Erica A. Green, Qiang Fu, Nelson Ndahiro, Thomas M. Leibiger, Yongdan Wang, Yongsuk Lee, Kelvin H. Lee, Michael Betenbaugh, Seongkyu Yoon, David J. McNally
{"title":"Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing","authors":"Erica A. Green, Qiang Fu, Nelson Ndahiro, Thomas M. Leibiger, Yongdan Wang, Yongsuk Lee, Kelvin H. Lee, Michael Betenbaugh, Seongkyu Yoon, David J. McNally","doi":"10.1002/bit.28980","DOIUrl":null,"url":null,"abstract":"<p>Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic-industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in-house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1640-1655"},"PeriodicalIF":3.6000,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28980","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology and Bioengineering","FirstCategoryId":"5","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/bit.28980","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic-industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in-house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.
期刊介绍：
Biotechnology & Bioengineering publishes Perspectives, Articles, Reviews, Mini-Reviews, and Communications to the Editor that embrace all aspects of biotechnology. These include:
-Enzyme systems and their applications, including enzyme reactors, purification, and applied aspects of protein engineering
-Animal-cell biotechnology, including media development
-Applied aspects of cellular physiology, metabolism, and energetics
-Biocatalysis and applied enzymology, including enzyme reactors, protein engineering, and nanobiotechnology
-Biothermodynamics
-Biofuels, including biomass and renewable resource engineering
-Biomaterials, including delivery systems and materials for tissue engineering
-Bioprocess engineering, including kinetics and modeling of biological systems, transport phenomena in bioreactors, bioreactor design, monitoring, and control
-Biosensors and instrumentation
-Computational and systems biology, including bioinformatics and genomic/proteomic studies
-Environmental biotechnology, including biofilms, algal systems, and bioremediation
-Metabolic and cellular engineering
-Plant-cell biotechnology
-Spectroscopic and other analytical techniques for biotechnological applications
-Synthetic biology
-Tissue engineering, stem-cell bioengineering, regenerative medicine, gene therapy and delivery systems
The editors will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science. Submission of papers dealing with routine aspects of bioprocessing, description of established equipment, and routine applications of established methodologies (e.g., control strategies, modeling, experimental methods) is discouraged. Theoretical papers will be judged based on the novelty of the approach and their potential impact, or on their novel capability to predict and elucidate experimental observations.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
