Micro-Flow Imaging and Flow Virometry as Preferred Methods to Monitor Aggregate Formation in the Submicron and Subvisible Size Range for Two Early-Phase Vaccine Candidates

Background: Unintended particle formation in vaccine samples has the potential to reduce product efficacy and impact immunogenicity. As a result, regulatory agencies routinely request tests to analyze particles in the micron size range for release. Particles may come in a variety of forms and can be inherent (e.g., from proteinaceous aggregates) or intrinsic (e.g., silicone oil from tubing or contamination from the manufacturing process) and can range in size from submicron to the visible size range. In recent years, there has been an increased interest in developing methods to detect and monitor aggregated particles as well as a desire to evaluate particle size and concentration of submicron particles in addition to the subvisible ranges routinely monitored by compendial methods. Methods: Typically utilized techniques include biophysical analytical methodologies that leverage light scattering or imaging. In this study, dynamic light scattering (DLS), micro-flow imaging (MFI), and flow virometry were evaluated as methods to detect and monitor submicron- to micron-sized particles. Results: These methods enabled assessments of stability and processability for drug substance (DS) and drug product (DP) samples of a Human Cytomegalovirus (HCMV) vaccine candidate. For this attenuated live virus vaccine candidate, MFI and flow virometry proved to be the most sensitive methods to monitor aggregate formation over the complete size range of potential aggregates. These established methods were applied to a coxsackievirus A21 vaccine candidate for process and formulation screening. Conclusions: MFI was most sensitive to changes in subvisible particle concentration of samples that were stressed via constant stirring, while flow virometry captured changes in submicron particles after stress from stirring and recirculation with a shear cell. This comprehensive screening approach combining multiple analytical methods to screen the full aggregate particle size range coupled with a broad range of forced aggregation conditions provides a framework for screening and development of vaccine formulation candidates and other modalities, such as biologics.