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Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

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Abstract

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The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

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Abbreviations

Ab:

Antibody

BSA:

Bovine serum albumin

Ch:

Chromosome

CT:

Cholera toxin

CTB:

Cholera toxin B-subunit

ELISA:

Enzyme-Linked ImmunoSorbent Assay

IgA:

Immunoglobulin A

IgG:

Immunoglobulin G

JP:

The Japanese Pharmacopeia

MSB:

Master seed bank

MS/MS:

Tandem mass spectrometry

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

RNAi:

RNA interference

SDS–PAGE:

SDS–polyacrylamide gel electrophoresis

WT:

Wild type rice

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Acknowledgments

We are grateful to Drs. Teruhide Yamaguchi, Nana Kawasaki, Eiji Goto, Yoshinori Yamamoto, Kunisuke Tanaka, and Takehiro Masumura and to Mr. Ushio Nakanishi for useful discussions and technical support. This work was supported by grants from the programs of the Ministry of Economy, Trade, and Industry (H.K.) and New Energy and Industrial Technology Development Organization (NEDO) (H.K.); the Programs of Special Coordination Funds for Promoting Science and Technology and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Y.Y., H.K.); the Ministry of Health, Labor and Welfare of Japan (Y.Y., H.K.); Translational Research Network Program, Seed C (H.K.); and the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-step) (Y.Y.).

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Authors and Affiliations

  1. Division of Mucosal Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Koji Kashima, Yoshikazu Yuki, Mio Mejima, Shiho Kurokawa, Yuji Suzuki, Satomi Minakawa, Natsumi Takeyama, Yoshiko Fukuyama, Tatsuhiko Azegami & Hiroshi Kiyono

  2. Engineering Headquarters, Asahi Kogyosha Co., Ltd., 3-13-12, Mita, Minato-ku, Tokyo, 108-0073, Japan

    Koji Kashima & Minoru Tamura

  3. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Yoshikazu Yuki & Hiroshi Kiyono

  4. Seto Center, Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, 4-1-70, Seto-Cho, Kanonji, Kagawa, 768-0065, Japan

    Mio Mejima, Shiho Kurokawa, Yuji Suzuki, Satomi Minakawa, Takeshi Tanimoto & Yasuyuki Gomi

  5. Research Department, Nippon Institute for Biological Science, 9-2221-1, Shin-machi, Ome, Tokyo, 198-0024, Japan

    Natsumi Takeyama

  6. Crop Development Division, NARO Agriculture Research Center, 1-2-1, Inada, Joetsu-shi, Niigata, 943-0193, Japan

    Masaharu Kuroda

Authors
  1. Koji Kashima
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  2. Yoshikazu Yuki
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  3. Mio Mejima
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  4. Shiho Kurokawa
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  5. Yuji Suzuki
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  6. Satomi Minakawa
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  7. Natsumi Takeyama
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  8. Yoshiko Fukuyama
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  9. Tatsuhiko Azegami
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  10. Takeshi Tanimoto
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  11. Masaharu Kuroda
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  12. Minoru Tamura
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  13. Yasuyuki Gomi
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  14. Hiroshi Kiyono
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Corresponding author

Correspondence to Yoshikazu Yuki.

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Additional information

Communicated by F. Sato.

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Kashima, K., Yuki, Y., Mejima, M. et al. Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants. Plant Cell Rep 35, 667–679 (2016). https://doi.org/10.1007/s00299-015-1911-9

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  • DOI: https://doi.org/10.1007/s00299-015-1911-9

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