Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD’s Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage.
Fig: Description of structure and morphology of rVSV-ZEBOV-GP. (A) MSD’s Ebola Vaccine was generated by recombinant engineering and genetic swapping of Vesicular Stomatitis Virus (VSV) surface glycoprotein with Ebola virus surface GP1 glycoprotein. (B) Cryo-EM of clarified virus harvest material from laboratory samples of rVSV-ZEBOV, scale shown for size reference. (C) Model representation of cathepsin-mediated cleavage in natural Ebola surface GP proteins, legend included. The Trypsin treatment performed during processing of ERBEVO is thought to simulate natural viral protein processing, increasing infectivity of the produced virions.
Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.
Ricci, G., Minsker, K., Kapish, A. et al. Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO. Sci Rep 11, 7432 (2021). https://doi.org/10.1038/s41598-021-86688-z