In-Depth Characterization of HCPs in AAV Downstream Processes Using a Quantitative LC-MS Approach

In recent years, considerable efforts have been made to detect host cell proteins (HCPs) in antibody drugs, but information on HCPs in gene therapy products remains limited and has not been comprehensively characterized and fully integrated into the downstream process (DSP). The detailed characterization of HCPs in gene therapy products and their screening through the DSP is necessary to identify and monitor critical HCPs as they represent a critical quality attribute (CQA). In the rapidly developing field of gene therapy, adeno-associated virus (AAV) represents one of the most prominent and promising platforms for therapeutic applications. However, there is still a knowledge gap regarding the composition of residual HCPs in AAV products and its depletion during DSP. In this study, we used a highly sensitive and quantitative LC-MS proteomics approach to characterize and elucidate the HCP proteome of an AAV2 product. We examined seven different process steps of the DSP, ranging from harvest to sterile filtrate. This resulted in the successful identification of over 3000 HCPs. In addition, depletion alongside the DSP and the effectiveness of the individual process steps were demonstrated. Even in the most purified process step, over 30 HCPs were detected and identified. The results of this study can be used as a basis for the generation of a database for critical AAV HCPs. The results of this study can be used as a basis for the generation of a database of critical AAV HCPs to improve patient safety.