Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography.
In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.
Center of Molecular Immunology, Havana, Cuba.
L E
LE
Matamoros Fernández
D E
DE
Kalume
L
L
Calvo
M
M
Fernández Mallo
A
A
Vallin
P
P
Roepstorff
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014357:Trypsin