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.

The discovery that many insect cell lines are contaminated with Sf-rhabdovirus raised obvious questions about the biosafety of biologics produced in these cells. In response, GlycoBac developed an Sf-rhabdovirus-negative cell line (Sf-RVN), which has now been licensed to several biologics manufacturers. In a turn of events, we are now using Sf-RVN cells to study Sf-rhabdoviruses as infectious agents. Recently, we isolated a wild-type strain of Sf-rhabdovirus from caterpillars. The genome of this strain indicates it is closely related to the progenitor of the Sf-rhabdovirus strains found in established cell lines. In this talk, I will discuss the ability of this Sf-rhabdovirus isolate to infect vertebrate cell lines, as well as key differences between the wild-type strain and cell line-adapted Sf-rhabdoviruses.

Christoph Geisler has worked in the baculovirus insect cell system since 2005. He has experience in both cell line development and baculoviral vector engineering, and specializes in the glycobiology of the system. Recently, he has developed new bioinformatics approaches to screen for adventitious viral contaminants in cell lines used to produce biopharmaceuticals.

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.

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.

Virus-like particles (VLPs) present viral antigens in a more authentic conformation than monomeric structural proteins. VLPs mimic the structure of virus particles they are derived from and display excellent adjuvant properties, being capable of inducing innate and cognate immune responses. Vaccines based on VLPs have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. In this communication we describe two baculovirus-based technologies that increase production yields for these kinds of vaccines. The first one is a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements acting in cascade. This novel cassette, denominated TB, conferred significant production improvements to the BEVS, including prolonged cell integrity after infection, improved protein integrity, and up to a 4-fold increase in the production yields. The second technology platform takes advantage of the insects, developing the CrisBio technology, based on a natural biocapsule that has the potential to displace stainless steel bioreactors in the recombinant protein production processes. These biocapsules (insect pupae) contain millions of cells in perfect physiological conditions, ready to produce any biotechnology medicament or vaccine. The insect pupae are placed in trays compatible with robots that may inoculate them with the recombinant baculoviruses producing the VLPs at a rate of more than 5,000 pupae per hour. Later, these inoculated trays, with the capacity to produce thousands of vaccine doses each, are incubated without any manipulation for around 5 days. Then, the vaccine antigen is ready to be purified from the pupae biomass by conventional means before its formulation. A 1,000 L conventional bioreactor is transformed in a simple incubation chamber the size of a home refrigerator. Anyone may produce by this technology the most sophisticated and complex protein-based vaccine, making the next generation of human and veterinary vaccines globally accessible. Data from production of two model VLPs in these baculovirus platforms will be illustrated in the presentation.

Romy has more than 17 years of experience in Molecular Biology and Virology and eleven scientific publications in peer-reviewed journals. She carried out her postdoctoral studies in the UK. Romy worked first as a researcher and then as a project leader at several biotech companies. She joined ALGENEX more than three years ago in the position of Chief Operations Officer. She will be the Manager of the CrisBio project, coordinating the Operation processes of the Research Team. She will also be responsible for the progress reports of the project and communications to the European Board.

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.

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. In this presentation, we will focus on our recent effort to create glycoengineered baculovirus vectors. We will describe new tools that will be of general interest, including a new bacmid, a new Cre-LoxP 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 61–100% of the total profile. Finally, we will discuss our current efforts to use these vectors to produce various influenza hemagglutinin (HA) glycoforms for studies on the impacts of N-glycan structure on glycoprotein functions.

Ajay Maghodia earned his PhD in Entomology at Anand Agricultural University and pursued his postdoctoral training at Agriculture and Agri-Food Canada, where he gained extensive experience in bacmid technology and baculovirus genome engineering. In 2014, Ajay joined GlycoBac, LLC at the University of Wyoming as a research scientist. His research is now focused on glycoengineering baculovirus vectors and creating virus-free insect cell lines as improved hosts for recombinant protein production in the baculovirus-insect cell system.

Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver disease, can cause neuropsychiatric complications, which in the worst cases result in brain damage, coma, or death. Diverse treatments exist for the treatment of hyperammonemia, but they have limited efficacy, adverse effects, and elevated costs. Gene therapy is a promising alternative that is explored here. Baculovirus and AAV vectors (serotypes 1 and 8) containing the glutamine synthetase (GS) gene were constructed and produced. Strategies for the production of both types of vectors will be presented. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a high expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia. When Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of ammonia in blood decreased 351 μM, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles from the rats transduced with Bac-GS. Bac-GS and AAV vectors were also tested in a chronic disease model with bile-duct ligated rats. Administration of Bac-GS or any of the AAV serotypes resulted in the reduction of ammonia blood levels to normal values. These results show that gene delivery for overexpressing GS in muscle tissue is a promising alternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy or UCD.

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.

Hemoglobinopathies are among the most prevalent monogenic disorders. Hematopoietic stem cells (HSCs) transduced with a lentiviral vector (LVV) encoding an anti-sickling globin are being evaluated in clinical trials for beta-thalassemia and sickle cell disease. To ensure drug product quality, it is important to demonstrate consistent LVV potency in manufactured lots. In this study, we present the development of LVV potency assays. qPCR was used to determine average LVV copy number from transduced healthy human donor CD34+ cells. Transduced CD34+ cells were cultured to stimulate erythroid differentiation, and liquid chromatography was used to determine the amount of β-A in erythroid differentiated cells (enucleated). Both assays report percent potency relative to a well-characterized LVV reference standard. These quantitative results demonstrate consistent manufacture of LVV.

Joyce Zhao is a Scientist in the Vector Analytics team at bluebird bio. She works on assay development of vector characterization and impurity analysis. Following her undergraduate education in Chemistry at Peking University, she completed her PhD studies in Biochemistry at Boston College in 2015, focused on the synthesis, analysis, and application of de novo designed peptides.

The ubiquitination pathway is utilized by numerous virus-host systems to support many aspects of the viral replication cycle. The archetype alphabaculovirus, Autographa californica nucleopolyhedrovirus (AcMNPV), encodes nine recognizable E3 ubiquitin ligases which include ie2, pe38, iap1, iap2, hcf-1, ac44, ac53, ac141, and cg30. The products of these genes are known to be involved in many replication processes critical to successful virus infection and gene expression. These include cell cycle regulation, gene expression, inhibition of apoptosis, budded virus production, midgut infection, and host range. Baculoviruses are also unique animal viruses as they express a gene that encodes a protein that is highly homologous (75% identity) to cellular-ubiquitin (cUbi) called viral-ubiquitin (vUbi). The vUbi gene is highly conserved and homologs have been identified in all alpha- and betabaculoviruses. The ubiquitination pathway therefore appears to play a major role in the baculovirus life cycle, and yet little is known about how this pathway regulates infection and gene expression. We have been revisiting the function of E3 ubiquitin ligases and show that IE2 has a greater impact on virus replication than previously reported. Also, critical to the function of the ubiquitination system is the specific lysine-linkage on ubiquitin that is used to form polyubiquitin chains. All seven cUbi lysines are conserved in vUbi. In this study we performed a mutational analysis of all the lysines in vUbi to determine which are essential for vUbi function. Our results show that vUbi uses atypical lysines for polyubiquitination which are infrequently used in other eukaryotic systems, yet have a significant impact on virus replication and budded virus production.

Dr. David Theilmann is a Senior Research Scientist at the Summerland Research and Development Centre. He received his PhD in Microbiology from Texas A&M University, College Station, Texas in 1988. He joined Agriculture and Agri-Food Canada (AAFC) in 1988 as a Research Scientist at the Vancouver Research Centre and later at the Summerland Research and Development Centre in Summerland, British Columbia, Canada. He is also an Adjunct Professor at the University of British Columbia at both the Vancouver and Okanagan campuses. His research interests encompass investigations into the molecular basis of baculovirus pathogenesis, host range, and virulence. This includes studies on the regulation of baculovirus gene expression and replication through structure-function studies of both viral and insect regulatory genes. In addition, he has an ongoing program studying baculovirus genomics, transcriptomics, and proteomics. These studies have also led to the development of the insect cell protein expression system, “Insect Select,” using baculovirus genes. Additional goals of the research program are using molecular methods for the development of insect viruses as sustainable viable alternatives to the use of chemical insecticides in field and greenhouse environments. The primary baculoviruses under investigation are those that infect Lepidoptera of agricultural economic importance. Registration of baculoviruses as biopesticides is also being undertaken in collaboration with other research groups and industries in Canada.

Beginning with a review of the challenges and complexities unique to vaccines production development, this presentation will highlight innovative and breakthrough bioprocess and analytical technologies. It also recognizes that innovation in vaccine product development not only requires technical innovations, but also that we change our mindset and approach to how we experience and use data.

Sangeetha Sagar is an accomplished leader, scientist, product development expert, communicator, and biochemical engineer, with over 25 years’ experience in vaccines and biologics. She has led transformational global product prioritization and technical modernization initiatives, as well as conceived, created, and led end-to-end CMC organizations, including management, governance technical reviews, and problem solving spanning pipeline and commercial products. In her recent roles, she worked at Merck & Co., Inc. in all aspects of vaccines and biologics development and is currently the Head of Innovation Technology and Quality at Sanofi Pasteur.

Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. Based on the experience in manufacturing biological therapeutics for routine use or clinical testing, we focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. The requirements for manufacturing, safety, and efficacy testing of MSC-EVs along their path from laboratory to the patient are comparable to requirements for various biological drugs. However, due to their complex molecular structure and their anticipated heterogeneity, development and standardization of EV-based drugs will be a major issue. The therapeutic potential of putative EV-therapeutics has to be investigated for each specific intended use and will be influenced by the source cell types or varying manufacturing strategies. While the scientific community, pharmaceutical companies, and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based therapeutics, the identification of the mode of action underlying a suggested potency in each therapeutic approach remains a major challenge to the translational path.

Eva Rohde is Vice Dean of the Paracelsus Medical University and Head of the Department of Transfusion Medicine at the University Hospital in Salzburg, Austria. She received her MD from the Karl-Franzens University of Graz, Austria in 1994, finished training in transfusion medicine in 2005, and spent several years in stem cell research as a postdoctoral fellow. Her research focuses on the application of stem cell-based therapies with a special emphasis on their extracellular vesicles (EVs). Eva Rohde is a Founding Member of the Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Austria and Director and Qualified Person of the SCI-TReCS GMP unit. The GMP unit in Salzburg achieved pharmaceutical manufacturing authorization for mesenchymal stromal cells (MSCs) and MSC-EVs in 2015. Currently, MSCs generated at the SCI-TReCS GMP Unit are investigated in an international multicenter clinical phase I/II study to evaluate their therapeutic effects in multiple sclerosis (MESEMS, PI: Antonio Uccelli, Genova, Italy). Recent efforts are directed towards the preclinical characterization and regulatory requirements for the clinical assessment and future application of stem cell-based products, including stromal cell-derived EVs. In vitro and in vivo potency assays to define the immunomodulatory, neuroprotective, neuroregenerative, and scarless healing support of MSC-EVs are currently being developed.

Vero cells are considered the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. However, presently, neither suspension-adapted Vero cells nor commercial culture media suitable for suspension Vero cultures are available to support the development of suspension Vero culture technology. In this talk I will discuss the adaptation of adherent Vero cells to grow in suspension using serum-free culture media developed in-house, and characterization of suspension-adapted Vero cells. The presentation will then highlight the development of batch and high cell density perfusion suspension Vero cell bioreactor culture processes to improve the productivity of viral vaccines.

Chun Fang Shen, PhD is senior research officer and project leader at the Human Health Therapeutics Research Centre, National Research Council of Canada in Montreal. He received his PhD in Biochemical Engineering from the Western University in Canada in 1993. He then joined Dr. Amine Kamen’s former group at the National Research Council as a research scientist. Dr. Shen’s work focuses on the development, optimization, and scale-up of batch, fed-batch, and perfusion cell culture processes for the production of viral vectors and vaccines. Dr. Shen also works on the development of cell culture media, feeds, and feeding strategies to improve culture performances. He leads and manages internal research projects, and also collaboration and contract work with biotech companies.

Virapur produces a variety of human influenza viruses in both eggs and Madin-Darby canine kidney (MDCK) tissue culture for the vaccine, antiviral, and diagnostic industries. The variability in the infectious titers, hemagglutination (HA) titers, and expression of viral proteins of different strains in different platforms is always striking. Culture conditions can be modified to affect titers, but the genetics and virulence of wild-type and reassortant viruses affect the production levels of flu. This presentation will describe production methods and examine production characteristics that play an important role in flu virus yield and subunit protein characteristics. We will outline our egg production protocol and highlight differences between egg and tissue culture produced material. We will also present a method to increase hemagglutination titer of low HA titer stocks as often found in the B virus strains. This method has reduced post-production concentration requirements of B virus strains for HA inhibition (HAI) testing.

Marylou Gibson has been the General Manager of Virapur in San Diego since 2002 when she co-founded the business based on her invention of the adenovirus purification kit. Virus related products have expanded to include many diverse virus families and highly quantified virus preparations. Marylou studied adenovirology at the University of Illinois. She went on to do postdoctoral training in the herpesviruses at the University of Michigan and the University of Chicago. Her career in the biotech industry began with developing veterinary and human herpesvirus vaccines. Before Virapur, Marylou held scientist positions at Bristol-Myers Squibb and Immunex (Amgen) where she was on the team that developed the cell line secreting the drug Enbrel. She also played an instrumental role in the startup of the University of California, San Diego’s Gene Therapy Clinical Manufacturing program, which later spun out to become Molecular Medicine Bioservices (MilliporeSigma).

There are two parts to any successful launch of a new therapy; first, the creation of a new methodology of attacking a disease; and secondly, the transition and commercialization of this therapy from the bench to a version of this new therapy that can be produced at a much larger scale of thousands or millions of doses, without losing any efficacy in the process. Today, genomic editing technologies are leading to a revolution in the repair and cure of the genetic basis of diseases, or of attacking cancer in new ways such as with CAR T-cells. Although viral transduction remains a useful methodology in this area, many are concerned with the long-term ramifications of viral DNA in the patient’s genome, and are searching for non-viral alternatives such as various types of electroporation for their genome editing needs. Compared to viral transduction, it is only with non-viral transfection that the benefits of promising substrates such as CRISPR Cas9 ribonucleic protein (RNP) or Cas9 mRNA transfection in genome editing can be utilized.

Dr. Gregory Alberts is currently the Global Subject Matter Expert for Lonza, and has worked at Lonza (and previously Amaxa) since 2003. He was the first field scientist hired by Amaxa in North America, and he has considerable experience in nucleofection, primary cells (including stem cells and iPSC generation); and is well versed in the use of CRISPR and other genome-editing technologies with nucleofection, and their increasing clinical use in cell and gene therapy. He received a PhD from the George Washington University in 2003 in Molecular Biology, and a MS in Bacterial Genetics from the University of Illinois-Chicago in 1986. On a practical note, Dr. Alberts worked at the American Red Cross Holland Laboratory for 14 years in basic vascular and cancer research, worked for three years at Oncor, Inc. in molecular cancer diagnostics, and spent four years working on bacterial genetics and DNA uptake in Streptococcus pneumoniae at University of Illinois in Chicago.

Advanced therapy medicinal products (ATMPs), i.e., cell and gene therapy products, is a rapidly evolving field of therapeutic development. A significant proportion of the products are being developed by academia or small/medium-sized enterprises (SMEs). How do we move from early development to first in human studies? The many challenges in this translation posed by this class of products include aspects ranging from manufacturing, non-clinical development plan as relevant to the clinical trial, marketing authorization, and reimbursement. In this context, the term translation refers to the relevance of non-clinical data in relation to how it impacts appropriate and efficient clinical development. In order to successfully overcome these challenges, a clear understanding of the requirements and expectations of all the stakeholders is critical. We will examine the potential challenges related to such translation, as well as suggested approaches to finding solutions based on experience and learnings from the perspective of US development, with some additional insight on differences/similarities in Europe.

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.

Baculovirus biopesticides are safe, stable, and highly selective alternatives to chemical pesticides capable of targeting many important crop pest caterpillars. Currently, baculovirus biopesticides are produced in vivo; occlusion bodies (OBs) are harvested from infected reared larvae. Although several companies have successfully commercialized the production process, it is not without limitations such as a lack of scalability. Using insect cell culture technology, it is possible to produce baculovirus OBs in vitro employing modern biotechnological processes. However the low yield of occlusion bodies per cell limits commercial feasibility at the bioreactor scale. Our recent research has focused on the genetic modification of host insect cell lines to improve their OB yields. We have demonstrated that yields of baculoviral protein and occlusion bodies can be improved by utilizing host insect cell lines that stably express various viral immune suppressor proteins. Extending the logic of this approach, we are currently undertaking gene knockouts using CRISPR/Cas9 to disable key genes involved in targeted pathways of the innate immune response within the insect host cell. Our aim is to show the potential of disabling these pathways to improve virus replication, ultimately paving the way for commercially viable in vitro baculovirus biopesticide production. In addition to the objective of boosting the OB yield of wild type baculoviruses, such an approach may also boost production of other biologics such as recombinant proteins, virus-like particles, and viral vectors using the baculovirus expression system.

Henry de Malmanche is a PhD candidate within the Australian Institute of Bioeningeering and Nanotechnology (AIBN) at the University of Queensland, Brisbane, Australia. He has a strong background in molecular biology, synthetic biology, and biotechnology commercialization. He has completed a Masters of Research in Synthetic Biology at University College London and holds bachelors degrees in commerce and science from the University of Auckland, New Zealand. In 2017, he joined the lab of Dr. Steven Reid to begin work on insect cell line engineering for the production of baculovirus biopesticides.

Combinations of osmolytes displayed distinct trends of post-thaw recovery, and a non-linear relationship between compositions and post-thaw recovery was observed, suggesting interactions not only between different solutes but also between solutes and cells. The post-thaw recovery for optimized cryoprotectants in different combinations of osmolytes at a cooling rate of 1 °C/min was comparable to that measured with 10% dimethyl sulfoxide. Statistical modeling was used to understand the importance of individual osmolytes as well as interactions between osmolytes on post-thaw recovery. Both higher concentrations of glycerol and certain interactions between sugars and glycerol were found to typically increase post-thaw recovery. Raman images showed the influence of osmolytes and combinations of osmolytes on ice crystal shape, which reflected the interactions between osmolytes and water. Differences in composition also influenced the presence or absence of intracellular ice formation, which could also be detected by Raman. These studies help us understand modes of action for cryoprotective agents in these osmolyte solutions.

Chia-Hsing Pi is a PhD candidate in the Department of Mechanical Engineering at the University of Minnesota. His current research topic is the general improvement of cryopreservation including developing DMSO-free cryoprotectant, understanding the mechanisms of cryoprotectants, and optimizing the formulations of cryoprotectants with computational algorithms. He received his MS from the University of Pennsylvania. His education and research path covers a wide breadth including mechatronics, micro-fabrication, microelectromechanical systems (MEMS), thermo-fluids, and biomedical engineering.

Predicting hit compounds early in drug development is important for large and small industry partners. Research over the past 20 years has demonstrated that early drug screening on 3D cell culture models is typically more predictive of the in vivo response compared to monolayer cell culture. Compared to monolayers of cells, 3D cell culture offers advantages in differentiation, gene expression, cellular function, viability, and responsiveness to stimuli. 3D models and assays have been used in various applications ranging from efficacy to ADME-Tox. At Likarda, we specialize in high-throughput 3D cell culture for drug discovery. Recently, we have developed and validated an immuno-oncology model for the evaluation of CAR T-cells and checkpoint inhibitors. The system mimics the interaction of immune cells with the solid tumor microenvironment and can be monitored via several assay endpoints and high-content imaging. In this presentation, we will provide an overview of different models utilized in 3D cell culture, including those with scaffolds and scaffold-free. We will provide examples of 3D cell culture assays utilized to assess efficacy and toxicity in oncology, immuno-oncology, hepatotoxicity, and diabetes.

With a PhD in bioengineering and an eye for innovation, Lindsey is the director of product innovation for Likarda. Lindsey received her BS in biological/agricultural engineering from Kansas State University, her PhD in bioengineering from the University of Kansas, and her postdoctoral training at the University of Kansas Medical Center. Her graduate research focused on biomaterials and tissue engineering, specifically developing biomaterial implants to treat tracheal stenosis in pediatric patients. Lindsey designed a synthetic nanofiber scaffold that could replace rib cartilage as an option to expand the trachea for infants and toddlers with a narrowed airway. In her role at Likarda, Lindsey expands Likarda’s patented 3D cell cluster technology into other preclinical drug discovery applications like oncology, immunotherapies, and ADME-Tox. She has published work in the fields of tracheal tissue engineering, biomaterial fabrication, adult mesenchymal stem cells for musculoskeletal tissue engineering, and preclinical drug discovery using 3D hepatic organoids. Lindsey has presented at numerous conferences including the Biomedical Engineering Society, Society for Biomaterials, and the American Association of Pharmaceutical Scientists.

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.

Olli Laitinen completed his doctoral studies at the University of Jyväskylä, Finland in 2001, the main subject being molecular biology and the study subject being protein engineering. Thereafter he did his postdoctoral training from 2002 to 2005 at the University of Kuopio and Ark Therapeutics Ltd. (Kuopio, Finland) focusing on gene therapy and the development of gene therapy vectors, especially baculovirus-derived vectors. Next he worked as director of the research department at Ark Therapeutics from 2005 to 2006. From there, he became CSO at Vactech Ltd. in Tampere, Finland to study and develop enterovirus vaccines. In 2012 Dr. Laitinen moved to Karolinska Institutet, Sweden as a Senior Researcher and his studies focused on the pathogenic mechanisms of enteroviruses. In 2014 he moved to the University of Tampere and was appointed as an adjunct professor in 2015. At Tampere his main study subjects have been the development of enterovirus vaccines and nature-based solutions to immune-mediated diseases.

Exosomes and other extracellular vesicles (EVs) have emerged as an immensely versatile source of biomarkers for molecular diagnostics, enabling and enhancing the field of liquid biopsy, from mutation detection to RNA profiling and/or protein analysis. The field of liquid biopsy and EV-based diagnostics has made substantial progress recently and we have now seen the first commercially available exosome-based tests help doctors and patients in their clinical decision-making process. The molecular content of EVs can be analyzed in isolation or in combination with other entities such as cell-free DNA (cfDNA). Unlike cfDNA, however, the EV fraction can be enriched using tissue-specific markers that enable disease-specific EV profiling. EVs are actively and continuously released from cancer cells and, importantly, other cells such as those that constitute the tumor stroma and immune response. The EV content of RNA, DNA, protein, and other disease-specific molecular analytes has expanded the applications of liquid biopsy tests both within and outside of oncology. Some of the recent developments in this area will be discussed.

Dr. Skog is the Chief Scientific Officer (CSO) and founding scientist of Exosome Diagnostics, now a Bio-Techne brand. He has pioneered discoveries about exosomes and other microvesicles and their vital role as cell messengers and disease proliferators. While at Massachusetts General Hospital/Harvard Medical School, he was the first to show that tumor-derived mutations can be detected in exosome RNA from serum and other biofluids, findings which were published in Nature Cell Biology in 2008. He also pioneered discoveries on how these vesicles can be used therapeutically by incorporating gene therapy vectors into microvesicles as a “stealth” vector with changed tropisms. In addition, he found that exosomes serve to deliver messages to other cells, inducing changes favorable to the proliferation of cancer. Dr. Skog has invented several novel exosome isolation platforms that are used clinically (cGMP manufactured) as well as an exosome isolation platform that was licensed to Qiagen. He continues to expand the field of exosome biology and lead critical advancements in diagnostics, including the launch of the first exosome-based diagnostic tests that are now helping clinicians and patients.

Increasingly, cell-based gene and immunotherapy has come to rely on the acquisition of large numbers of peripheral blood mononuclear cells collected by leukapheresis through a network of apheresis centers. Both quality and quantity of the apheresis product, and variance between products, has a direct bearing on the ability to reliably deliver and manufacture a consistent therapeutic product in a timely and cost-effective manner. Addressing risks across the supply chain from point of collection through manufacturing, and on to the hospital brings value to multiple stakeholders.

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. Mark brings more than 12 years of experience working in the field of immuno-oncology, cell-based gene and immunotherapy, and cell therapy manufacturing and bioprocessing. Previously, he served as the Executive Director of Sales and Marketing at Hitachi Chemical Advanced Therapeutics Solutions, a global CDMO for the cell and gene therapy industry, and as Senior Manager, Global Strategic Marketing, in the Therapeutic Systems business unit at Terumo BCT. In those roles he successfully launched multiple devices and technologies in the field of stem cell collection via leukapheresis, cell isolation, cell processing, and culture. Mark is a member of the International Society for Cell and Gene Therapy (ISCT) and the Society for Immunotherapy of Cancer (SITC). He graduated from the University of California, San Diego with a BS in Biological Sciences.

The large number of therapeutics that target one or more membrane proteins highlights the importance of this class of protein. However, membrane proteins have complex characteristics that have traditionally made working with them problematic. Technological advances over the last 20 years have led to an exponential increase in the numbers of solved membrane protein structures, but this still accounts for less than 2% of the unique protein structures in the Protein Data Bank. The Membrane Protein Laboratory operates as a user facility focused on overcoming the technical problems associated with working with membrane proteins. We accept users who wish to work on membrane proteins from anywhere in the world with the aim of solving the atomic resolution structure. We work on membrane proteins from any source organism and, as such, require a range of expression systems with which we can overexpress proteins from bacteria, yeast, plants, humans, and other mammals. Recently we have increased our efforts around eukaryotic membrane proteins and use a combination of transient transfection and baculo-based systems with which to produce these proteins. Here I will discuss the screening strategies that we use as well as highlighting the success of baculo-based systems for the production of membrane proteins of sufficient quantity and quality for structural studies.

Andrew completed his PhD at the University of Warwick where he studied the two-component system controlling glycopeptide resistance. Following on from this he joined the Structural Genomics Consortium in Oxford where he helped develop a pipeline to produce human integral membrane proteins that included ion channels, G protein-coupled receptors (GPCRs), transporters, and membrane enzymes. This pipeline has successfully led to 11 human membrane protein structures to date. Andrew joined the Membrane Protein Laboratory at Diamond Light Source in 2018 and is now focused on driving forward structure-based projects from visiting scientists and developing technologies to assist in the production of membrane proteins for structural studies.

Cell therapies and cell-derived products are the basis for the exciting and quickly growing field of regenerative medicine. Regenerative medicine holds great promise for cures from chronic and degenerative diseases rather than continual palliative care, thus reducing total healthcare costs. However, moving from a drug-based manufacturing process to cells as therapy presents many challenges, including the regulatory challenges of ensuring consistent and safe manufacturing processes. While only a few products have received full FDA approval to date, there are many more cell-based therapies at various stages of clinical trials and testing. As cell therapies become more mainstream, the manufacturing principles and approaches must reflect industry standards similar to those established for drug products. For the purposes of this presentation, we have grouped cell therapies into three broad categories including i) pluripotent stem cells, ii) multipotent (somatic) stem cells, and iii) unipotent cell-based therapies. Each cell therapy category has unique challenges in the manufacturing process, along with exciting possibilities. The FDA has provided guidelines on critical attributes of cell therapies and cell-based therapies to include a) identity, b) purity, c) potency, and d) safety of final product. Using these critical attributes, this presentation provides a comparative summary of assays and quality control measures used for each of the cell categories to address each of the critical attributes. While we are not providing a comprehensive catalogue of all assays that are currently in play, we highlight some of the assays that show agreement across the various cell therapies, and offer additional suggestions based on our experiences of those baseline assays that would offer additional benefits to help harmonize assays across cell therapies. We suggest that any critical quality control assays that are adopted must be broad, simple, reproducible, and collect as much data as possible with the smallest sample allowable. Only by comparing data with transparent reporting, including negative findings, will the optimal assays that relate to clinical outcomes be adopted.

Dr. Francis Karanu is Director for Animal Health and Senior Cell Therapy Researcher at Likarda. Francis’ current research efforts are aimed at developing a cell therapy-based solution for diabetic dogs and cats, which will be delivered as an injectable islet transplant and aimed at eliminating the need for daily insulin injections for diabetic pets. Beyond cadaveric islets 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 tasked with 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.

Viral contamination from upstream raw material components can severely impact pharmaceutical manufacturing processes, affecting cell performance and product quality. To address potential contamination issues, manufacturing processes must be halted for full site decontamination which can take several months and potentially result in a drug shortage. Biogen has taken a risk-based approach to upstream raw material contamination through elimination of animal-sourced raw materials, by identifying higher risk materials and using proven viral mitigation methods that minimize detriment to raw material performance. Outsourcing of raw material viral mitigation methods to suppliers with expertise in the field has allowed for quick transition to materials that provide a lower viral contamination risk to the manufacturing process. This talk will focus on the risk-based approach that Biogen used for determining which raw materials to include in its upstream raw material viral risk mitigation portfolio. High-temperature short-time (HTST) pasteurization at raw material suppliers is an integral part of this risk mitigation approach and should be applied in concert with upstream viral filtration when high risk heat labile raw material components are included in manufacturing processes. Treatment parameters have been detailed in publicly available peer-reviewed literature and confirmed in specific raw material components prior to implementation in commercial manufacturing processes. Additionally, benefits of decoupling materials with a high risk of viral contamination will be reviewed.

Mr. Mack is an Engineer at Biogen and is responsible for providing global support and technical raw material support for manufacturing sciences, and is focused on supplier capital projects and raw material control and monitoring strategies. He has a background in biomedical engineering, molecular science, and nanotechnology, and has led projects assessing cell culture media products’ amenability to viral risk mitigation (VRM) methods. He joined Biogen in April 2017 and brings expertise in pharmaceutical research and engineering, with over 13 years of experience in process development and seven years of experience in GMP manufacturing.

Determining the total concentration of capsid particles is a requirement for regulatory approval of viral therapies, and an important characteristic for gene therapy development. As the field continues to improve on established approaches (ELISA, EM), we are exploring alternatives for a means of determining capsid titer and other attributes such as capsid size, integrity, and levels of larger impurities (i.e., aggregates). In this talk we will cover recent investigations into the use of dynamic light scattering (DLS) and multi-angle static light scattering (MALS) to characterize AAV gene therapy materials for process development. Light scattering (LS) methods can provide a straightforward path for assessing the hydrodynamic radius, and thereby the size of AAV particles. When coupled with size-exclusion chromatography (SEC), MALS offers the potential for size, mass determinations, and capsid titer for each chromatographically resolved species. A newly developed system incorporating multi-angle dynamic light scattering (MADLS) generated accurate hydrodynamic diameters and total capsid titers without the need for chromatographic separation. Our investigation revealed good comparability between capsid titer determined by LS-based measurements and orthogonal methods such as cryo-electron microscopy and analytical ultracentrifugation coupled with infectious titer determinations. The wealth of data which can be extracted from a single LS experiment for AAV samples suggests biophysical light scattering to be an efficient and valuable tool when developing and refining virus production methods.

Darren Begley is a Senior Scientist within the Analytical Development group at Ultragenyx Gene Therapy, working on biophysical and analytical chemistry solutions for process and late-stage analytical method development. Darren holds a BSc from McGill University (joint honours, chemistry and English literature), and a PhD in chemistry with a focus on structural biology from the University of Washington in Seattle.

Adeno-associated viral (AAV) vectors have been reported to be a great promise in the field of gene therapies for the treatment of various human diseases. In parallel with AAV vector manufacturing, specific analytical assays were performed to assess vector productivity, vector purity, biological activity, and safety. To sustain this, reliable, fast, robust, GMP compliant analytical methods are needed. One of the major challenges is to deep characterize the impurities in AAV vectors to provide the most appropriate support to accelerate process development. This presentation will focus on product-related impurities (e.g. encapsidated DNA, empty particles), and their potential impact will be discussed as well.

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

The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multiprotein complexes emerged as vital catalysts of cellular function. To unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multiprotein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for highly efficient delivery of customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitates the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed.

Imre Berger was trained as a biochemist at Leibniz University and Medical School (MHH) in Hannover (Germany), MIT (Cambridge, USA), and ETH Zurich (Switzerland). He is a synthetic and structural biologist, researches multiprotein complexes in human health and disease, and develops enabling technologies for this purpose. Imre carried out his PhD work at MIT with the late Alexander Rich, a pioneer in molecular biology. He then joined Timothy Richmond at ETH Zurich as a postdoc and research assistant. In 2007, Imre was appointed Group Leader at the European Molecular Biology Laboratory (EMBL) in Grenoble, with a joint appointment at EMBL Heidelberg. Since 2014, he has been Professor and Chair of Biochemistry at the University of Bristol. In 2017, Imre became Director of the Bristol Synthetic Biology Centre and co-director of the Bristol Biodesign Institute (BBI). Imre Berger has pioneered synthetic viral nanosystems for DNA delivery, genome engineering, and complex biologics production. The tools he developed are accelerating research and development in academia and industry worldwide. He holds international patents for multigene delivery and expression technologies, has published more than 100 papers in leading periodicals, founded three biotech companies, and has received numerous distinctions including the Swiss Technology Award, the W.A. DeVigier Foundation Award, and recently a Wellcome Trust Senior Investigator Award for his innovative research.

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.

Jan Zitzmann is a bioprocess engineer, and graduated from Technische Universität (TU) Dresden, Germany. He is a former employee of the Leibniz Institute for Polymer Research in Dresden, where he participated in the development of biocompatible surface coatings. Since 2014, he has worked at the Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT) in Giessen. He is in the final year of his PhD studies, and as a research associate in the group of Prof. Dr. Czermak he is currently investigating the suitability of insect cell lines for the recombinant expression of antimicrobial peptides and proteins.

A critical bottleneck impeding the growth of regenerative medicine is the availability of cGMP-compliant cells for clinical development and translation. There have been over 900 registered clinical trials using mesenchymal stem/stromal cells (hMSCs) and hMSC-derived materials such as extracellular vesicles (hMSC-EVs) for therapeutic applications with billions of cells necessary per clinical trial. As these therapies continue to move through clinical trials and become commercial products, there is an urgent need for the development of economical biomanufacturing processes capable of generating these lot sizes. Innovative manufacturing technologies, such as suspension bioreactors, show great promise in reaching commercially-viable working volumes; however, scalability of cell production within such systems remain a challenge, hindering widespread adoption of this culture system for hMSCs. Therefore, a scalable xeno-free (XF) hMSC bioreactor process was developed using high volume XF hMSC cell banks, an optimized XF fed-batch bioprocess media system, and XF microcarriers. This process delivers therapeutically relevant hMSC lot sizes while maintaining a low final cell population doubling level and maintaining cell critical quality attributes, even as the bioreactor culture volume is scaled to hundreds of liters. Scalable bioreactor culture systems currently being developed to provide the lot sizes required for hMSC and hMSC-EV clinical trials and commercial therapies also offer significant time and cost savings compared to standard 2D systems, and can be offered as a plug-and-play standardized system to the fields of regenerative medicine, tissue engineering, and cell therapy.

Katrina Adlerz is a bioengineer by training. She received her Bachelor of Science in Biomedical Engineering from the University of Virginia, and her PhD from the University of Maryland. At RoosterBio, she is focused on product and process development for innovative products related to human mesenchymal stem/stromal cells in the fields of regenerative medicine, tissue engineering, and cell therapy.

We are now in the post-approval era of T-cell therapies, with the first two historic FDA approvals in 2017 and many more yet to come with hundreds of ongoing clinical trials globally. As incredible as this may have seemed a few decades ago, what follows next is standard in the evolution of new biomedical technologies — the ability to manufacture them simply, effectively, and affordably invariably lags behind the scientific and clinical advances, restraining their market growth potential and most importantly putting a limit on the number of patients that can be treated. Many of us are working hard to address this challenge, and my brief presentation (no doubt to be followed by a lively and informative discussion as in past ISBioTech meetings) will seek to outline the current state of the T-cell manufacturing industry, as well as offer some solutions and components which may take us closer to building the next generation of T-cell manufacturing platforms.

Cenk Sumen, PhD is Director of Business Development at Thermo Fisher. Cenk leads this function for the BioProduction Division, and is also Adjunct Professor at NYU Tandon School of Engineering. Most recently, Cenk was Senior Business Development Manager at Hitachi Chemical supporting Cell Therapy Manufacturing, Consulting, and Process Development. Prior to this role, he was Business Development Manager at Perkin Elmer where he supported PerkinElmer’s Drug Discovery Services business. Other prior positions included Technical Sales Specialist at STEMCELL Technologies, where he provided technical consultative sales for stem cell culture and differentiation systems, and Technical Consultant, Sales and Marketing at Life Technologies, where he served as scientific, strategic, sales, and marketing consultant for the Dynabeads technology platform based in Norway. Cenk received his BS in Biology from MIT, and a PhD in Microbiology and Immunology from Stanford University. He was also a Cancer Research Institute Fellow at Memorial Sloan-Kettering Cancer Center for two years with Nobel Prize winner James P. Allison.

Drosophila S2 insect cell expression is less known than the extensively used Spodoptera or Trichoplusia ni (Hi-5) insect cell-based baculovirus expression vector system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late-stage Drosophila melanogaster (fruit fly) embryos by Schneider in the 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D.mel. 2). The S2 expression system has been used for antigen manufacture up to Phase II clinical trials, and was used, among others, for production of Dengue antigens tested in a Phase I trial by Merck Inc. ExpreS2ion has developed S2-based production processes for two malaria vaccine clinical trails with the Jenner Institute, Oxford University (Rh5 1, blood-stage malaria) and Copenhagen University (VAR2CSA 2, pregnancy associated malaria). The placental malaria vaccine has successfully completed a Phase Ia trial in Germany, and a Phase Ib trial in Benin is nearing completion as well. The blood-stage malaria vaccine has recently successfully completed a Phase IIa malaria challenge study. In this talk, further improvements to the placental malaria vaccine will be presented based on the effects of altered glycosylation through engineering of the S2 production cell line, as well as virus-like particle display of the antigen.

Dr. de Jongh (South African) has a MSc in Chemical Engineering from the University of Stellenbosch, South Africa and was awarded a doctorate in Biotechnology from the Technical University of Denmark (DTU). Furthermore, Dr. de Jongh was awarded an affiliated associate professorship at DTU in 2017. Dr. de Jongh has 10 years' experience in the biopharmaceutical industry, with six years as CSO of ExpreS2ion Biotechnologies and one year as CEO of AdaptVac.

In July 2018, FDA published six draft guidances covering multiple topics pertinent to the development of gene therapy products. This talk will cover updates to the Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) guidance, as they pertain to adeno-associated virus (AAV) manufacturing, and updates to the Testing of Retroviral Vector-Based Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up guidance, as they pertain to retroviral vectors and cells modified ex vivo with retroviral vectors.

Dr. Kwilas received her PhD in Biomedical Science from the Ohio State University in 2010 with an emphasis in Molecular Virology & Gene Therapy and Translational Science. Her graduate research was performed at The Research Institute at Nationwide Children’s Hospital under the direction of Dr. Mark Peeples examining the potential application of respiratory syncytial virus (RSV) as a gene therapy vector for the treatment of cystic fibrosis. Dr. Kwilas performed her postdoctoral research at the National Cancer Institute in the laboratory of Dr. Jeffrey Schlom investigating the efficacy of modified vaccinia virus Ankara (MVA) and adenovirus-based cancer vaccines alone and in combination with approved and investigational cancer therapeutics. She designed and conducted proof of concept studies of MVA-TWIST-TRICOM supporting the clinical development of MVA-Brachyury-TRICOM. Dr. Kwilas received an Interagency Oncology Task Force Fellowship in 2015. She began conducting research at the FDA involving the generation of safer vector producing cells with the use of CRISPR/Cas9 genome editing technology, and participating in the regulatory review of gene therapy product chemistry, manufacturing, and controls (CMC). In May 2016, she accepted a full-time gene therapy CMC reviewer position at the FDA.

The Sf9 cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf21-AE, a commonly-used insect cell line in academic research and for biomanufacturing recombinant proteins using the baculovirus expression vector system (BEVS). Sf9 cells are known to constitutively produce low-level reverse transcriptase (RT) activity and a novel rhabdovirus containing a unique X gene. Recently, a variant of the Sf-rhabdovirus, with a partial X gene (minus 320 bp), was reported and a rhabdovirus-negative Sf cell clone was obtained by chemical-treatment that was used in infectivity studies to demonstrate infectivity of both rhabdovirus variants. We have investigated the presence of different cell clones in the Sf9 parent cell line and found a mixed population of cells including rhabdovirus-negative and rhabdovirus-infected cell clones. We have isolated rhabdovirus-negative cell clones and cell clones containing the intact and partial X gene variants of the rhabdovirus (designated as X+ and X-, respectively). This talk will describe the characterization of the different cell clones and development of a sensitive infectivity assay for rhabdovirus detection. Additionally, version 2.0 of the draft Sf9 genome will be described and ongoing efforts for characterizing the Sf9-RT activity be discussed.

Dr. Khan is a Principal Investigator/Supervisory Microbiologist 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). She joined the FDA in 1991, after working at National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) since 1979, where she became an expert on retroviruses and endogenous viruses. An important component of Dr. Khan’s current research program in CBER is focused on evaluating adventitious virus detection by next-generation sequencing for cell substrate and vaccine safety. Her regulatory responsibilities include review of Investigational New Drugs and Biological License Applications for a variety of candidate vaccines including influenza and HIV, and novel cell substrates across different product categories. Dr. Khan has been involved in licensure of several viral vaccines and development of various FDA, International Conference on Harmonisation (ICH), Public Health Service (PHS), and World Health Organization (WHO) guidance documents related to cell substrates, vaccines, therapeutics, and xenotransplantation. She is a lead in various working groups on next-generation technologies for virus detection, adventitious viruses, and cell substrate safety. Dr. Khan received her PhD in Microbiology from the George Washington University, Washington, DC.

Extracellular vesicles (EVs) represent an innovative alternative to cell therapy in regenerative medicine, holding promise to mitigate limitations associated with viable cell transplantation. Besides, EVs are multifaceted subcellular entities that may represent a new generation of bio-camouflaged drug/nanoparticle delivery system. However, the clinical translation of EV-based therapies is hampered by difficulties related to EV engineering, production, and characterization. My presentation will focus on our current research in the attempt to overcome these challenges. More particularly, our recent scalable method to produce EVs in high yield will be addressed as well as the application of the produced EVs for regenerative medicine. Recent results of fistula therapy data on a swine model using EVs in a gel carrier will be highlighted.

Amanda Silva obtained a degree in Pharmacy in 2005 and a PhD in Pharmaceutical Technology in 2008, both from Federal University of Rio Grande do Norte (UFRN), Natal, Brazil. She earned a second PhD in Cellular and Molecular Biology in 2010 concerning polysaccharides for thermo-controlled cell culture at Université d’Evry/Université Paris V, France. She worked as a postdoctoral fellow on drug-loaded magnetic extracellular vesicles (EVs) for image-guided therapy. She is currently a tenured CNRS researcher in Paris and works on EVs engineered with nanoparticles and drugs as drug delivery vectors for cancer therapy and regenerative medicine. She has supervised research on the concept of turbulence vesiculation leading to three patent applications in the last two years. She is the youngest elected board member of the French Society for Nanomedicine (SFNano). She has 47 publications, two book chapters, six pending patents, and an h-index of 19.

After years of relentless research and development coupled with unprecedented patient studies, gene therapy is making progress towards delivering safe and effective medicines to patients in need. In response to breakthroughs in gene therapy there is a growing need for scalable gene therapy processes, advanced analytical techniques specific to this modality, and GMP manufacturing capacity for these products. This presentation will highlight the manufacturing process for adeno-associated virus (AAV) products and discuss challenges regarding the development and technical transfer of high-dose rAAV products, as well as the manufacturing capabilities to meet patient safety, market demand, and regulatory standards. Furthermore, overarching challenges unique to gene therapy will be covered during the presentation.

Kevin is an Associate Process Engineer in the Gene Therapy Manufacturing Technical Services (MTS) group at Pfizer in Sanford, North Carolina. He is mainly responsible for providing technical support to the buffer preparation team and upstream and downstream manufacturing operations. Prior to his current role, he was an Associate Scientist in the Gene Therapy Downstream Process Development group at Pfizer's St. Louis site, developing purification processes for Pfizer's gene therapy products. Kevin also has upstream internship experience at MedImmune in Gaithersburg, Maryland. Kevin graduated from the University of Virginia, earning a BS degree in Chemical Engineering.

The insect cell-baculovirus expression vector system (IC-BEVS) has proven to be a versatile and promising production platform for virus-based biopharmaceuticals. Nevertheless, specific productivity drops abruptly as cell concentration increases (the cell density effect), thus hindering production at higher volumetric titers. Our work on metabolic characterization of insect cells has shown marked differences in metabolic signatures before and after baculovirus infection, which translates into different nutrient requirements. Based on this insight, several strategies were devised to circumvent the cell density effect, including additions of small molecules known to play a role in stress response (e.g., metallic co-factors, anti-oxidants) or virus production (e.g., pyruvate, cholesterol). Such supplementation strategies are able to support and modulate cell metabolic function during infection and thus improve productivity, even if the effect is somewhat product or baculovirus-dependent. Although much remains to be understood about the behavior of insect cell cultures under baculovirus infection, metabolism will undoubtedly be the critical source of information to allow the design of novel, intensified bioprocessing strategies.

Inês Isidro is a senior scientist in the Animal Cell Technology Unit of iBET working on computational biotechnology of animal cells. She holds a PhD in Bioengineering and her current work combines proven tools from systems biology, omics technologies, computational modelling, machine learning, and process analytical technologies to improve production processes and increase metabolic understanding in animal cell technology applications. Ongoing research includes the development of insect cell-based platforms for the production of viral vectors for gene therapy and the application of metabolomic approaches to human stem cell-derived and cancer cell models.

There are many bioprocessing challenges to the creation of regenerative medicines such as cell therapies and stem cell-derived exosomes. There are several reasons for this. For example, the products are often living and therefore responsive to and changeable by the process environment; second, unlike biopharma medicines, regenerative medicines are a class of very diverse products and so there is no standard or universal manufacturing process that can be learned from and improved upon. This talk will present some of our progress in bioprocess development for candidate regenerative cell and exosome medicines.

Ivan Wall is Professor of Regenerative Medicine, Cell and Gene Therapy Bioprocessing at Aston University in Birmingham, UK. He obtained his PhD in cell and molecular biology of wound healing from Cardiff University (2007) and subsequently took postdoctoral research posts at the Eastman Dental Institute and the Institute of Ophthalmology, both at University College London (UCL). He obtained his first academic post at UCL in the Department of Biochemical Engineering (2009–2017), progressing from Lecturer to Reader (Associate Professor) before taking up his current post. His research interests span bioprocess and analytical development for advanced therapy products and have been enabled through numerous collaborations with academic and industry partners around the world. He has published 70 papers to date and has been a visiting professor at Dankook University in South Korea since 2010. With partners in the UK and Ireland he has recently established a Centre for Doctoral Training that focusses on technologies for advancing regenerative medicine.

Chemically inactivated foot-and-mouth disease virus (FMDV) vaccines are used to control FMD around the world. However, three major drawbacks of traditional FMD vaccines are the fact that (1) large quantities of infectious, virulent FMD virus are necessary to produce vaccine antigen, with the associated risk of virus escape from manufacturing facilities or incomplete inactivation during the vaccine formulation process; (2) the vaccine strain must match the wild type FMDV responsible for the outbreak as vaccines provide little or no cross-protection; (3) traditional vaccines produced from wild type FMDV are not fully DIVA compatible, since small amounts of nonstructural proteins may still be present; and (4) they do not fully protect animals from persistent infection. A novel, antigenically marked FMDV-LL3B3D vaccine platform under development by Zoetis, Inc. and the US Department of Agriculture – Agricultural Research Service, consists of an attenuated virus platform containing negative antigenic markers in the non-structural proteins 3B and 3D^pol. This vaccine platform allows for custom design by virtue of the easy exchange of capsid coding sequences. In contrast to wild type FMD vaccine viruses, the recombinant FMDV-LL3B3D vaccine viruses induce no clinical signs of FMD and no shedding of virus in cattle or pigs when inoculated as a live virus (as part of the safety test for the platform). This vaccine platform may use existing FMD vaccine manufacturing technology and significantly lowers biosafety risks associated with FMD vaccine production. Upon exclusion from the Select Agent Program, the vaccine platform may be used to produce high potency, fully DIVA compatible FMD vaccines in the United States. There are two commercially available FMD DIVA diagnostic assays that support this vaccine. Cattle immunized with a variety of chemically inactivated FMDV-LL3B3D vaccine constructs were protected from challenge with parental virus. Two negative markers built into this vaccine allow the resultant FMDV-LL3B3D-based vaccines to be fully DIVA compatible. This vaccine platform, currently undergoing development in the United States, provides opportunities for safer and higher potency alternatives to current FMD vaccines in support of biodefense initiatives in the United States and the global control and eradication of FMDV.

Dr. Gay obtained a BSc in Chemistry and a DVM from Auburn University, and a PhD in Microbiology from the George Washington University. Dr. Gay has worked in the animal health research field for the last 25 years holding several positions of increasing responsibility in the federal government and the pharmaceutical industry. As Chief, Biotechnology Section, Center for Veterinary Biologics (CVB), US Department of Agriculture (USDA), Dr. Gay developed the procedures for licensing molecular vaccines that led to the first license for a live recombinant vectored vaccine. In the pharmaceutical industry (SmithKline Beecham Animal Health and Pfizer Animal Health) Dr. Gay led several cross-functional teams that successfully developed and licensed veterinary vaccines for companion animals and livestock. As Director, Global Product Development, Pfizer Inc., Dr. Gay developed strategic and tactical plans that interfaced R&D, clinical development, manufacturing, marketing, and product life-cycle management. Dr. Gay joined Agricultural Research Service (ARS), USDA, in 2002. Dr. Gay currently holds the position of Senior National Program Leader and provides program direction and national coordination for the Department’s intramural animal health research program, with a focus on eight research laboratories located in Ames, Iowa; East Lansing, Michigan; Clay Center, Nebraska; Athens, Georgia; Orient Point, New York; Beltsville, Maryland; Pullman, Washington; and Manhattan, Kansas. Dr. Gay provides technical support within the interagency in the implementation of the President’s National Strategy for Countering Biological Threats, including the National Bio and Agro Defense Facility (NBAF), and the President’s National Strategy for Combatting Antimicrobial Resistant Bacteria (CARB). Dr. Gay was the 2010 recipient of the USDA Secretary’s Honors Award for interagency response to the pandemic H1N1 influenza outbreak; the ARS Special Administrator’s Award for outstanding and rapid research support for pandemic H1N1; the USDA Secretary’s Honor Award for Emergency Response to the 2013 Chinese H7N9 avian influenza outbreak; and received a US Presidential Rank Award in 2017.

Chimeric antigen receptor (CAR) T-cell therapies have taken the medical field by storm in the past decade. The efficacy in some diseases is high while others trail behind. Associated with high efficacy is the phenomenon of cytokine release syndrome. Additionally, other toxicities are associated with this novel form of therapy. Lastly, while proof of concept has been delivered, we have much to do to streamline the manufacturing process to reduce cost and increase efficacy. My talk will address all these topics and look forward to novel developments in the field in the ensuing years.

Dr. Melenhorst graduated with a bachelor’s degree in continuing education from Tilburg Fontys Teacher’s College, a master’s degree in Medical Biology from the Nijmegen Catholic University, the Netherlands, and a doctorate of philosophy from the Leiden University, the Netherlands. After a postdoctoral fellowship followed by a staff scientist appointment at the National Institutes of Health he was recruited to the University of Pennsylvania in 2012 by Dr. Carl June, first as Deputy Director of the Clinical Cell and Vaccine Production Facility and later as the Director of Product Development & Correlative Sciences. In this role, he was at the cusp of the first ever CAR T-cell therapy approved by FDA: Kymriah. Dr. Melenhorst contributed significantly to the evolution of the CAR manufacturing process and to in-depth mechanistic studies into the immunobiology of CAR T-cell therapies; work that was published in high impact journals such as Nature, Nature Medicine, Cancer Discovery, Science Translational Medicine, and the New England Journal of Medicine. Dr. Melenhorst’s research aims to understand and improve the efficacy, safety, and product consistency of adoptive cell immunotherapy through biomarker, mechanistic, and product development studies.

Over the last decade, significant research and development activities have been targeted to design and optimize cell culture-based viral vaccine production processes. The high flexibility and scalability of cell cultures can cut weeks of production time and help to overcome limitations of traditional technologies, i.e., egg-based vaccine manufacturing platforms. Nevertheless, considering the diversity of host cell lines, the variety of viruses with their specifics in replication, and the large differences in cell-specific virus yields, the optimization of upstream and downstream processes is still a challenge for the production of potent and safe vaccines at low costs. To overcome existing limitations, process intensification in viral vaccine manufacturing needs to take into account current good manufacturing practice (cGMP) requirements. In addition, the transfer of solutions developed successfully for other biotechnological processes, e.g. in the production of recombinant proteins in CHO cells, should be considered. Besides cell line and medium design, the establishment of high cell density cultures can provide significant increases in productivity. Furthermore, the establishment of continuous processes should be further explored. In this presentation, examples for high-yield production of various viruses, including influenza virus, zika virus, and modified vaccinia Ankara (MVA) virus will be addressed. In addition, results for the use of steric exclusion chromatography (SXC) as a single-use purification platform for viral vaccines and gene therapy vectors will be presented. Finally, options for process monitoring and the control of antigen quality will be exemplified.

Udo Reichl is director at the Max Planck Institute for Dynamics of Complex Technical Systems (Department of Bioprocess Engineering) and holds the Chair of Bioprocess Engineering at the Otto von Guericke University in Magdeburg, Germany. Research of the group focuses on animal cell culture technology, downstream processing of vaccines and other biologicals, virus-host interaction, characterization of microbial communities, as well as mathematical modeling of bioprocesses and cellular systems. Currently, the following topics are covered: (1) optimization and scale-up of cell culture-based production processes (vaccines, vectors, recombinant proteins); (2) chromatographic methods for the purification of viral antigens and viral vectors; (3) mathematical modeling, monitoring, and control of bioprocesses; (4) mathematical modeling of cellular systems with a focus on animal cell growth, virus-host cell interaction, and microbial growth dynamics; (5) quantitative analysis of metabolic and virus-host cell networks; (6) (meta)proteomics for characterization of microbial communities (biogas, wastewater, gut microbiome); and (7) glycomics for characterization of protein glycosylation (viral antigens, monoclonal antibodies, complex carbohydrates).

A prerequisite for producing advanced therapy products requires appropriately controlled and standardized production and testing procedures that result in consistent safety and efficacy. Changes in production sites and manufacturing processes have become increasingly common, posing challenges to developers regarding reproducibility and comparability of results. Development of a first World Health Organization (WHO) international standard for gene therapy is especially important given the usual orphan nature of the diseases to be treated, hampering the comparison of cross-trial and cross-manufacturing results for the important lentiviral vector platform. The first WHO standard project is intended to standardize production process and product batch-to-batch comparability assessment and to enable comparison of cross-trial and cross-manufacturing results. The topics to be discussed include: 1) history of WHO standardization; 2) outline of WHO standard production processes; 3) historical efforts in standardization; 4) challenges in standardization of gene therapy; and 5) the concept and progress of developing the first WHO international standard for gene therapy.

Dr. Yuan Zhao is a principal scientist and head of the gene therapy section at National Institute for Biological Standards & Control (NIBSC)/Medicines and Healthcare Products Regulatory Agency (MHRA), United Kingdom. Dr. Zhao obtained her PhD at Manchester University and gained her postdoctoral experience at the University of Oxford, CR-UK Cancer Institute, and University College London (UCL). She has a background in gene therapy, virology, and cancer biology. Her current role includes developing WHO international standards and European official control tests for gene therapy products, and contributing to the development of European and international guidance on advanced therapies.

The emergence of new infectious human diseases, the need for improved protection against existing pathogens, and the existence of diseases for which there continue to be no approved products for prevention or treatment justify the significant efforts to develop new and improved vaccines. Nanoparticle vaccines are proving to be a safe and effective means of addressing these unmet needs. Protein subunits of nanoparticle vaccines can both improve the presentation of complex antigens and facilitate antigen uptake and processing by antigen presenting cells inducing a broad range of B- and T-cell responses. The baculovirus expression vector system (BEVS) is an excellent platform for the production of glycoprotein nanoparticle vaccines as 20–200 nm protein-protein, protein-detergent, or protein-lipid micelles (nanoparticles), or combined with other structural antigens and virus-like particles (VLPs). Protein-detergent nanoparticle vaccines based on Ebola virus glycoprotein (EBOV GP), influenza hemagglutinin (HA), and respiratory syncytial virus fusion protein (RSV F) are in early through late-stage clinical development. Advantages of the BEVS technology platform, physical and biological properties of nanoparticles including thermodynamic, 2D, and 3D analyses, and manufacture of nanoparticle vaccines at the 1000 L scale will be presented. The importance of the unique nature of Sf9 cell-produced nanoparticle immunogens, impact on neutralizing epitope presentation, and potential for protection in humans will also be discussed.

Dr. Smith is a leader in vaccine technology and holds numerous patents for the baculovirus-insect cell expression system, vaccines, and adjuvants. At Novavax, Dr. Smith has developed the first known commercial, scalable process for the manufacture in the insect cell system of virus-like particle and nanoparticle vaccines, including those for influenza, respiratory syncytial virus (RSV), Ebola, rabies, and other viruses. Prior to joining Novavax, Dr. Smith led a team at Protein Sciences Corp. that developed the first approved recombinant influenza vaccine. Dr. Smith also collaborated with the National Institutes of Health to produce the first experimental vaccines tested in man against HIV/AIDS and H5N1 avian influenza. Dr. Smith did his graduate work at the Baylor College of Medicine and has a PhD in Microbiology from Texas A&M University.

Adeno-associated virus (AAV) has a well studied safety profile and proven gene delivery capabilities. Sanofi uses a serum free, disposable, scalable manufacturing platform for AAV production, utilizing a producer cell line. Each AAV product requires generating a new clonal cell line. This presentation will cover cell line generation and selection including discussion of critical reagent control, manufacturability fit, stability assessment, and product characterization.

Lois Horton has over twenty years of experience in the field of gene therapy process development. Currently she is the Associate Director of Upstream Development in the Gene Therapy Development & Manufacturing group at Sanofi, responsible for cell line engineering and development of upstream processing to support clinical trials. She received her undergraduate degree in Chemical Engineering from the Massachusetts Institute of Technology.

Manufacturing is a well-recognized critical bottleneck for delivery of cell therapy products, and automation will no-doubt play a key role in the commercial cell therapy factory of the future. However, automation is a tactical solution to be applied, not a problem to be solved. In this talk, fundamental strategic drivers for manufacturing automation will be discussed in the context of other tactical solutions as well as unique considerations for patient-specific vs. universal allogeneic cell therapy products. Alternative approaches and practical considerations will be presented including case study examples across the different types of automation opportunities within a manufacturing environment.

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.

After decades of slow and steady progress, cell and gene therapies are continuing to experience important breakthroughs. Gene therapies, like Luxturna^TM, have proven that it’s possible to not just treat symptoms, but to correct and potentially cure the underlying genetic cause of disease. Similarly, cell therapies such as Kymriah^TM are proving the power that human cells have to treat disease in truly transformative ways. However, specifically because these therapies are so new and so cutting-edge, it can be challenging to navigate through the complex clinical, commercial, and regulatory hurdles that may be quite different than the challenges faced by traditional therapies. The paradigm has shifted in product development with respect to advanced therapies, particularly in the context of orphan indications and accelerated regulatory pathways. Translation of preclinical promise of efficacy to the reality of clinical efficacy is particularly challenging. There is an increasing need for bioprocessing and manufacturing to keep up with, if not exceed, the pace of clinical development. To meet this challenge it is important to acknowledge this shift and find synergies across scientific disciplines to successfully deliver groundbreaking medicinal products to patients in need. Important cross-cutting considerations include defining relevant potency measures that are predictive or at least correlated with the mechanism of action and potential efficacy; and development of a scalable manufacturing process in the face of the need to demonstrate safety and generate preliminary clinical evidence using the product that the sponsor intends to use for clinical development.

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.

Influenza virus infections cause significant morbidity and mortality on a global scale. Current seasonal influenza vaccines protect if well matched to circulating strains. However, the immune response that they induce is narrow due to its focus on the variable head domain of the viral hemagglutinin. Mismatches between vaccine strains and circulating strains occur and lead to a sharp drop in vaccine effectiveness. The second surface glycoprotein of the influenza virus, neuraminidase, has long been ignored for vaccine development. Current influenza virus vaccines contain variable amounts of neuraminidase, and its structural integrity in the vaccine formulations is suboptimal. Therefore, influenza virus vaccination does not induce a robust response against the neuraminidase. Neuraminidase is a validated target for antiviral drugs, its function is essential for the virus, and anti-neuraminidase antibody titers in humans correlate with protection. Furthermore, its antigenic drift is discordant with that of hemagglutinin and it drifts more slowly. Robust anti-neuraminidase immunity could therefore increase vaccine effectiveness and breadth. We have developed recombinant neuraminidase antigens expressed in the baculovirus expression system. Vaccination with these antigens leads to robust and broad protection in the mouse model. In the guinea pig model, vaccination with these immunogens leads to inhibition of transmission, specifically if animals are vaccinated mucosally. Data from murine and human antibodies that we have isolated provides further evidence for the extraordinary breadth and protection that neuraminidase-based immunity can provide. A neuraminidase-only vaccine or current vaccines supplemented with recombinant neuraminidase might increase protection against seasonal influenza and would also enhance our pandemic preparedness.

Florian Krammer, PhD graduated from the University of Natural Resources and Life Sciences, Vienna, Austria. He received his postdoctoral training in the laboratory of Peter Palese at the Icahn School of Medicine at Mount Sinai, New York working on hemagglutinin stalk-based immunity and universal influenza virus vaccines. In 2013 he became an independent principal investigator and is currently Professor at the Icahn School of Medicine at Mount Sinai. Dr. Krammer's work focuses on understanding the mechanisms of interactions between antibodies and viral surface glycoproteins and on translating this work into novel, broadly protective vaccines and therapeutics. The main target is influenza virus but he is also working on Zika virus, hantaviruses, filoviruses, and arenaviruses.

This presentation will deal with manufacturing issues during late-phase development of CAR T-cell products. A major focus will be on regulatory considerations for the commercial development of such products, including a discussion of the path toward a biologics license application (BLA). CAR T-cell manufacturing challenges, including product tracking and labeling, product consistency, and changes during late-stage development will also be highlighted.

Dr. Reiser received a PhD in biochemistry from the University of Basel in Switzerland, after which he obtained training in virology and protein chemistry as an American Cancer Society Fellow in the Department of Biochemistry, Stanford University School of Medicine. While at Stanford he studied proteins encoded by Simian virus 40 — work that supported development of the original Western blot technique. He also worked out a novel chromatin immunoprecipitation assay that maps binding sites of specific DNA binding proteins on viral chromatin. In 1994, he joined the National Institutes of Health (NIH) to work on viral vectors for gene therapy, focusing primarily on the design of novel vector systems for transgene delivery into nondividing cells. He was among the first to design lentiviral vectors based on HIV-1. His paper was one of the three original reports that emerged on the subject in 1996. In 1999 he joined the Louisiana State University (LSU) Health Sciences Center in New Orleans as an Associate Professor of Medicine and to serve as the Director of the LSU Gene Therapy Vector Core. His goal was to develop improved gene and protein transfer strategies for the central nervous system (CNS). His work dealing with lentiviral vectors and transgene delivery to the CNS was supported by R01 and R21 grants from the NIH, on which he was the principal investigator (PI). His research at FDA/CBER has focused on two of the major limitations of lentiviral vectors for clinical gene therapy, including the potential of such vectors to 1) activate oncogenes during random integration into the host genome, and 2) transduce cells in off-target tissues in vivo. To address these shortcomings, he has been developing safer HIV-1-based lentiviral vectors by 1) limiting their integration to well-defined sites in the human genome, and 2) narrowing their tissue tropism through targeted transduction.

The herpesvirus of turkeys (HVT) has been proven to be a convenient vector for the development of recombinant vaccines against poultry diseases. Several recombinant vaccines using HVT (rHVT) as a vector to express antigens of important poultry pathogens are efficacious and widely used. These vaccines are administered in the hatchery to chicken embryos (in ovo) or to day-of-age chickens (subcutaneous). The advantages of the rHVT vaccines include: (i) it is safe, (ii) it is not affected by the presence of maternal antibodies, (iii) it can be administered at the hatchery, (iv) it induces fast onset and long lasting immunity, (v) it allows the differentiation between vaccinated and infected birds (DIVA), and (vi) it is a technology easily applicable for the development of vaccines. The first generation rHVT vaccines expressed antigens of one singe poultry pathogen. Due to concerns related to replication interference of the vaccine vectors, rHVT vaccines are not administered concurrently, leaving to the producer the decision of which vectored vaccine to use at the hatchery. In order to overcome vector interference and allow for protection against three major poultry diseases with a single administration of an rHVT vaccine at the hatchery, a novel rHVT dual insert vaccine expressing immunogenic genes of the Newcastle disease virus and infectious bursal disease virus has been developed. This dual insert rHVT vaccine has been shown to be safe, stable, and efficacious against Marek’s disease, Newcastle disease, and infectious bursal disease.

Leticia Frizzo da Silva is a Project Leader at Ceva Animal Health Poultry R&D. She leads a team with the mission of developing a portfolio of innovative viral vectored vaccines and supporting the life cycle management/line extension of existing products. Her team has recently licensed two innovative recombinant vaccines: the ULTIFEND® prevents three main poultry diseases (Marek’s, Newcastle, and Gumboro) in a single shot; the Vectormune® AI (H7) prevents avian influenza. Leticia earned her PhD from the University of Nebraska–Lincoln (Nebraska Center for Virology). Her research interest during graduate school concentrated on understanding the mechanisms by which herpes viruses modulate the host immune response in order to establish productive infection and latency. Her doctoral research resulted in 11 peer-reviewed scientific articles and two distinguished student awards. Leticia obtained her DVM and master's degree from the Federal University of Santa Maria, Brazil. She started her scientific career during veterinary school supporting research investigating molecular markers for diagnostics of chronic obstructive pulmonary disease (COPD) in equines. After veterinary school, she obtained a master's degree in preventive veterinary medicine with a focus on virology.

AAV-based vectors represent a new type of drugs and are being developed for gene delivery in different therapeutic areas. Potency of the drug is the most critical quality attribute and needs to be assessed for lot release and stability. The potency assays must be specific, quantitative, and ideally reflective of the mechanism of action as well as stability-indicating. These assays are usually more difficult to develop and not possible to share between different programs. Potency of gene therapy drugs can be assessed in animal models and cell-based assays. The lack of relevant animal models for human diseases coupled with large variability and high cost of testing makes them a rare choice for drug release. The cell-based assays can measure three different activities of the viral vector: infectivity, target protein or mRNA expression, and functional potency. Our approach for two different programs will be presented and discussed.

Dr. Anthony Leyme is currently a Scientist II in Bioassay Development for Gene Therapy at Biogen. He has been with the company for two years. Anthony holds a PhD in cellular biology from Rennes University, France where his work focused on the activity of matrix metalloproteinases (MMPs) in liver fibrosis. Dr. Leyme underwent postdoctoral training in Cellular Biology, Molecular Biology, and Biochemistry at Boston University where his work focused on G protein signaling prior to joining Biogen.

The cell substrate used in the development and manufacture of a viral vaccine is one of the most critical raw materials in the manufacturing process. A high-quality cell substrate must be safe for its intended use and will lead to the optimal yield of a safe and efficacious antigen to be used in the final vaccine product. There are a wide variety of cell substrates used for production of US licensed vaccines and investigational products, ranging from live chicken embryos for influenza and yellow fever vaccines to engineered cell lines, such as CHO cells, that express an antigen. All cell substrates must be well-qualified from a safety perspective and shown to be appropriate for their intended use with regard to vaccine production. This presentation will cover a brief history of cell substrates used for viral vaccine manufacturing, current testing recommendations for qualifying cell substrates, and novel testing approaches under consideration.

Dr. Levis has worked at the US Food and Drug Administration (FDA) since 1995. She is currently the Deputy Director of the Division of Viral Products in the Office of Vaccines Research and Review at CBER/FDA, a position she has held since 2006. Prior to this position, she served as the Regulatory Coordinator for the Division of Viral Products (2002–2006) and served as a Senior Staff Fellow in the Laboratory of Vector Borne Viral Diseases (1995–2002). Her initial research work at the FDA related to dengue virus replication. She then transitioned to being the lead CMC reviewer for licensed rabies virus vaccine products and rabies vaccine and related products under development. In addition to her work on rabies, Dr. Levis served as the primary CMC reviewer for the Human Papillomavirus (HPV) vaccines to prevent cervical and related cancers. She was the chair of the licensing committee for the review and approval of Cervarix, an HPV vaccine made using the baculovirus expression system to produce viral antigens. Dr. Levis continues to be involved in the review and regulation of all baculovirus-expressed antigens.

This study was performed in order to identify variation in the process inputs of an antigen production process that correlate with the final antigen yield with multivariate data analysis (MVDA). The variation in antigen yield was marked as ‘special-cause variation’ in a CAPA and, as a result, additional parameters to monitor needed to be identified. In the project four sources of variation were identified: multiplicity of infection (MOI), cell health during cell maintenance, nutrient availability during infection, and the harvest criteria. The experimental design, measurements performed, data analysis, and model interpretation will be discussed, as well as recommended actions to reduce the variability in antigen yield.

Kevin Patrick Kent, PhD is a Scientist IV at Boehringer Ingelheim Animal Health (BIAH) in the Process Development group. He has been with BIAH for two years primarily working on development, evaluation, and implementation of new process analytical technology and off-line analytical methods within the process development group. Previously, he spent three years at MilliporeSigma developing analytical methods for characterization of complex raw materials, cell culture media, and high-throughput product quality characterization methods for therapeutic proteins, and one year at Dow Chemical primarily developing polymer characterization and identification methods using SEC and MS. He has a BS in Chemistry from Washburn University, Topeka, Kansas and a PhD in Biophysical Chemistry from Stanford University in California. At Stanford he worked primarily on characterizing a light activated protein-peptide interaction with several spectroscopic and chromatography based characterization methods.

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.

Michelet Dorceus is the Senior Bioprocess Application Specialist at Eppendorf North America. Michelet earned his bachelor's degree in biology and chemistry from Salem State University and has worked in process development and manufacturing in the biotechnology/biopharmaceutical industry for over 15 years.

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.

The world is facing an under-supply of some key vaccines due to poor synergies between growing market demands and aging production models. In this light, a proof of concept vaccine manufacturing platform aimed at increasing availability and affordability of vaccines has been developed. This simulated, continuous, and automated platform integrates both USP and DSP processes and is encapsulated into an isolator, making it a self-contained production unit (6m²). The technology relies on a single-use, high-density, fixed-bed bioreactor operated in perfusion chained with downstream filtration, clarification, and polishing steps to (a) decrease batch time, (b) reduce equipment utilization, (c) optimize utilities consumption, and (d) intensify operations. By optimizing single-use technologies we are able to drastically reduce capital expenditure (CapEx), cost of goods sold (COGS), and footprint and increase production capacity. Such a manufacturing platform can easily be implemented into flexible facilities with a simplified infrastructure, increasing adaptability in production and capacity for record time-to-market. This study will present the platform proof of concept on Vero cell line and trivalent Sabin-inactivated poliovirus vaccine (sIPV) production, achieving low COGS (0.28 $/dose for a trivalent sIPV) and large capacity. The presentation will not only feature the description of engineering development, but also results of cell growth, infections, and product quality, as well as a description of the CapEx, CoGS, and capacity calculations. This manufacturing platform is undergoing sIPV process scale-up and preclinical bulk production. This manufacturing system is expected to produce any type of viral vaccine at a very low cost and at large capacities to face global health challenges.

Dr. Meghrous received his MSc and PhD in Biochemistry from the University of Nancy (France) and has more than 18 years of experience in the pharmaceutical industry in process development for recombinant and therapeutic proteins, viral vaccines, and monoclonal antibodies. Dr. Meghrous is Manager, Upstream Process Development at Protein Sciences Corporation (PSC), a Sanofi company, in Meriden, Connecticut, and is currently in charge of developing, optimizing, and implementing the fed-batch process for hemagglutinin production for influenza vaccines. He has also held positions as fermentation specialist and Senior Scientist at PSC. Prior to joining PSC, he worked at the National Research Council - Biotechnology Research Institute (NRC-BRI) followed by DSM Biologics. He worked at NRC-BRI for eight years, first as a postdoctoral fellow then as a researcher in the Animal Cell Culture group. The focus of his work was the development of processes for the production of viral vaccines and other recombinant proteins using the baculovirus expression system.