Harvest Process and Affinity Resin Selection Impacts on Adeno-Associated Virus Residual Host Cell Protein Retention.

Abstract

Scalable purification platforms have been developed for adeno-associated virus (AAV) processing to support large-scale vector manufacturing. The ability of column chromatography to recover packaged vectors and remove empty capsids has been well-established, but knowledge gaps remain for understanding process parameter impacts on impurity retention. In this work, we examine the impacts of two key process parameters-the harvest method and affinity resin selection-on residual host cell protein (HCP) retention. Sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) proteomics is applied to comprehensively profile residual HCPs in affinity chromatography (AC) elution pools from four AAV serotypes (AAV2, -5, -8, and -9) produced by suspension HEK293 cells. Vectors were purified from cell culture lysates and from supernatants using POROS^™ CaptureSelect^™ AAVX (AAVX) and one additional serotype-specific affinity resin-Capto^™ AVB (AVB), POROS CaptureSelect AAV8 (PAAV8), or POROS CaptureSelect AAV9 (PAAV9). Significant divergence in residual HCP profiles was observed with the use of different affinity resins, with AVB and PAAV9 showing reduced residual HCP content in elution pools compared with AAVX and PAAV8. Processing of null culture lysates with fresh resins and resins with digested single-domain antibody fragments (sdAbs) shows that differences in resin performance are driven by variable nonspecific sdAb association with cellular impurities. Proteomic analysis of vector preparations from lysates compared with supernatants demonstrates product quality advantages of designing a media-only harvest process, specifically for AAV8, which was measured to contain an average of 66% of total vector genome content in the cell culture media. This work highlights the importance of serotype-specific tailoring of AAV downstream process design for improved product quality attributes to support clinical manufacture and small-scale analytics workflows.