title: Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.
abstract: To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. Electronic address: naiyu.zheng@bms.com.
authors.forename: Naiyu
authors.initials: N
authors.lastname: Zheng
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. Electronic address: jianing.zeng@bms.com.
authors.forename: Jianing
authors.initials: J
authors.lastname: Zeng
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Manney
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Lakenya
authors.initials: L
authors.lastname: Williams
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.affiliation: Discovery Chemistry, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Voronin
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Adela
authors.initials: A
authors.lastname: Buzescu
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Yan J
authors.initials: YJ
authors.lastname: Zhang
authors.affiliation: Discovery Chemistry, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Alban
authors.initials: A
authors.lastname: Allentoff
authors.affiliation: Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Carrie
authors.initials: C
authors.lastname: Xu
authors.affiliation: Drug Safety Evaluation, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Hongwu
authors.initials: H
authors.lastname: Shen
authors.affiliation: Drug Safety Evaluation, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: William
authors.initials: W
authors.lastname: Warner
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D000882:Haplorhini; D011092:Polyethylene Glycols / Q000032:analysis*; D011506:Proteins / Q000627:therapeutic use*; D011786:Quality Control; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001798:Blood Proteins; D004220:Disulfides; D011506:Proteins; D011092:Polyethylene Glycols
keywords: Disulfide-containing peptide; In vivo stability; Methionine oxidation; PEGylated protein; Protein quantitation; UHPLC-HRMS