Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

• Published in: Journal of the American Society for Mass Spectrometry Vol. 28; no. 5; pp. 827 - 838
• Main Authors: He, Lidong, Anderson, Lissa C., Barnidge, David R., Murray, David L., Hendrickson, Christopher L., Marshall, Alan G.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-05-2017; Springer Nature B.V
• Subjects: Accuracy; Analytical Chemistry; Antibodies; Bioinformatics; Biotechnology; Chains; Characterization of Protein Therapeutics by MS: Research Article; Chemistry; Chemistry and Materials Science; Focus: 28th Sanibel Conference; Immunoglobulins; Ions; Mass spectrometry; Monoclonal antibodies; Organic Chemistry; Proteomics; Quality control; Spectrometry; Collision-induced dissociation; Variable region; Middle-down; Multiple myeloma; Electron transfer dissociation; Fourier transform; Isotype; MS/MS; Electrospray; ETD; FTMS; Ion cyclotron resonance; CID
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-017-1602-6

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2–0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.
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