Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Area of Science:

• Virology
• Biotechnology
• Oncology

Background:

• Oncolytic adenoviruses offer a promising cancer treatment strategy by selectively destroying tumor cells and stimulating anti-tumor immunity.
• A significant hurdle in utilizing these viruses is the lack of scalable manufacturing processes for clinical-grade viral vectors.
• Promising early clinical results for ONCOS-401 necessitated the development of improved production methods.

Purpose of the Study:

• To establish and optimize scalable production methods for the oncolytic adenovirus ONCOS-401.
• To identify optimal cell density, multiplicity of infection (MOI), and incubation time for viral replication.
• To evaluate different harvesting methods for maximizing viral yield.

Main Methods:

• A549 cells were cultured to high confluency (220,000 cells/cm²) in T-175 flasks and HYPERFlasks.
• Cells were infected with ONCOS-401 at a multiplicity of infection (MOI) of 30.
• Viral production was optimized with a 66-hour incubation period, and the Lysis A harvesting method with benzonase was employed.

Main Results:

• Optimal production conditions involved A549 cells at 80% confluency, MOI of 30, and 66 hours of incubation.
• The Lysis A harvesting method yielded the highest viral titers, reaching 10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell in T-175 and HYPERFlasks, respectively.
• ONCOS-401 production reached up to 2.1 × 10⁸ ± 0.2 and 1.75 × 10⁸ ± 0.08 infectious particles/cm² in T-175 and HYPERFlasks, respectively.

Conclusions:

• A stepwise process for optimizing oncolytic adenovirus production was successfully developed and validated using ONCOS-401.
• The established methods demonstrate scalability and efficiency for generating clinical-grade oncolytic viruses.
• These findings provide a foundation for optimizing the production of other oncolytic viruses for cancer therapy.