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Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.
- Source :
-
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences [J Chromatogr B Analyt Technol Biomed Life Sci] 2015 Apr 15; Vol. 988, pp. 81-7. Date of Electronic Publication: 2015 Feb 16. - Publication Year :
- 2015
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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.<br /> (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Linear Models
Macaca fascicularis
Peptide Fragments analysis
Peptide Fragments chemistry
Peptide Fragments metabolism
Protein Stability
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Reproducibility of Results
Sensitivity and Specificity
Serum Albumin
Trypsin
Chromatography, Liquid methods
Recombinant Proteins blood
Tandem Mass Spectrometry methods
Subjects
Details
- Language :
- English
- ISSN :
- 1873-376X
- Volume :
- 988
- Database :
- MEDLINE
- Journal :
- Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
- Publication Type :
- Academic Journal
- Accession number :
- 25756208
- Full Text :
- https://doi.org/10.1016/j.jchromb.2015.02.007