Protein particle analysis

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

ACKNOWLEDGMENT

First, I want to express my deepest gratitude to my first supervisor from the university, Prof. Dr. Wolfgang Frieß, for his continuous and encouraging support of my work and for his inspiration to think “outside the box”. Furthermore, I would like to thank you for keeping up the great team spirit in the group which made me always feel as a part of the team although my regular work place was outside the university. My second supervisor from the university, Prof. Dr. Gerhard Winter, is kindly acknowledged for his valuable suggestions and input for our publications and for the project. I also want to thank you for the dedicated leadership of the chair concerning both scientific and social factors and for taking over the co-referee. I am most deeply indebted to my first supervisor from Coriolis Pharma Dr. Andrea Hawe for her outstanding and reliable support in every phase of the project. I enjoyed working with you a lot and am very grateful for all your valuable and honest advice – scientifically and personally. Dr. Sun Tantipolphan as my second supervisor from Coriolis Pharma is cordiall

How Subvisible Particles Become Invisible - Relevance of the Refractive Index for Protein Particle Analysis

The aim of the present study was to quantitatively assess the relevance of transparency and refractive index (RI) on protein particle analysis by the light-based techniques light obscuration (LO) and Micro-Flow Imaging (MFI). A novel method for determining the RI of protein particles was developed and provided an RI of 1.41 for protein particles from two different proteins. An increased RI of the formulation by high protein concentration and/or sugars at pharmaceutically relevant levels was shown to lead to a significant underestimation of the subvisible particle concentration determined by LO and MFI. An RI match even caused particles to become "invisible" for the system, that is, not detectable anymore by LO and MFI. To determine the influence of formulation RI on particle measurements, we suggest the use of polytetrafluoroethylene (PTFE) particles to test a specific formulation for RI effects. In case of RI influences, we recommend also using a light-independent technique such as resonant mass measurement (RMM) (Archimedes) for subvisible particle analysis in protein formulations