title: NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana.
abstract: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.
authors.affiliation: Faculty and Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yagi
authors.forename: Noriho
authors.initials: N
authors.lastname: Fukuzawa
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Tasaka
authors.forename: Kouki
authors.initials: K
authors.lastname: Matsuo
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Takumi
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Sachiko
authors.initials: S
authors.lastname: Kondo
authors.forename: Shiori
authors.initials: S
authors.lastname: Nakazawa
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Matsumura
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
mesh_terms: D000068879:Adalimumab / Q000235:genetics; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D000068879:Adalimumab