Abstract
Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. This review provides a comprehensive analysis of upstream, downstream and formulation unit operation challenges encountered during AAV vector manufacturing, and discusses how desired product quality attributes can be maintained throughout product shelf-life by understanding the degradation mechanisms and formulation strategies. The mechanisms of various physical and chemical instabilities that the viral vector may encounter during its production and shelf-life because of various stressed conditions such as thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH, excipients, and impurities on the stability of viral vectors is also discussed. As such, the aim of this review is to outline the tools and a potential roadmap for improving the quality of AAV-based drug products by stressing the need for a mechanistic understanding of the involved processes.
Highlights
Significant technological advancements have been made over several decades providing option to treat and control life threatening diseases
associated virus (AAV) viral vector consists of a protein shell protecting a small, single-stranded DNA genome of approximately 4.7 kilobases.[2,8,17,18,9−16] AAV viral vectors belong to the parvovirus family
Upstream process workflow Unit operations in upstream viral vector production include: (1) plasmid development and production where a cis-plasmid (that encodes a gene of interest (GOI) flanked by the AAV inverted terminal repeats (ITRs)), a trans-plasmid, and a helper plasmid (that encodes adenovirus (Ad) helper genes, E2A, E4, and VA RNA) are designed and produced, (2) cell expansion where E1 transduced cells are expanded to a desired cell density, (3) plasmid transfection where plasmids are introduced into cells after it has reached a desired cell density, and (4) viral vector production where transiently transfected cells are allowed to produce the virus for several days
Summary
Manufacturing challenges and rational formulation development for AAV viral vectors Arvind Srivastavaa,*, Krishna M.G. Mallelab,*, Nandkumar Deorkara, Ger Brophya a Biopharma Production, Avantor, Inc., 1013 US Highway, 202/206, Bridgewater, NJ, United States b Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East Montview Boulevard, MS C238-V20, Aurora, CO 80045, United States
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