Protein particle analysis - critical factors and new standards

The aim of this thesis was to identify and evaluate critical factors for protein particle analysis and to apply this knowledge for the development of novel standardized protein-like particles. Thorough analysis of particles in therapeutic protein formulations is crucial due to regulatory requirements, the potential immunogenicity of protein aggregates and particles, and the need for quality and stability control of the product. The introduction gives a comprehensive overview of analytical methods for particle characterization in therapeutic protein formulations based on the currently available literature. Within the thesis, the performance of novel techniques or instruments for (protein) particle counting, sizing, or characterization was assessed. Micro-Flow Imaging (MFI) and resonant mass measurement (RMM) were tested for the differentiation of protein particles and silicone oil droplets which is highly relevant especially for pharmaceutical products in prefilled syringes. Four different flow imaging microscopy systems (MFI4100, MFI5200, FlowCAM VS1, and FlowCAM PV) were subjected to a detailed investigation of particle quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. A material screening of proteinaceous and non-proteinaceous materials for the development of novel standardized protein-like particles revealed gelatin and PTFE particles as promising materials for light-based applications. The density of protein particles, as a crucial particle parameter for weight-based techniques like RMM, was determined by two newly developed methods. The relevance of the refractive index (RI), which is closely related to transparency, was investigated and a novel method for RI determination of protein particles was developed. As protein particles became “invisible”, i. e. not detectable anymore by light-based systems at increased RI values - e. g. due to high protein concentration and/or sugars as excipients - potential solution strategies were evolved. Taken together, this thesis provides new insight into the analysis of particles in therapeutic protein formulations. In this regard, potential candidates for the development of novel standardized protein-like particles identified in this study are very valuable and can help to improve protein particle analysis in the future.