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NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana

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Abstract

Key message

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.

Abstract

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 15N-enriched plant growth medium. The Fc fragment derived from the 15N-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.

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Acknowledgments

This work was supported, in part, by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and by Grants-in-Aid for Scientific Research (24249002, 25102008, and 25860053) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). This work was partly supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT. We gratefully acknowledge Drs. Minoru Tada and Akiko Ishii-Watabe (National Institute of Health Sciences) for providing ADM expression vector for mammalian cells. We also thank Ms. Kiyomi Senda and Ms. Kumiko Hattori (Nagoya City University) for their help in purification of IgG.

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Author notes

  1. Shiori Nakazawa

    Present address: Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba, Mie, 517-0004, Japan

Authors and Affiliations

  1. Faculty and Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan

    Hirokazu Yagi, Ying Zhang, Takumi Yamaguchi, Sachiko Kondo & Koichi Kato

  2. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, 062-8517, Japan

    Noriho Fukuzawa, Yasushi Tasaka, Kouki Matsuo & Takeshi Matsumura

  3. Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki, 444-8787, Japan

    Ying Zhang, Takumi Yamaguchi & Koichi Kato

  4. Medical & Biological Laboratories Co., Ltd., 2-22-8 Chikusa, Chikusa-ku, Nagoya, 464-0858, Japan

    Sachiko Kondo & Koichi Kato

  5. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kami-yoga, Setagaya-ku, Tokyo, 158-8501, Japan

    Shiori Nakazawa, Noritaka Hashii & Nana Kawasaki

  6. The Glycoscience Institute, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo, 112-8610, Japan

    Koichi Kato

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  1. Hirokazu Yagi
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Corresponding authors

Correspondence to Takeshi Matsumura or Koichi Kato.

Additional information

Communicated by E. Benvenuto.

Electronic supplementary material

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299_2015_1757_MOESM1_ESM.pptx

Supplementary material 1 Supplemental Fig. 1: Detection of glycopeptides in the chromatogram of a tryptic digest of nonlabeled and 15N-labeled IgG expressed in transgenic tobacco.Supplemental Fig. 2: Typical MS spectra of the observed peptides (HC1:VSYLSTASSLDYWGQGTLVTVSSASTK) derived from non-labeled (upper) and 15N-labeled (lower) IgGs produced in transgenic tobacco.Supplemental Fig. 3: Detection of purified Fc fragments derived from transgenic tobacco and CHO cells by Coomassie Brilliant Blue staining of SDS-PAGE gels under non-reducing condition.Supplemental Fig. 4: Superposition of 1H–15N HSQC spectra of the uniformly 15N-labeled IgG-Fc fragments derived from transgenic tobacco (black) and CHO cells (red). The HSQC spectrum of CHO-produced IgG-Fc was adapted from the literature (Yagi et al. 2014). (PPTX 854 kb)

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Yagi, H., Fukuzawa, N., Tasaka, Y. et al. NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana . Plant Cell Rep 34, 959–968 (2015). https://doi.org/10.1007/s00299-015-1757-1

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