title: Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b.
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 MS(n) (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.
authors.affiliation: Merck Research Laboratories, Kenilworth, NJ 07033, USA. yan-hui.liu@merck.com
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Wylie
authors.forename: Jia
authors.initials: J
authors.lastname: Zhao
authors.forename: Raymond
authors.initials: R
authors.lastname: Cure
authors.forename: Collette
authors.initials: C
authors.lastname: Cutler
authors.forename: Susan
authors.initials: S
authors.lastname: Cannon-Carlson
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yang
authors.forename: Tattanahalli L
authors.initials: TL
authors.lastname: Nagabhushan
authors.forename: Birendra N
authors.initials: BN
authors.lastname: Pramanik
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D020033:Protein Isoforms; D011994:Recombinant Proteins