Abstract
Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm2 (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 ± 100 and 14,559 ± 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 × 109 ± 0.2 and 1.75 × 109 ± 0.08 infectious particles of ONCOS-401 per cm2 of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses.
Highlights
Oncolytic viruses (OVs) selectively replicate and lyse cancer cells; spreading within the tumor mass; circulating into distant metastases; and not significantly harming normal cells
Common challenges for manufacturing clinical grade oncolytic adenoviruses include the optimization of growth conditions, viral productivity, and initial yields; scalable purification strategies; and formulations that provide long-term stability [11]
As contaminating wildtype E1A-driven viruses can replicate in healthy cells rather than being restricted to growth in cancer cells, the contaminating wildtype E1A-driven viruses may change the specificity of the administered viral product in vivo
Summary
Oncolytic viruses (OVs) selectively replicate and lyse cancer cells; spreading within the tumor mass; circulating into distant metastases; and not significantly harming normal cells. Optimization of the manufacturing process of Ad often includes evaluation of the type, confluency, and growth conditions of the host cell line; variation of multiplicity of infection; assessment of optimal harvesting time and initial harvesting method with cell disruption; often treatment with benzonaseTM to digest DNA and RNA; and passage through one or more chromatographic columns such as anion exchange, gel infiltration, size exclusion column; and formulation that fosters long-term stability [11] The purpose of these studies was to optimize the initial steps: conditions for ONCOS-401 production in A549 cells and the initial harvesting processes. Both the growth parameters and the harvesting methods can affect the viral yield.
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