Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 988; pp. 81 - 87
• Main Authors: Zhao, Yue, Liu, Guowen, Angeles, Aida, Hamuro, Lora L., Trouba, Kevin J., Wang, Bonnie, Pillutla, Renuka C., DeSilva, Binodh S., Arnold, Mark E., Shen, Jim X.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.04.2015
• Subjects: Albumin removal; Animals; Bioanalysis; Chromatography, Liquid - methods; LC-MS/MS; Linear Models; Macaca fascicularis; Peptide Fragments - analysis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Stability; Protein therapeutics; Recombinant Proteins - blood; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Reproducibility of Results; Sensitivity and Specificity; Serum Albumin; Tandem Mass Spectrometry - methods; Trypsin; Validation; Validation; LC-MS/MS; Albumin removal; Protein therapeutics; Bioanalysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2015.02.007

•A bioanalytical method for a protein drug candidate using LC-MS/MS was fully validated.•The method adopted a recently reported albumin removal approach to simplify sample preparation process.•Advantages of using the albumin removal step were investigated.•The method was used in a toxicology study and results were comparable to data generated using a traditional immunoassay.•LC-MS/MS was demonstrated to be a good complementary tool for protein bioanalysis. 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 30min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1mm×50mm, 1.7μm) 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 0.50–500μg/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.