Stability of human serum albumin during bioprocessing: denaturation and aggregation during processing of albumin paste.

Abstract

To assess the impact of various bioprocessing steps on the stability of freshly precipitated human serum albumin (HSA) obtained from pooled human plasma. After initial precipitation of HSA from plasma, the resultant paste is either (a) lyophilized or (b) washed with acetone and then air-dried in order to obtain a dry powder. The structure of HSA was examined using Fourier transform infrared (IR) spectroscopy. The extent of aggregation of redissolved HSA was measured using both dynamic light scattering and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both lyophilization and air-drying perturb the secondary structural composition of HSA, as detected by infrared (IR) spectroscopy. Upon dissolution of dried paste, most of the protein refolds to a native-like conformation. However, a small fraction of the protein molecules form soluble aggregates that can be detected by both dynamic light scattering and SDS-PAGE. The level of aggregation is so low that it could not be detected in the bulk by either circular dichroism or IR spectroscopy. The lyophilized protein, which appears to be more unfolded in the solid state than the acetone washed/air-dried material, exhibits a higher level of aggregation upon dissolution. There is a direct correlation between the extent of unfolding in the solid state and the amount of soluble aggregate present after dissolution. Moreover, the presence of the aggregates persists throughout the remainder of the purification process, which includes dissolution, chromatography, sterile filtration and viral inactivation steps. Analytical methods used to monitor the stability of biopharmaceuticals in the final product can be used to assess damage inflicted during processing of protein pharmaceuticals.