Speakers

Viral vectors are being increasingly utilized as therapeutics for delivery of a corrective gene, lysis of tumors by selective replication in cancer cells, or for introduction of antigens to the immune system in prophylactic and therapeutic vaccines. The Vaccine-Based Immunotherapy Regimen (VBIR) is a therapeutic cancer vaccine platform that employs a multi-antigen adenovirus (prime) and plasmid (boost) to generate an indication-specific cellular response with concomitant administration of immunomodulating monoclonal antibodies to sustain, expand, and enhance the effect. The multicomponent nature of this immunotherapy presents many challenges from development, to clinical trials and beyond. Additionally, FDA classifies this vaccine as a gene therapy, requiring familiarity with the evolving gene therapy regulatory environment.

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 five years and is currently analytical lead on viral and plasmid-based immunotherapeutics and gene therapies. Previously, he spent three years in small biotech at two different companies as the analytical lead in the development of serum-based cancer diagnostics, plus four years as a postdoc at the University of Tennessee investigating the binding interactions of serum proteins. He has a BS in Chemistry from the University of Tennessee, Chattanooga and a PhD in Biochemistry from Vanderbilt University. His education and research path covers a wide breadth including small molecule synthesis; protein cloning, expression and purification; enzyme structure/function determination; serum antigen identification and mAb production/selection; and virus/plasmid analytics from method development to characterization to critical quality attribute determination to overall control strategy. His work has generated a number of peer-reviewed publications and presentations at scientific conferences as well as internally within Pfizer.

The highly complex processes underlying in disease development and progress demand the development of emerging therapies that are effective, specific, and comprehensive in their therapeutic effects. Extracellular vesicles (EVs) are capable of generating multi-dimensional biological effects with ideal biocompatibility, by themselves or when loaded by therapeutic agents (e.g., drugs, nucleic acids, and proteins), for diverse therapeutic strategies. Key pivotal challenges in clinical translation of EV technology are high heterogeneity in their physical, chemical, and biological properties and limited scalability for production and purification in order to meet clinical needs. In contrast to naturally-occurring EVs, chemically-induced EVs offer significant advantages including an order-of-magnitude improved production, simple and fast separation and purification, easy and flexible loading of therapeutic modalities, and fine-tunable characteristics. The approach can also be applicable to a broad range of cells with high consistency in production yield and quality. This talk will present the rationales, up-to-date progress, and potential impact of the technology along with its proof-of-concept studies of chemotherapy and immunotherapy for cancer.

Young Jik Kwon is a professor at UC Irvine in the Pharmaceutical Sciences, Chemical Engineering & Materials Science, Biomedical Engineering, and Molecular Biology & Biochemistry departments. Following his undergraduate education in Biological Engineering at Inha University in South Korea, he received his PhD in Chemical Engineering from the University of Southern California with a focus on retroviral gene delivery in 2003, and did postdoctoral training in the department of chemistry at UC Berkeley on polymeric vaccine carriers. He started his academic career in Biomedical Engineering at Case Western Reserve University in 2005 and moved to UC Irvine in 2007. He currently oversees research on gene therapy, drug delivery, extracellular vesicle therapeutics, and cancer vaccines.

Mesenchymal stromal cells (MSCs) have been the subject of clinical trials for more than a generation, and the outcomes of advanced clinical trials have fallen short of expectations raised by encouraging pre-clinical animal data in a wide array of disease models. In this presentation, important biological and pharmacological disparities in murine pre-clinical research and human translational studies are highlighted, including cryoinjury. Analyses of clinical trial failures and recent successes provide a rational pathway to MSC regulatory approval and deployment for disorders with unmet medical needs.

Jacques Galipeau, MD, FRCP(C) is the Don and Marilyn Anderson Professor of Oncology within the Department of Medicine and UW Carbone Comprehensive Cancer Center at the University of Wisconsin in Madison, and is Associate Dean for Therapeutics Development at the University of Wisconsin School of Medicine & Public Health. Dr. Galipeau has an NIH-funded research program in the study and use of mesenchymal stromal cells as an immunotherapy of catastrophic illnesses including cancer and immune disease. He is an internationally recognized expert in translational development of cell therapies and the sponsor of a series of FDA-sanctioned clinical trials examining the use of autologous marrow-derived mesenchymal stromal cells for immune disorders, including Crohn’s disease and graft-versus-host disease. He has also developed the field of fusion engineered cytokines known as fusokines, as a novel pharmaceutical means of treating immune disorders and cancer. He is the director of the University of Wisconsin Advanced Cell Therapy Program whose mission is to develop personalized cell therapies for immune and malignant disorders and to promote and deploy first-in-human clinical trials of UW cell therapy innovations to improve outcomes for children and adults. Dr. Galipeau is the Chair of the International Society for Cell & Gene Therapy (ISCT) MSC Committee.

Exosomes bring a new set of challenges to purification; some in common with proteins, some in common with virus particles, but many unique to exosomes. It stands to reason that we need to apply the traditional purification tool box in different ways, and that we might benefit from development of tools to rationally exploit exosomes’ unique characteristics. One of those new tools is hydrogen bond chromatography. Experimental data will be shared showing that although hydrogen bonding is fairly weak for the majority of proteins (hydrodynamic diameter 2–15 nm), it supports very strong retention for large solutes (20–200 nm). Beyond enhancing removal of protein contaminants, some buffer systems support discrimination among different size classes of vesicles. Integrated in-line detection by multi-angle light scattering (MALS) enables direct visualization of vesicle populations on chromatograms, and thus identification of meaningful fractions for confirming analysis by immunoassay and other methods. Case study data will be shared illustrating the application of hydrogen bond chromatography and MALS to purification of vesicles from plasma and cell culture harvest. Selected multistep purification procedures will be shown illustrating complementary combinations of hydrogen bonding chromatography with other chromatography and precipitation methods. Removal data for DNA and endotoxins will be shared along with data for protein contaminants.

Pete Gagnon is a widely known and respected authority in the field of downstream processing. He holds more than 100 patents in more than a dozen countries, covering various aspects of purifying antibodies, other recombinant proteins, virus particles, and exosomes. He is the author of more than 100 articles, book chapters, and books; an editorial advisor for several journals and conferences; and a frequent conference presenter. He has held high level positions in several companies and is currently the Chief Scientific Officer of BIA Separations.

In the past few years, the view on macrophages has changed dramatically. Nowadays, macrophages are understood as a unique cell type of the hematopoietic system with high plasticity and regenerative potential. Given these specific functions, the presentation of Dr. Ackermann will provide recent insights into the therapeutic use of macrophages, which can be derived from various stem cell sources. In addition, she will also introduce the scalable generation of hematopoietic cells from pluripotent stem cells. Introduced in 2006, the technology of induced pluripotent stem cells (iPSC) holds great promise for various fields of modern medicine such as drug screening, disease modelling, and new cell-based treatment approaches. Given the potential of iPSC to differentiate also into cells of the hematopoietic lineage, Dr. Ackermann will present a recently developed, continuous hematopoietic differentiation process which is able to produce different hematopoietic cell subsets. Using this technology, Dr. Ackermann will present data on the use of iPSC and iPSC-macrophages for disease modelling and cell-based therapies. For future clinical translation of iPSC-derived cell subsets, Dr. Ackermann will provide an overview on scaling up macrophage production into industry compatible bioreactor systems and will shed light on the therapeutic use of generated cell types for rare and common diseases.

Dr. Mania Ackermann is a project leader in Experimental Hematology at Hannover Medical School, Germany and at the Dana Farber Cancer Institute, Boston, Massachusetts. She received her Master of Science in Biochemistry at Hannover Medical School and holds a PhD in Regenerative Sciences. Her research within the Cluster of Excellence REBIRTH/Hannover Medical School is dedicated towards the development of new gene and cell therapies for the treatment of rare and common diseases. She has a particular interest in the various possibilities of pluripotent stem cell (PSC) technology and has recently established a platform for the large-scale production of mature blood cells from PSCs in industry-compatible bioreactor systems. Given this powerful tool, her recent efforts focus on the application of this system for disease modelling, drug screening, as well as the development of novel cell-based therapies in a variety of diseases. Given her translational work, she has published several papers and was awarded with different national and international stipends/awards, highlighting her ambitious goal to drive PSC-derived macrophages towards clinical translation.

One long-term, overall goal of our research is to address a major limitation of the baculovirus-insect cell system (BICS), which is that it cannot produce higher eukaryotic glycoproteins with authentic glycosylation patterns. Our basic approach has been to add or subtract glycogenes, which are the genes encoding functions comprising recombinant protein glycosylation pathways. Given the BICS is a binary system, we can target the baculovirus vector, the insect cell host, or both in our efforts to modify endogenous protein glycosylation pathways. Our ultimate goal is to produce “glycoengineered” BICS, which can produce recombinant glycoproteins with pre-selected, relatively homogeneous, human-type glycosylation profiles. As the BICS is a binary system, we have the option to glycoengineer the baculoviral vector, the insect cell host, or both. While we originally targeted the baculovirus vector component to provide the first example of glycoengineering in the BICS, we have spent far more time on a broadly based, sustained effort to engineer the host component of the BICS. In this presentation, we will briefly review the results of those efforts, but then we will focus most of the talk on our re-focused effort to create glycoengineered baculovirus vectors. We will describe new tools that will be of general interest, including a new bacmid, a new LoxP/LR donor plasmid, and a new Tn7 transfer vector. We will show these can be used to isolate genetically stable recombinant baculovirus vectors carrying up to nine foreign genes and document the underlying basis for their genetic stability. We will describe the functional aspects of this new family of glycoengineered bacmids and baculovirus vectors, and show they can be used to produce nine distinct glycoforms of a recombinant glycoprotein in which the pre-selected N-glycan structure comprises 56-100% of the total profile. Finally, we will discuss our current efforts to use these vectors to produce various influenza hemagglutinin and IgG glycoforms for studies on the impacts of N-glycan structure on various glycoprotein functions.

Don Jarvis earned his PhD in virology at Baylor College of Medicine, performed postdoctoral studies with Max Summers at Texas A&M University, and established an independent research program as an Assistant and Associate Professor at Texas A&M. Don moved to the University of Wyoming in 1998, where he was promoted to Professor and continues to address protein processing and other issues in an effort to improve baculovirus-insect cell technology. In 2011, Don started GlycoBac, LLC to fine-tune, commercialize, and apply new BICS and BICS components created in his academic lab over the past 30 years.

Patient safety is at the core of biopharmaceutical manufacturing operations. To address virus safety assurance, regulatory guidance and industry best practices require a holistic approach that incorporates preventative and intervention strategies. The viral risk profile of any biological is contingent on multiple factors including: source of the biological, raw materials used, production systems, purification reagents, and excipients. The structural complexity and impurity profile of biologicals pose unique analytical challenges and, consequently, raw materials management is a cornerstone in risk mitigation and management of virus ingress. This presentation will address the current pragmatic approach with a focus on quality by design in virus safety assurance. Regulatory approaches and industry best practices will also be discussed.

Hazel Aranha is a biopharmaceutical industry professional with over 30 years’ experience in industry, academia, and consulting. She has provided consulting services in the US, Europe, and Asia. She teaches courses on ensuring virus safety of biologicals, as well as other topics including: good clinical practices (GCP), good manufacturing practices (GMP), downstream processing, navigating the drug development cycle, and effective biomedical writing. Hazel has a Master’s degree in Virology, a PhD in Environmental Microbiology, and holds a Regulatory Affairs Certification (RAC) for both the US and European Union. She has to her credit two books, over 45 publications, and five book chapters. Her past assignments have included positions at Catalent Pharma Solutions, Wyeth Vaccines (Pfizer), and Pall Corporation.

Influenza represents a global public health problem. Current seasonal vaccines are frequently updated to match circulating and emerging subtypes. Improved vaccines are needed that provide broad immunity to many influenza types/subtypes and do not require frequent updates. Recombinant multi-subtype virus-like particles (msVLPs) represent a novel approach to the development of broadly protective influenza vaccines. The msVLPs co-localize multiple subtypes of hemagglutinin (HA) within the VLP envelope and protect against multiple influenza viruses. Here we report a msVLP design that co-localizes seasonal A/H1, A/H3, B/Victoria, and B/Yamagata strains. In addition, we prepared msVLPs that co-localize potentially pandemic H5, H7, H9, and H10 subtypes. Vaccines were prepared using the baculovirus expression system and extensively characterized including quantitation of each subtype within the msVLPs. Expression of msVLPs was confirmed after several passages of baculovirus vector. Safety, immunogenicity, and efficacy of msVLPs were evaluated in animal challenge models. We conclude that msVLPs represent a feasible approach for preparation of broadly protective influenza vaccines.

Dr. Peter Pushko has over 25 years of experience in molecular biology, virology, and vaccine development. After receiving his PhD, Dr. Pushko completed postdoctoral studies at Cambridge University, UK and at the US Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland. He is the author of numerous publications on influenza vaccines. Dr. Pushko currently serves as President and Chief Scientific Officer at Medigen, Inc., Frederick, Maryland. His research interests include vaccines for emerging diseases including avian influenza.

A validated potency assay is required for approval and lot release of all cellular and gene therapy products. The assay must represent the product’s MOA and measure performance relative to a reference standard in a reproducible, controlled manner. This presentation will discuss steps for advancing an in vitro relative potency assay from development through validation.

Karen Doucette has worked for preclinical contract research organizations (CROs) for over 17 years. She joined Absorption Systems in 2003 as an Associate Scientist performing in vitro permeability and metabolism assays for ADME profiling of drug candidates. As Project Manager for Absorption Systems, she was instrumental in streamlining preclinical in vivo PK and toxicology studies, collaboratively driving research and expansion of Absorption Systems’ in vivo ocular services in San Diego, California, and increasing company-wide study throughput. In her current role as Director of Operations, Karen oversees a team of project managers as well as a wide variety of US-based and international accounts. Her account support spans consultative study design, internal and external project management, and study execution and reporting. She plays a key role in coordinating research innovation with customer need, particularly in the areas of in vitro DDI and BE testing, and potency assays for cell and gene therapy products. Prior to joining Absorption Systems, Karen was a Senior Research Pharmacologist at NovaScreen Biosciences where she developed and performed high-throughput receptor-binding assays for pharmacological profiling of new molecular entities. Karen has a bachelor’s degree in Animal Science from the University of Delaware and an MBA from Goldey-Beacom College.

This presentation will give an overview of the latest advancements in process intensification to support global demand for affordable vaccines; cell lines optimized for virus propagation, media capable of supporting high cell density cell growth, high cell density single-use bioreactors, and high efficiency and single-step purification technology. Together, these technologies enable vaccine yields to be significantly increased, which in turn allows commercial manufacturing in a small-footprint, low-cost micro-facility.

Alfred Luitjens is Director Cell Technology at Batavia Biosciences. He has a profound background in development and production of vaccines, with more than 30 years of experience. He worked for research & development at Solvay, DSM Biologics, and Crucell. He had a management position at the Animal Science Group and was senior manager, process architecture at GSK. At Batavia Biosciences, Alfred is responsible for all upstream development vaccine projects.

Mortality rates in late-stage cancers are exorbitant. An alternative to commonly applied cancer treatments is the use of oncolytic viruses. One promising candidate of oncolytic viruses is the measles virus due to its natural affinity to infect cancer cells. As the measles virus is enveloped and shows a high sensitivity against physicochemical parameters such as high temperature, low pH, and shear forces, the challenge is to generate active virus in a high-yielding purification process. The integration of suitable process analytical technologies, such as dielectric spectroscopy, and the associated possibility of detecting time of harvest (TOH), led to a drastic increase in measles virus yields. Compared to the static cultivation systems such as T-flasks, higher virus titers of up to 1010 TCID50 mL 1 in stirred tank could only be realized within narrow process limits. In a stirred tank reactor (STR), shear stress levels of < 0.25 N m 2 as well as aeration could be identified as critical processing parameters for the measles virus production in Vero cells. Having established a production process to generate high virus titer in a STR, we are now faced with the development of an efficient downstream process. Typically, the downstream process takes place in two essential steps. In the first step, microcarriers, cell debris, etc. are separated from the product by using membrane filtration or depth filtration. The performance of these separation processes is significantly influenced by the composition of the culture medium. For example, a substitution of serum containing medium by serum-free medium results in a yield reduction by more than a factor of 10. Similarly, results could be detected in the second step, chromatographic purification. In terms of selectivity, adsorptive materials are a potentially efficient method for final virus purification. Due to their separation mechanism based on electrical interactions, these methods are even more susceptible to changes in the United States Pharmacopeia (USP). Taking into account the specific characteristics of measles viruses produced in serum containing and serum-free media, we have developed an efficient purification method. With the development of a tailor-made production and purification process of an oncolytic measles virus, we are able to preserve high virus titers.

Vero cells are anchorage-dependent cells that are widely used as a platform for viral vaccine production. In stirred-tank bioreactors, they are ordinarily grown on microcarriers. Fibra-Cel® disks are a promising alternative attachment matrix with a high surface-to-volume ratio. They provide a three-dimensional environment that protects cells from damaging shear forces, helping to achieve high cell densities. In this study, we cultivated Vero cells in Eppendorf BioBLU 5p Single-Use Vessels pre-packed with Fibra-Cel. The process was controlled with a BioFlo® 320 bioprocess control station. We cultivated the cells in perfusion mode, which ensures a consistent supply of nutrients and the removal of toxic byproducts. We achieved the very high Vero cell density of approximately 43 million cells per mL, demonstrating great potential for Vero-cell-based vaccine production using Fibra-Cel packed-bed vessels.

Antimicrobial peptides (AMPs) from insects are valuable resources for the pharmaceutical industry and can serve as leads for the development of novel antibiotics. To access this potential, an effective recombinant expression process is mandatory, which includes the selection of a suitable expression host as well as process optimization during scale-up. Here, we describe the recombinant production of different AMPs using either the baculovirus expression vector system (BEVS), or stably transformed insect cells. Whereas BEVS enables fast production of AMPs, stable cell lines offer more flexibility in terms of further optimization and usable process modes. Based on a stable, polyclonal cell line that is obtained after transfection, it is possible to isolate highly productive single cell clones by limiting dilution. This achieves typically a two- to six-fold increase in cell-specific productivity. If an inducible promoter is used, further optimization on the cellular level can be achieved by employing statistically planned experiments to determine optimal conditions for induction. On the process scale, online measurement of the cell suspension's dielectric properties and turbidity enables efficient process control and monitoring of the cells' physiological status. Based on this information, typically 17–30 mg/L of the AMP can be expressed in batch mode, either using BEVS or stable cell lines. The current focus is further process intensification by the application of a high cell density perfusion process. First, perfusion results at the bench-scale already increased the final protein yield to 130 mg, using essentially the same equipment as for batch culture. Finally, the functional AMPs were recovered by affinity chromatography. The antimicrobial properties against E. coli were tested, indicating the successful isolation of active peptides.

Brian Hampson has over two decades of strategic and technical leadership experience driving innovative development of technologies, devices, systems, and automation for cell-based processes. He currently holds the position of Vice President, Global Manufacturing Sciences & Technology at Hitachi Chemical Advanced Therapeutic Solutions (“HCATS,” formerly PCT) where he provides executive oversight for Manufacturing Development and Innovation & Engineering teams dedicated to solving the challenges of cell therapy manufacturing. Previously, Brian held several executive and technical management positions at Aastrom Biosciences (now Vericel) and was a principal leader in the development and engineering of the AastromReplicell Cell Production System, a first-of-its-kind automated manufacturing platform for culture-derived cell therapy products. Prior to that, he was at Charles River Laboratories and Corning, Inc. where he managed a number of programs to develop and commercialize novel bioreactor systems to support large-scale biomolecule production. Brian has authored or co-authored a variety of abstracts, publications, invited presentations, grants, and patents. He holds a bachelor's and master's degree in engineering from Cornell University.

Imre Berger was trained as a biochemist and molecular biologist at Leibniz University and Medical School (MHH) in Hannover (Germany), at MIT (Cambridge, USA), and at ETH Zurich (Switzerland). His team develops enabling methods for researching large multiprotein complexes and investigates their structure and mechanisms in human health and disease. In 2007, Berger became Group Leader and Principal Investigator for eukaryotic complexes at the European Molecular Biology Laboratory (EMBL). In 2014, he was appointed Full Professor at the School of Biochemistry, University of Bristol, UK. Since 2017 he is Director of the BBSRC/EPSRC research centre for synthetic biology at Bristol, BrisSynBio, and Co-Director of the Bristol Biodesign Institute (BBI). Imre Berger has developed the MultiBac, ACEMBL, and ComplexLink systems for DNA delivery and multiprotein research. He holds international patents for DNA and protein technologies, co-founded three companies, and received numerous distinctions, including the Swiss Technology Award, the W.A. DeVigier Foundation Award, and a Wellcome Trust Senior Investigator Award for his innovative research.

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 United States 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. As a Principal Scientist in Global Regulatory Affairs and Product Development with Cardinal Health Regulatory Sciences, Dr. Webster is the Director of Advanced Therapy Medicinal Product Development. In this capacity, she provides guidance on clinical, nonclinical, and regulatory aspects of strategic product development, authors regulatory documents, and acts as the regulatory representative for sponsors in interactions with the FDA.

Modification of host behaviour is a widely adopted strategy of parasites to enhance their transmission. Fascinating examples of behavioural manipulation are known, covering a broad spectrum of parasites and hosts. Nevertheless, surprisingly little is known on the underlying causative physiological, neuronal, hormonal or molecular mechanisms. A typical case of behavioural manipulation is found in caterpillars infected with baculoviruses. Infected caterpillars show enhanced mobility (hyperactivity) and climb to the top of plants or the forest canopy prior to death (‘tree-top disease’ or ‘Wipfelkrankheit’). As a consequence, the virus is spread over a larger area, thereby increasing the chance to infect a new caterpillar. I will review the current knowledge on the mechanisms of baculovirus-induced behavioural manipulation, including an overview of viral genes found to modify host behaviour. I will explore which molecular pathways and processes in the host are involved and will elaborate on the role of light on the induction of tree-top disease.

Professor Monique van Oers, PhD is Chair of the Laboratory of Virology at Wageningen University in the Netherlands. The laboratory focuses on insect viruses, plant viruses, and arboviruses. Her main scientific interest lies in fundamental and applied aspects of insect viruses and how viruses in general interact with insects, as host or vector. Current topics include functional genomics and evolution of large insect DNA viruses, and the molecular mechanisms behind baculovirus entry and packaging. She has long term experience in the exploitation of fundamental data to optimize the baculovirus insect-cell expression system for the production of recombinant proteins, and more in particular for vaccine purposes. One of her research lines aims to develop a baculovirus-free system that aims to use baculovirus technology to produce recombinant proteins without contaminating baculovirus particles, for which she recently received a grant from the Netherlands Organisation for Scientific Research (NWO). She coordinates an ITN-European joint doctorate programme proposal in insect pathology to support the upcoming industry of insect mass rearing. Another intriguing research topic which she leads together with her colleague Vera Ros, is the unravelling of the molecular mechanism behind virus-induced changes in insect behaviour. This is the topic that was selected for her presentation at this year’s ISBioTech meeting. She currently supervises 15 PhD students; and several of the PhD students trained in her laboratory are/were involved in baculovirus-based vaccines against (emerging) human, cattle, and fish diseases. She has published over 100 research papers and reviews in peer-reviewed journals, as well as several book chapters. She is the Secretary of the Society for Invertebrate Pathology and recently guest edited a special issue of MDPI Insects, in which she published a review on baculovirus per os infectivity factors. Last year she published an overview paper describing the diversity of large invertebrate DNA viruses in the Journal of Invertebrate Pathology. Dr. van Oers received her PhD in 1994 from Wageningen University.

In the batch manufacture of recombinant products, harvest typically marks the transition between upstream production and downstream product recovery. In the manufacture of non-secreted viral vectors using the Sf9/baculovirus expression system, harvest commonly employs chemical lysis, followed by depth-filtration clarification, where the primary goal is to remove insoluble cell debris. In this work, various methods of cell lysis and harvest clarification for rAAV production with Sf9/baculovirus are described. Performance of numerous chemical and physical lysis methodologies are compared head to head, as are a variety of primary clarification approaches and filter train configurations. Product recovery, impurity clearance, process robustness, scalability, and statistical approaches to experimental design are also presented. In conclusion, a wide set of harvest and clarification approaches, their process performance, and ultimately their relevance for cGMP manufacturing of rAAV vectors with Sf9/baculovirus are discussed.

Dan is a Senior Associate Engineer at Voyager Therapeutics (Cambridge, Massachusetts) focused on rAAV purification process development, technical operations, and CMC. Dan earned his bachelor's in Physics and master's in Biological Engineering from Tufts University. Prior to Voyager, Dan worked on purification process development of recombinant proteins.

A chemically defined (CD) medium for HEK293 cells to produce AAV was developed by modifying our high-throughput cell culture medium development and optimization system which has been proven successful with Chinese hamster ovary (CHO) media development. This medium was developed to provide good cell growth and good AAV production using transfection. To improve the lot-to-lot consistency of the HEK293 medium, a CD formulation was developed using Design Expert software to blend proprietary media from the MilliporeSigma library. Prior to blending, the media were modified to support HEK293. The media were initially tested using high-throughput methods. All formulations supporting cell growth were expanded for multiple passages and were further optimized using multivariate analysis (MVA) software to model individual, critical components to evaluate. The final formulation is a highly performing CD medium capable of growing the HEK293 cells and supporting virus production.

Coxsackievirus B group (CVB) of enteroviruses are a cause of severe morbidity and mortality. They cause myocarditis, meningitis, pancreatitis, and severe sepsis-like syndrome of neonates. In addition, they have been associated with chronic diseases like type 1 diabetes. There are, however, no vaccines or antivirals against these viruses. In order to develop vaccines against CVBs we have developed a baculovirus expression system for CVB virus-like particles (VLPs). The enteroviral capsid is expressed as a polyprotein, which is then processed into individual capsid proteins by viral proteases, and both of these proteins are also needed to produce CVB VLPs. Originally, we utilized the Bac-to-Bac system, in which both the capsid encoding polyprotein and viral protease were expressed under strong promoters (polh and p10). The protease is, however, known to be toxic for the cells, and therefore we optimized the construct design by utilizing a weaker promoter (cmv) for protease expression, and changed the baculovirus expression system to flashBAC. With other modifications these changes increased the final yield of VLPs six-fold. The produced CVB VLP vaccines induced robust immune response in preclinical mice experiments.

Francis is Director for Animal Health and Senior Cell Therapy Researcher at Likarda. Francis' current efforts are aimed at developing a cell-therapy based solution for diabetic dogs and cats, which will be delivered as an injectable islet transplant aimed at eliminating the need for daily insulin injections for diabetic pets. Beyond cadaveric islet sources, Francis is also actively engaged in efforts to derive and establish adult and/or bona fide pluripotent stem cells from dogs and cats that can be efficiently differentiated into insulin producing cells for treating diabetes in pets. Prior to joining Likarda, Francis worked at Johnson & Johnson where he worked extensively in a stem cell and regenerative medicine venture group developing beta-like, insulin producing cells from pluripotent stem cells for the treatment of diabetes in humans. Francis has also worked extensively and published several original articles on human hematopoietic stem cells from cord blood and bone marrow focusing on developing assays for isolation, characterization, ex vivo expansion, and in vivo engraftment in mice.

Mark Flower is the Vice President of Business Development and Strategic Partnerships for Be The Match BioTherapies. He is responsible for aligning the organization’s objectives with a firm business strategy, overseeing research and development of new markets for products and services, proactively assessing existing sales organization support investments, and leading development initiatives to grow sales and new business development for the organization. Prior to joining Be The Match BioTherapies he held the role of Executive Director, Sales and Marketing at PCT Cell Therapy, a North American based CDMO supporting cell and gene therapy manufacturing. Prior to joining PCT, Mark worked at Terumo BCT as Senior Manager, Global Strategic Marketing in the Therapeutic Systems business unit. In this global role he successfully launched and maintained marketing responsibility for multiple devices and technologies in the field of stem cell collection (leukapheresis), cell isolation, cell processing, cell culture, and manufacturing. Mark is a member of the International Society for Cellular Therapy (ISCT), the Society for Immunotherapy of Cancer (SITC), and graduated from the University of California, San Diego with a BS in Biological Science.

As a Director Regulatory Science and Co-Head Complex Biologics at Voisin Consulting Life Sciences (VCLS), Mike is responsible for providing both regulatory strategy and CMC consulting services to clients for global product development ranging from small molecule drugs to biologics and advanced therapies. Mike has over 20 years of experience in the pharmaceutical/biopharmaceutical industry and consulting services, having worked on the review, preparation, and maintenance of numerous regulatory documents and submissions, including INDs, ANDAs, BLAs, and NDAs. Prior to joining VCLS, Mike was the Managing Director of CMC/Global Regulatory Affairs at Cardinal Health Regulatory Sciences. Whilst there, he was responsible for providing comprehensive regulatory and CMC global strategy to clients at various stages of their product development. This covered advising on drug formulation, as well as device and combination product development. He was also responsible for providing leadership and the development of regulatory/scientific staff. Over this time, Mike has led and attended a large number of meetings and interactions with FDA, including information requests, pre-IND, end-of-Phase II, pre-NDA, and Type C/Scientific advice. This has involved close collaboration with a number of FDA divisions including: the Division of Oncology Products, the Division of Neurology Products, the Office of Tissues and Advanced Therapeutics, the Office of Generic Drugs, and the Office of Combination Products. He has a thorough comprehension of modern techniques of analytical chemistry including the development and validation of methods, installation, operation, and performance qualifications of instrumentation. Additionally, Mike has significant experience in performing cGMP compliance audits. Mike received his PhD in Pharmaceutical Sciences and Chemistry from the University of Missouri-Kansas City. He also currently serves as adjunct faculty for University of Missouri-Kansas City, School of Pharmacy and is the Vice-President of the Pharmaceutical Technical Exchange Association. Mike is based in VCLS offices in Cambridge, Massachusetts USA.

Laura A. Palomares is a Biochemical Engineer from the Monterrey Institute of Technology, Mexico, and has a doctoral degree in Biochemical Engineering from the Universidad Nacional Autonoma de Mexico (UNAM). She pursued postdoctoral training at the School of Chemical Engineering at Cornell University. She is a Professor, Principal Investigator, and Group Leader at the Biotechnology Institute of UNAM. She works on the production of vaccines, vectors for gene therapy, and nanobiomaterials. She collaborates with several companies in Mexico and abroad on process development and participates in the evaluation of biological new products for COFEPRIS, the Mexican regulatory agency. Her work has been recognized with several prestigious awards in Mexico and abroad, such as the National University Award, the Award of the Mexican Academy of Sciences, the Mexican Society of Biotechnology and Bioengineering Carlos Campillo Award, the Government of the State of Morelos, and the award to outstanding young faculty (DUNJA) given by UNAM. The Interciencia Association (Canada-Latin America) recognized her with the Interciencia Award in Life Sciences in 2014. All these awards were presented for her work in technology and innovation in pharmaceutical biotechnology.