Dokument

Summary:
The biopharmaceutical sector is one of most promising and profitable sectors in medical treatment of a multitude of severe diseases. Biological molecules, however, lead to new challenges for the manufacturing companies. This thesis focused on one of the main challenges for therapeutic proteins: protein aggregation and the detection of the resulting particles. Within this thesis, three main tasks have been addressed: (1) the evaluation of emerging particle detection and characterization techniques, (2) the evaluation of novel technologies for aggregation and unfolding process characterization and (3) the investigation of the aggregation process of current therapeutic proteins (mABs). For the evaluation of particle detection and characterization techniques for the application on protein samples, the following three techniques have been selected: Nanoparticle tracking analysis (NTA), Tunable resistive pulse (TRPS) and STEP-technology® (space and time-resolved extinction profiles) applied in the LUMiSizer®. All techniques rely on different measurement principles and can be claimed as orthogonal methods. These techniques have been shown to be applicable for polystyrene particle suspensions and showed high agreement for the size and/ or concentration determinations of these particles. In the first step, all techniques have been evaluated in two comparative studies analyzing protein BSA standard particle suspensions and monoclonal antibody suspensions. In a second step, a more detailed evaluation of the NTA and the STEP-technology® was performed for monoclonal antibody samples. For the evaluation of novel technologies for the investigation and characterization of protein unfolding and aggregation processes, the following two techniques have been selected: Zetasizer Helix system and the SwitchSENSE®. The Zetasizer Helix system is an instrument combining dynamic light scattering and Raman spectroscopy. The three measurement modes (sample series, isothermal incubation, temperature ramp) have been successfully evaluated for selected therapeutic mABs. The outcome indicated differences in the aggregation mechanism. The same applies to the thermal ramp experiments. In addition, the obtained melting temperatures and aggregation onset fit to the results of orthogonal methods (DSC, ITF, SwitchSENSE). In conclusion, the Zetasizer Helix is an instrument that is not applicable in a high throughput manner, but it gives valuable information for better understanding the correlation between structural changes and aggregation behavior. The SwitchSENSE® technology is a chip-based analytical platform using a specific DNA-based biosurface system to investigate molecular interactions, such as binding kinetics and affinities or enzymatic activities. Out of the various applications of the SwitchSENSE technology, the following applications were evaluated for therapeutic proteins: protein sizing, protein interaction and thermal melting approaches. The most promising approach was the usage of the SwitchSENSE system as orthogonal method for the characterization of the thermal melting behavior. The combination of sizing step and thermal melting step gave information about the refolding potential. Comparative investigations with DSC and ITF showed good coincidence of unfolding start and melting temperature determinations. The system development and application identification is ongoing and continuous updates are necessary for further potential approaches. For the protein aggregation case studies, three monoclonal antibodies (mAB1, mAB2 and mAB3) have been selected. Following these model systems have been investigated considering their biophysical characterization, stability and aggregation behaviour using established and novel techniques and technologies. For the aggregation studies three approaches have been addressed, temperature ramp experiments, isothermal experiments and further protein stress factors, such as extreme temperature, mechanical stirring, extreme acidic pH, reducing stress. In summary, the results of these case studies describe the stability and aggregation processes of the three monoclonal antibodies. Depending on different stress factors (temperature, pH, reducing conditions, etc.) and different approaches (temperature ramp, isothermal incubation and extreme conditions), molecule specific aggregation mechanisms were postulated. The application of the evaluated technologies in the case studies outline their suitability for practical stability studies.

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