Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum.
In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.
Dubois
Mathieu
M
CEA, Service de Pharmacologie et d'Immunoanalyse, 91191 Gif-sur-Yvette, France, INRA, Laboratoire d'Immuno-Allergie Alimentaire, 91191 Gif sur Yvette, France.
Fenaille
François
F
Clement
Gilles
G
Lechmann
Martin
M
Tabet
Jean-Claude
JC
Ezan
Eric
E
Becher
François
F
D000911:Antibodies, Monoclonal / Q000097:blood* / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000097:blood* / Q000378:metabolism; D000068818:Cetuximab; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D000068818:Cetuximab