Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Abstract

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 depleted HCP 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 μg 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 Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…presented an evaluation of bottom‐up proteomic analysis of both HCP digests with and without depleted antibodies by UHPLC‐MS, CZE‐MS and SCX‐SPE‐CZE‐MS with an online pH gradient elution. . They reported a clear influence of the mAb depletion procedure on peptide identification.…”

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

“…Comprehensive analysis of host cell impurities (digest of host proteins) in preparations of monoclonal antibodies was performed by CZE‐ESI‐MS/MS . Separations were performed in LPA‐coated capillary (50/150 μm id/od, 85 cm long) using aqueous 1 M acetic acid as the BGE, and 0.5% formic acid in 10% v/v MeOH/water solvent as the ESI sheath flow buffer.…”

Peptide mapping of proteins by capillary electromigration methods

“…It allowed detection of all proteins spiked at 100 ppm level with respect to the Ab. Comprehensive analysis of host cell impurities in preparations of mAbs was performed by means of peptide maps of host proteins using the advanced CZE-ESI-MS/MS setup [239]. Separations were performed in linear polyacrylamide-coated FS capillary (50/150 μm id/od, 85 cm long) using aqueous 1 M AcOH as the BGE, and 0.5% FA in 10% v/v MeOH/water solvent as the ESI sheath liquid.…”