title: Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates.
abstract: The synthesis of a 16-residue, stable isotopically labeled peptide is described for use as a LC-MS/MS (Liquid chromatography-mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc-fusion protein drug candidates in animal species used in pre-clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave-assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable-labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc-[(13) C6 , (15) N]-l-leucine onto an 11-unit segment followed by automated microwave-assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long-term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave-assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side-chains.
authors.affiliation: Discovery Chemistry Platforms Radiochemistry, Bristol-Myers Squibb Research and Development, Route 206 and Province Line Road, Princeton, NJ, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Voronin
authors.forename: Alban J
authors.initials: AJ
authors.lastname: Allentoff
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Bonacorsi
authors.forename: Claudio
authors.initials: C
authors.lastname: Mapelli
authors.forename: Sharon X
authors.initials: SX
authors.lastname: Gong
authors.forename: Ving
authors.initials: V
authors.lastname: Lee
authors.forename: Douglas
authors.initials: D
authors.lastname: Riexinger
authors.forename: Nishith
authors.initials: N
authors.lastname: Sanghvi
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.forename: Jianing
authors.initials: J
authors.lastname: Zeng
mesh_terms: D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards*; D008872:Microwaves; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis*; D012015:Reference Standards; D060327:Solid-Phase Synthesis Techniques / Q000379:methods*
chemical_list: D002250:Carbon Radioisotopes; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009587:Nitrogen Isotopes; D010446:Peptide Fragments
keywords: LC-MS/MS bioanalysis; internal standard; pseudoproline; stable isotope; surrogate peptide