New Approaches for Analysing Various Aspects of Protein Aggregation in Biopharmaceutical Formulations

Abstract

Protein aggregation poses significant risks for formulation stability, efficacy and safety in biopharmaceutical formulations. This thesis focuses on three aspects of this major issue: screening extensive large aggregate formation, identifying and efficiently monitoring factors affecting aggregation, and, elucidating aggregation kinetics. Since these aspects hinge on effective methods, the overall objective was developing novel or improving existing methods to address them. The studies were based mainly on influenza split-virus vaccines but aimed to produce methods adaptable to other formulations. The thesis demonstrates and discusses in depth three methods that can enable more efficient industrial quality control and/or product improvement. The first method is a novel, effective test based on advanced “decomposition” analysis of Nile Red fluorescence spectra for rapid, reliable and user-friendly screening of large aggregate content in said vaccines. The second is an innovative, fairly accurate, preparation-free, rapid, user-friendly and inexpensive method based on “deconstruction” of UV-Vis absorption spectra for efficient routine screening of surfactant content (intricately linked to aggregation) during industrial processing of said vaccines. These two methods have great potential for high-throughput modification and adaptation to other formulations. The third method is a more holistic, realistic and robust kinetic analysis approach than those based on analysing only monomer loss or only aggregate formation data. Such one-sided analysis can lead to questionable mechanistic insights as different models can fit the data equally well. In “global analysis”, a model is fit to multiple kinetic profiles simultaneously and models that cannot simultaneously capture reactant consumption and product formation are easily eliminated. Here, a model protein formulation was analysed but the method can be used to analyse aggregation kinetics in actual biopharmaceutical formulations.