title: 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.
abstract: 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. Graphical Abstract ᅟ.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32310, USA.
authors.forename: Lidong
authors.initials: L
authors.lastname: He
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Dr., Tallahassee, FL, 32310, USA.
authors.forename: Lissa C
authors.initials: LC
authors.lastname: Anderson
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Barnidge
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55905, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Murray
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32310, USA.|National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Dr., Tallahassee, FL, 32310, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Hendrickson
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 32310, USA. marshall@magnet.fsu.edu.|National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Dr., Tallahassee, FL, 32310, USA. marshall@magnet.fsu.edu.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D000068879:Adalimumab / Q000032:analysis / Q000097:blood*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000097:blood*; D005583:Fourier Analysis; D006801:Humans; D010265:Paraproteinemias / Q000097:blood*; D011499:Protein Processing, Post-Translational; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000068879:Adalimumab
keywords: CID; Collision-induced dissociation; ETD; Electron transfer dissociation; Electrospray; FTMS; Fourier transform; Ion cyclotron resonance; Isotype; MS/MS; Middle-down; Multiple myeloma; Variable region