title: Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS(3).
abstract: Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS(3) analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS(3) analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product.
authors.affiliation: U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Testing and Research, Division of Pharmaceutical Analysis, 645 S. Newstead Ave., St. Louis, Missouri 63110, United States.
authors.forename: Michaella J
authors.initials: MJ
authors.lastname: Levy
authors.affiliation: U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Testing and Research, Division of Pharmaceutical Analysis, 645 S. Newstead Ave., St. Louis, Missouri 63110, United States.
authors.forename: Ashley C
authors.initials: AC
authors.lastname: Gucinski
authors.affiliation: U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Testing and Research, Division of Pharmaceutical Analysis, 645 S. Newstead Ave., St. Louis, Missouri 63110, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Boyne
mesh_terms: D000595:Amino Acid Sequence; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D016179:Granulocyte Colony-Stimulating Factor