A rapid cIEF–ESI–MS/MS method for host cell protein analysis of a recombinant human monoclonal antibody
- Resource Type
- Authors
- Norman J. Dovichi; Roza Wojcik; Dawn Kernaghan; Guijie Zhu; James B. McGivney; Liangliang Sun
- Source
- Talanta. 98:253-256
- Subject
- Spectrometry, Mass, Electrospray Ionization
Time Factors
Protein digestion
Molecular Sequence Data
Tandem mass spectrometry
Chromatography, Affinity
Analytical Chemistry
Magnetics
FLAG-tag
Tandem Mass Spectrometry
Animals
Humans
Trypsin
Amino Acid Sequence
Databases, Protein
Chromatography
biology
Myoglobin
Chemistry
Isoelectric focusing
Antibodies, Monoclonal
Cytochromes c
Serum Albumin, Bovine
Enzymes, Immobilized
Microspheres
Peptide Fragments
Recombinant Proteins
Protein L
biology.protein
Cattle
Protein G
Isoelectric Focusing
Protein A
Myc-tag
- Language
- ISSN
- 0039-9140
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 4 h. 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.