title: Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum.
abstract: An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.
authors.affiliation: Analytical and Bioanalytical Development and ‡Discovery Chemistry Synthesis, Bristol-Myers Squibb , Princeton, New Jersey, United States.
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.forename: Jianing
authors.initials: J
authors.lastname: Zeng
authors.forename: Craig
authors.initials: C
authors.lastname: Titsch
authors.forename: Kimberly
authors.initials: K
authors.lastname: Voronin
authors.forename: Billy
authors.initials: B
authors.lastname: Akinsanya
authors.forename: Linlin
authors.initials: L
authors.lastname: Luo
authors.forename: Hongwu
authors.initials: H
authors.lastname: Shen
authors.forename: Dharmesh D
authors.initials: DD
authors.lastname: Desai
authors.forename: Alban
authors.initials: A
authors.lastname: Allentoff
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.forename: Binodh S
authors.initials: BS
authors.lastname: Desilva
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000097:blood* / Q000378:metabolism / Q000627:therapeutic use; D001774:Blood Chemical Analysis / Q000379:methods*; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D005240:Feasibility Studies; D006801:Humans; D008252:Macaca fascicularis / Q000097:blood*; D011489:Protein Denaturation; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D014357:Trypsin