Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC–MS/MS analysis

• Published in: Journal of pharmaceutical and biomedical analysis 2011-06, Vol.55 (3), p.574-582
• Main Authors: Yamaguchi, Hideto, Hirakura, Yutaka, Shirai, Hiroki, Mimura, Hisashi, Toyo’oka, Toshimasa
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; Analysis; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Chromatography, Liquid; Cross-Linking Reagents - chemistry; crosslinking; Crystallography, X-Ray; desorption; drugs; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; General pharmacology; Interferon Type I - analysis; Interferon Type I - chemistry; Interferon-alpha; interferons; ionization; liquid chromatography; mass spectrometry; Medical sciences; Models, Molecular; Molecular Sequence Data; monitoring; Nuclear Magnetic Resonance, Biomolecular; peptide mapping; Peptide Mapping - instrumentation; Peptide Mapping - methods; peptides; Pharmaceutical Preparations - analysis; Pharmaceutical Preparations - chemistry; Pharmacology. Drug treatments; polyacrylamide gel electrophoresis; Protein Binding; Protein Folding; Protein folding/refolding; Protein structure; Protein Structure, Tertiary; protein tertiary structure; QC testing; quality control; Recombinant Proteins; sodium; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stability; Succinimides - chemistry; Tandem Mass Spectrometry; Tertiary structure
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2011.01.038

The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS 3)], followed by peptide mapping by LC–MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC–MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC–MS/MS analyses found that BS 3 formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control.