title: Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.
abstract: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 860-0082, Japan.
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Ohkuri
authors.forename: Eri
authors.initials: E
authors.lastname: Murase
authors.forename: Shu-Lan
authors.initials: SL
authors.lastname: Sun
authors.forename: Jun
authors.initials: J
authors.lastname: Sugitani
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D055550:Protein Stability; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D001216:Asparagine; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N
keywords: Pichia pastoris; deamidation; deterioration; humanized Fab; protein denaturatio