Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b |
| |
Authors: | Yan-Hui Liu David Wylie Jia Zhao Raymond Cure Collette Cutler Susan Cannon-Carlson Xiaoyu Yang Tattanahalli L Nagabhushan Birendra N Pramanik |
| |
Institution: | aSpectroscopy, Chemical Research, Merck Research Laboratories, Kenilworth, NJ 07033, USA;bDepartment of Biotechnology, Merck Research Laboratories, Union, NJ 07083, USA;cDepartment of Biotechnology, Merck Research Laboratories (formerly Schering–Plough Research Institute), Union, NJ 07083, USA |
| |
Abstract: | The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSn (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b. |
| |
Keywords: | E coli-derived recombinant human interferon α-2b and isoforms Pyruvic acid ketimine derivative of N-terminal cysteine S-Glutathionylated Cys98 Acetylated Cys1 LC/ESI&ndash MS LC/ESI&ndash MS/MS ESI&ndash MSn High-resolution LTQ Orbitrap MALDI&ndash TOF&ndash MS |
本文献已被 ScienceDirect 等数据库收录! |
|