Even after sophisticated purifications steps, low levels (1-100 ppm) of host cell proteins (HCPs) remain in the final purified drug substance. Some of the HCPs may cause immunogenic reaction in humans, therefore it is critical for patient safety that HCPs be identified and quantified. The analytical methods typically used for HCP quantification are based on immunoassays (ELISA), but ELISA cannot guarantee proteome-wide coverage.
In recent years, LC/MS-based assays have been adopted as orthogonal techniques to ELISA for HCP analysis due to their flexibility and potential for full proteome-wide applications. Here we describe an efficient analytical scale LC/MS workflow that allows the identification and quantification of HCPs during mAb purification in a CHO cell line.
The first step of the HCP identification and quantification workflow is the HCP discovery assay employing data-independent MSE acquisition using 90 min peptide separations. Following data processing with Progenesis QI for proteomics 4.2, HCPs are identified by a proteome-wide database search. In addition, LC/MS data can be assembled into spectral libraries, containing peptide precursors, charge states and retention times. In the second step of the HCP workflow, additional HCP samples derived from the purification of the same biopharmaceutical are analyzed by higher–throughput HCP monitoring assays using MSE acquisitions with 30 min peptide separations. Each purified sample can be analyzed by LC/MSE followed by searching against the spectral library for HCP identification and quantification. The HCP workflow described above was tested for identification of HCPs from the NIST mAb and then applied to identification and monitoring of HCPs from a different monoclonal antibody . Our results show that HCPs can be confidently identified, quantified and monitored in biopharmaceutical samples using the 1D LC-MS HCP workflow.