title: Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.
abstract: We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 μm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 μg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method.
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA. Electronic address: guowen.liu@bms.com.
authors.forename: Guowen
authors.initials: G
authors.lastname: Liu
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Aida
authors.initials: A
authors.lastname: Angeles
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Lora L
authors.initials: LL
authors.lastname: Hamuro
authors.affiliation: Drug Safety Evaluation, Bristol-Myers Squibb Co., 4601 Highway 62 East, Mt Vernon, IN 47620, USA.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Trouba
authors.affiliation: Drug Safety Evaluation, Bristol-Myers Squibb Co., 1 Squibb Drive, New Brunswick, NJ 08903, USA.
authors.forename: Bonnie
authors.initials: B
authors.lastname: Wang
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Renuka C
authors.initials: RC
authors.lastname: Pillutla
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Binodh S
authors.initials: BS
authors.lastname: DeSilva
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
authors.affiliation: Bioanalytical Sciences, Research & Development, Bristol-Myers Squibb Co., Route 206 and Province Line Road, Princeton, NJ 08543, USA.
authors.forename: Jim X
authors.initials: JX
authors.lastname: Shen
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D016014:Linear Models; D008252:Macaca fascicularis; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D012709:Serum Albumin; D014357:Trypsin
keywords: Albumin removal; Bioanalysis; LC-MS/MS; Protein therapeutics; Validation