Abstract
Monoclonal antibodies bind to Protein A/G resin with 100 nm-diameter pores, which orients the Fab toward the reaction solution. Then, they can be proteolyzed using trypsin immobilized on the surface of 200 nm-diameter nanoparticles. The difference between the two particle diameters allows Fab-selective proteolysis by limiting trypsin access to the antibody substrate. The specific signature peptide of monoclonal antibody is collected, which comprises the complementarity-determining regions (CDRs). Excess trypsin protease and peptide fragments from common sequences in Fc that inhibit the analysis can then be separated and removed. The resulting peptide samples are separated through high performance liquid chromatography on a 20 nm-diameter pore-size reversed-phase C18 column. These are then sequentially ionized with an electrospray interface and subjected to mass spectrometry (MS). In MS, peptide ions are trapped and fragment ions are generated by the collision-induced dissociation with argon gas. These are detected with multiple reaction monitoring measurements to perform a highly sensitive and accurate quantitative analysis.
By focusing on various physicochemical features at each analytical scene, such as characteristic structure and orientation of antibody, control of trypsin reaction field, carry-over on HPLC column, ionization suppression effect from endogenous proteins, and detection of amino acid sequence specificity of antibody, we optimized the overall conditions from the sample processing up to MS detection and developed analytical validation and clinical application of many therapeutic antibodies using our Fab-selective proteolysis technology that is based on the structure-indicated approach.
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Acknowledgments
nSMOL development and clinical application are part of the collaboration results with Akinobu Hamada, Ph.D. and Hitoshi Nakagama, M.D., Ph.D. of National Cancer Center (Tokyo, Japan), Atsushi Yonezawa, Ph.D. and Kazuo Matsubara, Ph.D. of Kyoto University Hospital (Kyoto, Japan), Bernard Fox, Ph.D., William Redmond, Ph.D., Eric Tran, Ph.D., Brian Piening, Ph.D., Yoshinobu Koguchi, M.D., Ph.D., and Walter Urba, M.D., Ph.D. of Providence Cancer Institute (Portland, Oregon), Naoe Yamane, Ph.D. of CMIC (Tokyo, Japan), and Ms. Misato Hara and Mr. Naohiro Hanyu of Tamagawa Seiki (Nagano, Japan). We would sincerely express our deeply appreciation to this collaboration and their colleagues for significant support and discussion.
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Iwamoto, N., Shimada, T. (2022). Structure-Indicated LC-MS/MS Bioanalysis of Therapeutic Antibodies. In: Houen, G. (eds) Therapeutic Antibodies. Methods in Molecular Biology, vol 2313. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1450-1_11
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DOI: https://doi.org/10.1007/978-1-0716-1450-1_11
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