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

Purpose. To assess the impact of various bioprocessing steps on the stabili ty of freshly precipitated human serum albumin (HSA) obtained from pooled h uman plasma. Methods, After initial precipitation of HSA from plasma, the resultant past e is either (a) lyophilized or (b) washed with acetone and then air-dried i n order to obtain a dry powder. The structure of HSA was examined using Fou rier transform infrared (IR) spectroscopy. The extent of aggregation of red issolved HSA was measured using both dynamic light scattering and SDS-polya crylamide gel electrophoresis (SDS-PAGE). Results, Both lyophilization and air-drying perturb the secondary structura l composition of HSA, as detected by infrared (IR) spectroscopy. Upon disso lution of dried paste, most of the protein refolds to a native-like conform ation. However, a small fraction of the protein molecules form soluble aggr egates 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 bul k by either circular dichroism or IR spectroscopy. The lyophilized protein, which appears to be more unfolded in the solid state than the acetone wash ed/air-dried material, exhibits a higher level of aggregation upon dissolut ion. Conclusions. There is a direct correlation between the extent of unfolding in the solid state: and the amount of soluble aggregate present after disso lution. Moreover, the presence of the aggregates persists throughout the re mainder of the purification process, which includes dissolution, chromatogr aphy, sterile filtration and viral inactivation steps. Analytical methods u sed to monitor the stability of biopharmaceuticals in the final product can be used to assess damage inflicted during processing of protein pharmaceut icals.