Viral Vector Manufacturing Research Articles

Overcoming the DNA Bottleneck

Genetic Engineering & Biotechnology NewsVol. 42, No. 6 Sponsored ContentFree AccessOvercoming the DNA BottleneckPowering nucleic acid–based therapies into the future with LinearDNA™Published Online:8 Jun 2022https://doi.org/10.1089/gen.42.06.01AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail The global demand for DNA continues to rise at a breakneck pace. This trend began with the introduction of redirected cell and gene therapies and was supercharged by the success of mRNA COVID-19 vaccines. While these transformative therapies come in a variety of forms, they all have a common thread—they rely on DNA.According to a May 2021 report from the American Society of Gene and Cell Therapy, there were approximately 1,745 gene/cell therapies and 600 RNA therapies in development.1 These numbers have continued to grow over the past year. The net result is an unprecedented demand for DNA for research, clinical development, and therapeutic manufacturing.Plasmid DNA—good enough, until nowTo date, nucleic acid–based therapies have used plasmid DNA (pDNA) as their source of DNA. Based on 1970's technology, pDNA was the only viable source of DNA when nucleic acid–based therapy development began. Thus pDNA became the industry standard.pDNA has several limitations and downsides, including the presence of antibiotic resistance genes and origins of replication, inclusion of unwanted DNA sequences, low yields, batch failures and inconsistencies, as well as sequence heterogeneity—the latter two issues being exacerbated by the complex DNA sequences needed for gene and mRNA therapies.Moreover, pDNA manufacturing is complex, and scaling its manufacture is both time consuming and expensive. These facts have resulted in a pDNA supply that has failed to meet current DNA demands.LinearDNA—breaking the DNA bottleneckLinearDNA is an enzymatically produced DNA that is substitutable for pDNA in nucleic acid–based therapy manufacturing. LinearDNA is produced via the polymerase chain reaction (PCR) in a cell-free manufacturing workflow, and unlike plasmids, contains no extraneous DNA sequences. LinearDNA can be produced from an existing pDNA construct or a de novo DNA sequence.LinearDNA is substitutable for pDNA in a wide range of nucleic acid–based therapy applications, including mRNA production, CAR-T and viral vector manufacturing, and DNA vaccines. LinearDNA is produced via a rapidly scalable manufacturing platform that can grow to meet the needs of virtually any customer.Gram-scale quantities of LinearDNA can be produced in mere weeks with high sequence fidelity and homogeneity—even with complex DNA sequences such as polyA and inverted terminal repeats (ITRs). Recently, a proof-of-concept study was conducted in which a commercial-scale batch of LinearDNA was produced from a de novo DNA template in 10 days—highlighting LinearDNA's unparalleled speed of production.Each batch of LinearDNA is produced to high purity according to strict quality specifications as shown in the table above.LinearDNA—powering mRNA therapies into the futureLinearDNA can accelerate and simplify mRNA research and production workflows that currently use pDNA. Unlike pDNA, which must be “linearized” via expensive restriction enzymes for in vitro transcription (IVT), LinearDNA is ready for IVT use without further modification. In addition, since LinearDNA is free of pDNA backbone sequences, there are no adventitious DNA sequences to remove.Furthermore, unlike plasmid-based DNA production methods that produce heterogeneous DNA sequences when faced with polyA sequences, large quantities of homogeneous LinearDNA can be produced that include a precise number of polyA nucleotides (ranging from 80 to 120).The combination of these unique advantages, coupled with LinearDNA's rapid production times and high purity, makes LinearDNA an ideal DNA template to continue the rapid growth of mRNA therapies.ConclusionpDNA, once the enabler of nucleic acid–based therapies, is now a problematic bottleneck. LinearDNA can rapidly meet all current and future DNA demands and remove DNA supply constraints on therapy developers and manufacturers.Learn more about LinearDNAadnas.com/lineardnaReference1. American Society of Gene and Cell Therapy. Gene, Cell & RNA Therapy Landscape: Q2 2021. Google ScholarFiguresReferencesRelatedDetails Volume 42Issue 6Jun 2022 Information© 2022 by GEN PublishingTo cite this article:Overcoming the DNA Bottleneck.Genetic Engineering & Biotechnology News.Jun 2022.7-7.http://doi.org/10.1089/gen.42.06.01Published in Volume: 42 Issue 6: June 8, 2022PDF download

Regulatory & supply chain implications for plasmids as critical starting materials in the manufacture of viral vector gene therapy products

Plasmid DNA is actively being used as starting material/intermediates, drug substance, and/or drug product in the manufacture of several therapeutics including DNA vaccines, viral-vector and non-viral-vector gene therapies, and mRNA vaccines. Since plasmids have a large range of uses, there have been various plasmid guidelines released from the different agencies. Specifically for gene therapy, recent guidelines have tried to provide clearer guidance, however there is still ambiguity around quality requirements. For example, the specifications for the release of plasmids from a plasmid manufacturer and the facility requirements for accepting these plasmids for use in viral vector manufacturing are not always aligned and can cause disconnect in the supply chain. For a sponsor managing plasmid manufacturing for use as starting material in viral vector manufacturing, it is important to be aware of these differences in requirements ahead of time. The current manuscript highlights these regulatory differences and calls out the importance for a sponsor to build a robust supply chain between the plasmid manufacturer and the viral vector manufacturer well in advance to minimize bottlenecks due to quality requirements and timelines.

Stepping foot into a successful partnership to support your viral vector therapy through commercialization

<p class="p2"><span class="s1"><span class="Apple-converted-space">&nbsp;</span></span><span class="s2"><b>Charlotte Barker, </b></span>Editor, <span class="s2"><i>Cell and Gene Therapy Insights</i></span>, <span class="s2"><b>speaks to Minh Hong, </b></span>Head of Commercial, Viral Gene Therapy, Merck, <span class="s2"><b>and Marc Gaal, </b></span>Director, Program Management at the Life Sciences Business Sector, Merck<span class="Apple-converted-space">&nbsp;</span> <div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Minh Hong copy.png" data-image="1"></div><div class="author-det"> <b>Minh Hong</b> leads the commercial team for Viral Gene Therapy contract manufacturing services within the Life Sciences Business Sector of Merck. He is responsible for account management, business development, and out-licensing activities of innovative viral vector manufacturing tools. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/MAG Profile pic small - Copy copy.jpg" data-image="1"></div><div class="author-det"> <b>Marc Gaal</b> heads Merck’s Commercial Project Management Office and is responsible for contract administration and project management of customer programs throughout the entire life cycle of the program. </div></div></div>

Advancing real-time monitoring of AAV vector processes

<h3>PODCAST INTERVIEW with:</h3><div class="authors authors-lg"><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/da6577c970bd400690b08cf6f1a9c72e.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Rachel Legmann</h3><h5 class="author-title">Director of Technology, Gene Therapy</h5> Rachel has more than 25 years of experience in the field of scalable biologics and gene therapy manufacturing of therapeutic products, viral vectors and proteins for gene therapy and biologics. She completed her Ph.D. in Food Engineering and Bio-technology at the Technion-Israel Institute of Technology, Israel. Rachel joined Repligen in 2021 as a subject matter expert leading the global gene therapy organiza-tion helping customers achieve their technical and operational objectives in their manufacturing of vector-based therapeutics and vaccines with a focus on gene therapy processes including upstream, downstream, analytics and scalability. In addi-tion to supporting global customers and building high level networks, Rachel is sup-porting various internal cross-functional activities and external collaborations. Prior to joining Repligen, Rachel held several scientific and leadership roles at Microbiology and Molecular Genetics department at Harvard Medical School, CRO SBH Sciences, Sea-horse Biosciences part of Agilent, CDMO Goodwin Biotechnology and Pall Corp part of Danaher. </div></div></div></div><h3>and&nbsp;</h3><div class="authors authors-lg"><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/55bf3e861c834564a324a7830ac4e694.png" data-image="1"></div><div class="author-det"><h3 class="author-name">Joe Ferraiolo</h3><h5 class="author-title">Joe FerraioloAssociate Director Bioanalytics, Repligen</h5> Joe is the Associate Director Bioanalytics for Repligen. He is in charge of the Bioana-lytics Applications department related to the SoloVPE Variable Pathlength UV solu-tion. He has been with the company for more than 20 years, with over 10 years of development and validation experience in analytical applications. He specializes in UV analysis and leads the development and commercialization of high-value products and flexible solutions that address critical steps in the production of biologic drugs, gene therapy solutions, and monoclonal antibodies. </div></div></div></div>

Merck boosts viral vectors in California

Merck KGaA has opened a second viral vector manufacturing facility in Carlsbad, California . The $116 million, 13,000 m 2 plant will more than double the site’s commercial-scale capacity. With the new facility, Merck will operate 30 clean rooms and reactors with production capacity of up to 1,000 L. Other large pharmaceutical services companies, including Thermo Fisher Scientific and Charles River Laboratories, are also ramping up viral vector services.

Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector

Viral vectors are a new class of biologics which facilitate gene transfer and modification in living cells, potentially treating a multitude of conditions with genetic causes. Scalable manufacturing technologies are critical to ensuring these cutting-edge medicines can be produced in sufficient quantities to meet the needs of process development, clinical trials, and ultimately commercial manufacturing Mol. Ther. 2016; 3: 15049.&quot; data-url=&quot;http://dx.doi.org/10.1038/mtm.2015.49&quot;&gt;[1]Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.Ayuso E. Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome. Mol. Ther. 2016; 3: 15049.. As viral vector-based products have only relatively recently received regulatory approval, public information on scalable optimization of these processes is very limited. Abeona Therapeutics is a gene therapy company developing novel gene replacement therapies for rare inherited diseases. These conditions can impact development and limit both quality of life and/or life expectancy et al. Estimation of impact of RPE65-mediated inherited retinal disease on quality of life and the potential benefits of gene therapy. Br. J. Ophthalmol. 2019; 103(11): 1610–14. &quot; data-url=&quot;http://dx.doi.org/10.1136/bjophthalmol-2018-313089&quot;&gt;[2]Lloyd A, Piglowska N, Ciulla T et al. Estimation of impact of RPE65-mediated inherited retinal disease on quality of life and the potential benefits of gene therapy. Br. J. Ophthalmol. 2019; 103(11): 1610–14.&#160;. These transformative medicines can be used to replace a defective gene with a functional copy, silence a defective gene or even directly edit genes Nat. Rev. Drug Discov. 2019; 18(5): 358–78. &quot; data-url=&quot;http://dx.doi.org/10.1038/s41573-019-0012-9&quot;&gt;[3]Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 2019; 18(5): 358–78.&#160;, J. Virol. 1999; 73(5): 3994–4003. &quot; data-url=&quot;http://dx.doi.org/10.1128/jvi.73.5.3994-4003.1999&quot;&gt;[4]Xiao W, Chirmule N, Berta SC, McCullough B, Gao G, Wilson JM. Gene therapy vectors based on adeno-associated virus type 1.&#160;J. Virol. 1999; 73(5): 3994–4003.&#160;. We evaluated the Pall Allegro™ STR bioreactor family as an rAAV vector production platform and evaluated the scalability of the PEI-mediated transfection manufacturing process for rAAV at the 50&#160;L and 500&#160;L working volume. Process scalability was evaluated based on cell growth, metabolic profile, and vector production. This testing demonstrates that control of key process parameters enables a scalable vector production process between the 50&#160;L and 500&#160;L scale using Allegro STR single use bioreactors.

Industrializing viral vector manufacture

Daniel Teasley has worked at Merck for over 6 years, serving in product management and product strategy roles across multiple portfolios including custom cell line engineering services, regenerative medicine products for research, cell therapy manufacturing, and viral gene therapy manufacturing. For the past three years, he has been a Senior Product Manager responsible for the development, launch, and marketing of products and services that enable at-scale viral vector manufacturing. He also supports cross-business strategic initiatives in novel modality manufacturing. Dr. Teasley holds a PhD in Molecular Genetics and Genomics from Washington University in St. Louis and a BS in Biology from the College of William and Mary.Katherine (Katie) Schewe has worked at Merck for over 4 years in various roles from product management in Viral &amp; Gene Therapy to quality assurance of Active Pharmaceutical Ingredient manufacturing. Katie holds a MS in Chemistry from Indiana University Purdue University – Indianapolis and a BS in Chemistry from Southern Illinois University – Edwardsville.

Scalable downstream process development for in vivo gene therapy products: understanding full/empty separation capabilities and challenges with ion exchange chromatography

Adeno-associated viruses (AAVs) for&#160;in vivo&#160;gene therapy are a dominant viral vector platform in the gene therapy landscape. A key challenge in viral vector manufacturing, particularly with AAVs, is effective and consistent separation of full particles (i.e. viral vector capsids containing the full recombinant cDNA of the gene of interest) from empty capsids. Monolith-based ion exchange chromatography (IEX) is a promising tool to achieve this separation but poses challenges with consistency upon scale-up. This paper outlines some of the scale-up challenges and potential mitigations to achieve consistent separation of full and empty particles. IEX is evolving to become the industry standard for downstream separations as industrialization of gene therapy manufacturing continues.

Scalable cell culture and transient transfection for viral vector manufacturing

Watch the video or read the poster to learn: Challenges for scalable viral vector productionConsiderations for choosing a cell culture platformBest practices for transient transfection of adherent culturesGuidance for transfection of Corning CellSTACK&#174;️and Corning CellCube platforms with Polyplus PEIpro&#174;️&#160;transfection reagentAnn Rossi Bilodeau graduated from the University of Rochester School of Medicine and Dentistry with a PhD in Pharmacology and received postdoctoral training at the University of Chicago. Ann began her career with Corning Life Sciences as the Applications Lab Manager where she utilized her strong academic and industry research experience to direct the activities of the Applications Laboratory. Currently, Dr Rossi Bilodeau is the Senior Bioprocess Applications Scientist, functioning as the technical lead for generating applications in support of Corning Life Sciences’ Bioprocess and Cell and Gene Therapy portfolios including the Corning&#174;️ CellCube&#174;️ and Corning HYPERStack&#174;️ platforms.

Breaking viral vector bioanalysis barriers with centrifugal force

Advances in viral vector gene delivery systems, particularly adeno-associated virus (AAV) and lentivirus (LV), have accelerated the development of new cell and gene therapies. Regulatory programs for accelerated review have added to the demand for the manufacture of viral vectors, exceeding capacity and creating backlogs. Improvements in analysis speed, accuracy, precision, and dynamic range are potential targets for accelerating production timelines. Plate-based enzyme-linked immunosorbent assays (ELISAs), commonly used in analysis of viral vector titer, purity, and potency, have laborious and time-consuming manual processing drawbacks as well as long processing times and poor precision. A novel automated microfluidic, compact disc (CD)-based immunoassay format that uses centrifugal force to precisely control the flow of sample and reagents has been notably effective in accelerating bioanalysis of antibody-based therapeutics with high-precision results. One-hour assay run times and wide dynamic ranges accelerate workflows, and 10 &#181;L sample requirements minimizes consumption of limited production material. These dramatic immunoassay improvements are expected to alleviate the analytical delays in viral vector manufacturing.

Viral vector production process intensification: analytics, automation, in-line testing and more

<div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/888145b9bcb9494d9523fe80a135ed5f.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Susan D’Costa</h3><h5 class="author-title">Senior Director, Technical Program Design, Viral Vector Services, Thermo Fisher Scientific</h5> In her current role, Susan functions as SME for VVS’ viral vector manufacturing platforms and systems providing scientific and technical input for customer programs. Prior to this role, she served as the Head of Process and Analytical Development at Viral Vector Services, Thermo Fisher Scientific, formerly Brammer Bio for over three years. In her role as head of PD/ADA, she provided critical scientific input for projects developed and manufactured at VVS. She has built a robust team for the development, customization, optimization, scale-up and tech transfer of gene therapy vector process and analytics. Susan was also integral to developing robust QC analytics for incoming starting and raw materials and for the release of the clinical vector products in her role as Director of Quality Control at Brammer Bio/Florida Biologix. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/1b65fe190645414eb14a9f6b56f12cbf.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Mike Delahaye</h3><h5 class="author-title">Head of Viral Vector Industrialisation, Cell and Gene Therapy Catapult</h5> Michael has almost 15 years of industrial experience within the cell and gene therapy sector. He leads a team of experienced scientists within the Viral Gene Delivery programme, responsible for managing the developmental output of the group within collaborative, commercial and supporting R&amp;D operations. The team’s key focus is the development and delivery of robust manufacturing processes, supported by rapid, high resolution characterisation tools to facilitate the production of AAV based gene therapies. His team is also supporting the development of new adaptive manufacturing approaches by integrating technologies to allow real-time product monitoring and enable feedback control during product manufacture. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/f89a9bee7b7f422f90e20f14ed51c093.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Michael White</h3><h5 class="author-title">Senior Scientist, ‎Affinia Therapeutics</h5> Michael is the Head of Viral Vector Industrialisation at the Cell and Gene Therapy Catapult and has almost 15 years of industrial experience within the cell and gene therapy sector. He leads a team of experienced scientists within the Viral Gene Delivery programme, responsible for managing the developmental output of the group within collaborative, commercial and supporting R&amp;D operations. The team’s key focus is the development and delivery of robust manufacturing processes, supported by rapid, high resolution characterisation tools to facilitate the production of AAV based gene therapies. His team is also supporting the development of new adaptive manufacturing approaches by integrating technologies to allow real-time product monitoring and enable feedback control during product manufacture. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/776b16a7bc2e4fbfbe681c777fe42fcd.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Franz Gerner</h3><h5 class="author-title">Vice President, Technical Operations, Sio Gene Therapies</h5> Franz Gerner is responsible for process development, manufacturing and analytical activities, including overseeing outsourced activities at CDMOs and CTOs. Prior to joining Sio Gene Therapies, Franz worked at Regenxbio, joining the company at an early stage and building up the Process Development team over the course of 5 years. He started to work in the AAV gene therapy field about 25 years ago. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/c0e2ab70fb724206bfe9ddbb85d6b59f.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Jessica Tate</h3><h5 class="author-title">Head of Science &amp; Technology Development, Viral Vector Services, Thermo Fisher Scientific</h5> Jessica Tate has spent the last 15 years producing, purifying, and characterizing various virus modalities, in a combination of industry and academic environments. Currently she leads Thermo Fisher Scientific’s Viral Vector Science and Technology team, who’s mission is to develop and/or employ novel state-of-the-art technologies in the manufacturing of viral vector gene therapies. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/23d387b3f3674a78b57c369bcbcdf517.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Jim Warren</h3><h5 class="author-title">Vice President, Pharmaceutical Development, Ultragenyx Pharmaceutical Inc.</h5> Dr. Warren has 27 years of experience in the pharmaceutical and biotechnology industry. At Ultragenyx, he leads a team of over 60 scientists and engineers, responsible for all early and late stage Process and Analytical Development of viral vector gene therapy products as well as Pilot Scale Manufacturing in the Woburn, MA Pilot Plant. Before coming to Ultragenyx Jim held senior CMC leadership positions at Homology Medicines, bluebird bio, and Shire. Earlier in his career, Jim spent 16 years at Merck &amp; Co., Inc., where he led process development, technology transfer, and manufacturing sciences teams responsible for development of several clinical vector and vaccine candidates and the licensure of multiple commercial vaccines (RotaTeq®, Varivax®, ProQuad®, and Zostavax®). </div></div></div>

Recent Advances of Chemical Definition of Cell Culture Media and Excipients for Virus and Viral Vector Manufacturing: A Review

A vital step in the manufacturing of vaccines and viral vector-based cell and gene therapies is virus production. Historically, virus production relies heavily on undefined and highly variable raw material for cell-based platforms to be successful. Yet due to supply chain constraints, poor reproducibility, and safety concerns attributed to potential adventitious agents present in serum and serum-derived proteins, regulatory agencies have encouraged replacement of these components. Recombinant serum proteins, specifically albumin and transferrin, can successfully replace serum-derived proteins in the expansion of VERO cells and subsequent production of multiple virus types. Furthermore, recent successes have demonstrated that recombinant human serum albumin can serve as an effective excipient in final formulations for the stabilization of virus in vaccines. Collectively, chemical definition of both upstream and downstream processes provides numerous advantages and is essential given the importance of virus and viral vector production for clinical applications.

Scalable AAV manufacturing: addressing challenges across the workflow

<!--StartFragment-->As the gene therapy field grows and evolves, adeno-associated virus (AAV) has emerged as one of the most significant vectors for its ongoing development. The number of approved therapies and the clinical pipeline continue to grow, making scalable and efficient solutions for AAV production ever more vital. A successful viral vector manufacturing process must be able to deliver a consistent, pure, and high-titer product that exhibits good safety and efficacy. In this article, both upstream and downstream solutions across the AAV production workflow are discussed. Scalable, high-titer AAV production is demonstrated in an insect-cell based Expression System. In addition, the benefits of implementing affinity chromatography in the downstream purification of AAV are also discussed, along with regulatory requirements for safety and purity testing during the AAV production process.<!--EndFragment-->

Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

BackgroundCell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed.MethodsThree batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.ResultsAll batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation.ConclusionsManufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

Viral vector manufacturing and transfection efficiency in the iCELLis® bioreactor system

Viral vector manufacturing and transfection efficiency in the iCELLis® bioreactor system

Generation of CAR-NK Cells Using a Notch-Mediated Cell Expansion and Differentiation Platform

An important challenge in the generation of therapeutic chimeric antigen receptor expressing natural killer (CAR-NK) cells is poor gene transfer efficiencies in mature NK cells requiring large quantities of viral vectors, significantly increasing the production cost. We have an established clinical stem cell expansion platform used to generate off-the-shelf hematopoietic stem/progenitor cell products that have been evaluated in 6 clinical trials to date. This proprietary Notch-mediated expansion platform has now been optimized to generate large numbers of functional NK cells by first ex vivo expansion and priming of CD34+ cells isolated from cord blood (CB), followed by directed differentiation to NK cells. The final NK cell product can be cryopreserved with post-thaw maintenance of viability (≥ 80%) and function. With respect to generation of CAR-NK cells, this manufacturing process is scalable and cost-effective and importantly, allows transduction using a viral vector early in the expansion phase of culture without compromising the robust expansion or phenotype of the developing cells. This significantly reduces both the number of cells to be transduced and the amount of viral vector needed to &amp;lt;5% of that required to transduce the final NK product. As proof of concept, we developed CB-derived CAR NK (CB-CAR-NK) cells with specificity against mesothelin (MSLN). Mesothelin is a cell surface adhesion molecule that is highly upregulated in 30-50% of AML cases in pediatric and adult cohorts and lacks expression in normal hematopoiesis. The AML-restricted expression of mesothelin makes it an ideal tumor-associated antigen for immunotherapeutic targeting in AML. CB derived CD34+ cells are transduced with a mesothelin-directed CAR vector during the expansion phase in serum-free expansion media supplemented with cytokines. Transduction efficiencies were assessed periodically in bulk, CD34+ and CD56+ cells throughout the culture duration by flow cytometry analysis of truncated CD19, which is linked to CAR expression by a cleavable T2A. We measured cytolytic activity of CB-CAR-NK and CB-NK cells against NOMO-1 AML cells, which express endogenous MSLN, or NOMO-1 MSLN knockout cells by flow cytometry analysis of cell viability in the target cells. Our manufacturing process generates over 2000 CD56+ NK cells per CD34+ cell initiated in culture. Following transduction and differentiation, we achieve a robust cell product that is &amp;gt;75% CD56+ NK cells and ~22% CD56+CAR+ using a multiplicity of infection of 10 virus particles per cell. The ex vivo generated CAR-NK cells demonstrate an immunophenotype similar to the unmodified NK cells, expressing the NK canonical surface markers NKG2A, NKG2D, NKp46, CD16, NKp44 and NKp30. Despite a modest transduction efficiency, the CB-CAR-NK cells demonstrate superior cytotoxicity against NOMO-1 cells over the unmodified CB NK cells (~4-fold increase). This enhanced killing is highly specific as CB-CAR-NK and CB-NK exhibited similar tumor-killing efficiency in NOMO-1 MSLN KO cells. Having demonstrated the functional potential of our CAR-NK cells in vitro, we are now working toward assessing their function in vivo in a mouse xenograft model. We will also explore other optimal leukemia-specific antigens as targets for our CAR-NK cells and optimize our CAR construct to further enhance transduction efficiency. The vast majority of anti-cancer cellular immunotherapies are patient specific, require apheresis, high volumes of viral vector and complex manufacturing processes that are expensive with a risk of delayed or no treatment if the production fails, all contributing to poor clinical adoption despite transformative outcomes. We have developed our platform to overcome these hurdles with the goal of delivering safe and cost-effective allogeneic off-the-shelf immunotherapies for improved clinical access. Disclosures Chandrasekaran: Deverra Therapeutics: Current Employment, Current equity holder in private company. Prieve:Deverra Therapeutics: Current Employment, Current equity holder in private company. Stoltzman:Deverra Therapeutics: Current Employment, Current equity holder in private company. Cook:Deverra Therapeutics: Current Employment, Current equity holder in private company. Blake:Deverra Therapeutics: Current Employment, Current equity holder in private company. Massey:Deverra Therapeutics: Current Employment, Current equity holder in private company. Delaney:Deverra Therapeutics: Current Employment, Current equity holder in private company.

Evolution from adherent to suspension: systems biology of HEK293 cell line development

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production.

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.

Expert Roundtable: getting a gene therapy product to market: pitfalls and how to prevent them

<div class="authors authors-lg"><div class="author"><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/5665fd4b0e0d43fd9457b130647e8679.png" data-image="1"></div><div class="author-det"><h3 class="author-name" style="color:#5D538A;">Hetal Brahmbhatt</h3><h5 class="author-title" style="color:#5D538A;"><strong><strong>Principal Investigator - Science and Technology Team, Pharma Services, Viral Vector Services, Thermo Fisher Scientific</strong></strong></h5> Dr Hetal Brahmbhatt is a Principal Investigator in the Science and Technology Team, Pharma Services, Viral Vector Services at Thermo Fisher Scientific. She has a PhD in Biochemistry and Biomedical Sciences from McMaster University where she characterized small molecule modulators of apoptotic proteins. In her present role, she contributes to the development of platform processes and technologies for the manufacturing of viral gene transfer vectors. </div></div></div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><strong><strong><img src="https://bioinsights.blob.core.windows.net/uploads/119afb6b1b5b4b119cd918769348eab8.jpg" data-image="1"></strong></strong></div><div class="author-det"><h3 class="author-name" style="color:#00A795;"><strong><strong>Elizabeth Simmons</strong></strong></h3><h5 class="author-title" style="color:#00A795;"><strong><strong>Associate Director QC Analytics, Homology Medicines Inc</strong></strong></h5> Beth has a Master’s degree from Lehigh University in Analytical Chemistry and Regulatory Affairs with 13 years of industry experience. She has managed small and large Quality Control laboratories including method transfer, qualification and validation teams in both small pharma and CMO settings. During that time Beth has supported 2 facility start ups (Commercial and Clinical) and managed technical teams focusing on small molecule, oligonucleotides, and now gene therapy. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><strong><strong><img src="https://bioinsights.blob.core.windows.net/uploads/thomas.png" data-image="1"></strong></strong></div><div class="author-det"><h3 class="author-name" style="color:#007589;"><strong><strong>Thomas Guarinoni</strong></strong></h3><h5 class="author-title" style="color:#007589;"><strong><strong>Manager, Downstream Process team, Viralgen</strong></strong></h5> Thomas Guarinoni, MSc was recruited in January 2017 as manager of the Downstream Process team at Viralgen. He was successful in transferring the entire purification process from Askbio to Viiralgen in record time. Thomas Guarinoni obtained his MSc in 2011 at ENSTBB (National Superior College of Biotechnology) in Bordeaux, France. </div></div></div>

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane.

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.