title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
authors.forename: Zhenbin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: BioPharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Albanetti
authors.affiliation: BioPharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Linkous
authors.affiliation: BioPharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Larkin
authors.affiliation: BioPharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD, USA.
authors.forename: Ronald
authors.initials: R
authors.lastname: Schoner
authors.affiliation: BioPharmaceutical Development, MedImmune LLC, One MedImmune Way, Gaithersburg, MD, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: McGivney
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D010446:Peptide Fragments
keywords: CZE-MS/MS; Cationic solid phase extraction; Electrokinetically pumped nanoelectrospray; Host cell proteins; pH bumps