Buffer exchange path influences the stability and viscosity upon storage of a high concentration protein

Abstract

High concentration protein solutions are generally produced by spin column concentration (SCC) during early development and by tangential flow filtration (TFF) during later stages, when greater quantities of protein become available. This is based on the assumption that the protein generated by the SCC process would be fairly similar to the TFF process material. In this study, we report the case of high concentration solutions of an Fc fusion protein produced by the two processes using the same upstream drug substance (DS) with very different storage stability. The TFF and SCC batches were characterized for aggregation, viscosity, and hydrodynamic radius before and after storage at different temperatures (5°C, 25 °C, and 40 °C). Aggregation and viscosity of the solutions processed by TFF were higher than those processed by SCC upon storage at 25 °C and 40 °C for three months. Differential scanning fluorimetry (DSF) revealed differences in initial protein conformation. Upon exposure to shear stress, protein solutions showed conformational instability and increased aggregation upon storage at 35 °C. In addition, protein solution showed higher aggregation upon shearing under mixed (downstream purification process and final formulation) buffer conditions - which are more likely to be encountered during the TFF, but not SCC, process. These results were further confirmed in an independent experiment by Fourier transform-infrared (FT-IR) spectroscopy and aggregation analysis. Taken together, these data indicate that shearing the protein in intermediate, unstable buffer conditions can lead to conformational perturbation during TFF processing, which led to higher rate of aggregation and viscosity upon storage. This study highlights the importance of testing shear stress sensitivity in the transitional buffer states of the TFF process early in development to de-risk process related product instability.

Keywords: Aggregation; Formulation; Protein; Stability; Tangential flow filtration; Viscosity.