in turn,
would assist large production scale. There is also
discussion of cell clone selection or cellular
modification to increase vector productivity at
the cellular level. It is entirely possible that these
approaches are being imported from the
monoclonal antibody industry.
On the clinical side, there is ongoing discussion
about AAV capsid design to increase specificity
toward target tissues. Improved tissue targeting
would result in lower dosing of vector, which in
turn would reduce manufacturing burden, lower
cost, and improve safety. Lower dosing could
also enable therapies that would otherwise
require prohibitively high titers of vector. Thus,
there is more work being done on the molecular
biology of gene therapy vectors and the cells
that produce them.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 20
Upstream Manufacturing
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
There has been a continued shift to the use of
fixed bed bioreactors over conventional flatware
culture for adherent cells. Fixed bed bioreactors,
such as the iCELLis bioreactor, provide a route to
increased scale of production up to 500L and a
quicker route to market.39 Their use has been
enabling for the industry as they can and move
production away from an unmanageable number
of flatware vessels into a single bioreactor.
There are also advancements in medium
scale transfection methods up to 60-70L. There
is greater understanding of the large-scale
In summary, we see continued advancement
and growth in an emerging industry that now
has several approved therapeutics on the
market and a bright future.
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Foreword
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Upstream
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Downstream
Manufacturing
Analytics
Reimbursement
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Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 22
Downstream Manufacturing
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Downstream Manufacturing of Gene
Therapy Vectors
By: Steve Pettit, Clive Glover, Tony Hitchcock, Rachel Legmann, Mark Schofield
Introduction
The downstream manufacturing process takes
the output from upstream operations and
transforms it through multiple steps into a
viable drug product ready for administration
to patients. The major challenge in downstream
processing is to maximize yield while meeting
both product and impurity specifications. Viral
vectors destined for clinical use must comply
with regulatory standards for product safety
and potency and this means contaminants
must be removed and impurities controlled.
Viral Vectors
Viral vectors vary greatly in physical
characteristics depending on the type of virus.
Approaches to vector purification exploit the
physical characteristics of the viral particle such
as size, surface charge and hydrophobicity. The
wide variation in physical characteristics between
various vectors presents a diverse set of
purification challenges. This diversity results in a
multiplicity of needed techniques for production
and purification, which also presents a challenge
in development and operation of multi-use
facilities, such as those routinely operated by
contract manufacturing organizations. Table 1
shows some similarities and differences for
commonly used virus vectors: adenovirus (AdV),
adeno-associated virus (AAV) and retroviruses
(which are primarily gammaretrovirus) (RV), and
lentivirus (a type of retrovirus) (LV).
Table 1. Differences and similarities in four common viruses
used as viral vectors
Virus
Adenovirus
AAV
Retrovirus/
Lentivirus
Size
(nm)
Envelope Stability
Buoyant
Density
~90
No
High
1.34
(CsCl)
~ 20
No
High
1.41
(CsCl)
~90120
Yes
Low(a)
1.16
(Sucrose)
a. Pseudotyping with VSV-g is reported to improve stability
to some extent.1
Further challenges arise in that there is
variation in physical characteristics not only
between the different vectors, but also
between vector serotypes. These differences
can be small, as in between AAV serotypes,
© 2020 The Cell Culture Dish, Inc.
however they can have major implications to
the downstream process conditions. Moreover,
physical differences can vary within the same
vector due to variation in the transgene insert
Process differences also present further
challenges. For example, AAV accumulates
both in the media and intracellularly via a
partially lytic process. The amount of cell lysis
is dependent on the AAV subtype. In order to
improve yield for some AAV subtypes, cells are
lysed via detergent. The cell lysis, in turn, can
significantly increase the quantities of host cell
contaminants including DNA and protein that
increase purification burden.
Another issue in AAV purification is the removal
of “empty” capsids devoid of transgene. The
high proportion of empty capsids, which can
be as high as 95%, makes this even more
difficult.2 Currently, the role of empty capsids
for a therapeutic is unclear: there are reports
that show that empty capsids in an AAV
therapeutic can either be clinically beneficial
or
detrimental
depending
on
the
circumstance.3-5 Despite the ongoing debate
over a possible benefit from the presence of
empty capsids in AAV therapies, they are still
considered a process related impurity, and as
such the level of empty capsids must be
controlled. Additionally, partially filled AAV
capsids are a potential safety concern.
RV/LV vectors present their own unique
purification challenges. The presence of a lipid
envelope makes these vectors less stable and
more shear sensitive. The instability of these
particles can lead to a loss of yield - potentially
up to 70%.6 It is not uncommon for LV to have a
half-life of just 8-12 hours.7 So designing the
downstream process to minimize time, for
instance reducing intermediate hold time, is a
critical consideration.
Difficulties in the purification of vectors can
arise because of vector type or choice of
upstream platform and process. For example:
1) vector supernatants that are produced by
transfection contain large amounts of
contaminating plasmid DNA or 2) use of a
suspension cell bioreactor can produce more
debris than a fixed-bed bioreactor or 3)
intracellular viruses produce more debris than
excreted viruses or 4) large viruses, such as
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herpes, may not be filter sterilizable, and may
require aseptic processing, and the validation
that goes with that.8-10 The use of serumsupplemented cell culture media can also
cause a greater purification burden and
increased viscosity from sera can affect
downstream process efficiency.11
chromatography (IEC) step for the separation
of empty and full capsids. LV purification, on
the other hand, employs mostly one,
sometimes two chromatography steps.
In this regard, upstream production systems
using serum-free media, which is common for
stirred tank bioreactors, can ease the
downstream purification burden. Another
consideration is that fixed bed bioreactors
generally produce output with reduced
turbidities. This can also reduce the downstream
burden, particularly upon clarification.
Cell lysis is often required to improve viral yield
when the vector is not fully secreted by the cells
(e.g. AdV, AAV). Lysis can be performed by
physical disruption via microfluidization or
heat-shock treatments. Alternatively, detergents
such Triton™ X-100 have proven efficient for lysis
in production of AdV and AAV and has been
utilized for insect cell vectors as well.12 However,
the European REACH list of banned substances
limits the use of alkylphenol ethoxylate (APE)
surfactants such as nonylphenol ethoxylates
(NPEs) and octylphenol ethoxylates (OPEs),
which include Triton X-100 and these are
scheduled for severe restriction in 2021.13
Despite the differences in physical characteristics
between vectors, current downstream protocols
generally have a similar workflow: A clarification
step (which may require additional depth
filtration due to cell lysis at time of harvest),
purification (typically ion exchange or affinity
chromatography), and a polishing step
(additional
chromatography
step/size
exclusion) (Figure 1). In addition to the above,
filtration, dialysis, or TFF steps are typically
utilized in order to concentrate, exchange
buffer, and prepare final formulation.
Secreted Viruses
Intracellular Viruses
Cell Collection (optional)
Harvest
Recovery
Clarification
Cell Lysis
Filtration/Microfiltration
Concentration
Buffer Exchange
Ultrafiltration/Diafiltration
Chromatography Step 1
Purification
Polishing
Chromatography Step 2
Polishing Step
Concentration
Buffer Exchange
Formulation
Ultrafiltration/Diafiltration
Figure 1. Example of a downstream process for secreted
viruses (retrovirus/lentivirus) and viruses released by cell
lysis (AAV/adenovirus).
The choice of viral vector will have an impact
of the purification steps that are required.
For example, AAV purification typically
involves an affinity chromatography step to
remove contaminants and an ion exchange
© 2020 The Cell Culture Dish, Inc.
Cell lysis, clarification, and
filtration
Alternative detergent lysis approaches can
usually be developed using Tween™ detergents14
and there are analytical methods for the
detection of Tween 20 in vector preparations
thereby providing a method to show that the
detergent
has
been
removed
during
downstream processing. Any cell lysis method
requires optimization as lysis is highly
dependent on the cell concentration, incubation
time, incubation temperature, cell type, and the
virus being released.14
Clarification involves the initial removal of cell
debris and impurities and typically involves a
combination of depth filters and sterile
filtration. This approach can also contribute to
the overall bioburden control strategy. To
improve recovery, direct flow solutions for
clarification have been utilized with some
vectors using 1 µm > 0.8 µm > 0.45 µm filters.15
Note that the sterile filtration of some viruses
may be challenging particularly for large
enveloped viruses. Another key consideration,
when the downstream harvest stream has high
turbidity, is the inclusion of a pre-filter with
relatively large nominal pore sizes to remove
larger cellular debris and protect subsequent
filters from clogging and creating high pressure
in the subsequent purification step.
Alternatively, tangential flow filtration (TFF)
has proven an effective method for both
clarification and concentration with reports of
recoveries as high as 90–100%, although flow
rate may need to be adjusted.16,17 Reduction in
viral
yield
during
concentration
and
diafiltration, due to loss of stability or loss of
function, is of upmost concern, particularly for
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enveloped viruses such as RV/LV vectors.
Strategies may need to be developed to
minimize shear throughout the purification
processes, such as limiting flow rates or
employing single-pass TFF, which is an area of
active development that has the potential to
improve yields. The duration of the process is
always a concern for unstable viruses
The limitation of classical
centrifuge methods
Purification of small quantities of virus for
research in academia or for small clinical
studies has classically relied heavily on
ultracentrifugation methods. Historically CsCl
has been commonly used for AAV and AdV
ultracentrifugation. However, iodixanol is now
preferred because it is easier to dialyze out,
leads to less aggregation, and run times are
shortened from 24 h to just a few hours.18
Because of the decreased stability of
enveloped viruses such as RV/LV, sucrose is
generally used for density purification of
these viruses.
A limitation of density ultracentrifugation is
scale up due to volume limitations,
operational logistics and cost. Since larger
volume ultracentrifuges are now commercially
available, it is possible to at least consider
ultracentrifugation at some scales. However,
any benefit must be weighed against the
disadvantages of labor, processing time, and
capital expenditure along with health and
safety issues associated with large scale
ultracentrifugation of infectious virus.11
Multiple rounds of gradient ultracentrifugation
may be required to highly purify particles.
Ultracentrifugation has been used by a
number of companies and academic
institutions to produce clinical material at a
small scale and ultracentrifugation remains in
use in current processes to some degree to
separate product related impurities such as
empty capsids and non-product impurities
such as HCP. Ultracentrifugation may have
some application in the later stages of an
overall purification scheme.11
materials. Chromatography is widely used and
may be applied in various downstream steps,
including capture, concentration, purification,
and polishing steps.20
Approaches that have been developed for
monoclonal antibodies (mAb) and recombinant
proteins have been modified for the purification
of viruses. However, resins that are the backbone
of mAb processing are inherently disadvantaged
by the diffusion limits of the pores in the media.
The pores are often so small that they exclude
viruses, limiting the binding capacity and
typically require longer residence times. This, in
turn, means operating at lower flow rates and
increasing processing times. Moreover, resins
typically require packing and validation, which
is laborious. This weakness has led to an
increased
adoption
of
chromatographic
approaches that are not limited by diffusion
and that can be operated convectively, this
includes monoliths and membranes.
Both monoliths and membranes are available
in pre-packed formats and may have “plug
and play” advantages. Typically process
economics are more challenging for monoliths
and this has furthered the adoption of
membrane
chromatography.
Membrane
adsorbers integrate easily with other unit
operations and deliver highly productive
downstream operations. Membrane adsorbers
are increasingly used in new flexible
manufacturing facilities, in which speed of
operations and robust performance are
essential production criteria. They can result
in a more productive downstream operation
which, in some applications, can be an order
of
magnitude
greater
than
resins.21
Furthermore, the membranes can come prepacked, such as the Mustang® Q Membrane
Mustang® XT Capsules
- Image courtesy of Pall Corporation
Chromatography
Chromatographic separation is a more versatile
and powerful method for large scale production
of recombinant adenovirus or AAV compared
to classical centrifugation19. In addition,
chromatography can be an effective means of
removing potential adventitious viruses as
they can have different physical properties
than the vector on many chromatographic
© 2020 The Cell Culture Dish, Inc.
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Downstream Manufacturing
Author Bios
(Pall Corporation), and have demonstrated
scalability due to their multilayer membrane
approach, which is held at a constant thickness
per bed depth.22 Moreover, the larger pore
size of membranes at ~0.8 µm facilitates the
purification of viruses and removal of DNA.22
Introduction
Affinity chromatography
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Affinity chromatography (AC) relies on the
interaction of the viral particle with a ligand
that has a high affinity for the target, but not
for contaminants.
For viral vectors two affinity approaches are
currently used. Heparin and affinity approaches
are used for AAV purification, which primarily
rely upon camelid-derived ligands. Heparin
affinity chromatography has also been used for
purification of LV and other viruses at scale.23
The use of low ionic strength solutions can help
preserve vector integrity.24 However, there can
be impurities that bind non-specifically, the
removal of which necessitates an additional
purification step.23 Other potential drawbacks
are animal derived ligands and the preferred
selectivity of some AAV serotypes.25 Cellufine™
sulphated cellulose (AMSBIO) is an alternative
to heparin with similar specificity for purification
of various viruses.26
For AAV, AVB Sepharose™ affinity resin (Cytiva)
utilizes a recombinant protein ligand and has
been shown to be an effective strategy for the
purification of AAV serotypes 1, 2, 3, and 5 as
recommended by the manufacturer.27 Others
have reported its successful use with other
serotypes, although efficiency may vary.28,29 AVB
resin has been successfully used in combination
with IEC to purify a variety of AAV serotypes.
Furthermore, POROS™ CaptureSelect™ AAVX
affinity resin (Thermofisher) has been developed
for a broad range of serotypes (AAVX for 1-8,
AAVrh 10, and some synthetic serotypes),.
Additionally there are Poros resins developed for
AAV8 and AAV9 specifically.
© 2020 The Cell Culture Dish, Inc.
such as ion-exchange chromatography for that
purpose.28; and 3) the protein-based affinity
ligand is not amenable to cleaning via NaOH.
This limits column regeneration through many
cycles and makes cycling of the resin a
challenge.7 Extensive studies must be conducted
just to obtain a few cycles of regeneration. Thus,
this lack of regeneration of current AAV proteinbased affinity resins adds cost to their use.
Ion exchange chromatography
Ion exchange chromatography (IEC) is a simple,
versatile, and cost-effective technique that has
emerged as a critical step in the purification of
many vectors. Anion (AEX) or cation (CEX)
exchangers can be used to bind either positively
or negatively charged viruses, respectively. IEC
can be used in both bind/elute mode (example:
virus capture or HCP removal) or flow through
mode (example: removal of DNA) and it has
found an important role for both DNA and HCP
removal. IEC has also found an important role in
virus reduction in the monoclonal industry. The
features of high capacity, high resolution, and
high salt tolerance make IEC especially suited
to bioprocessing applications.
AEX chromatography is applied to both AAV
and LV processing. Typically, LV purification
relies on a single AEX chromatography step.
Here there is a big advantage of membrane
chromatography as lentivirus is so large that is
cannot access resin pores. This results in lower
capacities for resins.
AEX can purify LV vectors of high purity
directly from the crude clarified harvest,
although the use of high ionic strength
solutions during the elution step can reduce LV
yield.31 IEC can also be used to separate helper
viruses such as AdV and baculovirus from AAV
preparations.32 IEC has also been applied to
the capture step for large scale production of
AAV. This method has been used for the
generation of GMP AAV vector.3,33,34
These affinity matrices reportedly also lead to
an increase in the overall yield3, and a lower cost
of goods.30 Genethon has reported purification
of AAV9 using CaptureSelect AAV9 resin at 10L
and 50L scale with >80% recovery.19
AEX is one of the few methods that can separate
empty from full AAV and AdV capsids because
of a slight physical difference from the presence
of full-length DNA.35 Some reports show >90%
elimination of empty capsids.35-37
These affinity resins provide a major step
forward in ease of development for AAV
purification, however there are a series of
limitations that remain. These include: 1)
possible leakage of ligand into the vector
preparation that may require additional
analytical testing or additional purification
steps; 2) lack of specificity between empty and
full capsids, which necessitates additional steps
To date, AEX has been demonstrated to be
effective across many AAV serotypes and it
could potentially allow the separation of empty
capsids of all AAV serotypes.28 AEX has also
proven to be an efficient method at larger
scale.3 However, the major challenge for the
use of AEX for the separation of empty AAV
capsids is that operating conditions need to be
optimized for each individual serotype, and
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sometimes even between different vectors of
the same serotype. Substantial development
time (up to many months) may be required to
optimize the gradients required to achieve the
separation. Anything that affects the charge of
a vector, such as serotype, vector design, or
length of the transgene insert, typically
requires re-optimization of the gradient
conditions. Thus, at the present time, one of
the major challenges in gene therapy
development is the effort required to optimize
the AAV full capsid enrichment. Furthermore,
there are often technical challenges for IEC to
fully remove empty capsids from preparations
with a high empty capsid load (i.e. 90%).
Recent advancements in empty/full AAV
separation include the use of Mustang Q
membrane to separate empty from full capsids.
In their patent pending approach, Pall Biotech
found that a step salt gradient provided greater
empty/full separation than a linear gradient. An
initial ~ 1 mS step on a small format XT Acrodisc
was used to initially probe separation of empty/
full capsids and examine the profile of the
process as shown by the differences in 280 nm
(protein) and 260 nm (nucleic acid) adsorption
profile. Absorbance is one of a handful of
methods commonly used as an assay for the
relative amounts of empty/full AAV capsids.38
The process was then scaled to the 5 ml capsule
with identical results (Figure 2). Furthermore,
Pall noted that the gradient could be reduced to
2-steps so that the process would be easier to
implement in a manufacturing environment.
Thus, work with Mustang Q AEX shows that it is
possible to implement separation of AAV
empty/full capsids using formats that can have
distinct advantages over resins.
Size exclusion chromatography
Size exclusion chromatography (SEC) can be
used to separate virus particles from
contaminants on the basis of size and shape.39
SEC has benefits in that it is very gentle on
particles since there is no binding or elution and
it is performed isocratically. However, SEC may
not be suitable for large-scale processing as it is
not usually possible for the load to be larger than
10% of the resin volume. Additionally, the SEC
resins used in bioprocessing are compressible,
this limits column size and flow rate. Nevertheless,
SEC has found use as a final polishing for many
viruses including AAV and LV.9,39,40
Hydrophobic interaction
chromatography
Hydrophobic interaction chromatography (HIC)
been used for viral capture/clearance in the
recombinant protein industry for years. In
purification schemes, HIC has been used for the
purification both AdV and AAV2.41,42 However, its
use with different serotypes of AAV has not been
extensively documented. HIC has challenges.
The use of high ionic strength solutions required
during binding can affect the stability of some
viruses41 and another step must be developed to
remove the high salt concentration. Generally,
HIC is not employed much in current day vector
downstream processes.
Figure 2. Separation of AAV5 on Mustang Q XT5
Separation of empty/full AAV5 capsids using Mustang Q XT5. Initial elution peaks shown by Absorbance at 280nm and 260nm
contain primarily empty AAV5 capsids while peaks with similar absorbance intensities (peaks 3 and 4) contain primarily full
AAV5 capsids.
© 2020 The Cell Culture Dish, Inc.
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Concentration/buffer exchange
Foreword
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Analytics
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Concentrating viruses by ultracentrifugation can
provide effective concentrations and high yields
for some viruses. However, ultracentrifugation
has potential disadvantages such as losses of
functional vector particles due to shear stress43
and co-concentration of impurities.40 The use of
low-speed centrifugation for longer durations is
a more gentle approach, which may prove
valuable with less stable vectors for higher
infectious particle recoveries.44 As discussed
above, centrifugation methods have impractical
scalability and alternative methods are typically
used for industrial production.
Tangential Flow Filtration (TFF) has advantages
for concentration or buffer exchange in that it
is typically scalable and allows for mild
processing conditions for use with less stable
vectors. Products are available in numerous
configurations based on the molecular weight
cutoff, the nature of the membranes, or the
filter surface. Although the achievable
concentration factors are often lower than for
centrifugation methods there is an additional
benefit in that some impurities may be removed
during the process of concentration.16,45
Tangential Flow Filtration (TFF)
TFF technology can be used in several places
throughout the downstream process. The
separation is mainly based on size, although
other molecular properties such as surface
charge and hydrophobicity play some role as
well. In some applications (e.g. continuous
harvest) filters are used with pore sizes that
allow passage of the product and retain larger
impurities (e.g. intact cells, cellular debris). In
other applications (e.g. concentrating pools
upstream of chromatography to limit load
times, or processing purified pools into final
formulation) the product is retained on the
upstream side of the filter and smaller
impurities and buffer components are filtered
out. Tangential flow filtration is a technology
that has transferred well from the recombinant
protein industry. Compared to alternative
techniques, such as centrifugation, it has
shown good scalability, process economics,
process robustness, and in many cases can
provide yields near or above 90%. The decades
of prior knowledge also aid in efficient process
development, process characterization, scaleup, and tech transfer.
Generally speaking, TFF filters are commercially
available in two main formats: flat-sheet
cassettes and hollow fiber modules. Both
formats are widely used in gene therapy
applications. An advantage of flat-sheet
© 2020 The Cell Culture Dish, Inc.
cassettes is that they tend to provide a higher
flux than hollow fiber modules. Flat-sheet TFF
also offers a ‘modular’ approach in that more
filtration area can be added by simply ‘stacking’
more cassettes together in the filter holder.
Hollow fiber TFF is thought to exhibit lower
shear than flat-sheet cassettes, which is an
important consideration for the downstream
processing of fragile biomolecules. Lentivirus
is especially susceptible to shear damage.
Most TFF membranes are caustic stable and
can be run in closed, aseptic processes – a key
consideration for gene therapy manufacturing.
There are several newer TFF products on the
market, in both flat sheet and hollow fiber
format, that are provided with a sterile claim,
and in some cases can be connected into the
system with aseptic connectors. This can
provide the same closed processing benefits
along with less required flushing and therefore
reduced operator time and footprint.
Finally, as gene therapy products are often
more sensitive to environmental conditions
such as shear and time compared to mAbs
and many recombinant proteins, the industry
will likely continue to investigate single-pass
TFF technology. There are products on the
market for single-pass concentration and
diafiltration such as the Cadence™ Inline
Concentrator and Inline Diafiltration Module
(Pall Corporation). These technologies can
offer reduced shear by limiting pump passes,
and reduced processing time by combining
unit operations continuously.
Bioburden Control
Bioburden
control
throughout
the
manufacturing processes is a critical part of
GMP compliance and minimizes the risk of
microbial contamination of the final product. In
most processes the final vector preparation is
filtered by one, or more 0.2 µm sterilizing grade
filters as a component of control, although
there may be variations in its placement in
downstream protocols.3 Some vector products
are not filter sterilizable at 0.2 µm because of
the size of the virus. It is possible to skip sterile
filtration provided that the process can be
certified as being fully aseptic; however, this
requires operation of a closed process and
operations must be performed in a clean room.8
TFF systems can provide increased throughput
for sterilization and can have benefit in that
regard. Nevertheless, sterile filtration currently
can be a huge challenge for some unstable
viruses as they can lose function.
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Insights on downstream
process issues
Moving a downstream process
to clinical and commercial
manufacturing
Transfer of a vector from a developmental
setting in academia, to manufacturing for
clinical use, presents several considerations for
downstream production development. Often
the objectives and consequently choice of
tools and technologies are different between
academia and clinical production. Areas
including consistency of operation and
bioburden control are not typically focused on
in most research production but are critical in
clinical manufacturing. Thus, there are several
areas that need further examination and
possible alternative solutions.
Scalability
One of the major considerations when
moving from research production is how to
achieve the increased production scale. At
larger scales, purification methods need to
accommodate large volumes with high
efficiency and speed. One approach toward
implementation of greater scale is to move
away from poorly scalable centrifugationbased techniques to systems with greater
throughput, such as filtration. Another
approach is the use of chromatographic
approaches as they are considered to be one
of the best technological methods for
manufacturing applications. The scalability
of IEC or AC methods can provide effective
purification of many vectors.11,35 When
considering
individual
chromatographic
approaches, efficiency is a huge aspect as it
can lead to a smaller, simpler process and
greater scale. It has been shown that VSG-g LV
vectors can be purified using Mustang Q
membrane (Pall Corporation) up to a volume
of 1,500 L/day46 and lead to vector
preparations of high purity; in contrast, LV
recovery may not be as efficient as with
ultracentrifugation due to the fragility of
these vectors.
reagents. Rational design of the downstream
process to simplify and increase efficiency can
reduce the number of required GMP reagents
and steps, which increases quality, safety and
significantly lowers costs.
Adventitious agent control is an aspect of
GMP manufacturing that is focused on once a
process has moved into clinical production.
Adventitious virus control can be particularly
challenging since the product is also a virus.
Use of sterile filtration can cause concern as
many viruses are unstable or are difficult to
filter because of viral size. Loss of yield during
filtration continues to be a significant
challenge for many vectors, including LV.
Development of alternative methods for
agent control, such as chromatographic
approaches, would benefit the industry.
Process development timelines
The timeline to develop a downstream process
is dependent on many factors such as the type
of
vector,
intended
use,
scale,
cost
considerations, and technology available.
Independent of the factors involved, a reduced
developmental timeline is a desirable goal as it
quickens time to market and lowers cost. Note
that time required for scientific work on a
downstream process can be extensive, several
months, depending on the resources available,
as there are few “platform-like” or “plug and
play” solutions currently on the market. There
have been helpful advances, however, such as
the rise of vector-specific AC solutions.19 For
AAV developing the process of empty/full
capsid separation takes a long time. It can also
be expensive as it can consume considerable
feedstock at scale.
GMP Compliance
Research material is not required to be GMP
compliant, thus this is a major change when
moving a product to clinical manufacturing.
Vectors for therapeutic use must meet
stringent standards for purity and safety with
product quality of upmost importance. This
transition to GMP requires examination of the
existing process including raw materials and
© 2020 The Cell Culture Dish, Inc.
Image courtesy of Pall Corporation
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Costs considerations
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Costs are a combination of many factors such
as, time, labor, operating expenses, and capital
expenses. The technology used in downstream
processing can impact the timeline of process
development, product quality, ease of process
scale-up, and cost of goods (COGs).19 Replacing
traditional, academic methods that are time
consuming, complex, and inefficient with
approaches that are more cost effective, such
as chromatography, can lower costs with the
tradeoff of required redevelopment time.20
Implementing newer strategies such as IEC or
AC that have greater potential for throughput
and scale up can significantly simplify process
strategy and increase efficiency. These
purification schemes significantly shorten
process development time and reduce COGs.
In
that
regard,
the
newer
affinity
chromatography approaches, if applicable to
the particular vector, can potentially simplify
the process architecture to a single
chromatography capture step accompanied
only by a clarification step and a polishing step
(AAV requires an additional empty/full
separation step). The use of purification and
polishing chromatographic systems can result
in high purity with many vectors.11
Development and use of technologies such
as (semi)-continuous processes can result in
footprint reduction, increased productivity,
automation, reduced inventory and storage,
and fewer unit operations - all of which can
lower costs.20,47 Other cost cutting strategies,
include reduction of the chromatographic
unit operations, through the use of multicolumn systems or single-use systems20 and
chromatographic
column
cycling
with
expensive resins. Another consideration is
implementing
scalable
solutions
for
downstream, such as depth filtration,
chromatography, etc.
The use of tangential flow filtration (TFF),
which can be up-scaled in manufacturing, can
provide cost savings. In that regard, automated
TFF systems are available to control both the
transmembrane pressure as well as the level of
concentration. Additionally, automation of TFF
can provide an ergonomic and secure
environment for the operator and facilitate the
speed and scale up of the process. Often a 20–
80x concentration factor can easily be
achieved using these systems.11
Risk Assessment
Another important consideration is an overall
evaluation of vector risks (risk assessment)
and subsequent design of the upstream and
© 2020 The Cell Culture Dish, Inc.
downstream process to minimize that risk with
available technology. For example, genotoxicity
and immunotoxicity are two theoretical risks,
depending on whether the cells used in
generation of the vector is of human or nonhuman origin. For AAV, empty capsid,
encapsidated host cell DNA, encapsidated
helper component DNA, replication competent
virus, non-infectious vector, and aggregated,
degraded, or oxidized vectors are all considered
potential risks.48 Thus, an overall risk
assessment followed by a mitigation design
should be considered in light of the regulatory
standards and patient safety that is required
for clinical manufacturing.48
Downstream process challenges
Yield
Yield is an important concern in downstream
as loss of viral yield puts increased pressure on
the upstream process to increase yield and
scale. Viral instability and loss of function can
be a huge problem for some vectors such as
LV and other enveloped viruses. Reducing the
number of steps, and time in process, and
reducing shear forces can improve overall yield
for unstable vectors. Storing of process
intermediates and how infectivity is impacted
by the hold conditions (temp, pH, etc.) can
also impact yield. Thus, the need to develop a
rapid process for unstable viruses is a key
consideration in process design.
Purity
Another major challenge in downstream is
obtaining sufficient purity without loss of
function. For example, in a typical production
of an AAV vector there are numerous impurities
to consider including host DNA/RNA, host cell
protein (HCP), plasmid DNA, helper viruses,
culture medium components, items introduced
during purifications (buffers, ligands, salts,
etc.), empty and partially full capsids,
replication competent AAV, noninfectious AAV,
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Analytics
Reimbursement
and other AAV forms such as aggregates.48 To
further complicate matters, residual DNA or
host cell protein can adhere to some vectors
and co-purify with them. Thus, the removal of
impurities is vital as insufficient purity impacts
product quality and safety and can ultimately
result in negative outcomes with regulatory
authorities. In particular, there are concerns
over possible immunogenicity and toxicity. In
addition, the process must meet regulatory
specifications for mycoplasma, adventitious
agents, microbial stability, and others.
Automation and closed systems
Overall process design and efficiency is key to
controlling impurities. Effort must be taken to
avoid introduction of contaminants in the
downstream process, unless necessary. Any
introduction of contaminants would then
require additional analytical and processing
steps for their removal. Benzonase treatment
can be used to remove residual DNA, however,
its use can add cost. Detergents can be used to
control enveloped viruses provided the vector
virus is non-enveloped. Again, anything that is
added to a process must be removed from the
final formulation.
Move toward more efficient methods
For AAV, the process development time for the
empty/full capsid separation can be a monthslong endeavor. There is no “platform” for
empty/full separation and the parameters for
empty/full separation by IEC must be carefully
developed for each vector. In addition, some
AAV vector preparations can enter downstream
containing, as much as 90% empty capsids,
and developing a process to remove high loads
of empty capsids can require significant time
and resources. It is helpful to optimize
feedstock from upstream so that it contains as
many full capsids as possible. Understanding
and defining the levels of required exclusion
for empty and partially full capsids can aid in
the development of a process that meets
regulatory requirements.
Areas for advancement
Affinity chromatography
AC shows great promise as it is effective, and
its use could potentially enable a platform-like
approach in portions of downstream simply by
switching AC columns. The advancement of
AC media across other viruses and serotypes
would be an advancement for the industry. The
resins can be expensive, and current proteinbased AAV resins cannot be regenerated
through many cycles without need to be
replaced. Perhaps, in the future, resins could
be developed to overcome the regeneration
issues and be available at a lower price point.
© 2020 The Cell Culture Dish, Inc.
Automation is needed in several areas to reduce
both labor and costs. Closed systems are
needed for larger vectors where sterilization by
filtration is an issue. Closed systems also serve
to protect both the product and the operators
and will be of large benefit by several measures.
In that regard, more development of processes
similar to those that have benefited the
recombinant protein industry is needed such as
disposable
technology
and
continuous
processing methods.
The continued movement away from older
methods such as ultracentrifugation to more
efficient processes such as chromatography will
benefit the industry. Nevertheless, much
ultracentrifugation is still being performed in the
industry, which indicates there are still
advancements needed in this area. Alternatives
to traditional chromatography resins show great
promise to increase efficiency and scale.
Moreover, the cost of some specific resins and
filters are quite expensive, especially at larger
scale. When chromatographic material is packed
into highly efficient, single-use columns, process
throughput can be further improved and
substantial savings in infrastructure, operations
and quality procedures can be realized.49
Analytics
On the analytics side, development of better or
faster at-line or inline analytics for intermediate
crude with lower viral concentration will
benefit the industry by enabling process
efficiency and lower costs. Because the
analytics can be quite imprecise and can take a
long time to perform, downstream processing
often operates “blind.” High throughput
analytics could reduce process development
timelines by providing a more comprehensive
look at the process and in identifying areas
for improvement. For example, analytical
ultracentrifugation for AAV, while good, can be
expensive and take time but is one of the
few
methods, together with capillary
electrophoresis, that can separate empty from
full capsid with good resolution.9,50. Alternate
analytics, such as HPLC or transmission
electron microscopy (TEM), can sometimes be
used inline to reduce costs, or improve
throughput. For more information, please see
analytics article in this publication.
The development of virus-specific tools
Many tools and technologies currently
employed in downstream production have been
developed for use with small molecules and
antibodies and not for viruses. For example,
some resins were developed for the purification
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of monoclonal antibodies from feedstock
containing 5 g/L antibody. Viral vectors are
much larger, more complex, and are found in
feedstock at 1000-fold less concentration than
antibodies. With viruses, column functionality,
the capacity to capture, and recovery are much
more critical than with monoclonal antibodies.
The needs are different.
Other items used at larger scales such as
concentration and use of membrane filters, can
be entirely different for viruses compared to
antibodies. For example, the concentration of
2000L of virus vs 2000L of antibody is very
distinct and physical re-engineering of available
systems may be helpful. Scale down tools are
also needed as many single use platforms were
Summary
In summary, there is an increasing range of
options to process and obtain high titers and
high-quality batches of viral vectors. We expect
continued advancement and the future is open
to new innovations and capabilities that should
reduce complexity, lessen the challenges, and
lower costs. In many ways, the industry is taking
a similar path to the monoclonal antibody
industry several years ago with expansive
growth and manufacturing capabilities.
References
1.
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Gijsbers, R. Upscaling of lentiviral vector production by
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Sommer, J. M. et al. Quantification of adeno-associated virus
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15. Merten, O. W. et al. Large-scale manufacture and
characterization of a lentiviral vector produced for clinical ex vivo
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designed for use with 100’s of gram of antibodycompared to viruses at < 1 g for small scale.
Thus increased virus-specific tools are needed
for both small scale and large scale processing.
19. Zhao, M. et al. Affinity chromatography for vaccines
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20. Silva, R., Mota, J., Peixoto, C., Alves, P. & Carrondo, M.
Improving the downstream processing of vaccine and gene
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21. Pall Biotech. Why membrane adsorbers? Accessed Oct. 3,
2020: Retrieved from https://pall.com/en/biotech/
chromatography/membrane-chromatography.html
22. Ball, P. & Gyepi-Garbrah, I. Membrane adsorbers as a tool for
rapid purification of gene therapy products. Accessed Oct. 9,
2020: Retrieved from https://www.pall.com/content/dam/
pall/laboratory/literature-library/non-gated/Mustang_
poster_841x1189mm.pdf
23. Summerford, C. & Samulski, R. J. Viral receptors and vector
purification: new approaches for generating clinical-grade
reagents. Nat Med 5, 587-588 (1999).
24. Segura, M. M., Kamen, A., Trudel, P. & Garnier, A. A novel
purification strategy for retrovirus gene therapy vectors using
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25. Mietzsch, M., Broecker, F., Reinhardt, A., Seeberger, P. H. &
Heilbronn, R. Differential adeno-associated virus serotypespecific interaction patterns with synthetic heparins and other
glycans. J Virol 88, 2991-3003 (2014).
26. Kanlaya, R. & Thongboonkerd, V. Cellufine sulfate column
chromatography as a simple, rapid, and effective method to
purify dengue virus. J Virol Methods 234, 174-177 (2016).
27. GE Healthcare. AVB Sepharose™ High Performance. Data file
28-9207-54 AB.
28. Nass, S. A. et al. Universal Method for the Purification of
Recombinant AAV Vectors of Differing Serotypes. Mol Ther
Methods Clin Dev 9, 33-46 (2018).
29. Mietzsch, M. et al. OneBac: platform for scalable and high-titer
production of adeno-associated virus serotype 1-12 vectors for
gene therapy. Hum Gene Ther 25, 212-222 (2014).
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Reimbursement
30. Thermo Fisher. Accessed Retrieved from http://
thermofisher.mediaroom.com/press-releases?item=122399
41. Shambram, P., Vellekamp, G. & Scandella, C. in Adenoviral
vectors for gene therapy (Academic Press, 2016).
31. Scherr, M. et al. Efficient gene transfer into the CNS by lentiviral
vectors purified by anion exchange chromatography. Gene Ther
9, 1708-1714 (2002).
42. Chahal, P. S., Aucoin, M. G. & Kamen, A. Primary recovery
and chromatographic purification of adeno-associated virus
type 2 produced by baculovirus/insect cell system. J Virol
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32. Khandelwal, P. (2015). (Bulletin 6807)Practical guide: selecting
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webroot/web/pdf/psd/literature/Bulletin_6807.pdf
33. Wright, J. F. & Zelenaia, O. Vector characterization methods
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34. Manno, C. S. et al. Successful transduction of liver in hemophilia
by AAV-Factor IX and limitations imposed by the host immune
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35. Okada, T. et al. Scalable purification of adeno-associated virus
serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange
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36. Urabe, M. et al. Removal of empty capsids from type 1
adeno-associated virus vector stocks by anion-exchange
chromatography potentiates transgene expression. Mol Ther 13,
823-828 (2006).
37. Davidoff, A. M. et al. Purification of recombinant adenoassociated virus type 8 vectors by ion exchange
chromatography generates clinical grade vector stock. J Virol
Methods 121, 209-215 (2004).
38. Wang, C. et al. Developing an anion exchange chromatography
assay for determining empty and full capsid contents in AAV6.2.
Mol Ther Methods Clin Dev 15, 257-263 (2019).
39. Rodrigues, T., Carrondo, M. J., Alves, P. M. & Cruz, P. E.
Purification of retroviral vectors for clinical application: biological
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40. Transfiguracion, J., Jaalouk, D. E., Ghani, K., Galipeau, J. &
Kamen, A. Size-exclusion chromatography purification of
high-titer vesicular stomatitis virus G glycoprotein-pseudotyped
retrovectors for cell and gene therapy applications. Hum Gene
Ther 14, 1139-1153 (2003).
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43. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. &
Yee, J. K. Vesicular stomatitis virus G glycoprotein pseudotyped
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44. Jiang, W. et al. An optimized method for high-titer lentivirus
preparations without ultracentrifugation. Sci Rep 5, 13875 (2015).
45. Buclez, P. O. et al. Rapid, scalable, and low-cost purification of
recombinant adeno-associated virus produced by baculovirus
expression vector system. Mol Ther Methods Clin Dev 3, 16035
(2016).
46. Slepushkin, V. et al. Large-scale purification of a lentiviral vector
by size exclusion chromatography or Mustang Q ion exchange
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47. Hernandez, R. (Apr. 1, 2015). Continuous manufacturing: A
changing processing paradigm. Accessed Oct, 22, 2020:
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view/continuous-manufacturing-changing-processingparadigm
48. Wright, J. F. Product-related impurities in clinical-grade
recombinant AAV vectors: characterization and risk assessment.
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Manufacturing of Affordable Biologics. Comput Struct
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Analytics
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Evolution of Viral Vector Analytics
for Gene Therapy Manufacturing
By Debbie King, Edita Botonjic-Sehic, Tony Hitchcock, and Mani Krishnan
With over 350 cell and gene therapies in
clinical trials in the US and more than 700
globally, the explosive growth in the field is
only expected to increase in the years to
come.1,2 This rapid progress from clinical
development towards product licensure has
resulted in a viral vector supply bottleneck.
The challenge in viral vector-manufacturing
capacity is estimated to be 1–2 orders of
magnitude lower than what is needed to
support current and future commercial supply
requirements.3 Concomitantly, regulatory
scrutiny
and
product
characterization
requirements are increasing, as more gene
therapy products reach commercialization.
To meet these demands, viral vector
manufacturing processes will need to evolve to
become more sophisticated and so too will the
analytical tools needed to evaluate them.
Continued improvements and adoption of
rapid, robust technologies for viral vector
analytics/characterization and rigorous quality
control assays will be essential to facilitate the
timely development of the gene therapies that
require them.2
how best to move forward to fill any existing
knowledge gaps, whether it is learning from
other, more mature bioproduction systems or
investing in novel, innovative technologies.
Current Challenges with Viral
Vector Analytics
As more gene therapy products are approved
and the technologies to produce them become
more
established,
increased
regulatory
expectations for greater sensitivity, specificity
and accuracy are exceeding the limits of
existing analytical technologies. There is a
need to shift away from lab-scale, “tried and
true” techniques that are slow, low throughput,
and often highly variability with poor
reproducibility and precision. These methods
should be replaced with assays that are more
robust, rapid and accurate.
The focus of PAT within the Quality by Design
(QbD) framework has driven an investment in
high quality analytics that can generate closer
to “real-time” data that can inform process
development decisions to achieve higher
Speed to market is the perhaps one of the
most crucial aspects in the cell and gene
therapy segment due to their potential as
curative therapies. Many of these therapies
qualify for orphan drug status and/or fast track
designation status, significantly shortening the
drug development timeline from 7 to 10 years
(for traditional biologics) to 3 to 5 years putting
even more pressure on manufacturers to
decrease production timelines.4 For viral vector
developers, the analytical methods required to
keep up with these accelerated timelines will
need to overcome the limitations of lab-based,
classical
technologies
for
product
characterization and quality testing to facilitate
in-process optimization efforts.
Here, we spoke with industry experts to get
their insight on how the landscape of analytical
tools has evolved over the past few years as
the implementation of process analytical
technology (PAT) by viral vector manufacturers
has become more commonplace. Analytical
processes have matured and become more
refined, but there is still much more that can be
improved. It is critical to evaluate as an industry
© 2020 The Cell Culture Dish, Inc.
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Author Bios
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Analytics
Reimbursement
yields and improve viral vector safety profiles.
The lag time between sampling and results
from traditional assays can sometimes be days
to weeks, which is prohibitive to effective
design of experiment (DOE). Additionally,
these assays may lack the resolution needed to
show subtle changes that may result from
stepwise
process
optimization.
While
incorporating novel analytical tools is a key
goal for viral vector production, it is recognized
that these methods require validation before
they are acceptable for use in clinical
manufacture by regulatory agencies. Therefore,
their usage in research and process
development is critical not only to enable
iterations to improve the manufacturing
process, but also to validate that these
methods can satisfy regulatory demands. Key
to this will be the usage of parallel orthogonal
methods with appropriate controls to ensure
the integrity of the data.
Evolution of Viral Vector
Analytical Assays
In January of 2020, the FDA published revised
regulatory guidelines for cell and gene therapy.
In case of viral vector analytics, changes were
made for analytical procedures specific to
impurities, replication, titer, infectivity.5 Overall,
the FDA requires more detailed information/
characterization
and
FDA
regulatory
documentation for these parameters, which is
only expected to increase as the field matures
and development continues.
The analytical tools to characterize viral
vectors continue to evolve as the needs of
the industry change, but the five main quality
pillars reflected in the current regulatory
guidance have not. The critical quality
attributes (CQA) as mandated by the FDA’s
chemistry, manufacturing and control (CMC)
guidelines for viral vector manufacturing
include: identity, strength/potency, purity,
safety and stability. These controls help to
quantify the product heterogeneity observed
with viral vectors to ensure safety and
efficacy, which is especially important for
cell-based gene therapies (Figure 1).
It’s clear that expertise in a wide variety of
analytical techniques is necessary to detect and
characterize biopharmaceuticals and their
impurities (both product- and process-related
impurities). In many cases, viral vector
manufacturers can leverage technologies that
have proven their effectiveness in the production
monoclonal antibodies (mAbs), some of which
will be discussed here. However, for most of
these newer, more complex molecules,
customized assays must be developed, keeping
in mind their speed, accuracy and robustness
(Table 1 on next page).4
Identity
Genetic Identity. Molecular methods, such as
PCR, and genome sequencing (high throughput
NGS), are utilized to confirm the identity of the
vector genome.
Figure 1. Generalized workflow for viral vector manufacturing.
© 2020 The Cell Culture Dish, Inc.
Insights on Successful Gene Therapy Manufacturing and Commercialization
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Analytics
Quality
Foreword
Identity
Attribute
Example Assays
Genetic identity
Genome sequencing (NGS)
PCR
Protein identity
SDS-PAGE
Mass spectrometry (MS)
Western blot (immunoblot)
CE-SDS
ELISA
qPCR
Optical density (A260/280)
NanoSight
HPLC (AAV packaging ratio)
MADLS
ddPCR
Functional viral titer
Plaque-forming assay
Fluorescence foci assay
TCID50 (end point dilution assay)
ECIS - impedance
Process impurities
(detergents, resin)
MS
Chromatography
TEM
LC-MS
Host cell-related impurities
Host cell DNA/RNA: Picogreen, DNA
Threshold assay, qPCR
Host cell proteins: SDS-PAGE, ELISA,
HPLC, TEM
Author Bios
Physical viral titer
Introduction
Regulatory
Strength/
Potency
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Purity
Reimbursement
Safety
Stability
Next Generation Assays
Automated Western blot
CE-SDS or CE-LIF (AAV
packaging ratio)
LC-MS, LC-MS/MS
Capsid content
(empty: full capsids)
ELISA/qPCR
HPLC
MS
TEM
AUC
Sterility
Standard sterility tests (EP 2.6.1, USP71)
Rapid microbial methods (RMM)
Endotoxin
LAL method (EP 2.6.14, USP85)
Rabbit pyrogen assay
Recombinant Factor C (rFC)
Mycoplasma
Cell-based assays
qPCR
Replication competent virus
(rep/cap sequences)
Southern blotting
qPCR
Adventitious agents
In vivo and in vitro cellular assays
pH
Potentiometry
Osmolality
Osmometry
Aggregate formation
Light microscopy
DLS
SEC-MALLS
TEM
AUC
FFF-MALS
CE-LIF, cIEF (isoelectric focus)
LC-MS
MADLS
TRPS
Table 1. Examples of current and next generation analytical assays used to assess viral vector CQAs.
Protein Identity. Comprehensive characterization
of the capsid proteins is critical for viral
infectivity and vector potency; therefore,
serotype identity, capsid protein stoichiometry
and integrity, contribute not only to expanding
product knowledge but also ensures product
and process consistency.6,7 The AAV serotype
can be determined using enzyme-linked
immunosorbent assay (ELISA), Western blot
(immunoblot) or mass spectrometry (MS)
methods.
Both
chromatographic
and
electrophoretic-separation techniques coupled
to MS like LC (liquid chromatography)-MS and
CE (capillary electrophoresis)-MS are used for
peptide mapping, where LC-MS is most
commonly used. Because CE-MS separates
peptide moieties differently than LC-MS, it
© 2020 The Cell Culture Dish, Inc.
serves as a valuable orthogonal and
complementary technique to provide a more
complete analysis of capsid proteins.8
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) is a commonly
used, well-characterized assay to confirm viral
vector stoichiometry (packaging ratios), but it
suffers from poor resolution and assay variability.
Capillary
electrophoresis-sodium
dodecyl
sulphate (CE-SDS), which is a routinely used and
well-established tool for mAb quality control, is a
more precise method compared to SDS-PAGE,
incorporating automation, improved resolution,
reproducibility, and throughput capabilities and
is a more appropriate instrument-based
replacement that is increasingly being
implemented in viral vector analytical workflows.9
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Novel methods, such as AAV-ID, where
differential thermostability is used to
distinguish AAV serotypes and HILIC-FLRMS method, which employs hydrophilic
interaction chromatography (HILIC), followed
by both fluorescence (FLR) and MS detection
are promising technologies for protein
characterization that will need further
analysis and validation to determine their
utility in QC workflows.7,10
Strength/Potency
According to the FDA 21 CFR 210.3(b)(16)
guidance, strength is defined as:
Downstream
Manufacturing
1.
The concentration of the drug substance (for
example, weight/weight, weight/volume, or
unit dose/volume basis), and/or
Analytics
2.
The potency, that is, the therapeutic
activity of the drug product as indicated
by appropriate laboratory tests or by
adequately developed and controlled clinical
data (expressed, for example, in terms of
units by reference to a standard).
Reimbursement
For biopharmaceuticals, potency is often
regarded as equivalent to strength defined as
“the specific ability or capacity of the product,
as indicated by appropriate laboratory tests or
by adequately controlled clinical data obtained
through the administration of the product in
the manner intended, to effect a given result.”11
Potency assays should be validated during the
product development process. Whenever
possible, a potency assay should measure the
biological activity of the expressed gene
product, not merely its presence.
Physical Titer. Physical titer is a measurement of
intact virus bulk – how much is present in a
given preparation, and is expressed as the
number of viral particles per mL (VP/mL), or
for AAV as genome copies per mL (GC/mL).
There are a variety of methods used to
determine the physical titer of virus based on
quantifying the concentration of viral genomes
or viral proteins including ELISA, optical
density assays (A260/280) and more sophisticated
visualization systems like the NanoSight device
(Malvern Instruments Ltd.) for viral particle
quantification.
High-performance
liquid
chromatography (HPLC) allows for the
visualization and quantitation of intact virus
particles in a heterogeneous preparation that
also contains cellular and process contaminants.
By far the most routinely used method for
physical titer measurement is real-time PCRbased assays because they are robust, easy,
fast, and convenient. In the industry, there is a
© 2020 The Cell Culture Dish, Inc.
shift now from quantitative PCR (qPCR) to
digital droplet PCR (ddPCR) to measure this
attribute. With ddPCR, the sample is
partitioned into thousands of individual
micro-reactions that are quantified as either
PCR-positive and PCR-negative at the
endpoint of Taq polymerase amplification.
This enables direct and absolute quantification
of target DNA offering greater sensitivity in
detection and less variability than qPCR,
which requires comparison to a standard
curve. ddPCR generates more precise and
reproducible data especially in the presence
of sample contaminants that can partially
inhibit Taq polymerase and/or primer
annealing.12,13 This makes it useful not only for
final product characterization but for inprocess
analytics
where
the
vector
preparations are more heterogeneous.
Infectious or Functional Titer. Functional titer, or
infectious titer, is a measure of infectivity - how
many of the virus particles can actually infect a
target cell. Infectious or functional titer is almost
always lower than physical titer by a factor of 10
to 100-fold because not all of the virus product
produced is functional/infectious. The infectious
titer of a viral vector stock can also be altered
by freeze-thaw of the viral solution, therefore
monitoring the strength/potency during
stability testing is pertinent.
Infectious titers are typically quantified by in
vitro cell transduction assays such as the viral
plaque (for cytopathic viruses), endpoint
dilution (TCID50), immunofluorescence foci
(IFA).14 For those vectors that express
fluorescent transgenes, FACS can be used to
quantify functional titer. While still considered
the “gold standard” for determining functional
titers, the cell-based assays are timeconsuming and can have high variability. For
example, some AAV serotypes do not
transduce well in vitro and can, therefore, result
in an underestimate of infectious titer.
The electric cell-substrate impedance sensing
(ECIS) or real-time cell analysis (RTCA) method
used to study cell growth and migration has
shown utility in determining virus infectious titer,
particularly in vaccine development. This labelfree method is based on measuring the real-time
changes in the electrical impedance of a circuit
formed in a tissue culture dish plated with cells
that are infected with virus.15,16 Biosensors
monitor changes in host cell morphology, and
cell-substrate attachment that occur as part of
the virus cytopathic effect. As cells are infected
with virus, they become more permissive to
electrical current passage, which is measured as
a decrease in impedance over time. The assay
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greatly improves time-to-result as well as being
a more robust and rapid real time quantification
of virus infectivity over traditional cellular assays.
Purity
Viral vector purity demands are high and will
only increase as gene therapies become more
established and are developed for more
common indications and larger patient
populations. Impurities from both the upstream
and downstream operations as well as from
the production cell lines can be introduced
into vector preparations and must be carefully
monitored and quantified since they can have
unintended immunogenic effects.
Process Impurities. Examples of upstream
impurities
include
cell
culture
media
components and additives whereas detergents,
residual chromatography resin, and other
chemicals can be introduced through
downstream purification processes. Typically,
downstream clarification removes the large
impurities but <1000nm molecules are still
present in trace amounts, therefore, regulatory
bodies require their quantification, which is
typically achieved using MS and HPLC methods.
Production Impurities. Included in this category
are unwanted host cell proteins (HCP) and
nucleic acids from both the production cell
lines as well as from any helper viruses and
plasmids required to produce the therapeutic
vectors. Host cell DNA/RNA can be detected
using assays like PicoGreen, DNA Threshold
assay and qPCR.
Classical HCP detection using ELISA provides an
estimation of total HCP amount but the results
are variable and can be influenced by buffer
compositions and more. The main challenges for
HCP analytics are the measurement of low
concentration HCPs and the reproducibility of
the assays. Therefore, health authorities are also
requesting methods like CE, CE-MS, LC-MS and
LC-MS/MS as orthogonal methods to ELISA for
HCP characterization.17 These methods can also
provide rapid analysis of capsid protein ratio,
capsid protein structure and transgene length to
facilitate rapid decision-making during process
development and for in-process testing.18
In addition, impurities related to the viral
vector product itself are of particular
concern for viral vector developers and thus
separate characterization of capsid content
is a major focus.
Capsid Content. As emphasized by industry
experts, capsid content characterization is a
challenge and an area that requires the most
© 2020 The Cell Culture Dish, Inc.
development in terms of analytics. During the
production of viral vectors, there will be a
population of viral particles produced that fails
to package the vector DNA properly. Vectorrelated impurities include:
• Empty capsids
• Capsids that contain nucleic acid sequences
other than the desired vector genome
» Illegitimate, non-vector DNA (from
transfection plasmids, host cells, or
helper viruses)
» Truncated vector genomes
» Replication competent virus
One challenge in characterizing this attribute
is that there is little change in size and shape of
the capsid between empty, full, and partially
full capsid particles. It is estimated that
encapsidated DNA impurities may exceed 10%
of vector genome-containing AAV particles in
crude harvests.19 Because they closely resemble
the actual vector product, separating vectorrelated impurities from legitimate vectors in
downstream purification can be difficult (for
empty capsids) to almost impossible (for some
encapsidated
DNA
impurities).
These
unwanted vector-related impurities represent
a population of heterogeneous, potentially
immunogenic material in the clinical vector
product that can impact patient safety and
thus necessitates increased scrutiny.
A commonly used tactic to determine the
percentage of the full capsids in a vector
preparation is to divide the number of genome
vectors obtained from qPCR data by the total
capsid number obtained from ELISA or optical
density (A260/280) data.20,21 Other approaches
including MS, HPLC, transmission electron
microscopy
(TEM),
size
exclusion
chromatography
(SEC)
and
analytical
ultracentrifugation (AUC). However, many of
these methodologies are unable to resolve
partially full capsids, which drive a need for a
technique with higher resolution capabilities.13,22
SCIEX has developed a capillary isoelectric
focusing (cIEF) method using the PA 800 Plus
Pharmaceutical Analysis System, which utilizes
the different isoelectric points (pI) to resolve
full, empty and partial capsids to determine
their ratios in a vector preparation (Figure 2).
The sample analysis time is less than 1 hour per
sample, and is able to produce reliable,
reproducible, high-resolution results across
multiple AAV serotypes.23 Capsid ratios
calculated using cIEF were comparable to
orthogonal methods HPLC and AUC. The
SCIEX platform is routinely used for QC of
other biologics, making it an easy transition to
viral vector manufacturing workflows.
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#1 Enriched Empty
0.004
Full
Empty
0.002
#2 Enriched Full
0.000
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21.0
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22.0
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Figure 2. cIEF profiles of two samples of the same AAV
product with different amounts of full and empty capsids
were analyzed. Sample #1 was enriched with empty capsids,
while sample #2 was enriched with full capsids. For AAV full
and empty capsids, the full capsids have lower pI values than
the empty capsids due to negative charges contributed from
the ssDNA encapsulated inside the shells. Some potential
partial capsid peaks appeared to sit between those empty
and full capsid peaks because of their moderate pI values.
(Figure courtesy of SCIEX)
Sterility. The standard two-week testing period
is a challenge to reconcile with the timely
release of viral vectors necessary for cell-based
gene therapies, such as CAR T, with
manufacturing pipelines that often have a
narrow window for clinical application. The
FDA and other regulatory bodies (EMA, PMDA,
and ICH) have acknowledged this changing
landscape by allowing the use of alternate,
rapid microbiological methods (RMMs) for
these new technologies (Table 2) through
amendments to the 21 CFR 610.12 regulations
in 2012 and the US Pharmacopeial Convention
is developing a new chapter for rapid sterility
testing.24
Safety
RMMs are an increasingly common alternative
to the manual test and while none of these
methods will be universally applicable to all
products, they can help significantly reduce
testing and product release time as well as
having a higher sensitivity, throughput and
digital data output.24
Compendial safety testing of viral vector
products includes sterility (USP 71), endotoxin
and mycoplasma testing, as well as detection
of any replication-competent viruses, and
other adventitious agents.
Mycoplasma.
Conventionally,
mycoplasma
contamination is detected by culture and cell
indicator methods, which are time-consuming
- a minimum of 28 days to complete. Rapid
real-time PCR-based testing systems can
Reimbursement
Assay
Method
Instrument
ATP
Bioluminescence
Luciferase assay: measures the
ATP output of different
microorganisms
Biotrace 2000
Pallcheck Rapid System
Milliflex Rapid System
Celsis RapiScan
BioMAYTECTOR
Flow Cytometry
Detection of fluorescently
labeled viable microbial cells
after an initial 24–48 hrs
enrichment step
Bact-Flow
FACSMicroCount
Isothermal Microcalorimetry
Measures the thermal output
resulting from microbial
metabolism.
TAM III Calorimeter
Biocal 2000 Isothermal Calorimeter
48-challen isothermal
microcalorimeter
Nucleic Acid
Amplification
RT-PCR amplification of
cellular RNA using universal
primers; can be used with or
without growth-based
enrichment phase.
Multiple thermocyclers and
amplicor analyzers
Respiration
Instrumentation such as
respirometers, gaseous
headspace analyzers and
automated blood culture
systems can be used to detect
and enumerate respiring
microorganisms.
Solid Phase
Cytometry
Combines fluorescent labeling
and solid-phase laser scanning
cytometry to rapidly enumerate
viable microorganisms in
filterable liquids.
LOD
(cfu/mL)
Duration
103
30 mins
10-100
6-8 hrs
104
2-7 days
10-1000
2-4 hrs
Promex Microrespirator 4200
BACTEC System
BioLumix BacT/ALERT System
eBDS Pall System
TDLS
1-10
Overnight
– 7 days
ScanRDI® System
BioSafe PTS
MuScan System
1-10
2-3 hrs
Table 2. Summary the six proposed analytical platforms as alternatives to traditional assays for compendial rapid sterility
testing (adapted from Bonnevay, 2017).
© 2020 The Cell Culture Dish, Inc.
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provide results in a much shorter time-frame,
suitable for in-process testing as part of a
quality by design (QbD) approach for process
development workflows.
Endotoxin. The USP currently recognizes two
endotoxin tests, the Rabbit Pyrogen Test and
the Limulus Amebocyte Lysate (LAL) Test. The
Recombinant Factor C (rFC) test is available as
an alternative method and is currently under
evaluation by the USP.25
Replication Competent Virus (RCV). Viral vectors
are engineered to be replication defective,
therefore the risk of generating replicationcompetent virus is low. While the incidence is
low, these events can occur during any stage
of vector manufacturing by means of
recombination events within the production
cells and can increase with each successive
amplification. Therefore, RCV testing is
mandated by regulatory bodies to ensure the
biosafety of the final product. Cell lines
permissive to retro-, lenti- or AAV infection are
used to test for RCV in vitro. Southern blotting
or qPCR are also used to detect presence/
amplification of rep or cap sequences.
Stability
Stability studies are conducted during all
phases of drug development typically starting
at the preclinical stage of drug development
and continue through Phase I–Phase III clinical
trials to support formulation development, and
to satisfy the regulatory requirements for
clinical trials. In general, the shelf-life
specifications should be derived from the QC
release criteria, with additional emphasis on
the stability-indicating features of tests used
and tests/limits for degradation products.
Vector integrity, biological potency (including
transduction capacities) and strength are
critical product attributes which should always
be included in stability studies. A common
problem is a tendency for viral vector
aggregation and particle formation upon
extended storage after filling that can affect its
functionality. Techniques such as AUC, size
exclusion chromatography with multi-angle
laser light scattering (SEC-MALLS), TEM/CryoTEM and dynamic light scattering (DLS) to
study aggregation states of viral products as
part of stability testing.14
Multi-Angle Dynamic Light Scattering (MADLS)
is an advancement to DLS that is becoming
increasingly employed for aggregate detection.
Whereas DLS measurements occur at a single
fixed detection angle, MADLS measures the
sample at three angles. This combinatorial
© 2020 The Cell Culture Dish, Inc.
data results in less noise, improved resolution
and improved size accuracy for particle
distribution analysis.26
Tunable Resistive Pulse Sensing (TRPS) is
another high throughput platform that could
facilitate more accurate measurements of
viral vector aggregation. Particle size
measurement is based on impedance as they
pass through a nanopore, to generate an
accurate size distribution output (relative to a
known calibration standard) comparable to
TEM results.27
Looking Forward: Knowledge
Gaps to Address
The consensus amongst our experts that viral
vector manufacturing is essentially where
monoclonal antibody development was 20
years ago. While the uptake of PAT has been
slow, it is definitely gaining momentum with
stakeholders heavily invested in driving
analytical tool development forward. Certainly,
we can use the knowledge gained through the
antibody bioproduction scale-up process as a
roadmap for how to proceed with viral vectors.
The ability to leverage applicable technologies
can be advantageous to streamline process
development time.
Having real-time or near real-time analytical
assays to monitor key attributes like titer, capsid
content, and aggregation is needed to support
PAT. These rapid analytical tools will allow for
in-process monitoring that, when combined
with predictive analytics, can provide real-time
feedback controls to decrease in-process
development cycle times for product quality
improvements. Ultimately these methods can
increase the speed to market for a new
therapeutic. This can also be immensely helpful
in making decisions about the forward progress
of a viral vector batch if a particular attribute is
out of specification. The in-process data can
determine whether or not to continue a batch
run, which can help to minimize batch failures
and decrease the cost of goods.
Predictive analytics brings together data mining
(current and historical data) and machine
learning to generate predictive models about
what will happen during manufacturing
processes. As well, the incorporation of
automation with traditional analytical tools, such
as automated Western blots and ELISAs,
produces digital outputs that allow automatic
tracking and processing of data. This automation
not only decreases labor costs, but also enables
faster iterations during in-process development.
Management of the automated bioprocess, data
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collection and analysis by commercially available
computerized platforms has the potential to
advance the speed of process development and
optimization by providing real-time feedback
control. This is more suited to continuous,
scalable manufacturing workflows. Taken
together, these next generation analytical tools
can facilitate the creation of a flexible, efficient,
and cost-effective development supporting
flexible and streamlined manufacturing process
allowing therapeutics to reach the market faster
to meet patient needs.
References
1.
PhRMA (Mar. 10, 2020). Medicines in Development for Cell
and Gene Therapy 2020. Accessed Oct. 12, 2020:
Retrieved from https://www.phrma.org/Report/
Medicines-in-Development-for-Cell-and-GeneTherapy-2020
14. King, D., Schwartz, C., Pnicus, S. & Forsberg, N. (Oct. 10,
2018). Viral vector characterization: a look at analytical tools.
Accessed Oct. 12, 2020: Retrieved from https://
cellculturedish.com/viral-vector-characterizationanalytical-tools/
2.
Vicaire, F., Makowiecki, J. & Houts, C. Strategies To
address the viral vector manufacturing shortage. Accessed
Oct. 12, 2020: Retrieved from https://www.cellandgene.
com/doc/strategies-to-address-the-viral-vectormanufacturing-shortage-0001
15. Pennington, M. R. & Van de Walle, G. R. Electric
cell-substrate impedance sensing to monitor viral growth and
study cellular responses to infection with alphaherpesviruses
in real time. mSphere 2 (2017).
Analytics
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3. van der Loo, J. C. & Wright, J. F. Progress and challenges
in viral vector manufacturing. Human molecular genetics 25,
R42-52 (2016).
4. Lundgren, M. Meeting the process development challenges
of a diverse biologic pipeline. Accessed Oct.12, 2020:
Retrieved from https://www.cellandgene.com/doc/
meeting-the-process-development-challenges-of-adiverse-biologic-pipeline-0001
5.
US FDA. Cellular & gene therapy guidances. Accessed Oct.
12, 2020: Retrieved from https://www.fda.gov/
vaccines-blood-biologics/biologics-guidances/
cellular-gene-therapy-guidances
6. Jin, X. et al. Direct liquid chromatography/mass spectrometry
analysis for complete characterization of recombinant
adeno-associated virus capsid proteins. Hum Gene Ther
Methods 28, 255-267 (2017).
7.
Liu, A. P. et al. Characterization of adeno-associated virus
capsid proteins using hydrophilic interaction chromatography
coupled with mass spectrometry. Journal of pharmaceutical
and biomedical analysis 189, 113481 (2020).
8. Sastre Toraño, J., Ramautar, R. & de Jong, G. Advances in
capillary electrophoresis for the life sciences. J Chromatogr B
Analyt Technol Biomed Life Sci 1118-1119, 116-136 (2019).
9. Li, T., Malik, M., Yowanto, H. & Mollah, S. Purity analysis of
adeno-associated virus (AAV) capsid proteins using CE-SDS
method. Accessed Sep. 20, 2020: Retrieved from https://
sciex.com/Documents/tech%20notes/applications/
pharma/AAV-purity.pdf
10. Pacouret, S. et al. AAV-ID: A rapid and robust assay for
batch-to-batch consistency evaluation of AAV preparations.
Mol Ther 25, 1375-1386 (2017).
11. US FDA (Jan. 2011). Potency tests for cellular and gene
therapy products final guidance for industry: January 2011.
Accessed Sep. 9, 2020: Retrieved from https://www.
fda.gov/regulatory-information/search-fda-guidancedocuments/potency-tests-cellular-and-gene-therapyproducts
12. Taylor, S. C., Laperriere, G. & Germain, H. Droplet digital
PCR versus qPCR for gene expression analysis with low
abundant targets: from variable nonsense to publication
quality data. Sci Rep 7, 2409 (2017).
13. Dobnik, D. et al. Accurate quantification and characterization
of adeno-associated viral vectors. Frontiers in microbiology
10, 1570 (2019).
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16. Charretier, C. et al. Robust real-time cell analysis method
for determining viral infectious titers during development of a
viral vaccine production process. J Virol Methods 252,
57-64 (2018).
17. Moleirinho, M. G., Silva, R. J. S., Alves, P. M., Carrondo, M. J.
T. & Peixoto, C. Current challenges in biotherapeutic particles
manufacturing. Expert Opin Biol Ther 20, 451-465 (2020).
18. LI, T., Yowanto, H. & Mollah, S. (2019). Purity analysis of
adeno-associated virus (AAV) capsid proteins using CE-LIF
technology. Accessed Sep. 20, 2020: Retrieved from
https://sciex.com/Documents/tech%20notes/2019/
AAV-Purity-CE-LIF.pdf
19. Wright, J. F. Transient transfection methods for clinical
adeno-associated viral vector production. Hum Gene Ther 20,
698-706 (2009).
20. Grimm, D. et al. Titration of AAV-2 particles via a novel capsid
ELISA: packaging of genomes can limit production of
recombinant AAV-2. Gene Ther 6, 1322-1330 (1999).
21. Sommer, J. M. et al. Quantification of adeno-associated virus
particles and empty capsids by optical density measurement.
Mol Ther 7, 122-128 (2003).
22. Dorange, F. & Le Bec, C. Analytical approaches to
characterize AAV vector production & purification: advances
and challenges. Cell Gene Ther. Insights 4, 119-129 (2018).
23. LI, T. et al. (2020). Determination of full, partial and empty
capsid ratios for adeno-associated virus (AAV) analysis.
Accessed Sep. 20, 2020: Retrieved from https://sciex.
com/Documents/tech%20notes/2019/AAV-FullPartial-Empty.pdf
24. Bonnevay, T., Breton, R., Denoya, C. & Cundell, A. The
development of compendial rapid sterility tests.
Pharmacopeial Forum 43 (2017).
25. Pharmacopoeia, E. Recombinant factor C: new Ph. Eur.
chapter available as of 1 July 2020. Accessed Sep. 20,
2020: Retrieved from https://www.edqm.eu/en/news/
recombinant-factor-c-new-ph-eur-chapter-available-1july-2020
26. Shrourou, A. (Jun.24, 2020). Introducing MADLS as a new
approach to light scattering particle size analysis. Accessed
Sep. 20, 2020: Retrieved from https://www.azom.com/
article.aspx?ArticleID=18197
27. Yang, L. & Yamamoto, T. Quantification of Virus Particles
Using Nanopore-Based Resistive-Pulse Sensing Techniques.
Frontiers in microbiology 7, 1500 (2016).
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Securing Sustainable Reimbursement
for Innovative Gene Therapies
By: Jane F. Barlow
2017 was a watershed year, yielding approval
and launch of the first of an anticipated wave
of gene and genetically-engineered cell
therapies. These innovative treatments hold
the promise of a durable cure for a wide range
of rare to more common conditions, from
blindness and blood disorders to treatment of
Alzheimer’s and hyperlipidemia. Along with
the clinical innovation comes a hefty price tag.
In May 2019, Zolgensma launched at $2.1 million
for a one-time drug to gain the distinction as
the most expensive drug ever.1
But gene therapies create multiple financing
challenges for U.S. health care payers beyond
their price tag. Identifying and addressing these
hurdles is critical to fostering an environment of
sustainable coverage and reimbursement.
Consistent with the introduction of disruptive
technologies, innovative solutions are rising to
address concerns.
The insights into reimbursement of gene
therapies presented in this article explore
challenges and potential solutions based on the
U.S. healthcare landscape. While not directly
applicable to other nations, risks and solutions
identified here may be a starting point for
exploration of short and long-term sustainability
discussions for other countries.
Overview of the Financial
Challenges
Payers are confronted with a robust pipeline of
genetically-engineered cell and gene therapies
with over 700 products in development for over
180 indications. Estimates are that as many as
60 or more therapies will be approved for the
US market by the end of 2030.2 Many of these
treatments target an unmet need, providing
clinically meaningful treatment for conditions
that previously had no treatments or at least no
disease-modifying treatments. While these
breakthrough treatments may yield long-term
medical and societal cost off-sets, they will
undoubtedly carry a high upfront cost and
exacerbate financing challenges for payers.
At least three major financing challenges exist
for payers.
© 2020 The Cell Culture Dish, Inc.
Payment timing – Most treatments for chronic
conditions are administered and paid for over
time in periodic, mostly monthly, increments as
prescriptions are filled. And, the benefits of
these treatments accrue over time as well,
coupling in some respects, the treatment cost
with the benefit derived. Durable gene and cell
therapies are administered one time and
accrue benefits over time, creating a mismatch
between cost and the benefit derived.
In May 2019, Zolgensma
launched at $2.1 million for
a one-time drug to gain the
distinction as the most
expensive drug ever.1
Two concerns derive from this. The first is
planning for and absorbing the high one-time
cost. For some payers, this is feasible, but for
others, particularly smaller payers, unexpected
high cost treatments can disrupt the income
statement. The second concern is related to
patient mobility. The payer that reimbursed
the high cost treatment may not see the
benefit of cost off-sets over time if the member
moves to another health plan. The acquiring
payer reaps the benefit, without the cost,
creating an imbalance. Granted, that imbalance
may be corrected over time. As more patients
are treated with gene therapies, the likelihood
increases that the initial payer will enroll a
member that was treated by another plan. This
assumes that other plans are covering and
reimbursing gene therapies. And, for the most
part, the payer will need to take this on faith,
because there is no systematic means for them
to know of the prior treatment.
Performance uncertainty – Inherent in the high
cost of gene therapies is the promise of a
durable cure. Uncertainty regarding real-world
clinical efficacy and long-term durability of the
therapeutic benefit presents challenges for
payers. This is a particular concern as current
treatments have been studied in relatively
small numbers of patients over a relatively
short period of time. There is a real concern
that after some period, payers will be facing
additional costs for treatment or retreatment.
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Actuarial risk – The uncertainty of predicting
the number of patients receiving a cell or gene
therapy during a given benefit cycle introduces
risk to plan design and premium pricing. This is
exacerbated since the new therapies treat rarer
conditions. If the plan overestimates the
impact, they may price such that they are not
competitive in a market. If they underestimate,
they may not be able to cover their costs. More
importantly, these treatments may result in
volatility in the income statement.
The final concern, adverse selection, is not
unique to gene therapies but has ramifications
as payers define their coverage policies.
Adverse selection occurs when plans attract a
disproportionate share of sicker members or in
this case, patients eligible for gene therapies.
This may occur if one plan covers the treatment
or covers it more generously than other plans
in the market. Adverse selection ultimately
leads to higher costs and premiums, making
the plan less competitive.
Payers Experience Gene
Therapies Differently
The impact of high cost gene therapies and the
tools they employ vary by payer segment and
size. Most payers can be segmented into 4
groups, risk-bearing self-insured employers,
insurers and Managed Care Organizations
(MCOs), Medicare and government-stewarded
health systems, and Medicaid. Payers face
different challenges depending on their size,
financial strength and regulations that govern
their operations.
The scale of national insurers and Federal
Medicare plans with millions of members
reduces the impact of actuarial risk to their
plan. Further, these plans have the means to
absorb the impact of high one-time costs. But,
nearly half of the roughly 400 payers in the US
spread their risk over less than 50,000 lives.3
For self-insured employers, a 50,000-life plan is
a large plan. 61% of covered workers are enrolled
in a self-insured employer plan and many of
these plans are enroll 5,000 lives or less.4
Employer self-insured plans are able to define
what is covered or not covered by their plan.
Most plans cover FDA approved therapies, with
the exception of some categories like cosmetic
treatments. Plan sponsors can choose not to
cover medical or prescription drug charges for
gene therapy whether the therapy has received
FDA approval or not. Without new solutions to
address the concerns posed by gene therapies,
coverage and reimbursement may be stifled.
© 2020 The Cell Culture Dish, Inc.
State Medicaid plans compete for funding with
other mandated or obligated services. State
governments
do
not
generally
make
commitments outside their 1-2-year budget
cycle. And if unanticipated costs, like the cost
of a gene therapy are realized, other plan
services may need to be reduced to balance
the budget in a particular cycle.
Several states following the example of the
Oklahoma Health Care Authority, have gained
approval for state plan amendments that allow
them to engage in value-based agreements.
This gives the plan the opportunity to collect
supplement value-based rebates if they
contract with pharmaceutical companies for
outcomes based agreements.
Precision Financing Solutions5
address specific challenges
The unique challenges of gene therapies have
given rise to innovation in financing intended to
alleviate some of the concerns inherent in these
treatments. These are evolving as the industry
evolves, but generally fall into two categories.
Value-based solutions – These solutions
primarily address the risk of performance
uncertainty. They involve sharing risk based on
tying payment between the pharmaceutical
company and the payer to indicators of value
such as product performance and patient
outcomes. Examples include:
Milestone-based contracts – The payer pays
the agreed upon cost of the treatment at the
time of administration. The pharmaceutical
company refunds an agreed upon amount
for non-performance if specific milestones
or outcomes are not met. These contracts
may be short term (one year or less) or
multi-year and incorporate one or more
outcome measures. This solution addresses
performance uncertainty.
Performance-based annuities – This solution
spreads payment over several years and ties
future payment to the performance of the
therapy. The payer makes a partial payment
at the time of treatment. Further payments
are tied in whole or in part to pre-defined
outcomes or performance measures. Due to
its multi-year time period, additional patient
tracking, pricing regulation and accounting
issues may limit its use. This solution
addresses performance uncertainty and
aspects of payment timing and actuarial
uncertainty by spreading the payments over
multiple years.
Insights on Successful Gene Therapy Manufacturing and Commercialization
Page 43
Reimbursement
Warranty solutions - Under this model, the
pharmaceutical company purchases a
warranty for the product at the time of
treatment. Much like a car warranty, if over
time the product under performs, the
warranty would kick in to cover needed
treatments to address the gap in
performance. This solution addresses
performance uncertainty for the payer and
establishes a certain price for the developer
as the discount accounted for by the
warranty purchase is known up-front.
Subscription7 or capitated solutions –
Under a subscription or capitated payment
model, the developer would provide
treatment for a set fee regardless of the
number of patients treated or a set price
per patient. This model is associated with
more prevalent conditions like treatment
of hepatitis C or hyperlipidemia. It
addresses the actuarial risk by fixing costs
and may address payment timing
depending on the time period of the
agreement.
Risk carve-out solutions – These solutions
address actuarial risk, payment timing and
performance uncertainty by carving out
these patients or treatments from the plan.
The plan may pay a set fee, usually per
member per month, to cover the cost of
managing these patients. While attractive in
concept, payers generally pay a premium for
shifting risk to others, making these solutions
over time more costly on average than
bearing the risk themselves.
Examples of both value-based and risk-based
carveout solutions have emerged with approval
of the earliest products. Although not always
publicly disclosed, milestone-based and
performance-based annuity agreements for
cell and gene therapies are in place today. At
least 4 national insurers are offering programs
to their members to carve out gene therapy
treatments for a per member per month
charge. Another intermediary has developed
the warranty approach. Finally, third party
administrators are stepping up to assist
pharmaceutical companies and payers with
management of the data.
Foreword
Author Bios
Introduction
Regulatory
Upstream
Manufacturing
Downstream
Manufacturing
Analytics
Reimbursement
Reinsurance and Stop loss – These products
exist today. Reinsurance is purchased by an
insurance company to pass on all or part of
their risk to another insurance organization.
Self-funded employers may purchase stoploss insurance to protect against large
individual claims or higher than expected
claims overall. These products help payers
manage actuarial risk and smooth the
impact of very high cost treatments.
Companies that offer reinsurance and stop
loss are actively evaluating their offerings in
light of these new treatments.
Orphan Reinsurer and Benefit Manager
(ORBM) – The ORBM is a risk pooling,
service solution proposed by MIT FoCUS6
to manage actuarial risk and executional
challenges associated with making gene
therapies available to patients, including
risk pooling, contracting, reimbursement
and care coordination.
Conclusion
The vast majority of payers cover and reimburse
gene therapies today. They are actively engaged
in understanding the challenges generated by
these innovative treatments and as a result, new
paradigms in contracting and benefit design
have resulted. As payers continue to assess this
space, price and value must be linked. Payers
will continue to seek and implement new
solutions that ensure sustainability for all plan
members as they transition payment models to
address the challenges of one-time high cost
gene therapy treatments. The good news is that
clinical innovation is spurring reimbursement
innovation in products and services to address
the uncertainties inherent in coverage of gene
therapies and provide new avenues to ensure
sustained reimbursement.
References
1.
Stein, R. (2020). At $2.1 Million, New Gene Therapy Is The
Most Expensive Drug Ever. Accessed Sep. 24, 2020:
Retrieved from NPR.org,
5.
2.
MIT NEWDIGS research brief 2020F207-v51 pipeline analysis.
Accessed Sep. 25, 2020: Retrieved from https://
newdigs.mit.edu/sites/default/files/NEWDIGSResearch-Brief-2020F207v51-PipelineAnalysis.pdf
6. MIT NEWDIGS research brief 2018F205-v021-ORBM.
Accessed Retrieved from https://newdigs.mit.edu/sites/
default/files/FoCUS Research Brief 2018F205v021.pdf
3. MMIT payer data Accessed Jan. 2020:
4. Kaiser family foundation 2019 employer health benefits
survey. Accessed Sep. 25, 2020:
© 2020 The Cell Culture Dish, Inc.
7.
MIT NEWDIGS precision financing solutions toolkit. Accessed
Sep. 25, 2020: Retrieved from www.payingforcures.org/
toolkit-overview/precision-financing-solutions/
Trusheim, M., Cassidy, W. & Bach, P. Alternative state-level
financing for hepatitis C treatment—The “Netflix model”
JAMA 320, 1977-1978 (2018).
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