Associate Professor Biotechnology, Centre for Medicines Discovery (CMD), NDM
Screening and Production of Human Proteins Using Baculovirus and BacMam
The Centre for Medicines Discovery (CMD), established in August 2020, comprises a number of disease-focused groups and small research facilities (SRF). The biotech facility provides protein production services for academic and industrial customers. In this talk, our well established expression platforms for production and validation of intracellular, secreted, and membrane proteins will be presented, with a highlight on production of SARS-CoV-2 proteins for serological assay development.
Nicola Burgess-Brown is an Associate Professor and Head of the Protein Production Small Research Facility (SRF) in the Centre for Medicines Discovery (CMD). Nicola leads a research group experienced in all aspects of biotechnology, molecular biology, protein biochemistry, and technology development who support the CMD and provide protein production and mass spectrometry services for internal and external academic and industrial customers. Nicola obtained a First Class degree in Applied Biochemical Sciences from the University of Ulster in 1997, then worked as a molecular biologist for SmithKline Beecham. She received her PhD in Molecular Microbiology at the University of Nottingham in 2001 and then moved back to industry to work on high-throughput cloning and validation of therapeutic cancer antigens for Oxford Glycosciences and subsequently Celltech R&D. For the past 16 years, she has worked in the field of Biotechnology at the Structural Genomics Consortium (SGC) at the University of Oxford.
Developing a Potency Assay for a Proprietary AAV-Based Genome Editing Platform Technology
The GeneRide platform utilizes the natural process of homologous recombination to achieve targeted genome editing without the use of exogenous nucleases or promoters. Developing a potency assay for this unique technology poses additional challenges over those for canonical gene therapy products, including the requirement of a highly sensitive detection method to measure low levels of genome integration. The first GeneRide candidate, LB-001, is currently under clinical development for the treatment of methylmalonic acidemia. LB-001 targets site-specific integration into the albumin locus to allow the gene of interest (MMUT) to be expressed concomitantly with albumin. In order to control LB-001 product activity and assess lot-to-lot consistency, a cell-based assay was developed to measure fused mRNA expression as a surrogate of biological activity. The assay was developed in a cell line that naturally expresses albumin and can thus drive expression of the MMUT gene upon site-specific integration. Fused mRNA is quantified using primers overlapping the host genome and the transgene DNA to ensure that only the integrated product is detected. The results show that AAV-driven homologous recombination is reproducible in vitro, which allows for the qualification of assay control material. The method was tested for linearity, repeatability, and specificity. Examination of the assay data demonstrates that this method is suitable for assessing the relative potency of integrating GeneRide vectors.
Matthias Hebben has been serving as VP Technology Development at LogicBio Therapeutics since February 2019. Before that, he served as Director of AAV Technology Development and Head of Bioprocess Development at Genethon for six years where he managed the design and scale-up of manufacturing processes for AAV and LV vectors. Prior to his role at Genethon, Matthias was Director of the Virology Unit at Vivalis for four years. Before that, he occupied several roles in the animal health industry at Intervet Schering Plough and Virbac between 1999 and 2008. Matthias has a PhD in molecular biology from the University of Nice Sophia Antipolis (France) and a bioprocess engineer degree from the University of Strasbourg (France).
Biophysicist Research Scientist, Molecular Biophysics and Integrated Bioimaging, Structural Biology
New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multi-Protein Complexes
MacroBac is a multigene baculovirus system that uses ligation-independent cloning for efficient cloning and assembly that is equally well-suited for either single or high-throughput cloning reactions. MacroBac vectors are polypromoter to minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have observed significant increases in expression levels and quality of large, multiprotein complexes over traditional coinfection with multiple, single-gene baculoviruses.
Jill O. Fuss, PhD is a Research Scientist at Lawrence Berkeley National Laboratory and Founder and Chief Technology Officer of Cinder Biological, Inc. Dr. Fuss earned a BA in environmental science from Wesleyan University, and a PhD in molecular and cell biology from the University of California, Berkeley. Her postdoctoral fellowship was performed at Lawrence Berkeley National Laboratory (LBNL) where she received a National Institutes of Health National Research Service Award and was named a US Department of Energy Outstanding Mentor. She received two LBNL Director’s Awards for Exceptional Achievement in 2013 and 2016 and was recognized as a Berkeley Visionary in 2015.
Baculovirus-Cell Interactions in the Insect Midgut: Transcriptome Profiling and Envelope Protein Trafficking
In nature, the midgut is the first tissue encountered during baculovirus infection of the animal. The virus must navigate through the midgut cells and bud into the hemocoel to achieve successful systemic infection. To better understand the complex interactions between virus and host, we have used transcriptomic approaches to examine both viral and host transcription profiles in the midgut (the primary phase of infection) in comparison with similar studies in cultured insect cells (which represent the secondary phase of infection). Also, to better understand how many viruses navigate through the midgut, we have recently developed several new experimental systems for broad screens of host cell proteins and pathways involved in viral envelope protein trafficking in cultured insect cells and in the insect midgut.
Gary Blissard is a Professor at the Boyce Thompson Institute at Cornell University, and an Adjunct Professor in the Departments of Microbiology and Immunology, as well as Entomology, at Cornell University. His studies have focused on the structure, function, and trafficking of viral envelope proteins, viral entry and egress, and viral and host cell gene expression.
Professor of Virology, Department of Pathogen Molecular Biology
Use of Recombinant Proteins and Particles for Molecular Studies and Novel Vaccines
Large-scale interactome maps of eukaryotic systems have found that biologically active proteins are often part of large multi-subunit complexes. Therefore in order to properly study protein activities in vitro, or for vaccine purposes, it is essential that all the subunits of large protein complexes are functional and correctly assembled. Baculovirus expression is an excellent system for the generation of multi-subunit protein complexes and we have been successful in recovering enzymatically active complexes for a variety of proteins. The protein complexes that we have tested range from relatively simple enzymatic systems with small numbers of subunits to large multi-layered complexes and other large multi-subunit particulate structures. In particular, we used an architecturally complex model virus with a view to understanding the role of each viral protein in the virus replication cycle. The model virus is composed of seven discrete proteins in a specific but non-equimolar ratio that are organised into four layers of two shells, the inner core and outer capsid and enclosed genome of 10 double-stranded (ds) RNA segments. Bluetongue virus (BTV) is an insect vectored emerging pathogen of wild ruminants and livestock, causing disease in sheep, goats, and cattle with mortality reaching 70% in some breeds of sheep, with high economic consequences.
We expressed each viral protein, then purified and analysed the role of each in the virus life cycle. Data obtained from these early studies have defined the key players in BTV entry, replication, assembly, and egress. Specifically, it has been possible to determine the complex nature of the virion through three-dimensional structure reconstructions; atomic structure of proteins and the internal capsid; the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of three nucleoid structuring (NS) proteins in virus replication, assembly, and release. Based on information gained from structural and molecular studies, a novel technology has been developed to produce highly efficacious safe vaccines for BTV and related viral diseases.
We undertook a series of vaccination trials in animals. We found that the recombinant receptor binding protein VP2, alone or in combination with membrane penetration protein VP5, could afford protection against virulent virus challenges. This led to the observation that presenting VP2 in the appropriate conformation would reduce the amount of protein required for vaccination. This was taken to a logical conclusion and the two BTV outer capsid proteins together with scaffolding core proteins, VP3 and VP7, were expressed by a recombinant baculovirus expression system to generate double-capsid virus-like particles (VLPs). The BTV VLPs, the first in the field (1990), had the characteristic icosahedral structure, the most complex protein assembly produced, with the four proteins in non-equimolar ratios which assemble into a particle with a total of 1440 subunits. This particle acts to mimic the overall structure and immunogenicity of authentic virus and efficiently stimulates both B-cell and T-cell responses, conferring protection against virulent virus challenge. Further, it was possible to co-express the outer capsid proteins from different serotypes onto the conserved inner core and these heterologous particles, as expected, were highly protective against virulent virus challenges in sheep and generated multiple vaccine strains for BTV. The same technology was then adopted for related viruses and also for many other viruses that are currently commercialized.
Professor Polly Roy began her education in India, where she won a fellowship to New York University for her PhD under the supervision of the renowned molecular biologist, Sol Spiegleman. She continued with a three-year postdoctoral position in virology at the Waksman Institute, Rutgers University and then joined the University of Alabama, Birmingham. There she established her own virology research group, becoming a full Professor in 1986.
In 1987, she received a senior International Fogarty fellowship to study at the University of Oxford where she established a UK-based research group. In 2001, Professor Roy took the Chair of Virology at the London School of Hygiene and Tropical Medicine, where she continues to lead a large research group working on basic virology and applied vaccine research. Her work focuses on the molecular biology of RNA viruses, using Bluetongue virus (related to human rotavirus) as a model. These studies have contributing to many areas of virology such as virus structure and assembly, RNA replication, and novel vaccine development. Professor Roy was the first to demonstrate the assembly of virus-like particles (VLPs), a technology which has since been applied to many other viruses including successful vaccine development for human papillomavirus (HPV), influenza, and SARS. Her research group still continues to use the baculovirus expression system for both basic research and application. She recently pioneered the synthesis of infectious viruses solely from synthetic genes, leading the way for the development of yet newer vaccines and therapies.
Professor Roy has supervised over 150 postdoctoral and post-graduate researchers and published over 350 papers in various high impact journals, as well as a number of chapters in medical, veterinary, and virology books including Field’s Virology and various encyclopaedias. She is also an editor of several books. Her research has been funded by many agencies including the National Institutes of Health (NIH), National Science Foundation (NSF), United States Department of Agriculture (USDA), Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council (MRC) and the Wellcome Trust, UK, as well as several European Commission (EC) grants.
Professor Roy has served on various international scientific organizations, committees, and boards. She has organized several highly successful international conferences, particularly on the subject of virus structure and assembly, double-stranded (ds) RNA viruses, and several viral vaccine symposia.
In 2006 Professor Roy was elected a Fellow of the Academy of Medical Sciences, UK and in 2012 she received the Gold Medal for her contribution to science and technology from the then Indian Prime Minister Manmohan Singh. She is one of the three BBSRC Scientific Innovators of the year and in 2014 received the “Order of the British Empire” for her achievements in Virus Research.
Considerations for the Development of Gene-Edited Stem Cell Therapies
The era of gene editing has unlocked the potential to cure diseases that previously had few treatment options. In particular, gene-edited stem cell therapies have shown great promise in the treatment of β thalassemia and sickle cell disease, and also offer the opportunity to change the treatment landscape in hematological malignancies. This talk will discuss some of the points to consider when designing and developing a gene-edited stem cell manufacturing process.
Brent Morse is the Head of Process and Analytical Development at Vor Biopharma. Prior to Vor, he served as Director of Analytical Development at CRISPR Therapeutics where he established the Analytical Development group and supported multiple regulatory filings for gene-modified stem cell and CAR T-cell therapies. Before that, he was Director of Bioanalytical Development at EPIRUS Biopharma where he developed biosimilarity protocols for multiple products. Brent has also held several R&D positions within the biotech and pharmaceutical industry, including roles at Abbvie and Adnexus (Bristol-Myers Squibb).
Development of a GMP Relevant Process for Gene Circuit Engineered Allogeneic CAR-NK Cell Therapies in Oncology
Gene circuit technologies allow for the programming of sophisticated functions in cell and gene therapies, including logic gated recognition of cancer cells and controlled regulation of immune effector expression. We are developing a proprietary portfolio of allogeneic CAR-NK products to address key challenges in oncology. Here, we will discuss key considerations for developing a scalable allogeneic cell process for GMP manufacturing.
Philip is an entrepreneur and technology platform builder specializing in cellular systems. He has over a decade of life science leadership experience spanning product development, technical operations, marketing management, and innovation. He is a co-founder of Senti Biosciences, a biotechnology company pioneering the use of gene circuits to program next generation cell and gene therapies, where he leads Technical Operations and Manufacturing. Prior to Senti, he was the co-founder and CEO of CellASIC, which was acquired by Merck KGaA, and served as the former Head of Cell Culture Systems at Merck KGaA. He received his PhD in Bioengineering from UC Berkeley/UCSF and BS degrees in Chemical Engineering and Biology from the Massachusetts Institute of Technology.
Regents Distinguished Professor, Diagnostic Medicine Pathobiology & KBA Eminent Scholar, College of Veterinary Medicine
Baculovirus-Based Vaccines for Rift Valley Fever Virus
Rift Valley fever (RVF) is a zoonotic disease that causes severe epizootics in ruminants, characterized by mass abortion and high mortality rates in younger animals. The development of a reliable challenge model in target animals is an important prerequisite for evaluation of existing and novel vaccines. We conducted studies aimed at comparing the pathogenesis of RVF virus (RVFV) infection in US sheep and cattle using two genetically different wild-type RVFV strains, SA01-1322 and Kenya-128B-15. The Kenya-128B-15 strain manifested higher virulence compared to SA01-1322 by inducing more severe liver damage, and longer and higher viremia in both sheep and cattle. Genome sequence analysis of both isolates revealed sequence variations between the two isolates, which potentially could account for the observed phenotypic differences. These results demonstrate the establishment of virulent target host challenge models for vaccine evaluation based on the RVFV strain Kenya-128B-15. There is currently no fully licensed RVFV vaccine suitable for use in livestock or humans outside endemic areas. Therefore, we evaluated the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. The vaccine elicited strong virus neutralizing antibody responses in sheep and cattle and is DIVA (differentiating infected from vaccinated animals) compatible. Furthermore, a group of sheep was vaccinated subcutaneously with the Gn/Gc-based subunit vaccine candidate, and then challenged with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever, and absence of RVFV-associated histopathological lesions. We conclude that the subunit Gn/Gc vaccine represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts.
Dr. Richt came to Kansas State University in 2008 as the Regents Distinguished Professor and Kansas Bioscience Eminent Scholar. In 2010, he became Director of the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD). He received his doctorate in veterinary medicine (DVM) from the University of Munich and his PhD in Virology and Immunology from the University of Giessen, both in Germany. After coming to the United States in 1989, he completed three years of postdoctoral/residency studies at the Johns Hopkins University in Baltimore, Maryland, and later served for eight years as a Veterinary Medical Officer at the National Animal Disease Center (USDA-ARS) in Ames, Iowa. He has edited several books, published more than 240 peer-reviewed manuscripts, and raised more than $40 million in grants for veterinary research since 2008.
Dr. Richt is a pioneer in veterinary science, most notably in the “One Health” field. His work on high consequence pathogens with zoonotic and transboundary potential led to strategies to identify, control, and/or eradicate such agents. His basic and applied research includes studies on animal influenza viruses, animal prion diseases including bovine spongiform encephalopathy (BSE), Rift Valley fever virus (RVFV), Schmallenberg virus (SBV), African swine fever virus (ASFV), vesicular stomatitis virus (VSV), and Borna disease virus (BDV). Dr. Richt established the first reverse genetics system for swine influenza viruses (SIV), made seminal contributions to the development of a modified live SIV vaccine (sold in the US as Ingelvac Provenza™), and to understanding the virulence of the reconstructed 1918 “Spanish Flu” virus in livestock. He identified an atypical BSE case with a causative mutation (“genetic BSE”), used gene-editing approaches to develop the first prion protein knock-out cattle which are resistant to prion infection, and provided valuable information on the host range of animal prions essential for risk analysis. Dr. Richt’s RVFV work led to the development of novel domestic and wild ruminant models for RVF and a safe, efficacious, and DIVA-compatible subunit vaccine. For ASFV, he is developing subunit and modified live virus vaccine candidates as well as point-of-need diagnostic tools to protect swine from this devastating disease. As founding Director of a Department of Homeland Security (DHS) CEEZAD and the NIH CEZID Centers, he is supporting NIH, DHS, and USDA in protecting public health and US agricultural systems from devastating animal and zoonotic diseases.
Optimizing Cell Manufacturing Processes for the Treatment of Severe Genetic Diseases and Cancer
The cell and gene therapy space has seen a great deal of growth with an increasing number of ongoing and newly-initiated clinical trials for different disease indications. The speed at which these cell therapies move through the clinic and the advancements in manufacturing technology have brought forth both unique challenges and opportunities in the last decade. Two case studies will be presented to demonstrate some of the challenges with developing a robust process for commercial manufacturing of cellular therapies. The first case study examines the relationship between the different inputs in the transduction operation for hematopoietic stem cells and identifying some of the critical outputs which determine clinical success. The second case study investigates the different media formulations necessary for T-cell expansion to manufacture the clinical doses required for CAR T-cell or TCR-based therapies. Both case studies will explore how a process development team develops and optimizes an autologous cell manufacturing process, which helps gain better process understanding overall.
Liz is currently the Associate Director of Cellular Process Development at bluebird bio managing the clinical programs in severe genetic diseases (SGD) and oncology from preclinical to commercial. Before bluebird bio, Liz started her industry career at Novartis Pharmaceuticals in New Jersey working on the development of Kymriah as one of the first approved CAR T-cell products for CD19. Liz was part of the analytical and process science team involved in process characterization and development for biologics license applications (BLAs), supplemental BLAs (sBLAs), and marketing authorization applications (MAAs). She was then promoted to process lead at Novartis for their pipeline projects and worked on the investigational new drug (IND) application filing for the next-generation CAR T-cells. Liz received her PhD in Biochemistry at the University of Illinois and then did her postdoctoral work at Duke University under the guidance of Dr. Bruce Sullenger and Dr. Smita Nair.
Routine Size, Mass, Aggregate Level, Total Concentration and Empty:Full Ratio Determinations for AAV Particles by SEC-MALS
Mass and size data can be advantageous in assessing the overall quality of adeno-associated virus (AAV) materials. Recent improvements in multi-angle light scattering (MALS) data analysis methods suggest SEC-MALS can be applied to estimate both the total capsid titer and the extent of DNA encapsulation (i.e. empty:full ratio) for well-behaved AAV samples. We developed and tested a SEC-MALS approach using AAV "empties," "fulls," and mixtures of the two samples obtained from a single batch of cesium chloride (CsCl) gradient-purified AAV. After refining our acquisition and data processing methods, we found the empty:full ratio determined by SEC-MALS comparable to that obtained from both non-staining cryo-transmission electron microscopy as well as analytical ultra-centrifugation (AUC). We also found the total capsid titer determined by SEC-MALS for these samples to be consistent with the total titer back-calculated from AUC data and vector genome concentration (i.e. active titer). Preliminary tests using SEC-MALS for empty:full ratio show the method is fast, robust, reproducible, and executable across a useful concentration range for AAVs. The method neither performs nor requires separation of empty from full AAV species, meaning little to no adaptation for successful application with differing serotypes. Though caveats apply for anomalous materials which may co-elute with the main peak, we find this method highly suitable for routine empty:full ratio determinations, and highly advantageous given the additional information (mass confirmation, size check, level of aggregates) collected simultaneously.
Darren Begley is a senior scientist working in the Analytical Development (AD) group at Beam Therapeutics. Beam is pioneering the use of base editors to develop a new class of precision genetic medicines. Darren has helped build and grow the AD team, providing biophysical testing capabilities both internally and through strategic external collaborators. for a variety of materials, including small and large oligonucleotides, protein-nucleic acid complexes, lipid nanoparticles (LNPs), and adeno-associated virus (AAV) particles. Prior to joining Beam, Darren worked with AAV and virus-like particles at Ultragenyx and Wolfe Laboratories. He obtained a PhD in chemistry with a structural biology focus on RNA and small molecules at the University of Washington in Seattle, and has a Bachelor of Science degree from McGill University in Montréal.
Development and Optimisation of Virus-Like Particle Vaccines Against Pathogenic Arboviruses
Chikungunya virus and Mayaro virus are emerging pathogens that cause debilitating arthritic disease in humans, whereas Zika virus is associated with brain malformations in the unborn child. These viruses are transmitted to humans by mosquitoes, and no licensed vaccines or antiviral drugs are currently available for human use. We developed enveloped virus-like particle (VLP) vaccines against chikungunya virus, Mayaro virus, and Zika virus using the scalable baculovirus-insect cell expression system. Moreover, we aimed to increase VLP production yield using methods that inhibit RNA interference in insect cells and enhance VLP budding from insect cells. High-level secretion of VLPs into the culture fluid of insect cells was achieved at volumes reaching bioreactor-scale, and particles with correct diameters were observed after purification. Challenge experiments to assess the ability of the different VLP vaccines to confer protection in mice are currently ongoing.
Sandra Abbo is a PhD candidate at the Laboratory of Virology at Wageningen University, the Netherlands. Within the ZikaRisk project she investigates the vector competence of mosquitoes from the Netherlands for Zika virus, and she tries to understand the molecular mechanisms underlying the vector specificity of Zika virus. Moreover, she also works on the development and optimisation of VLP vaccines against multiple pathogenic arboviruses. Sandra obtained her MSc degree in biotechnology cum laude at Wageningen University in 2016. During her MSc internship, she worked on cancer cell metabolism at the MRC Cancer Unit of the University of Cambridge, United Kingdom. She then continued with her MSc thesis research about VLP vaccine development at Wageningen University, where she received the Van der Want award for the best MSc thesis in Virology.
Associate Professor of Biochemistry & Molecular Biology
Ongoing Work for Expression of Difficult-to-Express Proteins and Protein Complexes
Development of recombinant protein expression technologies has been one of the cornerstones in modern molecular biology. Recombinant protein expression is based on cloning genes encoding the protein of interest into the vector(s) followed by delivering the cloned genes into the host cells for heterologous protein expression. Despite an array of recombinant protein expression systems, one fundamental problem remains practically unsolved — to express large and often problematic proteins in a reasonable quantity in soluble form in a consistent fashion. We developed a new technology that enables us to express large and often problematic proteins in insect cells. Our system uses a synthetic artificial protein that enhances the solubility of proteins, thereby enabling expression of large and often problematic proteins.
Yuichiro Takagi, PhD is Associate Professor at the Indiana University School of Medicine. He received a BS degree from Ibaraki University, an MS degree from the University of Tokyo, and his PhD in biochemistry from the University of Oklahoma. He conducted postdoctoral research in the laboratory of Dr. Roger Kornberg at Stanford University. The Takagi laboratory is interested in understanding the mechanisms of assembly, structure, and function of large multi-protein complexes involved in eukaryotic gene regulation. His lab is particularly interested in the development of new expression technologies which enable production of large and often problematic proteins and protein complexes for structural (x-ray crystallography and cryo-EM) and functional studies.
Development and Validation of a Potency Assay for a Viral-Based Gene Therapy Product
Viral-based gene therapy is relatively new and gaining momentum with promising early successes in clinical and regulatory approval. The potency assay is typically the most customized and unique among the battery of product release and stability indicating assays. Unlike other biological drugs, the potency assay for a gene therapy product involves multiple steps including infectivity, transcription, translation, protein modifications, localization of the protein product, and protein function. The potency assay for a gene therapy product is extremely challenging, especially for AAV viral vectors, as it has poor infectivity and limited sensitivity. A successful potency method must evaluate infection, transcription, and function of the resulting protein. Here we present our approaches and strategies used in the development and validation of a potency assay for a viral-based gene therapy product.
Rashmi Prasad, PhD is an experienced scientist with a demonstrated history of working in the biotechnology industry. She is skilled in protein analytical biochemistry, protein purification, and molecular biology tools. Dr. Prasad works as Scientist II in the Quality Control team at MassBiologics in Massachusetts. Her major focus area is the qualification and validation of bioanalytical assays for viral vectors (AAV) targeted to characterize the products and in-process samples of gene therapy. She also supports her team as the Technical SME of major bioassays. Rashmi is a strong research professional who earned her PhD in Biochemistry for work on yeast chromatin remodeling enzymes and their crosstalk with histone chaperones, from Southern Illinois University in Carbondale.
Building Effective Control Strategies for Cellular Therapy and Other Complex Biopharmaceutical Products
The best method of developing any control strategy is to use two straightforward paradigms for achieving the three simple goals that every control strategy must achieve. This presentation outlines the three basic goals of a control strategy and describes the two paradigms required to efficiently achieve those goals. Since every control strategy is basically a process, the first paradigm is Lifecycle Process Development and Validation (LPDV) that answers four basic questions that describe the control strategy’s functional lifecycle. The second paradigm is System Risk Structures (SRS) that provide a straightforward way of managing important risks to the control strategy’s performance. Although many control strategies required for developing and manufacturing cell therapy products are complex, the basic methods for developing the control strategies can be relatively straightforward and simple to use.
Mark has 35 years of experience in the biopharmaceutical industry in a wide variety of executive, consulting, and engineering roles. Prior to joining Exyte, Mark was a member of NNE’s Strategic Manufacturing Concept Group after working at Integrated Project Services (IPS) on feasibility and conceptual design studies for advanced biopharmaceutical manufacturing facilities. Before and after working for engineering companies, Mark was an independent consultant in the biopharmaceutical industry for more than 25 years on operational issues related to: process and product development; strategic business development; clinical and commercial manufacturing planning; tech transfer; and facility design and construction. For many years, he taught an International Society for Pharmaceutical Engineering (ISPE) Biopharmaceutical Process Validation course. He was previously the Sr. Vice President, Manufacturing Operations for the CMO Covance Biotechnology Services. At Covance, he was responsible for the design, construction, start-up, and operation of Covance's $50MM contract manufacturing facility. Prior to joining Covance, he was Vice President of Manufacturing at Amgen, Inc. Mark was with Amgen for nine years and held positions as Engineering Manager, Plant Manager, and Director of EPOGEN® Manufacturing. He has published more than forty articles related to process validation, risk management, advanced facility designs, and operational strategies. Mark received his PhD in Chemical Engineering from the University of Massachusetts.
Machine Learning Applications for the Characterization of Particle Profiles of Therapeutic Products
Flow imaging is a proven method for characterization of particulates in therapeutic products. It is routinely performed alongside the USP 788/787 Light Obscuration methods to more accurately quantify and characterize the particle subpopulations in drug products (silicone oil, protein aggregate, extrinsic material, etc.). Typical classifications of imaging data use single parameter filters such as aspect ratio to quantify silicone oil compared to protein. However, machine learning provides a sophisticated approach to more accurately classify particles in therapeutic products by leveraging the information present in the raw particle images. We will demonstrate how various machine learning algorithms facilitate improved classification compared to the traditional approach, leading to superior sample descriptions. We provide examples of the benefits that machine learning provides for cell therapy products. Flow imaging has tremendous potential to monitor particle size distributions, aggregates/agglomerates, and extrinsic contaminants from batch to batch. Applying machine learning to flow imaging of pharmaceutical products can assist in defining the criticality of product quality attributes, as well as establishing an integrated control strategy for characterization and control of drug products.
Amber currently holds the position of Director at KBI Biopharma where she manages the Particle Characterization Core that specializes in analytical methods for quantifying, characterizing, and identifying particulates. She received her PhD in Chemical Engineering within the Pharmaceutical Biotechnology Program with a specialization in the field of protein stability at the University of Colorado at Boulder. Previously, at Amgen, Amber was a Scientist within the biomolecular structures and interactions group where she supported biophysical characterization of protein products with a specialty in subvisible particle characterization and identification. She has over 12 years of experience with analytical method development and validation, formulation strategy, and protein biophysical characterization.
Automation in Delivery Technologies to Empower Breakthrough Therapies
With the advent of personalized medicines, the ability to deliver payloads in a precise and efficient manner into cells becomes more integral to the success of the therapy. Although the existing strategies of viral vectors and electroporation allow for mass manipulation of cells, they lack the ideal precision to be used on fragile cell types for certain applications. Thus, many emerging technologies are focusing on strategies to improve accuracy by navigating delivery at the single cell level. The goal of any delivery technology is to create a system of automation that can be easily scalable and translatable to manufacturing of these new therapies. We will discuss the strategies behind creating a platform that can be universally adapted and automated for a variety of applications that can make manufacturing of personalized therapies much more cost-effective and efficient.
Dr. Anil Narasimha is the co-founder and CEO of Mekonos, a San Francisco, California-based company developing a platform for universal delivery of payloads into cells. After receiving his BS from the University of California, Berkeley, he completed his PhD at the University of California, San Diego. He then completed a postdoctoral fellowship at Stanford University in Dr. Michael Snyder’s group, where he co-founded Mekonos.
Advanced Therapies, Clinical Holds, and Strategies to Avoid
The global landscape for cell therapies is robust with a reported 1.6 billion dollars in financing and more than 200 ongoing clinical trials as of the third quarter of 2019. These cell therapies include allogeneic, autologous, and primary cells. The global landscape of approved therapies includes six cellular immunotherapies and 20 cell therapies. The number of clinical trials that rely on cells is rapidly evolving from those isolated from perinatal sources to those relying on mesenchymal/stromal cells. Many of these products are being developed under expedited approval designations such as the US FDA Regenerative Medicine Advanced Therapy (RMAT), European Medicines Agency (EMA) PRIME, or Japan’s Sakigake. Recently clinical holds have been reported for several advanced therapies under clinical investigation under an approved investigational new drug application (IND). The reasons for clinical hold have included adverse events, delivery device used, and manufacturing. What constitutes a clinical hold is specifically codified in the US Code of Federal Regulations under 21 CFR 312.42. Of these, insufficient information and unreasonable risk are particularly relevant with respect to manufacturing, as problems with manufacture can convey a risk leading to a clinical hold. This is particularly important for cellular therapies where quality, safety, and efficacy are all intertwined. FDA can issue a clinical hold for an initial IND via the issuance of a complete response letter or place an ongoing clinical trial on hold by issuing a clinical hold letter. While news regarding clinical holds of ongoing investigations is typically public information, there is less visibility with respect to holds placed on initial INDs. Three publications by FDA staff have examined the reasons for INDs placed on clinical holds. CMC or quality issues have ranked first or second as the primary reason cited for the clinical hold and submitting with incomplete information was the cited reason in greater than 90% of all clinical holds. Important strategies for avoiding a clinical hold include beginning with the end in mind, maintaining alignment among quality, nonclinical, and clinical stakeholders, maintaining data integrity, and accurate, clear communication.
Debra Aub Webster, PhD has over 20 years of experience in pharmaceutical research and the regulatory environment. She received her undergraduate degree from Virginia Polytechnic Institute and State University, and her graduate degree in pharmacology and toxicology from the Virginia Commonwealth University’s Medical College of Virginia. After leaving academia and bench research, Dr. Webster joined the US Food and Drug Administration (FDA) as a reviewing toxicologist. Dr. Webster was an inaugural member of the FDA’s Division of Anti-Viral Drugs in the Center for Drug Evaluation and Research. Here she was responsible for critical evaluation of the nonclinical pharmacology and toxicology sections of investigational new drug applications (INDs) and new drug applications (NDAs). She authored reviews, advisory opinions, and executive summaries, and was awarded a Medal of Appreciation from the Commissioner for her work. Prior to leaving the FDA, she held a rotation as Assistant to the Director for Pharmacology/Toxicology. Dr. Webster is the Director of Advanced Therapy Product Development at Cardinal Health Specialty Solutions. She is responsible for providing regulatory strategy consulting support across clinical, nonclinical, and CMC to lead product development from proof-of-concept through to approval. Dr. Webster has provided support and leadership for the development of regenerative medicines and advanced therapies, including CAR T-cell and other genetically engineered cells, stem cell therapies, gene therapies, genome editing therapies, oncolytic virus therapies, therapeutic cancer vaccines, recombinant human proteins, antibody-based therapies, biosimilars, and bioengineered tissue constructs.
PhD Candidate, Biological & Biomedical Engineering, Viral Vectors and Vaccine Bioprocessing Group
rVSV-Vectored Vaccine Production in Vero Cells
Viral vector-based vaccines are receiving increased attention, especially in light of the recent Ebola virus epidemic in West Africa and the COVID-19 pandemic. Their cell culture-based manufacturing, however, is lengthy and cumbersome; mostly using conventional production technologies. Our work aims to contribute to the field of vaccine bioprocess engineering by further developing a cell culture platform to establish more efficient, scalable, and cost-effective manufacturing technologies.
Applied to the recombinant vesicular stomatitis virus (rVSV) vaccine platform, and in particular the Ebola virus disease vaccine rVSV-ZEBOV, we study bioreactors for Vero cell processes. Based on small-scale optimization studies, we have developed microcarrier and fixed-bed bioreactors for adherent Vero cell cultures and bioreactors for a suspension-adapted Vero cell line under serum-free conditions. Further, we compared critical process and product characteristics, such as yield of infectious particles, cell specific productivities, as well as ratio of total to infectious particles, and determined the optimal time of harvest. In addition, we have demonstrated the bioreactor production of a highly relevant vaccine candidate against COVID-19 in the Vero suspension system.
Sascha Kiesslich is a PhD candidate at McGill University in Montreal, Canada. His current research focuses on developing the Vero cell line as a platform for rVSV vector production. In his work, he compared adherent and suspension Vero cell bioreactors, optimized critical process parameters, and developed analytical techniques for rVSV quantification. Sascha obtained a BSc and MSc in biotechnology from the Technical University of Braunschweig, Germany. During his master’s degree program, he spent one year at the University of Waterloo, Canada, as part of an academic exchange, and one year as a visiting graduate researcher at the University of Western Ontario in London, Canada.
Functional Roles of the Novel AAV Membrane-Associated Accessory Protein
Adeno-associated virus (AAV) has evolved overlapping genes to maximize the usage of its genome. An example is the recently-discovered ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. To study the largely unknown role of MAAP in wild-type (wt)-AAV and recombinant (r)AAV context, we made point mutations along the MAAP ORF while keeping overlapping capsid protein ORFs intact. In cells co-transfected with plasmids encoding wt-AAV genome and adenovirus helper genes, MAAP localized not only in the plasma membrane but also to intracellular membranes. Both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. Importantly, while MAAP was beneficial for wt-AAV replication/infection, it was not essential for rAAV generation as opposed to some MAAP-modifications which resulted in substantially improved virus yields and capsid integrity. Altogether, our results reveal some surprising MAAP functions with important implications for better-quality and quantity production of AAV vectors for therapeutic purposes.
Dr. Galibert has accumulated over 15 years of experience in the adeno-associated virus gene therapy field. While on the team of Professor Otto-Wilhelm Merten at Genethon in France, they developed a single baculovirus for the production of rAAV8 in Sf9 cells. They demonstrated the involvement of the baculovirus protease on the degradation of capsids of certain AAV serotypes. He then worked in the group of Professor Michael Linden for Pfizer in London. He was involved in the development of the rAAV production system in fixed-bed bioreactors using mammalian cells. For the last four years, he has worked in Kuopio´s biotech environment with positions in FinVector and its sister company Kuopio Center for Gene and Cell Therapy. In the group of Professor Kari Airenne, he has been leading the AAV research with a particular focus on wild-type AAV. He will be presenting the work they performed on the novel AAV "membrane-associated accessory protein” (MAAP).
Scale-Up of rAAV Production Using Stable Producer Cell Lines
In the context of viral vector production for gene therapy applications, our work focuses on process development for recombinant adeno-associated virus (rAAV) production. Recently, we have established a novel platform based on human suspension producer cells. The producer cells have stably integrated all components necessary to produce rAAV and therefore do not require any helper virus or transiently transfected plasmid DNA. Production in this system is initiated by simple induction via doxycycline. Stable rAAV production using this platform has been proven with different serotypes as well as different GOIs. In this work, upstream process development was carried out using a proof-of-concept clone for production of AAV8-GFP. Screenings were done, followed by scale-up to 10 L. Subsequently, the process scale-up to 50 L and then to 200 L using a single-use stirred tank bioreactor, in collaboration with Pall Corporation, was successfully completed. Additionally, to further increase rAAV yields, we established an intensified process in perfusion mode using the same producer clone. The perfusion set-up consisted of a lab-scale stirred-tank bioreactor connected to an ATF-2 system. Perfusion was used both for cell growth to high cell density and, after induction, for rAAV production. The perfusion process resulted in significantly higher volumetric production, cell specific yield, and higher percentage of full particles.
Dr. Juliana Coronel has extensive experience in upstream process (USP) development in the fields of biopharmaceuticals, viral vaccines, and more recently viral vectors. Her main expertise is animal cell culture processes in bioreactors. Since 2019, she has held the position of Head of USP at Cevec Pharmaceuticals in Cologne, Germany. Previously, she held scientific positions at the Max Planck Institute in Germany and the Federal University of Rio de Janeiro in Brazil. She received her doctorate degree, DSc, in chemical engineering, a master's degree, MSc, in biomedical engineering, and a bachelor's in biosciences from the Federal University of Rio de Janeiro.
Senior Vice President of Manufacturing
New Nanotechnology for Efficient Non-Viral Gene Delivery to Cells Ex Vivo
Essentially all technologies designed to deliver molecular payloads to cells suffer from stochastic variations that limit the ability to optimize delivery but also result in difficulty achieving uniformity and targeted thresholds. The result is a delivery distribution where significant percentages of cells will get no payload or get too much payload. This is true regardless of the delivery approach, such as viral, chemical, mechanical, or electrical. Our novel deterministic mechanoporation (DMP) device creates a single pore in each cell with a defined diameter. It also creates a pathway through the internal cellular compartments which can result in direct ingress of payloads to the nucleus. By utilizing a massively parallel array of needle-like penetrators integrated into cell capture ports on a silicon chip-based substrate, the technology is easy to scale. The device operation is also quite simple as it relies solely upon microfluidics to operate. In this presentation our DMP device will be introduced as an alternative approach for efficient, optimizable molecular payload delivery to cells ex vivo.
Chris Ballas is co-inventor of the SoloPore technology, and is an experienced adult stem cell and gene therapy research scientist with expertise in regenerative processes and wound repair. He is currently Senior Vice President of Manufacturing at Innovative Cellular Therapeutics. He has additional expertise in process development, manufacturing, testing, clinical trial management, new technology development, along with significant experience in R&D and cGxP environments. He specializes in gene therapy, non-viral delivery, viral vectors, stem cells, transplantation biology, regulatory, and microinjection within the CGT discipline. He received his PhD in Cellular and Molecular Pathology from the Vanderbilt University School of Medicine.
BacMam-Mediated In Situ Modification of Recombinant Proteins
Viral transduction is one of the most efficient mechanisms for the introduction of foreign genes into mammalian cells. Advantages associated with this method of gene transfer include ease of gene amplification, reproducible efficiencies of gene transfer and the ability to tightly regulate the number of gene copies introduced into the host cell. This latter characteristic is of particular interest in the formation of protein complexes such as VLPs and the ability to successfully produce difficult to express proteins like equine encephalitis E1 and E2 antigens in soluble form. BacMam viruses are BSL1 entities that have the ability to carry large inserts (at least 15 kb) and are extremely easy to amplify and use. A potential use of the BacMam technology that we have been working with is its use to drive modifications of overexpressed proteins in situ. In this presentation we describe the development of a BacMam viral construct carrying the gene for BirA to catalyze the biotinylation of rIgG produced in HEK-293 cells transfected using PEI. In a second application a BacMam virus with the gene for human furin inserted was used to overexpress furin in HEK-293 cells transfected with genes carrying furin cleavage sites. Data related to the development of the two processes and their functionality will be presented.
Chris Kemp is President of Kemp Proteins, LLC, a contract gene to protein expression company located in Frederick, Maryland. Dr. Kemp has over 25 years of experience in the scale-up of recombinant protein expression systems including baculovirus, mammalian, and BacMam expression platforms. Chris founded the protein expression service company Kemp Biotechnologies, Inc. in 1992, the molecular biology product company GeneChoice in 2000, and Kempbio, Inc. in 2008. The current company was formed after the purchase of Kempbio by 6.02 Bio in 2018. The company is focused on hybridoma development, IgG and rIgG production, viral glycoprotein production and purification, and the production and purification of virus-like particles (VLPs) and nanoparticles.
Head of Molecular Biology & Upstream Development
Process Intensification for a Human Recombinant Influenza Vaccine
Flublok is the first licensed recombinant hemagglutinin (HA) influenza vaccine. The HA proteins produced in insect cell culture using the baculovirus expression system technology are exact analogues of wild-type circulating influenza virus HAs. The universal manufacturing process for making highly purified HAs has been shown to be robust and scalable. The process has been successfully scaled to 21,000 L and contributes to rapid delivery of a substantial number of doses of seasonal or pandemic influenza vaccines. Approaches for improving the manufacturing process and yields of HAs will be presented.
Dr. Khramtsov has a PhD in Bioorganic Chemistry. He has 17 years of academic and 20 years of industrial experience in discovery, upstream development, process scale-up, and technology transfer. He directed all research and development activities (molecular biology, protein expression and purification, strain development, analytical assays, and fermentation) in a fast-paced environment. Dr. Khramtsov has a strong background in small-scale bioreactor experimentation and scale-up to production levels; extensive experience expressing proteins in insect, mammalian, yeast, and bacterial systems (batch, fed-batch, chemostat, solid-state fermentation, aerobic, and anaerobic); co-authored over 30 peer-reviewed articles and 17 patent applications; a proven record of successful grant writing; and strong management and leadership skills with proven ability to direct and lead functional teams and to collaborate effectively with internal and external clients to meet project milestones.
Autologous Cell Therapy Manufacturing Innovation and Industry 4.0 Solutions
Autologous cell therapy has shown unprecedented clinical efficacy leading to enhanced rapid approval of the product with limited process development. In turn, the challenges of manufacturing are significantly higher due to donor-to-donor variation, batch size, different products, dose range, protocols, and the clinical state of the cell source donor. Conventional manufacturing solutions are limited and are highly qualified labor intensive. In order to meet the demand and treat patients there is a critical need for automation that can not only reduce labor and cost, but significantly reduces the high qualification needs and can handle donor-to-donor variation. Additionally, scaling out results in a large volume of data and sophisticated quality assurance, chain of custody, and control. Therefore, there is a need to approach automation in a holistic manner by integrating solutions that include machine learning and artificial intelligence. The ADVA X3 platform is an innovative solution allowing flexible manufacturing with sensor activated automation, and in turn enabling industry 4.0 solutions into challenging decentralized autologous cell therapy manufacturing. The talk will present the challenges and the innovative approaches taken to move cell therapy into the 21st century of manufacturing.
Dr. Karnieli is a well-known international expert in cell therapy with extensive knowledge of the industry. Dr. Karnieli is the founder and former President of Atvio Biotech, a leading Innovation center for cell and gene therapy. He is also the former VP of Technology and Manufacturing at Pluristem Therapeutics, and the former VP of Medical Devices at Goji Solutions. Dr. Karnieli served as the chair of several industry committees including the process and product development committee of the International Society for Cellular Therapies (ISCT), expert member in the ISO TC276 biotechnology standard committee, and the former chair of the science and technology committee of the Alliance for Regenerative Medicine (ARM). Furthermore, Dr. Karnieli serves as an advisor and board member to several lead developing cell therapy companies. Dr. Karnieli earned his PhD in Cell and Gene Therapy from the Sackler School of Medicine at Tel Aviv University. He also holds an MBA from the Haifa University business management school.
Vaccine and Viral Vector Production Using the Baculovirus-Insect Cell Expression System: AAV Case Study
The insect cell-baculovirus expression system (IC-BEVS) has been extensively used as a platform for production of vaccines and viral vectors for gene therapy applications. Our lab has dedicated significant efforts to improve the production of influenza-like virus particles as a potential influenza vaccine candidate. In this regard, major manufacturing challenges have been identified including the difficulty to separate baculovirus from VLPs in the final formulation. Furthermore, we have explored overcoming bottlenecks known to affect vaccine production in the insect cell platform by using RNAi-mediated silencing. A critical review and understanding on the topic guides us in future directions using this approach. On the other hand, our research work has also been focused on the optimization of the production process of adeno-associated viral vectors (AAV) in the IC-BEVS system. Recently, the implementation of an efficient production process using the OneBac system to produce AAV5 has been achieved. The volumetric yields of AAV genomic particles were significantly increased by applying a refined fed-batch strategy achieving high cell densities in bioreactors. Additionally, substantial improvements to the downstream processing of the AAVs and separation of empty and full capsids at preparative-scale has been attained. These findings support the feasibility and promise of the IC-BEVS system as a remarkable platform to support the industrial manufacturing of biological products.
Impact of rAAV Titer Accuracy for Translational Studies
Dr. Zhu Zhen Pirot is the vice president of translational science at Kriya Therapeutics. She currently leads AAV gene therapy vector development, characterization, and analytical science in the company. Dr. Pirot previously worked at Sangamo Therapeutics as the head of the analytical department to develop methods for gene therapy vector/product characterization and release in support of the company’s gene editing and gene and cell therapy programs. She has developed extended experience and knowledge in AAV gene therapy development since early 2000 at Avigen. Her industry experience also includes cancer drug discovery and development at Chiron /Novartis, and bioanalytical assay and biomarker development at Geron Corp. She was trained as a medical doctor in China and earned her PhD in cell and molecular biology from the University of Turin in Italy.
Principal Investigator/Supervisory Microbiologist, Office of Vaccines Research and Review
Dr. Arifa S. Khan received her PhD in Microbiology from the George Washington University, Washington, DC. She is currently a Supervisory Microbiologist and Principal Investigator in the Division of Viral Products, Office of Vaccines Research and Review in the Center for Biologics Evaluation and Research (CBER), US Food and Drug Administration (FDA). Dr. Khan joined the FDA in 1991 after working in Dr. Malcolm Martin’s laboratory at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) since 1979, where she contributed significantly to the field of murine leukemia retroviruses, endogenous retroviruses, and simian immunodeficiency virus (SIV). In CBER, Dr. Khan established and maintains a rigorous research program on the development of sensitive, state-of-the art assays for adventitious virus detection, with a focus on safety of novel cell substrates and vaccines. Dr. Khan’s current research efforts include standards and standardization of next-generation sequencing for adventitious virus detection in biologics. Her regulatory responsibilities include review of candidate viral vaccines for HIV and emerging viruses such as SARS-CoV-2, and to provide expert consultation on novel cell substrates and adventitious viruses in CBER and CDER. Dr. Khan has been involved in licensure of several viral vaccines and development of FDA, International Conference on Harmonisation (ICH), Public Health Service (PHS), United States Pharmacopeia (USP), and World Health Organization (WHO) guidance documents related to cell substrates and viral safety. She is the FDA lead on adventitious viruses and next-generation sequencing for cell substrates and product safety.
Development and Validation of Analytical Methods for GTx Commercialization
Baculovirus-Sf9 System for the Manufacture of SARS-CoV-2 Vaccine
Product CMC Reviewer, Gene Therapy Branch, Division of Cellular & Gene Therapies, Office of Tissue & Advanced Therapies
Recommendations for the Development of Allogeneic CAR T-Cell Products — A CBER Perspective
Spearheading a Paradigm Shift in Cell Therapy Manufacturing
Professor of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine; Director of Technology Innovation, Center for Definitive and Curative Medicine, School of Medicine
AAV Vector and Manufacturing Process Design Considerations: A 2021 Perspective
Head of Cell Therapies, Pharmaceutical Sciences
Senior Account Program Manager, Lentigen Technology