Evaluation of peptide fractionation and native digestion as two novel sample preparation workflows to improve HCP characterization by LC-MS/MS

Host cell proteins (HCPs) are the predominant class of impurities during manufacturing of therapeutic proteins. Previous reports have successfully shown that HCP characterization by LC–MS/MS ultimately leads to drug products of superior safety and quality. Here, we present two sample preparation strategies to approach the wide dynamic range required and compared them systematically to a standard protocol. First, we describe PreOmics fractionation as an effective 2D offline strategy. Second, we evaluate an alternative digestion approach specifically designed for purified antibodies – native (nondenaturing) digestion. Both protocols increased detection sensitivity as shown by two low level HCP models. Out of a 5 ppm spike of eight common HCPs into antibody product, all spiked proteins were positively identified. Additionally, by Universal Proteomics Standard 1 (UPS-1) spiking we obtained a comprehensive coverage of 77% below 10 ppm for the native digestion. Furthermore, we were able to detect 27% to 173% mo...