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Production and application of high quality stable isotope labeled monoclonal antibody for mass spectrometry analysis

Amsler, Philipp, Heudi, Olivier, Lanshoeft, Christian, Bettighofer, Anja, Eisfeld, Jochen, Moenius, Thomas, Probst, Claudia Monika and Etter, Coralie (2017) Production and application of high quality stable isotope labeled monoclonal antibody for mass spectrometry analysis. Journal of labelled compounds and radiopharmaceuticals. ISSN 03624803


Quantitation of recombinant monoclonal antibodies (mAb) using liquid chromatography tandem mass spectrometry LC-MS/MS for pharmacokinetic (PK) studies is becoming a complementary approach to traditional antibody-based ligand binding assays (LBA). Here we describe the production of stable isotope labeled human immunoglobulin G1 (hIgG1) mAb for use as an internal standard (IS) for LC-MS/MS-based antibody quantitation. Stable isotope labeling with amino acids in cell-culture (SILAC) allowed production of [13C6-L-Lysine (Lys),13C6-L-Arginine (Arg)]-mAb. A fermentation process was run in shake flasks containing defined medium, so that the targeted stable isotope labeled amino acids were incorporated into the recombinant monoclonal antibody (mAb). The mAb was then purified and label incorporation was determined to be >99% of all Lys and Arg moieties by mass spectrometry. Sequence coverage was confirmed by peptide mapping. The full length antibody was then used as IS to assist the development of a generic LC-MS/MS method for the quantitative analysis of human IgG1 in rat serum. Four tryptic peptides (GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), FNWYVDGVEVHNAK (FNW) and VVSVLTVLHQDWLNGK (VVS)) originating from the Fragment crystallizable (Fc) region of a selected IgG1 were used for quantitation and they were chromatographically resolved. In addition, the calibration curves resulting from the peak area ratio of each peptide to its respective labeled IS over the range of 1-1000 µg/mL (GPS, VVS and TTP) or 5-1000 µg/mL (FNW) were reproducible and good accuracy precision data were obtained on calibrators (Cs) and quality control samples (QCs). The produced full length stable isotope labeled monoclonal antibody was found to be a suitable IS to support the development of generic method for IgG1 quantitation in rat serum. Our data demonstrated that this antibody, used as a universal IS, can allow for more rapid, accurate and cost effective quantitation of fully human and human IgG1 in preclinical species.

Item Type: Article
Date Deposited: 02 Feb 2017 00:45
Last Modified: 02 Feb 2017 00:45


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