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Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII

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Abstract

Human blood coagulation factor VIII (hFVIII) is used in hemostatic and prophylactic treatment of patients with hemophilia A. Biotechnological innovations have enabled purification of the culture medium of rodent or human cells harboring the hFVIII expression cassette. However, cell lines express hFVIII protein derived from an exogenous expression vector at a lower level than most other proteins. Here, we describe hFVIII production using piggyBac transposon and the human-derived expi293F cell line. Use of a drug selection protocol, rather than transient expression protocol, allowed cells harboring hFVIII expression cassettes to efficiently produce hFVIII. In heterogeneous drug-selected cells, the production level was maintained even after multiple passages. The specific activity of the produced hFVIII was comparable to that of the commercial product and hFVIII derived from baby hamster kidney cells. We also applied codon optimization to the hFVIII open reading frame sequences in the transgene, which increased production of full-length hFVIII, but decreased production of B-domain-deleted human FVIII (BDD-hFVIII). Low transcriptional abundance of the hF8 transgene was observed in cells harboring codon-optimized BDD-hFVIII expression cassettes, which might partially contribute to decreased hFVIII production. The mechanism underlying these distinct outcomes may offer clues to highly efficient hFVIII protein production.

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Acknowledgements

This work was partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to Takuji Yoshimura (grant number 17K08632), Keiji Nogami (grant numbers 18K07885 and 21K07804), and Investigator-Research Support grant (Sanofi AS).

Funding

This study were funded by Japan Society for the Promotion of Science (Grant Nos. 17K08632, 18K07885, 21K07804), Sanofi AS (Investigator-Research Support grant).

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Authors and Affiliations

  1. Department of Pediatrics, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara, 634-8522, Japan

    Takuji Yoshimura, Kaoru Horiuchi, Naruto Shimonishi, Kenichi Ogiwara, Midori Shima & Keiji Nogami

  2. Department of Physiology II, Nara Medical University, Kashihara, Nara, Japan

    Kyoji Horie

  3. The Center of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara, Japan

    Midori Shima

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  1. Takuji Yoshimura
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  2. Kaoru Horiuchi
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  3. Naruto Shimonishi
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  4. Kenichi Ogiwara
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  5. Kyoji Horie
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  6. Midori Shima
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Contributions

TY conceived and designed the research, performed experiments, analyzed and interpreted the data, prepared figures, and wrote and edited the manuscript; KaH performed experiments; NS helped in vector construction; KO supplied resource; KyH supplied resource; MS supervised the research; KN supervised the research and edited the manuscript.

Corresponding authors

Correspondence to Takuji Yoshimura or Keiji Nogami.

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Conflict of interest

TY has received research funding from Bioverativ Inc./Sanofi S.A.; KaH, KO, and KyH have no conflict of interest; NS teaches a course endowed by CSL Behring; MS and KN have received research funding from Bioverativ Inc./Sanofi S.A.

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Yoshimura, T., Horiuchi, K., Shimonishi, N. et al. Modified expi293 cell culture system using piggyBac transposon enables efficient production of human FVIII. Int J Hematol 117, 56–67 (2023). https://doi.org/10.1007/s12185-022-03468-9

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  • DOI: https://doi.org/10.1007/s12185-022-03468-9

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