BioMAN Summit - December 11-12, 2019

The following abstracts have been accepted to be presented in the 2019 BioMAN Summit's Poster Session:

1.	Using the AppliFlex ST to Investigate the Effect of CO2 Concentration on the Expansion of T-cells
	George Barringer
	Applikon Biotechnology, Inc.
	Adaptive T-cell therapy (ACT) has emerged as a promising way to treat systemic cancers such as Acute Lymphoblastic Leukemia. However, robustness and reproducibility of the manufacturing process remain challenging. It is therefore pivotal to understand the effect that cell culture conditions have on expansion and differentiation of T-cells. Here we invetigate the effect of CO2 on the expansion of T-cells using a SUB due the advantages of this single-use system such as lower initial investments, faster setup and reduced cross-contamination risks. The influence of CO2 is of particular interest with regards to potential allogeneic T-cell therapies and the associated cultivation at the large scale, where CO2-removal may become insufficient. Several studies have demonstrated for other mammalian cell types that elevated concentrations of dissolved CO2 can severely affect the bioprocess (e.g. Brunner et al., 2018, Nguyen Dang et al., 2019), but no such studies exist for the expansion of T-cells.

2.	Risk-Informed Manufacturing Decisions: Tradeoffs in the Location and Allocation of Capacity
	Donovan Guttieres
	Research Scientist, MIT Center for Biomedical Innovation
	While the biologics market and manufacturing industry have historically been concentrated in a few geographic locations (i.e. USA and Western Europe), several factors are leading to more decentralized patient populations and manufacturing operations. New processes (e.g. continuous manufacturing) and facilities (e.g. pre-fabricated pods) allow for smaller footprints, while emerging products (e.g. ATMPs) have shorter shelf-life and higher levels of personalization that lead to unique supply-chain requirements. The number and relative location of facilities in a network is modeled in the context of cost-effectively meeting global demand for a commercial biologic product. The model presented investigates trade-offs regarding location-and-allocation of manufacturing capacity by a) calculating cost-of-goods for monoclonal antibody production and b) measuring the impact of location-dependent risks on long-term financial cost. Of particular interest are catastrophic (low-probability, high-impact) events that can have detrimental consequences on manufacturing cost, supply chain integrity, and patient access. Applications extending to emerging ATMPs are demonstrated.

3.	High-Density Perfusion Bioreactor Platform for Suspension HEK293 Cells
	Lindsay Hock
	MassBiologics, Upstream Process Development
	The emergence of gene therapy for genetic diseases has rapidly evolved with the approval of several gene therapy products. New research suggests that ex-vivo transfection may deliver corrected genes more efficiently and safely than historical methods. Cell lines such as HEK293 and HT1080 are generally accepted to reproducibly produce high-quality viral vectors, yet are often adherent, requiring dated labor-intensive techniques. As an alternative, suspension cell lines are being evaluated to improve infective titers and produce viruses. Additionally, adapting high-density cell culture production from mAb technology can increase output to potentially treat more patients with less equipment, time, and material cost. Using perfusion, we have reliably grown suspension HEK293 cultures up to 80x106 viable cells/mL. This platform sustains high-density cultures that can be transfected and continually harvested to ensure high-quality viral vector production. This next-generation platform has potential to transform gene therapy manufacturing into a cost-effective, highly efficient, and low-waste technology.

4.	Label-Free Recognition of Non-Activated and Activated Human T Cells by Quantitative Phase Imaging
	Igor Andreyev
	Future Targeted Healthcare Manufacturing Hub, Department of Biochemical Engineering, University College London
	There is a gap in current methods for evaluating quality and durability of cells in immunotherapy products like CAR-T. Ability to identify and characterize immune cells on single-cell level without labelling would be highly valuable for biomedical research and clinical trials where unmodified cells are required. We are developing analytical methods for label-free monitoring of human T cells activation and viral transduction. A method for detection of T cells activation status by Quantitative Phase Imaging (QPI) was developed. We showed that stimulated cells could be statistically distinguished from non-stimulated by optical morphology fingerprints extracted from their phase images. Miniaturization of cell bio-processing platforms and performing cell QC analysis in a fast and low-cost way would be a benefit. Microfluidic devices were tested and QPI was applied for detecting an activation of T cells on a chip. This presents the first step towards high throughput analytical platforms for CAR-T cell immunotherapy.

5.	Scalable Education & Training to Grow the Cell Therapy Manufacturing Workforce
	Talia Wolfson
	Department of Materials Science and Engineering, Massachusetts Institute of Technology
	As the cell therapy field grows rapidly, there is a need for a well-trained workforce to develop and produce these treatments. Due to the field’s relative youth, there are limited informational resources already available, and companies or labs are left to spend valuable time and resources bringing newcomers “up to speed.” The goal of this project is to develop a course to teach the fundamentals of cell therapy manufacturing, using both online learning and hands-on training workshops. The primary audience is industry scientists or academics interested in entering into cell therapy development or manufacturing. Benefits include accelerating the training of the cell therapy manufacturing workforce and reducing the amount of required resources spent on training. This course hopes to help the continued advancement of the regenerative medicine field and these potentially life-saving medical technologies.

6.	Direct 3D Printing of Perfusable Scaffolds for Liver Mesophysiological System
	Pierre Sphabmixay
	Mechanical Engineering, Griffith Laboratory, Massachusetts Institute of Technology
	Animals studies are an essential tool for in-vivo modeling for clinical research. Nonetheless, many biological findings from these studies are confronted with limited or even contradicting outcomes when compared to humans, raising the issue of the ability to translate animal to human clinical data. The rise of various organ-on-chips also known as microphysiological systems (MPS) offers promising perspectives for the development of more insightful systems using human-derived tissues. The major obstacle for these MPS remains the ability to recreate the native environment of the organ of interest at a scale that allow quantitative studies. In order to maintain the physiology of the “organ” in vitro, nutrients and oxygen need to be provided locally while waste products cleared at a rate that maintaining elevated metabolic activity. Here we show a direction approach of 3D printing perfusable microcapillary networks allowing the healthy culture of hepatocytes at mesoscale for more direct translational studies.

7.	Development and Scale-Up of a Bench-Scale Bioreactor Process for Transient Lentivirus Production using a Suspension-Adapted HEK293T Clone
	Dongli Guan
	MilliporeSigma
	In recent years, the use of lentiviral vectors for gene therapy applications has become increasingly popular. As a result, there has been an increased demand for manufacturing of these viral vectors in large volumes for clinical trials. Current production processes are labor intensive and make use of adherent, flat stock cultures. Bioreactors enable production of large volumes of viral vectors with improved environmental control (pH, dissolved oxygen, mixing and temperature) and reduced production costs. Here, we describe the development and scale-up of a bench-scale bioreactor suspension-based, transient lentivirus production process. The effects of pH, dissolved oxygen and mixing on growth of the cells and viral vector production were tested in Mobius^® 3L Single-use Bioreactor. Optimal process parameters were defined for growth and lentivirus production phases and the bench-scale bioreactor lentivirus production process has been scaled up in Mobius^® 50L Single-use Bioreactor.

8.	Critical Factors in Adeno Associated Virus Tangential Flow Filtration
	Matthew Burak
	MassBiologics
	Tangential flow filtration has been widely adopted for concentration and buffer exchange of various biologics, including adeno-associated virus (AAV). AAV based vectors are an increasingly relevant commercial biologic. Therefore, it is crucial to determine parameters critical for AAV bio-manufacturing. Here, we evaluated the impact of TFF load density (viral genomes per membrane area) and shear rate. Samples were collected throughout the process and evaluated for recovery, aggregation and infectivity. We established that load density and shear rate play a critical role in product stability and recovery during TFF of AAV.

9.	rAAV Large-Scale Manufacturing using BEVS Technology: Scale-Up to 500L Single-Use Bioreactor
	Ashutosh Gupta
	uniQure
	Recombinant Adeno Associated Virus is becoming a vector of choice for a variety of human gene therapy applications. This leads to an increased demand for AAV vectors of high quality and quantity to advance new therapies to large patient populations. In order to exploit the full potential of the Baculovirus Expression Vector System uniQure initiated the development of a Single-Use Stirred Tank Reactors (STR) process for rAAV manufacturing. Here we demonstrate similarity between the small-scale (50L) wave-based and STR processes. Furthermore, the feasibility of scaling to 500L scale is shown.

10.	Optimizing the Clarification Process for Viral Vectors Feed Streams for Use in Gene Therapy
	Mike Collins
	Pall Corporation
	Viral-based vector systems such as lentivirus (LV) and Adeno-associated virus (AAV) are more widely used and show great potential for delivery of genetic material to the target cells in gene therapy. The objective of this work is to identify an efficient clarification step for the separation of viral vectors from the range of impurities found in typical adherent and suspension based viral vector cell culture. The clarification step needs to combine high capacity, high product yield and ease of scale-up to prepare for downstream operations. Our strategy includes evaluating various depth and membrane filters made up of different materials for their virus clarification performance. Both AAV and LV processes can be adherent or suspension, each having their own challenges and in this work we show an optimized improvement in throughput and recovery for these viral vector feed streams. Thus, making the overall downstream process more efficient, scalable and economical.

11.	Next Steps in AAV Characterization via Light Scattering Measuring AAV Critical Quality Attributes
	John Champagne
	Wyatt Technology Corporation
	Adeno-associated virus (AAV) is a popular vector for gene therapy due to its mild immunogenicity and ability to deliver its genetic payload to a wide variety of host cells. Because more stringent quality requirements are being implemented by regulatory agencies and to ensure the safety of AAV-based therapeutics, it is imperative to implement robust characterization methods throughout the production and manufacturing processes. This poster is focused on the overview of different AAV characterization methods such as size exclusion chromatography (SEC) coupled with UV, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors to measure the following AAV quality attributes (QAs): total number of viral capsid particles, relative capsid content (e.g., ratio of empty and full capsids), and percentage of monomer or aggregates. Additionally, orthogonal methods using dynamic light scattering (DLS) and field flow fractionation (FFF) will be compared.