title: Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?
abstract: Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody. We have used this method routinely for over 1 year to support bioprocess development and test production lots for clinical trials. Herein we summarize the precision and ruggedness of the testing procedure and the main findings with respect to 'coverage of amino acid sequence' and limits-of-detection for various hypothetical structural variants. We also describe, in more detail, two unanticipated insights into the method gained from the validation study. The first of these is a potentially troublesome side-product arising during the reduction/alkylation step. Once the cause of this side-product was identified, it was easily prevented. We also report on subtle changes to the peptide map upon extended storage of the digest in the autosampler. These findings helped us to develop a 'robust' method for implementation in a quality control laboratory.
authors.affiliation: Department of Analytical Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA. jacob_s_bongers@sbphrd.com
authors.forename: J
authors.initials: J
authors.lastname: Bongers
authors.forename: J J
authors.initials: JJ
authors.lastname: Cummings
authors.forename: M B
authors.initials: MB
authors.lastname: Ebert
authors.forename: M M
authors.initials: MM
authors.lastname: Federici
authors.forename: L
authors.initials: L
authors.lastname: Gledhill
authors.forename: D
authors.initials: D
authors.lastname: Gulati
authors.forename: G M
authors.initials: GM
authors.lastname: Hilliard
authors.forename: B H
authors.initials: BH
authors.lastname: Jones
authors.forename: K R
authors.initials: KR
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Mozdzanowski
authors.forename: M
authors.initials: M
authors.lastname: Naimoli
authors.forename: S
authors.initials: S
authors.lastname: Burman
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015704:CD4 Antigens / Q000276:immunology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D007074:Immunoglobulin G; D014357:Trypsin