Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depletedHCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 mu g of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE-MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE-ESI-MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q ExactiveHFmass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.