Biopharmaceutical developers and manufacturers are augmenting their cell and gene therapy (CGT) capabilities in response to promising clinical data, high demand for novel treatment approaches to rare diseases, and significant commercial milestones achieved by currently marketed products. However, manufacturing the viral vectors required to deliver such therapies remains complex, cost-prohibitive, and inefficient. This issue’s featured report describes barriers that continue to hinder production of viral vectors despite the rapid clinical development of therapies based upon them. The authors and contributors included herein also highlight potential solutions such as careful selection of raw materials for associated cell-culture operations, improvements to transfection reagents and processes, increased implementation of suspension cell-culture systems, improved understanding of mechanisms for vector immunogenicity, and aseptic technologies for combined formulation and fill–finish steps.

Optimizing the AAV Transfection Process in Suspension Cells
Matthias Hebben and Alengo Nyamay’antu
Typically, vectors based on adenoassociated virus (AAV) are expressed in cell culture, then purified chromatographically. In many cases, capture steps are followed by ultracentrifugation or anion-exchange chromatography to reduce partially filled and empty viral capsids. Tangential-flow filtration (TFF) is used to transfer vectors into a desired buffer solution. That is subjected to sterile filtration and aseptic filling. Despite all that processing, however, most vector production systems show low yields and recoveries. The greatest opportunity for process improvement lies upstream because titer and yield increases of several logs could augment overall process yields significantly. Herein, authors from LogicBio Therapeutics and Polyplus-transfection show how the combination of enhanced plasmids with high-quality transfection reagents can boost vector production significantly.
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Viral Safety of Viral Vectors: Special Considerations Arise When the Virus Is the Product
Cheryl Scott, with Hazel Aranha and S. Anne Montgomery
Viral safety is a critical aspect of downstream processing for biopharmaceuticals, especially those derived from mammalian cell lines. These days, the most promising and popular candidates for gene therapy vectors are AAV and lentivirus (LV) species — both relatively small as viruses go. Being small makes them more difficult to purify downstream; it’s a lot easier to catch bacteria and big viruses with a filter that lets proteins and smaller viruses move through, but some adventitious agents of concern are relatively small, too. Viral safety also includes prevention of contamination from adventitious agents and inactivation of what contaminants persist through upstream steps. BPI’s editor in chief and senior technical editor sat down with editorial advisor Hazel Aranha in fall 2021 to learn best practices for ensuring the viral safety of viral vector products.
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Combined Formulation and Fill–Finish of ATMPs Based on Lentivirus Vectors
Leslie Southam and James L. Drinkwater
Based on a presentation from the March 2021 ISPE Aseptic Conference, this article describes Oxford Biomedica’s efforts to improve its “Oxbox” facility for the combined formulation and fill–finish of advanced therapies. The writers focus on a case study involving a CAR T-cell cancer therapy manufactured using LV vectors, describing what the company has learned about developing and documenting a holistic contamination control strategy (CCS) and a more specific aseptic containment strategy (ACS) for operator protection and control of cross-contamination risks between products and batches. The writers showcase several innovative technologies for viral containment and aseptic processing, including those enabling “no-touch” transfer of process materials into filling isolators.
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Spurring on Innovation in Gene Therapy Development
Brian Gazaille, with Roland W. Herzog and Nagendra Venkata Chemuturi
Gene therapies based on AAV vectors hold promise for treating myriad conditions. Immunogenicity remains a challenge for such products, however. With support from PerkinElmer, Roland W. Herzog (professor of pediatrics and Riley Children’s Foundation professor of immunology at the Indiana University School of Medicine) joined Nagendra Venkata Chemuturi (scientific director of global research for drug metabolism and pharmacokinetics, DMPK, at Takeda Pharmaceuticals) to deliver a BPI “Ask the Expert” presentation exploring strategies for minimizing immune responses to AAV vectors. Herzog described emerging research on the role of innate immune signaling in adaptive immune responses to AAV. Chemuturi highlighted the increasing importance of clinical pharmacological tools for evaluating AAV biodistribution. Together, the presentations show how gene therapy companies can improve AAV-product safety and efficacy.
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