The Chromatography Forum

of Delaware Valley

Join us on March 25th for two exciting talks on alternate separation techniques:

AF4 Method Development for Vaccine Characterization

Speaker: Troy Halseth, Merck

Abstract: Vaccines include a variety of complex modalities such as protein-polysaccharide conjugates, virus-like particles, and lipid nanoparticles, giving rise to challenges in product characterization. Controlling for aggregation, an important attribute for vaccine quality and efficacy, requires analytical methods capable of evaluating the analyte in its native state. Size exclusion chromatography (SEC) is typically used for this analysis due to its widespread availability, robustness, and ease of use. However, interactions with the column stationary phase as well as high shear forces experienced when moving through narrow pores in the column can potentially alter the sample, leading to differences in parameters such as weight-averaged molecular weight and degree of aggregation. Asymmetric Flow Field-Flow Fractionation (AF4) is a size-based separation technique with no stationary phase, wherein an analyte’s diffusion against a cross-flow determines its retention time, leading to a reversed elution order compared to SEC.  The gentler approach of differentiating molecules provided by AF4 is an ideal method for characterizing large, complex particles that may form loosely associated aggregates or be prone to secondary actions with a column matrix. This presentation will cover basic AF4 theory, practical considerations for optimizing an AF4 separation, and case studies applied to different vaccine modalities.

Capillary Gel Electrophoresis for Multivalent Recombinant Vaccine Purity Determination 

Speaker: Ashley Prout, Merck

Abstract: Merck produces multivalent recombinant vaccines which consist of adjuvanted particles assembled from monomers of the main structural protein expressed in yeast. The critical quality attributes (CQAs) of purity and intact monomer are measured for product release and stability by SDS-PAGE. The goal was to explore capillary gel electrophoresis (CGE) as a modern replacement given that the platform has become well-established in the commercial manufacturing space for other large biomolecules. Vaccines are particularly complex analytes due to multivalency, adjuvants, and a tailored bioprocess which encompasses intermediates that vary widely in protein concentration, ionic strength, and matrix composition. The complex nature of vaccine samples posed specific challenges for CGE analysis and necessitated sample treatments for desalting and adjuvant dissolution to achieve sufficient sensitivity for minor impurity detection. The CGE method demonstrates close alignment with the benchmark SDS-PAGE method and passed late-stage method validation, meaning that there’s a viable path forward for implementation in commercial release and stability testing.