Premium
Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE ‐ ESI ‐ MS / MS system
Author(s) -
Zhu Guijie,
Sun Liangliang,
Linkous Travis,
Kernaghan Dawn,
McGivney James B.,
Dovichi Norman J.
Publication year - 2014
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300545
Subject(s) - chemistry , chromatography , monoclonal antibody , mass spectrometry , peptide , tandem mass spectrometry , recombinant dna , capillary electrophoresis , quantitative proteomics , antibody , biochemistry , proteomics , gene , immunology , biology
We report the first use of CZE for absolute characterization of host cell proteins ( HCP s) 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 HCP s. The target proteins were present at the 7–30 ppt level in the purified HCP samples.