(205b) Role of Shear and Extensional Stress on the Degradation of Proteins During Downstream Processing | AIChE

(205b) Role of Shear and Extensional Stress on the Degradation of Proteins During Downstream Processing

Authors 

Simon, S. - Presenter, University of Erlangen-Nuremberg
Peukert, W. - Presenter, University of Erlangen-Nuremberg
Weber, C. - Presenter, Abbott GmbH & Co.KG


Therapeutic proteins are often exposed to mechanical stress during their production. Especially during pumping, filtration and mixing mechanical forces act on the molecules which can lead to changes in the protein structure and the formation of aggregates. In literature often the synergistic effect of shear stress and adsorption phenomena to interfaces is described to induce unfolding and/ or aggregation of proteins. In addition to shear forces this work also investigated the impact of extensional forces. It is known that extensional forces lead to an increase in cell damage compared to applied shear stress at the same level of energy dissipation. Different proteins were subjected to a range of mechanical stress in a two-dimensional homogeneous extensional and shear flow in a four-roll mill. The flow pattern was investigated by numerical (Computational Fluid Dynamics) and experimental (Particle Image Velocimetry) methods and the hydrodynamic stress environment of the molecules was characterized by the local energy dissipation. By the use of in-situ UV-Vis spectroscopy the formation of protein aggregates in the generated shear and extensional flow was monitored. The increase in absorbance in the visible range due to Mie scattering of formed particles fit to the amount of counted particles by light blockage (1-100 µm). Flow-induced conformational changes of the proteins were detected by in-situ fluorescence spectroscopy. In addition to intrinsic tryptophane fluorescence, different fluorescence dyes were used to monitor the exposure of hydrophobic groups of single molecules. By changing the lid material of the fluid flow device, the role of interfaces on the degradation behaviour was investigated. The stressing conditions occurring in manufacturing processes were reproduced in the lab scale under well-defined flow and stressing conditions. We found out that the so far mostly neglected extensional flow configuration is largely responsible for the denaturation and appears to be much more important than the intensively studied shear stressing. From there, we derive guidelines how mechanical stress (type and intensity) can be minimized during the manufacturing and processing of proteins.

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