The conformational and colloidal stability of proteins is considered as a bottleneck in the biopharmaceutical process and formulation development. The unique surface structure of biomolecules as well as changing solution conditions during processing and formulation result in very complex protein-protein and protein-solvent interactions which cause a stabilization or destabilization of the active molecules. Due to the complexity of influencing parameters decision-makers hesitate to recommend one single analytical method to predict protein long-term stability. In this work a toolbox based on experimental and in silico methods was applied to understand and predict the long-term stability of biopharmaceutical relevant proteins. The here presented case study explores the impact of a fusion tag (Cherry-TagTM) on the long-term stability of Glutathion-S-Transferase (GST). Therefore, the microscopic surface properties of GST and Cherry-GST were investigated with the help of molecular dynamic simulations and connected to high frequency rheological measurements. Here the strength of protein-protein interactions and protein network formation were examined on a macroscopic level. Furthermore, zeta potential measurements and thermal stability tests were conducted. With these tools in hand, we were able to precisely predict the long-term conformational and colloidal stability of both proteins at various conditions. So that the better robustness of the fusioned protein towards high protein concentration and changing buffer conditions could be demonstrated directly after sample preparation. The accuracy of the gained results were verified by long-term stability tests and FTIR measurements. Concluding, the presented toolbox enables buffer optimizations and fast decisions during pharmaceutical process development and formulation so that product stability can be ensured.

Toolbox of experimental and in silico methods to predict protein long-term stability

253rd National Meeting (2017)

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