title: A rapid cIEF-ESI-MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody.
abstract: A rapid and reproducible system that couples capillary isoelectric focusing to a high-resolution mass spectrometer was developed for on-line analysis and identification of protein digests. Magnetic microsphere-based immobilized trypsin was used for protein digestion to reduce the digestion time to 10 min, with a total analysis time of 4h. A three-protein-mixture (myoglobin, BSA, cytochrome c) with a molarity ratio of 1:10:50 was successfully digested and identified. This system was also used to analyze host cell protein impurities in a recombinant humanized monoclonal antibody product in which the sample was product-depleted using affinity capture on protein A/protein L columns prior to analysis. A database search identified 37 host cell proteins with peptide and protein identity probability greater than 0.9.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Roza
authors.initials: R
authors.lastname: Wojcik
authors.forename: Dawn
authors.initials: D
authors.lastname: Kernaghan
authors.forename: James B
authors.initials: JB
authors.lastname: McGivney
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D045304:Cytochromes c / Q000032:analysis; D030562:Databases, Protein; D004800:Enzymes, Immobilized / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D009211:Myoglobin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D014357:Trypsin