title: Direct quantitative analysis of a 20 kDa PEGylated human calcitonin gene peptide antagonist in cynomolgus monkey serum using in-source CID and UPLC-MS/MS.
abstract: PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented. CGRP[Cit, Cit] is an investigational human calcitonin gene peptide receptor antagonist with amino acid sequence Ac -WVTH[Cit]LAGLLS[Cit]SGGVVRKNFVPT DVGPFAF-NH(2). In-source collision-induced dissociation (in-source CID) of 20 kDa PEGylated peptide was used to generate CGRP[Cit, Cit] fragment ions, among which the most abundant b(8)(+) ion was selected and measured as a surrogate for the 20 kDa PEGylated peptide. A solid phase extraction (SPE) method was used to extract the PEGylated peptides from the biomatrix prior to the UPLC-MS/MS analysis. This method achieved a lower limit of quantitation (LLOQ) of 5.00 ng/mL with a serum sample volume of 100 μL, and was linear over the calibration range of 5.00 to 500 ng/mL in cynomolgus monkey serum. Intraday and interday accuracy and precision from QC samples were within ±15%. This method was successfully applied to a pharmacokinetic study of the 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkeys.
authors.affiliation: Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. hongyanl@amgen.com
authors.forename: Hongyan
authors.initials: H
authors.lastname: Li
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Rose
authors.forename: Jerry Ryan
authors.initials: JR
authors.lastname: Holder
authors.forename: Marie
authors.initials: M
authors.lastname: Wright
authors.forename: Les P
authors.initials: LP
authors.lastname: Miranda
authors.forename: Christopher A
authors.initials: CA
authors.lastname: James
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016018:Least-Squares Analysis; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D011092:Polyethylene Glycols; D015740:Calcitonin Gene-Related Peptide