title: Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE-ESI-MS/MS system.
abstract: We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic-labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope-labeled lysine or arginine. The heavy-labeled peptides were spiked in the HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the peaks of native and isotopic-labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic-labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R(2) values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7-30 ppt level in the purified HCP samples.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Travis
authors.initials: T
authors.lastname: Linkous
authors.forename: Dawn
authors.initials: D
authors.lastname: Kernaghan
authors.forename: James B
authors.initials: JB
authors.lastname: McGivney
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D011994:Recombinant Proteins
keywords: CZE; Electrokinetically pumped sheath flow nanospray; Host cell proteins; Recombinant antibodies