Development of process analytical tools for rapid monitoring of live virus vaccines in manufacturing.

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between "good" and "damaged" virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO.

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. 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.