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Integrated and Continuous Upstream and Downstream Processing: Integrated and Continuous Upstream and Downstream Processing
04:30pm - 06:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Lars Pampel, Organizer, Novartis Pharma AG; Nihal Tugcu, Organizer, Merck Co Inc; Timothy Tully, Organizer, Pfizer Inc; Thomas Villiger, Presider, ‍ ; Jiuyi Lu, Presider; Mehdi Ghodbane, Presider, GSK; Dr. John Erickson, Presider, GlaxoSmithKline
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Recent advances in perfusion processes have enabled novel approaches to manufacture a plethora of biologics. Along with the increased prevalence of GMP implementation, the Quality-by-design concept for perfusion processes has matured significantly over the past several years. This session mainly focuses on case studies of scale-up/scale-down, innovative process characterization, and cutting-edge PPQ strategies. Topics such as process economics, cell line stability, operational complexity, and batch to continuous conversion are encouraged. In addition, this session will also capture advances in the areas of cell line development, media development, and novel cell retention devices for integrated and continuous processes. Applications of the aforementioned aspects to new modalities are also of high interest.

Thursday
Surge tanks as critical enablers of continuous processing for production of monoclonal antibodies
04:30pm - 04:50pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Garima Thakur, Presenter, IIT Delhi; Dr. Anurag S Rathore, Indian Institute of Technology Delhi
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Surge tanks are critical but often overlooked enablers of continuous bioprocessing. They provide multiple benefits including dampening of concentration gradients and allowing process resumption efforts in case of equipment failure or unexpected deviations, which can occur during a continuous campaign of weeks or months. They are also useful in enabling steady-state operation across a continuous train by facilitating mass balance between unit operations such as chromatography which have periodic loading and elution cycles. In this paper, we propose a design of a system of surge tanks for a monoclonal antibody (mAb) production process consisting of cell culture, clarification, capture chromatography, viral inactivation, polishing chromatography, and single-pass ultrafiltration and diafiltration. A Python controller has been developed for robust control of the continuous train. The controller has four layers, namely data acquisition, process scheduling, deviation handling, and real-time execution. A set of general guidelines for surge tank placement and sizing have been proposed together with process control strategies based on the design space of the individual unit operations, failure modes analysis of the different equipment, and expected variability in the process feed streams for both fed-batch and perfusion bioreactors. The control system has been successfully demonstrated for several continuous runs of up to 36 hours in duration, and is able to leverage surge tanks for robust control of the continuous train in the face of product variability as well as process errors while maintaining critical quality attributes. The proposed set of strategies for surge tank control are adaptable to most continuous processing setups for mAbs, and together form the first framework that is able to fully realize the benefits of surge tanks in continuous bioprocessing.

Thursday
Method development, validation, and implementation of Raman spectroscopy as an in-situ process analytical technology for fermentation process development and commercial manufacturing
04:50pm - 05:10pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Microbial fermentation processes are widely used in industry as production methods for the development and manufacture of numerous vaccine candidates. The rapid nature of some microbial fermentations renders both quantitative and qualitative process analytics challenging. Glucose, a carbon source necessary to drive fermentation metabolism, is a common analyte of interest for quantification throughout a fermentation process. Traditional off-line analysis methods for metabolites such as glucose consume valuable time, require costly resources, and present unnecessary hazards to personnel when working with pathogenic organisms. The necessity for a robust and in-situ analytical method capable of delivering real-time measurements was evident. Here we discuss the successful method development, validation, and implementation of Raman spectroscopy as an in-situ Process Analytical Technology (PAT) for glucose quantification during process development and commercial scale fermentations. Utilizing the Kaiser Raman system with a 785nm laser, raw spectral data was collected throughout fermentation batches in bioreactors, pre-processed, and correlated to off-line glucose reference data. Using chemometric techniques and multivariate analysis, a Partial Least Squares (PLS) model was created to yield real-time and accurate glucose predictions. Through this work, the Raman PAT glucose method was validated for commercial use to improve process robustness, reduce operating costs, and limit off-line sample manipulations. The value and feasibility of Raman spectroscopy as an in-situ PAT method is demonstrated and recognized as an enabling technology suited to mitigate several challenges fundamental to vaccine process development and commercial manufacturing.
Thursday
Developing an innovative strategy to reprocess permeate in continuous process to improve efficiency and sustainability of perfusion process
05:10pm - 05:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Perfusion process provides many benefits over traditional fed-batch process. Intensified perfusion process can achieve far higher titer compared to typical fed-batch culture. As a result of the higher productivities, continuous processes are more economical and enable flexible and smaller facility footprints. However, one of the biggest operational challenges with perfusion processes is the high media demand required to support high cell density cultures. Moreover, significant nutrients remain unused in the permeate which become waste once product protein is captured. Developing a strategy to reprocess permeate provides opportunities in reducing perfusion process water usage and potentially improving material and operational efficiency. In this study, we evaluated the feasibility of processing and recycling permeate in perfusion culture.
Protein A column flow-through (ProA FT) was collected from continuous processing runs that produce monoclonal antibody (mAb). The ProA FT was also further treated to investigate its matrix suitability for cell culture use. The original permeate was used as a control. Composition of these materials are assessed by residual host cell protein, host cell DNA analysis and untargeted metabolite analysis. We then evaluated the impact of culture performance of these types of materials using mock perfusion culture and then demonstrated its preliminary feasibility in ambr 250 perfusion bioreactor system. Opportunities for media development were also identified by the metabolite analysis. Process Mass Intensity (PMI) analysis estimated that recycling 60% of ProA FT translates to favorable reduction in water consumption and cost of water. In summary, we provided an initial feasibility assessment of permeate reuse for perfusion process and presented a roadmap for the development of integrated permeate reuse continuous process.

Thursday
Multi-column continuous protein A capture of a monoclonal antibody at kilogram scale
05:30pm - 05:50pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Multi-column continuous chromatography (MCC) offers significant economic advantages over traditional batch methods for purification of monoclonal antibodies (mAbs), including increased resin capacity utilization, smaller columns, reduced buffer consumption, and faster processing time. The Protein A capture step is a primary target to apply MCC due to its high cost, which is driven even higher as improvements in upstream processing have produced a steady increase in mAb titers. A humanized IgG1a was expressed in a CHO cell line using a 250 L Single-Use Bioreactor (SUB). Two separate SUB runs were harvested by filtration that resulted in titers of 5.6 and 6.2 g/L of expressed mAb. Initially, process development (PD) at gram scale was accomplished using a bench-top Octave® BIO System and 6 x 5 ml prototype SkillPak® rProtein A columns (TOYOPEARL® AF-rProtein A HC-650F). Key factors in the optimization of the MCC process included mAb titer, column size and number, residence time, mAb binding capacity, and flow properties of the resin. Use of 3 columns in the capture zone enabled 90% resin utilization even at short residence times down to 0.5 minutes, while the 6-column process relieved scheduling constraints imposed by insufficient column number. The optimized PD method file was transferred and scaled-up approximately 150-fold on the single-use ProGMP™ System. The mAb was purified using 6 SkillPak 0.79-L rProtein A prototype columns. Over 1 kilogram mAb was obtained from approximately 200 L feed in less than 4 hours using only 4.7 L of rProtein A resin in each of two separate runs. The yield of both runs averaged 92% and the overall productivity exceeded 80 g mAb/L resin/h with quality and purity attributes similar to prior batch-purified mAb.
Thursday
Continuous depth filtration in perfusion cell culture
05:50pm - 06:10pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Nicholas Marchand, Presenter, Pall Corp; Mike Collins
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
As the biopharmaceutical industry embraces continuous manufacturing there has been significant progress made in converting standard unit operations from batch to continuous. Upstream processing has led the way with key developments including more stable cell lines and perfusion technologies. Downstream processing is making progress with continuous chromatography and continuous tangential flow filtration (TFF) technologies now on the market. Cellulose-based depth filters are used at several points in traditional batch processing due in part to their high capacity when loaded with complex feeds. However, they have been slow to transition into continuous processing, and there remains a question of how these depth filters perform when run for long periods of time. It is a particularly interesting question in applications with unlysed cells in the feedstream. Here, we use a secondary clarification application to improve this understanding. In this work, Pall® P-series depth filters were sterilized and connected inline with a continuous CHO cell culture and perfusion system. We demonstrate that cellulose-based depth filters can maintain performance for up to nine days of continuous operation and start to build a relationship between key input parameters such as run duration and outputs of filter capacity, filtrate quality, and impurity clearance.
Thursday
Panel Discussion
06:10pm - 06:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Cell & Gene Therapy: Development & Manufacturing of Cell Therapy Products: Cell and Gene Therapy Session IV: Lentiviral Vector Focus
04:30pm - 06:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
William Kelly, Organizer, Villanova; Mercedes Segura, Organizer; Steven Cramer, Presider, Rensselaer Polytechnic Inst; Meisam Bakhshayeshi, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Cell and gene therapies have demonstrated their efficacy to treat or be curative in severe diseases, chronic diseases, and cancers. The development and manufacturing of these cell therapy products remains a key part of the overall drug product development lifecycle. Multiple avenues have been explored in the cellular therapy space to increase the potency but also simplify the process of generating cell therapy products, for example CAR-T, TCR-based therapies, and other gene-edited cells such as engineered NKs. A multiple of new cellular drug products will be approved in the next five to ten years. Oftentimes the clinical process initiated for first in human Proof-of-concept studies is not the same process that is later approved for commercial and beyond. A key problem to solve for future development of cell therapies is how to incorporate improvements during development and life cycle management (LCM); when is the drug product the same or a new product for LCM. In this session, the organizers would like to explore avenues to improve the efficacy of cell therapy products as well as improvements to the manufacturing process.

Thursday
Engineering HEK293T cell lines to express nuclease for DNA clearance in lentiviral vector production
04:30pm - 04:50pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
To improve the purity of lentiviral vectors (LVV) for cell and gene therapy applications manufacturers’ currently remove residual DNA using a DNase treatment step with endonucleases such as Benzonase. This is a prohibitively expensive but necessary step in the manufacturing process as regulators require concentrations of less than 10 ng/dose at sizes less than 200 base pairs in the final product. As an alternative we have engineered serum-free adapted HEK293T cells to express nuclease enzymes to remove DNA impurities from LVV supernatant. Three nuclease expression cassettes were designed by fusing the N-terminus of the Staphylococcus aureus nuclease B (nucB) open reading frame to: the native S. aureus nuclease signal peptide, the mammalian murine Igκ chain leader sequence, and a virus-encoded transport protein. The Tet repressor system was selected to regulate expression of nucB to reduce cell cytotoxicity and any negative effects on lentivirus production from potential intracellular nuclease expression and constitutive nuclease secretion. All three nuclease expression cassettes demonstrated levels of activity equivalent to or better than Benzonase at 250 units in growth medium harvested from engineered cell lines 24 hours post tetracycline induction. When treating 1.5 µg 1 kb DNA ladder (500 base pairs to 10 kilobase pairs), 2-hour incubations at 37C with 10 µL of growth media reduced DNA ladder to non-visible sizes on 1% agarose gel. To verify that a nuclease secreting cell line can support LVV production, a LVV encoding for green fluorescent protein was prepared by transient polyethylenimine transfection. Nuclease activity was demonstrated in fractions collected during LVV production and had no measurable effect on infectious titre of the clarified supernatant when transducing both HEK293T and AGF-T cells under serum-free conditions. This provides a holistic approach to remove residual DNA early in the process stream and improve the purity of the final product, whilst avoiding the addition of exogenous nuclease and its associated costs at scale. This may also potentially reduce any deficiencies of downstream processing attributed to the viscosity of residual DNA.
Thursday
Development and scale-up of a bioreactor process for transient lentivirus production using a suspension-adapted HEK293T clone
04:50pm - 05:10pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Eva Fong, Presenter, MilliporeSigma; Sonal Patel; Henry George
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
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 process control (pH, dissolved oxygen, mixing and temperature) and lower production cost for process intensification. Here, we describe the development and scale up of a 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 3 L Mobius® Single Use Bioreactors. The optimal process parameters for cell growth and lentivirus production were developed at the bench scale in 3 L Mobius® Single Use Bioreactors and the process was scaled up in the 50 L Mobius® Single-Use Bioreactors.
Thursday
Setting up a perfusion process in ambr®15 to produce lentiviral vectors
05:10pm - 05:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Alexander Tappe, Presenter; Ms Katrin Frenzel, Sartorius Stedim Biotech GmbH
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Background:
The increasing importance of viral vectors for gene therapy purposes has brought into question the efficiency of the manufacturing process. A higher space/time yield is one of the key factors for an increase in productivity and thus a broader adaption of a cell culture-based process to industrial scale. Recently, a suspension-based process operated in batch mode using HEK293T17SF cells was developed. High infectious viral particle titers of 4-5 x 107 TU/mL and a total of 7 x 108 transducing units were achieved. However, production was thought to be limited due to a lack of nutrients and aggregation of toxic metabolites. One potential solution to reduce metabolites is the switch to perfusion mode. A semi-continuous production procedure was developed in ambr®15 as a multi-bioreactor platform.
Results:
As part of a DoE, we focused on enhancer concentration, stirring speed and perfusion rate as representatives of volumetric productivity. Both process modes, batch and perfusion, were compared in terms of the resulting responses. These included physiological cell parameters, metabolic analysis, and a total and infectious virus titer. Increasing the cell concentration at time of transfection from 1 x 106 to 5 x 106 and setting a 50% perfusion rate leads to a titer of 6-7 x 107 TU/ml, corresponding to a total production of 2 x 109 transducing units. Compared to the batch mode, this is a 5- fold increase in viral vector production.
Conclusions:
These results confirm that the perfusion mode increases the space/time yield and the relevance for industrial scale production. However, the production process still shows potential for optimization. Further investigations relate to the establishment of a cell-specific perfusion rate and factors influencing up-scaling to several liters.

Thursday
Realizing the full potential of gene therapy: Upstream viral vector manufacturing process intensification and integration
05:30pm - 05:50pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
While already approved gene therapies meet important unmet therapeutic needs for a small patient population, the new gene therapies currently being developed could address larger patient population. The manufacturing processes for viral vectors used for the so far approved treatments were developed in research laboratories with technologies not suitable for large-scale industrialization. Major manufacturing improvements are therefore needed to realize the full potential of gene therapy.
Low upstream viral vector productivity represents the main limitation of current processes to meet the demand. We used process intensification and integration to overcome this challenge. AAV8 viral vectors were produced in bioreactor from HEK293 cells grown at high cell density in perfusion mode with two cell retention technologies (XCellTM ATF® and KrosFlo® TFDF®). The performance of the perfusion cell culture processes was compared to that of a standard batch bioreactor process. A 2 to 5-fold increase virus production per liter of bioreactor was achieved with the perfusion processes. The cell retention filters enabled the continuous harvest of AAV8 particles present in the media during the virus production, demonstrating the potential for continuous upstream-downstream processing of secreted AAV vectors. The post-lysis recovery of the intracellular AAV8 particles was achieved with the same cell retention filters using an integrated clarification process. A high flux (up to 900 LMH) and throughput (>500 L/m2) clarification step resulted from the use of the TFDF filter. Finally, the cost benefit at commercial manufacturing scale from the measured process improvements will be discussed.

Thursday
Lentiviral vectors purification: improving current processes and exploring new strategies
05:50pm - 06:10pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Lentiviral vectors (LVs) have become the vector of choice for the treatment of acquired and inherited diseases, since they can stably integrate the genome of dividing and nondividing cells. The recent approval of LV-based therapies has increased the industry’s interest and demand for more efficient manufacturing processes. However, the bioprocess of LVs poses several challenges that can result in the loss of biological activity. Despite all the advances, improved bioprocesses are required before lentivirus can be routinely used in preclinical and clinical research. LVs liability, arising from the membrane envelope fragility, pH sensitivity and salt variation, is hampering the clinical-to-market transition. Here, we report an improved four-step LVs purification process and present the use of 3D cellulose chromatographic columns, as a replacement for traditional techniques.
We implemented a scalable protocol for LVs purification that combines clarification, anion-exchange chromatography (AEX) and ultrafiltration membrane technologies towards the maximization of infectious virus recovery. Various clarification filters were investigated and compared in terms of virus recovery yield and throughput. Design of Experiments was used to build up a comprehensive analysis of the AEX step and determine the best operating conditions. The process comprises only four steps and can be performed in less than 5 hours with a global recovery yield of 45%.
Aiming to extent the portfolio of chromatographic media available for LVs purification, we have also been exploring 3D cellulose printed columns functionalized with different ligands. The ability to design flow channels and tailored larger porous bed structures to the targeted product, constitute an advantage for the purification of enveloped viruses. Overall, LVs were purified with a recovery yield of 57% transducing units, suggesting the power of 3D printing technologies on the viral vector manufacturing.
<b>Lentiviral vectors purification: improving current processes and exploring new strategies</b>

Lentiviral vectors purification: improving current processes and exploring new strategies


Thursday
Discussion
06:10pm - 06:30pm USA / Canada - Eastern - August 26, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual