Viral vector manufacturing is experiencing significant changes as the industry transitions from patient-specific ex vivo production to scalable in vivo supply, according to an Aug. 10 interview with AGC Biologics’ Upstream Vector Development Manager Silvia Ungari and Analytical Methods Development Manager Chiara Boroni.
Ungari said that while the core process for viral vectors remains largely unchanged between ex vivo and in vivo applications, the way vectors are managed, controlled, and qualified—as well as production scale—differs significantly. For ex vivo treatments, cells are modified outside the body before reinfusion, making the vector a tool rather than the final therapeutic entity. "The vector is essentially a manufacturing tool," Ungari explained, "and the final drug product is the patient’s modified cells." In contrast, for in vivo therapies where vectors are injected directly into patients without further purification opportunities, purity requirements become much stricter.
Regulatory approaches also differ by region and application. In Europe, regulators typically classify ex vivo vectors as starting materials; in the United States, they are considered drug substances but may be evaluated using a risk-based approach because engineered cells are ultimately administered. However, all regions agree that for in vivo therapies purity standards must match those of any injectable biologic.
Ungari highlighted scaling challenges unique to in vivo therapies: “Bioreactors must grow from 200 liters up to 2,000 liters,” she said. Process scale-up involves more than increasing equipment size—it requires dedicated procedures for steps like mixing transfection complexes and early planning for facility layout and workflow optimization. Another bottleneck identified was reliance on HEK293T producer cell lines containing SV40 large T antigen (LTA), which raises regulatory concerns due to its oncoprotein properties. While downstream purification can mitigate risks for ex vivo products, even trace LTA contaminants pose greater safety issues for in vivo applications.
Immunogenicity remains a key challenge; innate immune responses can be triggered by impurities such as residual plasmid DNA or host cell proteins, while adaptive immunity can reduce efficacy or cause toxicity at high doses. Mitigation strategies include engineering envelope proteins or capsids through directed evolution or artificial intelligence-driven design and adopting plasmid-free DNA systems such as doggybone DNA or minicircles.
Boroni described efforts toward stable producer cell lines: “The transition to stable cell lines provides analytical benefits but also poses challenges in developing and validating new analytical methods,” she said. These include genomic integration site analysis and genetic stability testing throughout production cycles. Downstream purification requirements diverge sharply between LVV (lentiviral) and AAV (adeno-associated) platforms—especially given higher purity demands for fragile LVVs used in direct administration—and AAV’s need to separate full from empty capsids during processing.
Boroni concluded: “AGC Biologics is committed to being at the forefront of this transition... Our manufacturing expertise, analytical capabilities, and regulatory track record make us the right partner for developers bringing in vivo therapies to patients.” The company continues investing in proprietary technologies aimed at addressing these evolving industry challenges.
