insights on successful gene therapy manufacturing and
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Insights on Successful Gene Therapy Manufacturing and CommercializationInsights 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.
Insights on Successful Gene Therapy Manufacturing and Commercialization Page i© 2020 The Cell Culture Dish, Inc.
Foreword
Author Bios
Introduction
Regulatory
Upstream Manufacturing
Downstream Manufacturing
Analytics
Reimbursement
Foreword
Gene Therapy - An Industry Coming of AgeIn 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.
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 ii© 2020 The Cell Culture Dish, Inc.
Foreword
Author Bios
Introduction
Regulatory
Upstream Manufacturing
Downstream Manufacturing
Analytics
Reimbursement
Forewordsuccesses, 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 iii© 2020 The Cell Culture Dish, Inc.
Foreword
Author Bios
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Downstream Manufacturing
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Reimbursement
Author BiosJane F. Barlow, MD, MBA, MPHChief Clinical Officer Real Endpoints
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-FrickeHead 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, PhDDirector, 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.
Tony Hitchcock Technical Director Cobra Biologics
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 KingScientific 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 KrishnanVice 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 iv© 2020 The Cell Culture Dish, Inc.
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Downstream Manufacturing
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Reimbursement
Author BiosRachel Legmann PhDDirector, Technical Consultancy - Gene Therapy and Viral Vectors 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, PhDScientific 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 SargentEditor 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.
Mark Schofield PhDSenior R&D Manager Pall Corporation
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 StarttDirector 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.
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Foreword
Author Bios
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Upstream Manufacturing
Downstream Manufacturing
Analytics
Reimbursement
Introduction
Insights on Successful Gene Therapy Manufacturing and CommercializationBy: Brandy Sargent, Jane Barlow, Clive Glover, Tony Hitchcock
Industry OutlookThe 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
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
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.
IndicationsPerhaps 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
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.
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Introductionantigen 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 antigen-expressing 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 CommercializationAs 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 ManufacturingGene 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
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 vector-manufacturing 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 ProcessMany 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
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Introductionsubstrates 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.
ScalabilityScale 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 ManufacturingIt 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.
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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IntroductionCompanies 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 ProcessesViral 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 ControlsAseptic 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 ConsiderationsCurrently 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
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IntroductionHowever, 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.
Reimbursement StrategiesGene 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.
Supply Chain SecurityThe 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.
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
Image courtesy of Cobra Biologics
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Introductionin 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.
Provider and Patient Education and AccessIt 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
framework for a suitable Target Product Profile. Initial and ongoing stakeholder education is a critical part of any commercialization plan.
In ClosingIt 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-annual-report/
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/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics
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. 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-of-contract-development-and-manufacturing-for-cell-and-gene-therapies#:~:text=With%20traditional%20biologics%2C%20approximately%2035,65%25%20or%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%202019_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-chain-risks-cell-gene-therapy
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A Remarkable Year for Gene TherapiesMaturing 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.
FDA Prepares for Increasing Reviews and ApprovalsTo 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 MaturingIn 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 ProgressThere has been a reasonably widespread perceived leniency, particularly for the review of one-time therapies for rare diseases.
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
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.
High-dose AAV Toxicity Called into QuestionWhile 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 ChallengesPerhaps the most nerve-wracking aspect of gene therapy development is that while product and process understanding and regulatory frameworks are maturing, there are
Scott Gottlieb, the former FDA commissioner, predicted that by 2025, the US would be approving between 10 and 20 gene therapies each year.
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Regulatorystill 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 ValidationAs 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 ChallengesIn 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.
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 ValidationProcess 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
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Regulatoryprocess 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)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.
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
titer, infectivity, HCP, residual DNA, aggregates, etc. Like all product attributes, product characterization must continue to become more robust as product development advances.
ConclusionThe 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 much-needed regulatory framework is rapidly developing. Ultimately, gene therapy developers will continue to make progress in product consistency and understanding so that the life-enhancing and life-saving promises of gene therapies can be realized.
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/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products
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-gear-2020-despite-hurdles-experts-say
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/press-announcements/fda-continues-strong-support-innovation-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/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics
5. 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-therapy-guidances-unveils-a
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-gene-therapy-raises-durability-questions
7. 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/gene-therapy-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-guidance-documents/potency-tests-cellular-and-gene-therapy-products
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.
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Upstream Manufacturing of Gene Therapy Viral VectorsBy: Steve Pettit, Clive Glover, Tony Hitchcock, Rachel Legmann, Don Smartt
OverviewThe 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 re-infusion 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 vivo-directly 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
for convenience, or by necessity in order to avoid toxicity of a vector component to producer cells (Figure 1).
293 Cell
pTransgene VectorpHelper Vectors
AAV ssDNA
AAV Extraction Solution
AAV2 Particles
Transcription & TranslationReplication
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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 useRetroviral (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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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 doubled-with 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 long-lasting depending on immunogenicity
Table 1. Overview of common viruses used for generating gene therapy viral vectorsa) Recent AAV engineering efforts have demonstrated that AAV capacity can be doubled via the use of split vectors.9,10
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Upstream ManufacturingOverview of viral vector manufacturingThe 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
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
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 time-consuming 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
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Clarification
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Harvest
Formulation
Concentration
Purification
Purification
Isolate & Enrich
Viral Vector Manufacturing
CellProcessing
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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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Upstream Manufacturinggenerally 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
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 lineMany 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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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 vectorThe 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
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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Upstream Manufacturingproduction of virus at small scale. When larger scales are required, processes developed in the iCELLis Nano bioreactor can be easily scaled-up 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
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.
Bed Size Surface Area/m2 Equivalent Units
Bioreactor Bed Diameter (cm)
Bed Height(cm)
Carrier Compaction Low-High
850-cm2 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
Table 2. Scalability options for the iCELLis Nano and 500+ bioreactors.
Image courtesy of Pall Corporation
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Upstream ManufacturingRocking 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 vectorWhile 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.
Figure 4. Allegro STR 50, 200, 500, 1000, and 2000 L bioreactors.
Image courtesy of Pall Corporation
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Upstream ManufacturingScaling of suspension cells for manufacturing vectorA 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 small-scale bioreactors.62,63
Current challenges and limitations of suspension cell cultureIt 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 cell-specific 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
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 considerationsVector 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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Upstream Manufacturingsystems 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 replication-competent 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 determinantProduction 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
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 long-term 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 considerationsRequired 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.
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.
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%
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Upstream ManufacturingFor 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 considerationsThe 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 well-established 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
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.
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Upstream ManufacturingRoom for ImprovementThere 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 processingOne 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.
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.
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Upstream ManufacturingThere 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
transfection process as gentle mixing and delivering DNA rapidly to the cell within a few minutes are critical parameters.39 New pump and mixing technology has allowed transfection at some increased scale, without a loss of productivity.39 Challenges remain for larger multi-hundred liter 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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28. Madhan, S., Prabakaran, M. & Kwang, J. Baculovirus as vaccine vectors. Curr Gene Ther 10, 201-213 (2010).
29. Wang, S. & Balasundaram, G. Potential cancer gene therapy by baculoviral transduction. Curr Gene Ther 10, 214-225 (2010).
30. Volkman, L. E. & Goldsmith, P. A. In vitro survey of Autographa californica nuclear polyhedrosis virus interaction with nontarget vertebrate host cells. Appl Environ Microbiol 45, 1085-1093 (1983).
31. Tani, H. et al. In vitro and in vivo gene delivery by recombinant baculoviruses. J Virol 77, 9799-9808 (2003).
32. Ho, Y. C., Chung, Y. C., Hwang, S. M., Wang, K. C. & Hu, Y. C. Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells. J Gene Med 7, 860-868 (2005).
33. Ramachandra, C. J. et al. Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors. Nucleic Acids Res 39, e107 (2011).
Insights on Successful Gene Therapy Manufacturing and Commercialization Page 22© 2020 The Cell Culture Dish, Inc.
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Upstream Manufacturing34. Luo, W. Y. et al. Development of the hybrid Sleeping Beauty:
baculovirus vector for sustained gene expression and cancer therapy. Gene Ther 19, 844-851 (2012).
35. Urabe, M., Ding, C. & Kotin, R. M. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 13, 1935-1943 (2002).
36. Lesch, H. P. et al. Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors. Gene Ther 18, 531-538 (2011).
37. Park, J. et al. Progressing from transient to stable packaging cell lines for continuous production of lentiviral and gammaretroviral vectors. Curr Opin Chem Eng 22, 128-137 (2018).
38. Sanber, K. S. et al. Construction of stable packaging cell lines for clinical lentiviral vector production. Sci Rep 5, 9021 (2015).
39. Pall Biotech. Current observation.
40. Gama-Norton, L. et al. Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells Hum Gene Ther 10, 1269-1279 (2011).
41. Thermo Fisher. Accessed Retrieved from https://www.thermofisher.com/order/catalog/product/R70007
42. National Inst. for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) (Nov. 2018). Gene Therapy Roadmap. Accessed Oct. 10, 2020: Retrieved from https://niimbl.force.com/s/gene-therapy-roadmap
43. Bae, D. H. et al. Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region. Mol Ther Methods Clin Dev 18, 631-638 (2020).
44. US FDA. Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs)-Guidance for industry. Accessed Retrieved from https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug
45. Segura, M. M., Mangion, M., Gaillet, B. & Garnier, A. New developments in lentiviral vector design, production and purification. Expert Opin Biol Ther 13, 987-1011 (2013).
46. Spier, R. in Animal Cell Biotechnology, Vol. 1 (eds Spier, RE & Griffiths, JB) (Academic Press, 1985).
47. Merten, O. W. et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 22, 343-356 (2011).
48. Allay, J. A. et al. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther 22, 595-604 (2011).
49. Okada, T. et al. Large-scale production of recombinant viruses by use of a large culture vessel with active gassing. Hum Gene Ther 16, 1212-1218 (2005).
50. Pakos, V. & Johansson, A. Large scale production of human fibroblast interferon in multitray battery systems. Dev Biol Stand 60, 317-320 (1985).
51. Kutner, R. H., Puthli, S., Marino, M. P. & Reiser, J. Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography. BMC Biotechnol 9, 10 (2009).
52. Ghani, K., Cottin, S., Kamen, A. & Caruso, M. Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media. Gene Ther 14, 1705-1711 (2007).
53. Merten, O. W. State-of-the-art of the production of retroviral vectors. J Gene Med 6 Suppl 1, S105-124 (2004).
54. Sheu, J. et al. Large-scale production of lentiviral vector in a closed system hollow fiber bioreactor. Mol Ther Methods Clin Dev 2, 15020 (2015).
55. Lennaertz, A., Knowles, S., Drugmand, J.-C. & Castillo, J. Viral vector production in the integrity® iCELLis® single-use fixed-bed bioreactor, from bench-scale to industrial scale. BMC Proc. 7, 59 (2013).
56. Lennaertz, A., Knowles, S., Drugmand, J.-C. & Castillo, J. Viral vector production in the integrity® iCELLis® single-use fixed-bed bioreactor, from bench-scale to industrial scale. BMC Proc. 7, 59 (2013).
57. Pall Biotech. Application Note: iCEllis 500+ single-use Q series 333 m2 bioreactor cell culture validation study.
58. Gene therapy company (name not disclosed). Internal data.
59. Barrett, P. N., Mundt, W., Kistner, O. & Howard, M. K. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines. Expert Rev Vaccines 8, 607-618 (2009).
60. Snyder, R., Glover, C. & Vaidya, R. Accelerating The Development of Viral Vector Manufacturing Processes. Accessed Retrieved from https://www.pharmasalmanac.com/articles/accelerating-the-development-of-viral-vector-manufacturing-processes
61. Dormond, E. et al. An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection. Biotechnol Bioeng 102, 800-810 (2009).
62. Bauler, M. et al. Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media. Mol Ther Methods Clin Dev 17, 58-68 (2020).
63. Manceur, A. P. et al. Scalable lentiviral vector production using stable HEK293SF producer cell lines. Hum Gene Ther Methods 28, 330-339 (2017).
64. Pall Biotech. Cost modeling of upstream production process of lentiviral vectors in HEK-293 cells comparing multi-tray 10 stacks and fixed-bed bioreactor. Poster ESACT 2017 (2017).
65. McCarron, A., Donnelley, M. & Parsons, D. Scale-up of lentiviral vectors for gene therapy: advances and challenges. Cell GeneTherapy Insights 3, 719-729 (2017).
66. White, M., Whittaker, R., Gandara, C. & Stoll, E. A. A guide to approaching regulatory considerations for lentiviral-mediated gene therapies. Hum Gene Ther Methods 28, 163-176 (2017).
67. Ausubel, L. J. et al. Production of CGMP-Grade Lentiviral Vectors. Bioprocess Int 10, 32-43 (2012).
68. Rodrigues, T., Carrondo, M. J., Alves, P. M. & Cruz, P. E. Purification of retroviral vectors for clinical application: biological implications and technological challenges. J Biotechnol 127, 520-541 (2007).
69. Gao, K. et al. Empty virions In AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side-efffects. Mol Ther Methods Clin Dev 1, 20139 (2014).
70. Zeltner, N., Kohlbrenner, E., Clément, N., Weber, T. & Linden, R. M. Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors. Gene Ther 17, 872-879 (2010).
71. Joshi, P. R. H. et al. Achieving high-yield production of functional AAV5 gene delivery vectors via fedbatch in an insect Cell-One baculovirus system. Mol Ther Methods Clin Dev 13, 279-289 (2019).
72. Adamson-Small, L. et al. A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform. Mol Ther Methods Clin Dev 3, 16031 (2016).
73. Piras, B. A. et al. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector. Mol Ther Methods Clin Dev 3, 16015 (2016).
74. CRS. CRS-Chemical inspection and regulation service -REACH Restriction. Accessed Retrieved from http://www.cirs-reach.com/REACH/REACH_Restriction.html.
75. Cameau, E. & Capone, J. Evolution of culture production systems for viral vector production: advantages, challenges and cost considerations. Cell Gene Ther. Insights 6, 225-230 (2020).
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Downstream Manufacturing of Gene Therapy VectorsBy: Steve Pettit, Clive Glover, Tony Hitchcock, Rachel Legmann, Mark Schofield
IntroductionThe 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 VectorsViral 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
VirusSize (nm)
Envelope StabilityBuoyant Density
Adenovirus ~90 No High 1.34 (CsCl)
AAV ~ 20 No High 1.41 (CsCl)
Retrovirus/Lentivirus
~90-120 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,
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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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.
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.
Harvest Recovery Clarification
Secreted Viruses Intracellular Viruses
Concentration Buffer Exchange
Concentration Buffer Exchange Formulation
Purification Polishing
Cell Collection (optional)
Cell Lysis
Filtration/Microfiltration
Ultrafiltration/Diafiltration
Ultrafiltration/Diafiltration
Chromatography Step 1
Chromatography Step 2
Polishing Step
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
chromatography (IEC) step for the separation of empty and full capsids. LV purification, on the other hand, employs mostly one, sometimes two chromatography steps.
Cell lysis, clarification, and filtrationCell 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
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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The limitation of classical centrifuge methodsPurification 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
ChromatographyChromatographic 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
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 pre-packed, such as the Mustang® Q Membrane
Mustang® XT Capsules - Image courtesy of Pall Corporation
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Downstream Manufacturing(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
Affinity chromatographyAffinity 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.
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
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
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 protein-based affinity resins adds cost to their use.
Ion exchange chromatographyIon 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
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
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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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 chromatographySize 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 chromatographyHydrophobic 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.
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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, scale-up, 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
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 ControlBioburden 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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Downstream ManufacturingInsights on downstream process issuesMoving a downstream process to clinical and commercial manufacturingTransfer 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.
ScalabilityOne 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 centrifugation-based 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.
GMP ComplianceResearch 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
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.
Image courtesy of Pall Corporation
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Downstream ManufacturingCosts considerations 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 multi-column 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 AssessmentAnother important consideration is an overall evaluation of vector risks (risk assessment) and subsequent design of the upstream and
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 non-human 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 YieldYield 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.
PurityAnother 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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Downstream Manufacturingand 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.
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.
For AAV, the process development time for the empty/full capsid separation can be a months-long 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 advancementAffinity chromatographyAC 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 protein-based 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.
Automation and closed systemsAutomation 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.
Move toward more efficient methodsThe 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
AnalyticsOn 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 toolsMany 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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Downstream Manufacturingof 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
designed for use with 100’s of gram of antibody-compared to viruses at < 1 g for small scale. Thus increased virus-specific tools are needed for both small scale and large scale processing.
SummaryIn 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
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3. Merten, O. W. AAV vector production: state of the art developments and remaining challenges. Cell Gene Ther. Insights 2, 521-551 (2016).
4. Gao, K. et al. Empty virions In AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side-effects. Mol Ther Methods Clin Dev 1, 20139 (2014).
5. Wright, J. F. AAV empty capsids: for better or for worse? Mol Ther 22, 1-2 (2014).
6. Carmo, M. et al. Relationship between retroviral vector membrane and vector stability. J Gen Virol 87, 1349-1356 (2006).
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8. Ausubel, L. J. et al. Production of CGMP-Grade Lentiviral Vectors. Bioprocess Int 10, 32-43 (2012).
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11. Benchaouir, R. Recent advances in the purfication of adeno-associated virus. Cell Gene Ther. Insights 2, 287-297 (2016).
12. Dias Florencio, G. et al. Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors. Mol Ther Methods Clin Dev 2, 15024 (2015).
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14. GE Healthcare (2018). Application Note: Optimization of midstream cell lysis and virus filtration steps in an adenovirus purification process. Accessed Oct. 20, 2020: Retrieved from https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid= 10061&destinationid=10016&assetid=26781
15. Merten, O. W. et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 22, 343-356 (2011).
16. Geraerts, M., Michiels, M., Baekelandt, V., Debyser, Z. & Gijsbers, R. Upscaling of lentiviral vector production by tangential flow filtration. J Gene Med 7, 1299-1310 (2005).
17. Cooper, A. R. et al. Highly efficient large-scale lentiviral vector concentration by tandem tangential flow filtration. J Virol Methods 177, 1-9 (2011).
18. Potter, M., Chesnut, K., Muzyczka, N., Flotte, T. & Zolotukhin, S. Streamlined large-scale production of recombinant adeno-associated virus (rAAV) vectors. Methods Enzymol 346, 413-430 (2002).
19. Zhao, M. et al. Affinity chromatography for vaccines manufacturing: Finally ready for prime time? Vaccine (2018).
20. Silva, R., Mota, J., Peixoto, C., Alves, P. & Carrondo, M. Improving the downstream processing of vaccine and gene therapy vectors. Pharm. Bioprocess. 3, 489-505 (2015).
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 heparin affinity chromatography. Biotechnol Bioeng 90, 391-404 (2005).
25. Mietzsch, M., Broecker, F., Reinhardt, A., Seeberger, P. H. & Heilbronn, R. Differential adeno-associated virus serotype-specific 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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Downstream Manufacturing30. Thermo Fisher. Accessed Retrieved from http://
thermofisher.mediaroom.com/press-releases?item=122399
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).
32. Khandelwal, P. (2015). (Bulletin 6807)Practical guide: selecting theoptimal resins for adenovirus process purificatioin. Accessed Oct. 21, 2020: Retrieved from https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_6807.pdf
33. Wright, J. F. & Zelenaia, O. Vector characterization methods for quality control testing of recombinant adeno-associated viruses. Methods Mol Biol 737, 247-278 (2011).
34. Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12, 342-347 (2006).
35. Okada, T. et al. Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes. Hum Gene Ther 20, 1013-1021 (2009).
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 adeno-associated 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 implications and technological challenges. J Biotechnol 127, 520-541 (2007).
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).
41. Shambram, P., Vellekamp, G. & Scandella, C. in Adenoviral vectors for gene therapy (Academic Press, 2016).
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 Methods 139, 61-70 (2007).
43. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. & Yee, J. K. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90, 8033-8037 (1993).
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 capsule. Bioproc J 2, 89-95 (2003).
47. Hernandez, R. (Apr. 1, 2015). Continuous manufacturing: A changing processing paradigm. Accessed Oct, 22, 2020: Retrieved from https://www.biopharminternational.com/view/continuous-manufacturing-changing-processing-paradigm
48. Wright, J. F. Product-related impurities in clinical-grade recombinant AAV vectors: characterization and risk assessment. Biomedicines 2, 80-97 (2014).
49. Jacquemart, R. et al. A Single-use Strategy to Enable Manufacturing of Affordable Biologics. Comput Struct Biotechnol J 14, 309-318 (2016).
50. Zolotukhin, S. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6, 973-985 (1999).
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AnalyticsEvolution of Viral Vector Analytics for Gene Therapy ManufacturingBy 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
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
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 AnalyticsAs 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
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Analyticsyields 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 AssaysIn 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
IdentityGenetic 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.
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Table 1. Examples of current and next generation analytical assays used to assess viral vector CQAs.
Quality Attribute Example Assays Next Generation Assays
Identity
Genetic identity Genome sequencing (NGS)PCR
Protein identity SDS-PAGEMass spectrometry (MS)Western blot (immunoblot)
CE-SDS
Automated Western blot
Strength/Potency
Physical viral titer ELISAqPCROptical density (A260/280)NanoSightHPLC (AAV packaging ratio)
MADLSddPCR
CE-SDS or CE-LIF (AAV packaging ratio)
Functional viral titer Plaque-forming assayFluorescence foci assayTCID50 (end point dilution assay)
ECIS - impedance
Purity
Process impurities (detergents, resin)
MSChromatographyTEM
LC-MS
Host cell-related impurities Host cell DNA/RNA: Picogreen, DNA Threshold assay, qPCRHost cell proteins: SDS-PAGE, ELISA, HPLC, TEM
LC-MS, LC-MS/MS
Capsid content (empty: full capsids)
ELISA/qPCRHPLCMSTEMAUC
CE-LIF, cIEF (isoelectric focus)LC-MS
Safety
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 blottingqPCR
Adventitious agents In vivo and in vitro cellular assays
Stability
pH Potentiometry
Osmolality Osmometry
Aggregate formation Light microscopyDLSSEC-MALLSTEMAUCFFF-MALS
MADLSTRPS
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
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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AnalyticsNovel methods, such as AAV-ID, where differential thermostability is used to distinguish AAV serotypes and HILIC-FLR-MS 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/PotencyAccording to the FDA 21 CFR 210.3(b)(16) guidance, strength is defined as:
1. The concentration of the drug substance (for example, weight/weight, weight/volume, or unit dose/volume basis), and/or
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).
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 PCR-based assays because they are robust, easy, fast, and convenient. In the industry, there is a
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 in-process 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 time-consuming 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 label-free 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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Analyticsgreatly improves time-to-result as well as being a more robust and rapid real time quantification of virus infectivity over traditional cellular assays.
PurityViral 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
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. Vector-related 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 vector-related 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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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)
SafetyCompendial 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.
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
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
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
Assay Method Instrument LOD (cfu/mL) Duration
ATP Bioluminescence
Luciferase assay: measures the ATP output of different microorganisms
Biotrace 2000Pallcheck Rapid SystemMilliflex Rapid SystemCelsis RapiScan BioMAYTECTOR
103 30 mins
Flow Cytometry
Detection of fluorescently labeled viable microbial cells after an initial 24–48 hrs enrichment step
Bact-Flow FACSMicroCount 10-100 6-8 hrs
Isothermal Micro-calorimetry
Measures the thermal output resulting from microbial metabolism.
TAM III Calorimeter Biocal 2000 Isothermal Calorimeter 48-challen isothermal microcalorimeter
104 2-7 days
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 10-1000 2-4 hrs
Respiration
Instrumentation such as respirometers, gaseous headspace analyzers and automated blood culture systems can be used to detect and enumerate respiring microorganisms.
Promex Microrespirator 4200 BACTEC System BioLumix BacT/ALERT System eBDS Pall System TDLS
1-10 Overnight – 7 days
Solid Phase Cytometry
Combines fluorescent labeling and solid-phase laser scanning cytometry to rapidly enumerate viable microorganisms in filterable liquids.
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).
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Analyticsprovide 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 replication-competent 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.
StabilityStability 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/Cryo-TEM 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
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 AddressThe 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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Analyticscollection 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-Gene-Therapy-2020
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-vector-manufacturing-shortage-0001
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-a-diverse-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-guidance-documents/potency-tests-cellular-and-gene-therapy-products
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).
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-characterization-analytical-tools/
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).
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-Full-Partial-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-1-july-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 TherapiesBy: 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 ChallengesPayers 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.
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.
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.
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
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ReimbursementActuarial 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 DifferentlyThe 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.
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 challengesThe 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.
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ReimbursementWarranty 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.
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.
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 stop-loss 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.
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.
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.
ConclusionThe 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,
2. MIT NEWDIGS research brief 2020F207-v51 pipeline analysis. Accessed Sep. 25, 2020: Retrieved from https://newdigs.mit.edu/sites/default/files/NEWDIGS-Research-Brief-2020F207v51-PipelineAnalysis.pdf
3. MMIT payer data Accessed Jan. 2020:
4. Kaiser family foundation 2019 employer health benefits survey. Accessed Sep. 25, 2020:
5. MIT NEWDIGS precision financing solutions toolkit. Accessed Sep. 25, 2020: Retrieved from www.payingforcures.org/toolkit-overview/precision-financing-solutions/
6. MIT NEWDIGS research brief 2018F205-v021-ORBM. Accessed Retrieved from https://newdigs.mit.edu/sites/default/files/FoCUS Research Brief 2018F205v021.pdf
7. Trusheim, M., Cassidy, W. & Bach, P. Alternative state-level financing for hepatitis C treatment—The “Netflix model” JAMA 320, 1977-1978 (2018).