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Otto-Wilhelm Merten, PhD - Généthon
Amine A. Kamen, PhD - McGill University

Lawrence C. Thompson, PhD
Principal Scientist and Group Leader, BioTherapeutics Pharmaceutical Sciences,
Analytical Research and Development, Pfizer, Inc.
Characterization of Stability-Indicating Assays for Viral Vectors


Assay development for control of biotherapeutic product quality must include methods used to demonstrate stability of the product during manufacturing, shipping, storage, and administration. Said methods must be capable of detecting product deterioration and therefore be recognized as stability-indicating assays. For viral vectors, these are often cell-based bioassays which evaluate critical quality attributes like infectivity and/or protein expression. It is desirable to have at least one physicochemical technique in place that gives an analogous assessment of product quality but also quantifies the molecular attributes that are being modified. For adenoviruses, this is often an ion-exchange high-performance liquid chromatography (IEX-HPLC) method which can detect changes in the intact particle as well as quantify product related impurities/degradants. Because these methods have the ability to monitor multiple attributes that change concurrently, they are very powerful analytical tools; however, linking these attributes to alterations in the adenovirus structure and/or its proteins can be challenging. Here we present a characterization strategy for the identification of adenovirus molecular modifications and product related impurities that are correlated with the IEX-HPLC readout. Furthermore, these heightened characterization tools can reveal additional, unpredicted amino acid degradation hot-spots that are crucial to capsid stability.


Lawrence (Larry) C. Thompson, PhD is a Principal Scientist in Analytical Research and Development within BioTherapeutic Pharmaceutical Sciences at Pfizer. He has been with Pfizer for four years and is currently analytical lead on viral and plasmid-based immunotherapeutics. Previously, he spent three years in small biotech at two different companies as the analytical lead in the development of serum-based cancer diagnostics. He is a graduate of the University of Tennessee (Chattanooga) with a BS in Chemistry in 2001, and Vanderbilt University with a PhD in Biochemistry in 2006. He did his postdoctoral work at the University of Tennessee (Knoxville) from 2006–2010. His education and research path covers a wide breadth including small molecule synthesis; protein cloning, expression and purification; enzyme structure/function determination; protein-protein interaction investigation; serum antigen identification and mAb production/selection; and virus/plasmid method development and characterization (to name a few), generating a number of peer reviewed publications and presentations at scientific conferences as well as internally within Pfizer.

Steven E. Pincus, PhD
Associate Vice President Virology and Analytical Methods Development
FUJIFILM Diosynth Biotechnologies Texas, LLC
A Suspension Vero Cell Line for Production of Viral Vaccines and Viral Therapeutics


As the acceptance of viral vectors as a delivery system for therapeutics grows, biomanufacturers are looking for an alternative to the classical adherent cell production models. Adherent cell lines such as Vero and Madin Darby canine kidney (MDCK) are licensed for vaccine production, but they require large volumes of media, specialized large-scale adherent culture vessels with a large working footprint, and the equipment to support them. Suspension cell lines, such as Chinese hamster ovary (CHO) and HEK-293 are licensed for antibody production, but have not yet been licensed for vaccine production. A mammalian cell line that can simultaneously grow in suspension, support many types of viruses and viral vectors, and produce high virus titers would be of great value for production of vaccines and viral therapeutics.

Fujifilm Diosynth Biotechnologies Texas (FDBT) is a contract development and manufacturing organization that specializes in the production of viral therapeutics and vaccines. We have employed the use of a Vero cell line, which is permissive to a wide array of viruses, to create a platform for virus and viral vector growth. We have adapted this adherent Vero cell line to a serum-free, fast growing suspension culture. We have evaluated the ability of the adherent and suspension cell lines to amplify viruses and viral vectors such as influenza virus, adeno-associated virus, and adenovirus. This proprietary suspension Vero line will be an excellent asset to viral vector and vaccine manufacturing, as it will allow the growth of commonly difficult-to-scale vectors. FDBT has also developed a wide variety of analytical methods to evaluate the growth of virus and viral vectors which are commonly used to support the numerous manufacturing stages of vaccine production. These include analyses designed to identify (PCR, ELISA), quantify (FACS, immunostaining, plaque assay), and verify the purity (HPLC, SDS-PAGE/Western, ELISA) of our products.


Steven Pincus is Associate Vice President of Virology and Analytical Development at Fujifilm Diosynth Biotechnologies Texas where he leads teams involved in process development and analytical development for client driven programs in viral therapeutics and vaccines. He obtained his BS and PhD in Biochemistry from the State University of New York at Buffalo. His PhD thesis project involved studies on the mechanism of adenovirus DNA replication, and he gained experience growing, purifying, and titering multiple adenovirus serotypes. He obtained his postdoctoral training in the microbiology department at the State University of New York at Stony Brook under an NIH fellowship studying inhibitors of picornavirus replication, and gained experience in the growth, purification, and titration of poliovirus and the growth of other picornaviruses.

He then joined Virogenetics where he was Senior Scientist and Platform Leader in Molecular Biology and Microbiology. At Virogenetics he was involved in the development of highly attenuated poxvirus vaccine vectors (NYVAC, ALVAC, TROVAC) and developed vaccine candidates for measles virus, flaviviruses (JEV, dengue, yellow fever), human cytomegalovirus, and therapeutic vaccines against several cancers along with development of molecular assays necessary for releasing clinical lots. At Elusys Therapeutics he was Senior Director Virology and Animal Biology and received $1.6 million NIH grant funding to investigate the use of the Elusys antibody heteroplymer technology as a therapeutic for vaccinia vaccination complications. He developed animal models and release assays for heteropolymer clinical trials and supported the development of an anthrax anti-toxin monoclonal that is funded by the Biomedical Advanced Research and Development Authority (BARDA). He then joined Novavax, Inc., a biotech focused on the development of vaccines against influenza and respiratory syncytial virus (RSV), based on virus-like particle (VLP) platform vaccine technology. There he was responsible for analytical development, quality control and quality assurance, and recombinant baculovirus production. He was a key member of the team that secured a contract award valued at up to $179 million by BARDA for the advanced clinical and manufacturing development of recombinant vaccines for the prevention of seasonal and pandemic influenza. He has consulted for several companies in the areas of assay development, transfer, qualification, and validation for monoclonal antibody and vaccine projects.

Francesca Bellintani
Downstream Vector Development Manager, MolMed S.p.A.
Retroviral and Lentiviral Production in Disposable Bioreactor: Development
and Scaling Up of Upstream and Downstream Process Steps


MolMed is a medical biotechnology company focused on research, development, and clinical validation of innovative therapies to treat cancer and rare genetic diseases. MolMed's portfolio in cell and gene therapy includes anti-tumor drugs in clinical and preclinical development:
- Zalmoxis® (TK) is a cell-based therapy enabling bone marrow transplants from partially compatible donors, in absence of post-transplant immune-suppression, currently in Phase III in high-risk acute leukaemia and approved by European Medicines Agency (EMA) for conditional marketing authorization;
- CAR-CD44v6, an immuno-gene therapy project potentially effective for many haematological malignancies and several epithelial tumours, currently in preclinical development.

Moreover, MolMed offers high level expertise to develop, perform, and validate custom studies, optimize and scale-up manufacturing projects, devise innovative testing procedures, and address the unique test specifications required for novel therapies. In order to meet vectors manufacturing demands for both clinical and commercial phases, MolMed is developing modular innovative processes (24 L, 48 L, 200 L) in cell factories and in disposable bioreactors that permit an increase in lentiviral/retroviral (LV/RV) vector productivity, reducing cost of goods and preserving high vectors quality. MolMed is also investigating LV production using suspension cell lines cultured in the absence of animal-derived components to ensure a higher level of safety and unlimited possibilities for scale-up. MolMed is improving hematopoietic stem cell and T-cell production processes in order to develop closed systems with a high level of cell transduction, lower number of manipulations to increase sterility assurance levels, and process reproducibility.


Francesca Bellintani is Downstream Process Development Manager at MolMed. She holds a degree in Biology from the University of Milan for her work on overproduction and purification of recombinant proteins. She specialised in biochemistry working on phage display while at Cambridge University. She joined MolMed in 2007 and has worked in the Development Unit with growing responsibilities. She has strong operations expertise in the development of processes for the purification of proteins and retroviral/lentiviral vectors using chromatography and tangential flow filtration techniques. Francesca supervises a multidisciplinary team of scientists on projects from development to early phase GMP manufacturing. Her knowledge includes ICH guidelines for development and qualification of analytical methods to assess identity, potency, impurity profile, and stability (UPLC, SDS-PAGE, Western blot, and ELISA).

Thomas Wesley Powers
Senior Scientist, BioTherapeutics Pharmaceutical Sciences,
Worldwide Research & Development, Pfizer, Inc.
Pushing the Boundaries of Adeno-Associated Virus Characterization
for Enhanced Product and Process Understanding


Adeno-associated virus (AAV) is emerging as a major modality in biotherapeutics development for gene therapy. The current presentation discusses the implementation of state-of-the-art analytical techniques, including liquid chromatography-mass spectrometry (LC-MS), to better assess AAV product quality attributes such as empty/full capsid ratio, capsid identity, capsid purity, and capsid protein modifications. Heightened characterization, which involves interfacing routine biochemical LC-based methods with ultrahigh-resolution MS have provided novel information regarding both global and site specific capsid characteristics. When combined with in silico modeling, these characterization approaches can be applied to identify sequence hotspots that could potentially impact capsid stability, receptor interactions, or immunogenicity. In addition to the general biochemical characterization of AAV material, several case studies that document the importance of heightened characterization will be discussed. In one instance, routine methods were applied to monitor the impact of a bioprocess change, which resulted in the observation of an unknown impurity. Heightened characterization approaches were able to identify the impurity and support bioprocess refinement, ensuring the absence of the impurity and enhancing process understanding. In another instance, advanced analytics were used to support a forced degradation study, helping to both elucidate the impact of the formulation buffer and the degradation pathways of AAV. The development and implementation of both routine and advanced analytical methods for AAV is essential to improve attribute understanding and drive product and process understanding in the AAV field.


Thomas Powers is a senior scientist at Pfizer working in the Analytical Research and Development group at the Chesterfield, Missouri location. Thomas obtained his BS in chemistry and BA in economics at Furman University in Greenville, South Carolina. He then attended the Medical University of South Carolina in Charleston where he completed his PhD under the guidance of Dr. Richard Drake. His dissertation research focused on developing methods for MALDI imaging of N-linked glycans and the discovery of glycan and glycoprotein disease markers for pancreatic cancer.

Thomas joined Pfizer almost three years ago and works in the Mass Spectrometry and Biophysical Characterization (MSBC) group, which is responsible for heightened product characterization and bioprocess support. Since joining Pfizer, he has developed a wide range of experiences working with multiple modalities, including mAbs, recombinant proteins, and ADCs. More recently, Thomas has been working to develop methodologies capable of analyzing viral vector capsid proteins, specifically those of adeno-associated virus and adenovirus. In addition to supporting several programs, Thomas is involved in the implementation of multi-attribute monitoring by mass spectrometry at Pfizer and in exploring new software to assist heightened characterization of Pfizer products.

Haifeng Chen, PhD
CEO, Virovek, Inc.
Novel Purification Process to Obtain Pure AAV Vectors of 1e+16 vg
in a Single Run of Conventional Ultracentrifuge


Purification of adeno-associated virus (AAV) has mainly been carried out by chromatography and density gradient methods. However, purification of AAV vectors with chromatography is expensive and complicated. Different resins/purification processes are required for different AAV serotypes. The density gradient purification process with either iodixanol or cesium chloride is limited by the volume of each centrifuge tube. Here we report that three-phase partitioning (TPP) can be used as an economical and scalable purification process to purify AAV vectors. TPP is a non-chromatographic technology for the separation of bioactive proteins from natural sources. Recently it has been used as a scalable method for purification of several proteins and viral antigens. Our results indicate that two rounds of TPP could yield 90% purity of bulk AAV vectors at a recovery rate exceeding 70%. In the first round of TPP, more than 70% of cellular impurities such as proteins, lipids, and nucleic acids formed a solid interphase and could be easily removed, leaving most of the AAV vectors in the aqueous phase. In a second round of TPP, the majority of AAV vectors with some cellular impurities formed a solid interphase and could be easily collected and dissolved in aqueous solution. We have successfully purified AAV2, AAV5, and AAV6 vectors using the TPP method with more than 90% purity. Our results indicate that TPP purified AAV vectors were as infectious as those purified by the cesium chloride method in vitro cell culture assays.

We further report that by combining TPP with density gradient centrifugation methods, not only could we remove the remaining impurities and empty AAV capsids from the bulk AAV sample, but also greatly increase the capacity of conventional centrifuges. Our results indicate that a 10-liter AAV production culture could be easily processed with the TPP method into a mere 120 mL of bulk AAV sample with 90% purity and further purified by overnight centrifugation in a single Beckman or equivalent ultracentrifuge, which could yield more than 1e+16 vg of pure AAV vectors devoid of empty AAV capsids. By this calculation, five conventional centrifuges running twice could easily purify up to 1e+17 vg of AAV vectors, enough for 100 to 10,000 patient-doses depending on the gene therapy applications. This novel process should provide a useful tool for large-scale purification of AAV vectors devoid of empty AAV capsids, an impurity that is difficult to remove with the column chromatography method.


Dr. Haifeng Chen obtained his PhD in 1992 from the University of Saarland in Germany. He was then awarded the Marion Merrell Dow Postdoctoral Fellowship to perform research on adenovirus replication at the University of Kansas Medical Center in Kansas City, Kansas. In 1996, he joined Cell Genesys, Inc. and continued his postdoctoral work focusing on the development of adenovirus and adeno-associated virus (AAV) as gene therapy vectors. In 1997, he took a Research Scientist position at Genovo, Inc. and developed two AAV production systems, one based on baculovirus and the other based on adenovirus. In 2000, he joined Avigen, Inc. as a research scientist focusing on the development of novel AAV vector production technologies. In June 2005, he joined Asklepios BioPharmaceutical, Inc. as Vice President of Production. In June 2006, Dr. Chen started his own company, Virovek to provide AAV vector production services to the academic and biopharmaceutical research communities. Besides providing services, he has invented two novel technologies, one for large-scale AAV vector production and the other for production of baculoviral and AAV vectors harboring toxin genes in Sf9 cells; and both have been granted patents. Under his leadership, Virovek has now developed the capacity to provide purified AAV vectors exceeding 3e+16 vg per production run. Dr. Chen is currently a member of the editorial board for Molecular Therapy - Methods and Clinical Development, and a committee member of the Viral Gene Transfer Vector Committee of the American Society of Gene and Cell Therapy (ASGCT). He is also a member of Society for Neuroscience (SfN) and the American Association for Cancer Research (AACR).

Marian Bendik
Head of Gene Therapy, Process Development , Shire
Biological Activity of Additional AAV Subpopulation in AAV Gene Therapy Product


The Factor IX (FIX) gene therapy (BAX335) was investigated in a Phase I clinical study for hemophilia B patients. The BAX335 product for clinical study was produced in small scale and in the course of internal BAX335 process development, the small-scale clinical product was compared with Shire’s product from a large-scale process. During comparability an additional AAV subpopulation was identified by analytical ultracentrifugation only in the small-scale product, and it was further characterized.

The goal of the study was to investigate the AAV subpopulation in small-scale BAX335 product, and whether or not it bears active transgene with biological activity.

The BAX335 was produced by the HEK293 cell line with triple transient transfection. The small-scale production process employed downstream purification with iodixanol gradient in a benchtop ultracentrifuge, whereas the large-scale production process employs a proprietary large-scale ultracentrifugation step. The AAV subpopulation detected in the iodixanol process was separated from the main full AAV fraction and further enriched in order to obtain an isolated AAV subpopulation fraction for analytical characterization (qPCR, analytical ultracentrifugation, agarose gel electrophoresis, in vitro potency (FIX), in vivo potency FIX gene therapy vector in FIX ko mice, SDS PAGE).

Analytical ultracentrifugation of BAX335 from a small-scale process detected the following fractions: full AAV capsids at 80S (sedimentation coefficient), AAV subpopulation at 70S, and empty AAV capsids detected at 50S. The biological activity measured by in vitro potency and in vivo potency was significantly lower in the AAV subpopulation in comparison to full AAV capsids, and confirmed that the AAV subpopulation is undesired and has to be removed during the downstream process.

Shire’s proprietary large-scale ultracentrifugation process, in contrast to the small-scale iodixanol ultracentrifuge process, is capable of removing the AAV subpopulation which does not possess desired biological activity.


Marian Bendik is heading the gene therapy process development team in Orth, Austria. In 2014 he joined the gene therapy process development team to provide a commercial-scale process perspective to speed up the scale-up and commercialization of the AAV manufacturing platform. Marian has 10 years of relevant process development and manufacturing experience including gene therapy and cell-based vaccines. Before joining Shire, he worked as an immunoassay research scientist in academia and industry. Marian received his master's degree in Biochemistry from the University of Chemistry and Technology, Prague, Czech Republic. He actively participates as a speaker in gene therapy conferences and has co-authored patent applications for AAV technology.

Nathalie A. Clément, PhD
Associate Director, PGTC Vector Core Laboratory, University of Florida
Large-Scale Manufacturing of Clinical-Grade rAAV in an Academic Setting: How Efficacy, Versatility, Space, and Cost Drive Process and Development Toward Sustainable Methods


The Powell Gene Therapy Center has been a pioneer in the development and use of recombinant adeno-associated vectors for human gene therapy applications. Over the past 15 years, the center has supported hundreds of proof-of-concept research studies, multiple pre-clinical studies, and more than ten investigational new drug (IND) manufacturing and clinical trials worldwide. Due to the increasing demand for high quality and high titer rAAV preparations, our focus has significantly shifted toward the implementation of large-scale manufacturing methods, both for upstream and downstream processes, with a clear goal of supporting an amenable and affordable platform to support a versatile demand both from academic and industry sponsors. The talk will present our current methods for the production of research and clinical-grade rAAV with a special emphasis on the HSV-based suspension method capable of generating high titers of improved rAAV quality. Specifically, yields and biological activities of rAAV prepared in this new platform with be presented in detail and compared to traditional transfection-made preparations with regard to bio-potency, vector genome, full and empty capsid ratios, VP ratios and purity, among others. Up-to-date in vitro, in vivo, and clinical data will be shown, and pros and cons of the method will be discussed in comparison to the two other most common methods, transfection and the baculovirus system.


Dr. Clement has more than 20 years of experience in the field of gene therapy and has dedicated most of her research to the development of novel adeno-associated vectors, their production, and testing. She obtained her PhD at the University Libre of Brussels, Belgium on the development of vectors using the autonomous parvovirus MVM, then focused on AAV vector design for several years during her postdoc at Mount Sinai School of Medicine, New York. Her interest led her to direct a small vector core team there, and subsequently at the University of Florida where she moved as the Associate Director of the Powell Gene Therapy Center. She currently overviews the manufacturing of research and pre-clinical grade AAV vector preparations, assists with GMP manufacturing, and directs the quality control of GMP-grade products. Her research is largely focused on process and development toward the implementation of optimized processes supporting large-scale production of high quality rAAV stocks and their implementation into GMP settings.

Hanna P. Lesch, PhD
Research and Development Director, FinVector Vision Therapies OY
Viral Vector Manufacturing: Challenges and Solutions


The number of clinical programs moving towards Phase III and beyond have been increasing. This trend is exciting, but the way to reach market approval might not always be straightforward. Many challenges have been related to manufacturing of the product on a large scale. We have developed both adherent and suspension commercial-scale production processes. Our large-scale adherent production system is based on the iCELLis 500 fixed-bed bioreactor. We have highly optimised production processes for adenovirus type 5, lentivirus, and adeno-associated virus. The suspension serum-free production system is scalable and uses a disposable stirred tank bioreactor. The alternating tangential flow (ATF) cell retention device is used for bioreactor perfusion. All therapeutics intended for human use should be extensively characterised before clinical trials. Detailed characterisation during early development provides insight to support development and formulation and should be done sooner rather than later. In addition to traditional methods, we utilise several modern analytical techniques such as light scattering, high-throughput sequencing, and automated electron microscopy for early product characterization. The talk will cover issues related to the large-scale viral vector manufacturing systems and analytical innovations. Also, new funding options will be discussed.


Hanna P. Lesch has over 15 years’ experience in the field of gene therapy. As a Research and Development Director she is responsible for the gene therapy research programs of Trizell group's Finvector Vision Therapies and FKD Therapies. In addition, she is working as a Gene Therapy Unit Director in the Kuopio Center for Gene and Cell Therapy, a recently established research center. Her interest is in gene therapy research with viral vectors and their translational development. She has extensive knowledge of all the most commonly used viral vectors (lentivirus, AAV, adenovirus, and baculovirus). She has a PhD in Molecular Medicine from the University of Kuopio, and did a postdoc at the University of California, San Diego (UCSD) and the University of Eastern Finland, Kuopio concentrating on the understanding of gene regulation and the role of enhancer RNAs in macrophages and vascular biology. She has several patents for vector manufacturing.

Pepijn Burgers, PhD
Scientist, Product Characterization, Janssen Vaccines & Prevention B.V.
Many Ways Lead to Rome for Assessing the Genetic Stability of a Viral Vector


There are two main reasons as to why we wish to assess the genetic stability of viral seeds for manufacturing of viral vectors. First, it is a regulatory expectation. However, more importantly, your commercial supply relies on the stability of your viral seed. Dependent on the product demand after commercialization, the current seeding strategy may need to be adjusted, requiring for example more passaging, which could, pending the genetic stability of the seed, lead to product differences.

FDA and EMA have different requirements and expectations regarding seed genetic stability studies. Nevertheless, all regulatory guidelines clearly state that suitable methods and models must be used in order to get a clear understanding of the genetic stability, both genotypically and phenotypically, of the viral seed. Based on the regulatory expectations and our own wish to gain more knowledge about the genetic stability of our viral seeds, a plan was proposed for characterizing genetic stability. This includes choosing the most appropriate scale to carry out the study, choosing which quality attributes need to be tested in order to best demonstrate the genetic stability of the seed and its suitability to consistently produce a safe and efficacious drug product. In addition, it is important to choose the right analytical methods for assessing these quality attributes. In the presentation, some results from a genetic stability study of a viral vector will be shared.

Nicole Faust, PhD
Chief Scientific Officer, Cevec Pharmaceuticals GmbH
CAP-GT, a Platform Addressing the Production Challenge


An increasing number of gene therapies are based on AAV. Attractive features of AAV as a gene therapy vector are e.g., its lack of pathogenicity and its ability to transduce dividing and non-dividing cells. Moving away from mainly targeting ultra-rare diseases and taking more common indications into focus will need to see significant improvements concerning productivity and consistent quality of AAV vector production in order to ensure supply. Efforts are ongoing to develop efficient suspension cell systems for transient production. However, a major step towards cost efficient and reliable large-scale vector production will be the availability of stable producer cell lines.

To address this gap in AAV production technology, we have developed a stable AAV packaging system based on the human CAP-GT suspension cell line. The system works independently from helper virus or transient transfection. All components are stably integrated and AAV production is induced after cell expansion. Data on the development of the system and efficiency of AAV production will be presented.


Nicole Faust is Chief Scientific Officer and Managing Director at CEVEC Pharmaceuticals, overseeing the company´s activities in gene therapy vector production and recombinant glycoprotein expression. Over the last 18 years she has held scientific management positions with several biotech companies. Before joining CEVEC in 2011, she worked with Lonza as Director R&D combining her expertise in cell biology and gene transfer technology to develop cell-based assays. Prior to Lonza she was heading the Molecular Technology Department at Artemis/Taconic Biosciences, using site-specific recombination and gene editing technologies for the development of pharmaceutically relevant animal models. Nicole holds an MBA degree from Educatis University, Switzerland and she received her PhD in Molecular and Cell Biology from University of Freiburg and spent her postdoctoral period at EMBL, Heidelberg, where she worked on embryonic stem cells and cell differentiation within the hematopoietic system.

Helen Maunder, PhD
Principal Scientist, Oxford BioMedica plc
The Use of Automation in the Development of
Lentiviral Vector Packaging and Producer Cell Lines


Large-scale production of lentiviral vectors (LV) for therapeutic applications in gene therapy is challenging with current transient transfection processes. The development of packaging (PaCL) and producer cell lines (PCL) enabling the generation of large quantities of vectors will not only reduce the cost of raw materials but provide consistency between manufacturing runs and downstream processing. To generate PaCLs and PCLs, isolation of stably transfected clones by cloning by limiting dilution (LDC) in antibiotic selective media is required. Oxford BioMedica (OXB) has developed a bespoke Automated Cell Screening System (ACSS) which uses state of the art automation. The ACSS enables the isolation of up to 3000 clones by automated LDC in either 96- or 384-well format. Furthermore, the ACSS can perform routine passage and high-throughput (HTP) clonal LV production and evaluation of clonal LV productivity using various screening methods.

HIV-1 PaCLs were generated at OXB by stably transfecting an HEK293T.TetR cell line which constitutively expresses the Tet Repressor (TetR) protein, with inducible plasmids encoding HIV-1 GagPol, HIV-1 Rev, VSV-G envelope, and selectable antibiotic resistance makers. Following antibiotic selection, more than 1000 HIV-1 PaCL clones were isolated and screened for their ability to produce LV using the ACSS. The best LV producing candidate PaCL clones were selected and expanded, then were screened further to select the best five clones. The best candidate PaCL clones underwent a second round of LDC to ensure monoclonality and were further screened using the ACSS before scale-up and selection of the best sub-clones. The best sub-clones produced higher titre LV than obtained using the manual clone selection process, and also demonstrated higher titres than those achieved using the current HEK293T transient transfection process. In conclusion, the isolation and screening of larger numbers of clones (>1000) using the ACSS leads to the selection of higher titre LV producing clones than previously achieved when screening smaller numbers of clones (100–200) manually.


Helen is a Principal Scientist within Oxford BioMedica’s (OXB) Cell Engineering Group, which is part of OXB’s Research Department. Under the direction of the Group Lead, the Cell Engineering Group is involved in the development, optimisation, and characterisation of lentiviral vectors (LV), and is responsible for cell line development which includes the generation of LV packaging and producer cell lines. She is responsible for managing a small team of scientists which oversees the development of the cell engineering projects.

She completed a PhD in Virology and Vaccinology at the University of Warwick before moving into the biotech industry. Dr. Maunder has eight years of biotech industry experience which includes working for Lonza Biologics specialising in vector engineering and cell engineering for antibody and viral vectors therapeutics. Following her position at Lonza Biologics she moved into her current role at OXB.

Ana Sofia Coroadinha, PhD
Head of Cell Line Development and Molecular Biotechnology Laboratory
Instituto de Biologia Experimental e Tecnológica (iBET)
Novel Stable Lentiviral Vector Producer Cells: Overcoming Viral Vector Cytotoxicity


Lentiviral bioproducts have gained renewed attention over the past five years where we witnessed the approval of several gene therapeutic products. In addition to gene therapy, lentiviral particles are also being explored in the clinic as a scaffold for antigen presentation and protein delivery. Capable of transducing non-dividing cells and presenting safer integration profiles, self-inactivating lentiviral vectors have progressively undertaken gammaretroviral vector use in ex vivo gene therapies. However the knowledge on gammaretrovirus, particularly its manufacturing, is far more mature. While the production of gammaretrovirus relies on stable producer cell lines and perfusion systems, enabling high cell density and longer term productions, most of the bioprocesses for lentiviral bioproducts rely on transient transfections and short-term batch productions.

Many of the challenges lentiviral bioproducts present in manufacturing are related to the apoptosis-leading cytotoxicity of some of the vector components. Supported by our long track experience and enabling tools developed for gammaretrovirus manufacturing, we undertook the challenge of establishing a constitutive stable lentiviral producer cell line. To address this challenge, we proposed to eliminate or reduce the cytotoxicity of the lentiviral vector expression components. Several strategic novelties were introduced in the development of the cell line, namely: (i) the use of a mutated Gag-Pro-Pol, (ii) introduction of all the third generation lentiviral expression cassettes by chemical transfection, and (iii) performing only one clone screening step (enabling the use of the ‘single step cloning screening’ protocol developed by our group). A lentiviral producer cell line was established, constitutively producing titers above 106 TU/mL/day. Moreover the developed protocol to generate the cell line enables its development in less than six months. The cell line was shown to be stable, consistently maintaining vector productivity over one month in the absence of antibiotics. At the bioreaction it was possible to maintain the cells continuously producing over 10 days. These results validate the transition of continuous large-scale production systems using stable cell lines. Herein we will present and discuss the challenges on the production of lentiviral vectors as well as the strategies and novel technologies to be adopted in order to enable a faster development of gene therapy products.


Ana Sofia Coroadinha is currently the leader of Cell Line Development and Molecular Biotechnology at the Health & Pharma Division of iBET. Her research is focused on viral-based biopharmaceuticals for vaccine and gene therapy purposes. Through the development of novel molecular biology systems, her group has generated novel expression systems and enabling tools for research and development in the area of viral-based biopharmaceuticals. Her research spans a wide collection of bioproducts for gene therapy including gammaretroviral, lentiviral, adenoviral, and AAV vectors, as well as eVLPs for vaccination and research (e.g., HCV, influenza). After completing a degree in Biochemistry, Ana Sofia Coroadinha specialised in animal cell technology and biopharmaceutical research and development. She performed her PhD in Gene Therapy in a tripartite fellowship between Généthon, Helmholtz Centre for Infection Research (HZI), and iBET (PhD degree granted in October 2005). In July 2009 she become director of the Cell Line Development and Molecular Biotechnology Laboratory (iBET and ITQB-UNL). She has published more than 40 papers in reputed journals and has served as an editorial board member of respected journals such as Biotechnology Letters and Scientific Reports.

Lesley Chan, PhD
Scientist II, Vector Process Development & Manufacturing, bluebird bio Inc.
The Development and Intensification of a
Lentiviral Vector Manufacturing Process Using Stable Cell Lines


Production of lentiviral vectors (LVV) by transient transfection has limited scalability. As such, stable cell line production systems are an attractive alternative for LVV manufacturing. Unfortunately, the development of high-titer LVV-producing stable cell lines (PCL) is challenging due to the cytotoxicity of the viral proteins. The PCL used in this study initially had yields 10-fold lower than a transient transfection process in batch mode. To address this, we focused on increasing the LVV production period and infectious LVV yield in a bioreactor-based production system. To that end, cell separation technologies were evaluated for their ability to both increase active LVV recovery and retain viable cells in the reactor. In conjunction, we devised and tested culture media formulations to improve culture growth, cell specific productivity, and total product yield. We identified a method for continuous harvest and a new production medium. In combination, these process improvements increased average LVV titer by 3.5-fold, and extended the LVV production period by three days. This translated to a 14-fold increase in total LVV yield, which eliminated the gap with the transient transfection process. This work highlights the potential of PCL LVV manufacturing systems.


Lesley received a Bachelor of Applied Science in Biomedical Engineering from the University of Toronto and a PhD in Bioengineering from the National University of Singapore. For the past five years Lesley has worked in process development, first in the field of regenerative medicine at the Centre for Commercialization of Regenerative Medicine (CCRM), and now in the field of gene therapy at bluebird bio. Her work has focused on designing processes for complex therapeutics using novel technologies at small scale.

Aziza P. Manceur, PhD
Research Officer, National Research Council Canada
Scalable Lentiviral Vector Production Using Stable HEK293 Suspension Cells


A critical step for successful gene or cell therapy consists of transducing cells with a lentiviral vector (LV) carrying the gene of interest. The amount of LV required to treat one patient or for a small clinical study is achievable by transfecting adherent cells in traditional cell factories. However, depending on the application and disease, up to 4x1010 infectious units of vector per patient are needed; therefore, larger clinical studies and commercialization will require higher yields and more efficient production processes. To address these issues, we propose to use suspension cells in serum-free media. Starting with an inducible packaging cell line that expresses Rev, VSV-G, and Gag-Pol, an inducible producer cell line can be obtained within three months. The expression of the LV genes is induced by adding cumate and doxycycline, which turn on two molecular switches. The double switch system is key because some elements, such as VSV-G, are highly cytotoxic. Next, using a stable producer cell line that produces LV expressing GFP, we have compared different modes of production in a bench-scale bioreactor taking into account yields, costs, and effects on downstream purification. Results suggest that the yield can be improved in fed-batch or with different types of media. Also, up to a 15-fold increase in LV titer was obtained when the bioreactor was operated in perfusion mode compared to batch mode. As a perfusion device, an acoustic cell filter and an ATF system were compared.

Despite the advantages of perfusion mode compared to batch in terms of total process yield, batch mode remains more attractive due to its operational simplicity. In this case, we tested nucleic acid digestion prior to bioreactor harvest, followed by a clarification step to remove the cells and cell debris. Different types of filters were tested with recovery yields varying from 17% to 63%. In summary, using an LV that expresses GFP as a model, we will describe a production process that can easily be amenable to large-scale production.


Aziza Manceur is a research officer at the National Research Council Canada, a research and technology organization funded by the government of Canada. She is part of the Cell Culture Scale Up team that specializes in the large production of viral vectors and proteins using mammalian cells. Their focus is on lentiviral vectors, adenoviruses, and adeno-associated viruses. With her colleagues, Rénald Gilbert and Sven Ansorge, she is developing packaging cells and stable producers, and optimizing the production process. She earned her PhD in biomedical engineering from the University of Toronto and went to the University of Pennsylvania for postdoctorate training.

Leyla Diaz, PhD
Research Officer, MilliporeSigma BioReliance® Services
Testing Approaches for Viral Vectors Used in Gene Therapy:
Novel Methods and Regulatory Expectations


Ensuring the biosafety and quality of viral vectors used in gene therapy is achieved through a multi-tiered approach that examines several factors to establish product safety and manufacturing consistency. The manufacture of viral vectors is complex and challenging with a number of key process goals that need to be maintained to achieve scalable processes and ensure reproducibility of product. The steady increase in the use of viral vectors to produce ground-breaking gene-based therapies has intensified the need for novel approaches to both manufacturing and virus testing that improves upon well-established techniques and streamlined testing.

Based on the innovative nature of many gene therapy processes, customization of test methods has been critical for success. The use of state-of-the-art techniques to improve and expand existing testing methods that examine process product related impurities, identity, and viral safety provides a level of quality assurance that addresses current regulatory expectations. We provide an analysis of the regulatory requirements for cell substrate testing and characterization of gene therapy viral vectors. Current testing methods are reviewed and testing challenges for viral vectors are discussed. This presentation will focus on innovative techniques, address critical quality attributes, and address the limitations of small lot size, lack of terminal sterilization, limited availability of starting materials, and continued supply of reference standards.


Dr. Leyla Diaz is a Principal Scientist on the Field Development Services team at BioReliance responsible for providing scientific leadership for customers based in the United States. Leyla has 18 years' research and product development experience spanning both human and animal health. Leyla’s areas of expertise are virus/host pathogen interaction, therapeutic antibody development, and the design and implementation of molecular and bioassays for large molecules and vaccines. Leyla is a graduate of the University of Maryland, College Park with a PhD in Microbiology.

Sergei Zolotukhin, PhD
Professor, Division of Cellular & Molecular Therapy, University of Florida
OneBac rAAV Scale-Up Production Platform with Serotype-Specific
Modulation of AAV Capsid Protein Stoichiometry


We describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid protein stoichiometry. By way of example, rAAV3, rAAV5, and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. The data will be presented demonstrating a superior infectivity and higher genetic identity of OneBac-derived rAAV vectors providing a scalable platform for good manufacturing practice (GMP)-grade vector production.


Sergei Zolotukhin graduated from Kiev State University, Ukraine and pursued his graduate studies and PhD at the Institute of Molecular Biology and Genetics, Ukraine. His postdoc was completed at the State University of New York, USA, and he is now Associate Professor in the Department of Pediatrics at the University of Florida (UF), USA. Dr. Zolotukhin’s research program comprises two goals: to develop a better recombinant adeno-associated virus (rAAV) vector for tissue-specific delivery of therapeutic transgenes, and to develop gene based therapies for obesity. He has authored more than 80 publications in refereed journals as well as 12 patents. His track record includes 18 uninterrupted years of multiple NIH funding.

Christine Le Bec, PhD
Head of CMC Analytical, Généthon
Analytical Issues for AAV Gene Therapy Products: Vector Genome
Titration and Full/Empty Viral Particles Quantification


Adeno-associated viral (AAV) vectors have been reported to be a great promise in the field of gene therapies for the treatment of various human diseases. In parallel with AAV vector manufacturing, specific analytical assays were performed to assess vector productivity, vector purity, biological activity, and safety. To sustain this, reliable, fast, robust, GMP-compliant analytical methods and characterization protocols are needed. As vector genome (VG) concentration is used to define the dose effect and the therapeutic dose, an accurate measure of VG titer is necessary for efficacy and safety considerations. Real-time quantitative PCR (qPCR) is the gold standard method widely used in research and GMP laboratories. Droplet digital PCR (ddPCR), a new technology that has been designed for accurate DNA quantification, could improve AAV genome titer determination. Comparison of these two methods in terms of precision (intra-assay and inter-assay precision), accuracy, robustness, ease of use, and high throughput will be presented. Besides the dose/strength of the vector, the most important concern is also improving the methods for characterization and quantification of empty, partially full, and full AAV particles. I will present our experience in using the analytical ultracentrifugation (AUC) technique to support process development and characterize preclinical and clinical lots.


Christine Le Bec joined Genethon in 1997 as a scientist and currently heads the CMC Analytical Department. She is responsible for the analytical activities in the characterization and release testing of gene therapy products at early-stage development, stability studies, and interfacing with a CMO for method transfer and validation, and analytical/QC testing. She has strong expertise in the development and qualification of analytical methods based on biochemical, biophysical, and cell-based assays to assess identity, potency, impurity profile, and safety. Before joining Genethon, she obtained her PhD in bio-organic chemistry from Université Pierre et Marie Curie (Paris VI) in 1993. She worked as a postdoctoral researcher at Thomas Jefferson University (Philadelphia, Pennsylvania) and then at Institut Pasteur (Paris, France) in the field of synthesis, structural analysis, and in vitro evaluation of antisense DNA as therapeutic agents for cancer and AIDS.

Etienne Boutry
AVP Head of Bioprocess R&D, Sanofi Pasteur SA
Challenges in Process Development and Industrialization for Live Virus Vaccine:
Flavivirus Experience Using a Vero Cell Line


Sanofi Pasteur has developed a proprietary Vero cell line capacity to support new vaccine product development with the ability to implement it at the industrial scale. With this platform, Sanofi Pasteur was able to submit and support a licensing dossier submission.

The presentation will give an overview of the approaches taken to address some CMC challenges faced during the development and commercial production of live recombinant vaccines. As an illustration, we will use our experience on recombinant Flaviviridae such as dengue and yellow fever.

We will share the approach taken to balance expected productivity and process implementation at industrial scale in regards to regulatory requirements in particular for purity, robustness, viral contamination, and genetic stability. The results from specific scale-down models and full-scale approaches will be presented, along with the associated characterization package.

Expectations from health authorities and patients on the final product stability remains a big challenge during product development. To prevent any issues upfront, the formulation must be anticipated to minimize the impact on clinical development. We will present some early step formulation components screening using stress conditions, long-term stability testing, and modelisation.

Xiaohui Lu, PhD
Senior Scientist, Bioassay and Gene Therapy, Analytical Development, Biogen
Analytical Strategies on Quantification of Adeno-Associated Virus (AAV)
Empty Capsids to Support Process Development


Recombinant adeno-associated virus (AAV) for human gene therapy is a fast-evolving field in the biotech industry. One of the major challenges throughout process development is how to well characterize empty capsids and remove them effectively. Throughout process development, samples usually have wide ranges of titers. There are different analytical methods to quantify empty capsids. Understanding the specifications of different analytical methods is crucial in order to provide the most appropriate support to accelerate process development. In this study, varieties of analytical methods are applied on the same set of samples and compared with their specifications. In our finding, anionic exchange (AEX)-HPLC demonstrates very good correlation with transmission electron microscopy (TEM), and the results of AEX-HPLC are also close to analytical ultracentrifugation (AUC). Finally, strengths and weaknesses of different methods are summarized and discussed.

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