Development of peptone-based serum-free media to support Vero CCL-81 cell proliferation and optimize SARS-CoV2 viral production

Abstract

Background: Developing an optimal media for Vero cell lines is crucial as it directly influences cell survival, proliferation, and virus production. The use of serum in cell culture raises safety concerns in biological production. The United States Food and Drug Administration (FDA) and the European Medicines Agency have implemented stricter regulations on the use of animal-derived components in commercial protein manufacturing to ensure patient safety. Objective: This study aims to develop new chemically defined media using peptone hydrolysate as a serum-free alternative. Methods: A rational experimental design was employed to screen and optimize potential serum substitutes. Six types of peptones were tested at varying concentrations to determine the top three candidates. These candidates were subsequently combined into five mixtures to identify the two most effective serum-free media for Vero CCL-81 cells. The evaluation criteria included morphology observation and proliferation assay. The two most promising serum-free formulations were further evaluated for their ability to support virus production and using TCID50 and absolute quantification by RT-qPCR. Results: The results of this study indicate that Mixture 4, consisting of 0.5 g/L Phytone™ Peptone, 0.5 g/L Difco™ Soytone, and 2 g/L Bacto™ Malt, significantly enhanced Vero CCL-81 cell proliferation and produced high viral titers. The developed medium demonstrated comparable performance to commercially available serum-free media. Conclusion: Peptone-based serum-free media provide a robust alternative for supporting Vero CCL-81 cell growth and are suitable for virus production.

Keywords

• Serum-free media
• Protein hydrolysate
• Vaccine
• Manufacture process
• COVID-19