한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제32권2호
  • /
  • Pages.142-148
  • /
  • 2004
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Sialic Acid 함량 증가 배양기술에 의한 재조합 인간 다당쇄 에리스로포이에틴의 생산

Production of Recombinant Human Hyperglycosylated Erythropoietin Using Cell Culture Technology by Improving Sialylation.

  • 박세철 ((주)유한양행 중앙연구소 바이오텍연구실) ;
  • 이승오 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 박만식 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 김승훈 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 김준환 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 송무영 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 이병규 ((주)유한양행 중앙연구소 바이오텍연구) ;
  • 고인영 (춘천 바이오산업진흥) ;
  • 강희일 ((주)유한양행 중앙연구소 바이오텍연구실)
  • 발행 : 2004.06.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

에리스로포이에틴은 인간 적혈구분화의 조절인자로 작용한다. 유전자 재조합 인간에리스로포이에틴(rhEPO)은 동물세포에서 생산되고 있는 재조합 당단백질의 하나이며 당쇄부분이 전체 분자량의 40％를 차지한다. 시알산 함량은 체내약물 투여 지속기간과 직접적인 연관이 있어 시알산 함량은 의약용 당단백질의 중요한 성질로 여겨진다. 본 연구에서는 CHO세포 배양액에 시알산 생합성 전구물질인 N-acetylmannosamine(ManNAc)과 sialidase 저해제인 2-deoxy-2,3-hyo-N-acetylneuraminic acid(NeuAc2en)를 첨가하여 rhEPO의 sialic acid함량을 증가시킬 수 있었다. 특히, 배양액에 20 mM ManNAc/0.5 mM NeuAc2en를 첨가할 때 대조구에 비하여 약 10배의 시알산 함량이 증가하였으며 세포성장이나 배양액의 rhEPO생산량에는 영향이 없었다. rhEPO의 정제시 시알산 함량이 11∼15％인 다당쇄 rhEPO분획을 얻었으며, 배양액 내에 20 mM ManNAc와 0.5 mM NeuAc2en를 동시에 첨가함으로 대조구에 비하여 시말산함량이 높은다당쇄 rhEPO의 생산성이 50％ 증가하였다.

Erythropoietin is a main regulator of human erythropoiesis. Recombinant human erythropoietin (rhEPO) is one of the glycoproteins produced in animal cells, and it has oligo saccharides chains which comprise about 40％ of its molecular mass. Because the content of sialic acid can extend circulatory lifetime, the high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the sialylation of rhEPO produced by chinese hamster ovary cell culture was maximized by supplementing the culture medium with N-acetylm-annosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a sialidase inhibitor. Feeding of 20 mM ManNAc/0.5 mM NeuAc2en into culture medium increased the sialic acid content by nearly tenfold compared with unsupplemented medium. This effect was achieved without affecting the cell growth or product yield. Six erythropoietin fractions differing in sialic acid content, ranging from 11∼15％ of EPO, were identified from chinese hamster ovary cell-derived rhEPO by mono Q column chromatography. It was found that, at 20 mM ManNAc/0.5 mM NeuAc2en feeding, productivity of hyper-glycosylated EPO increased up to 50％, compared with the unsupplemented medium.

키워드

참고문헌

  1. British J. Cancer v.84 Development and characterization of novel erythropoietin stimulating protein(NESP) Egrie, J. C.;J. K. Browne https://doi.org/10.1054/bjoc.2001.1746
  2. Blood v.89 Mapping of the active site of recombinant human erythropoietin Eliott, S.;T. Lorenzini;D. Chang;J. Barzilay;E. Delorme
  3. Pro. Drug. Res. v.41 Recent advances in erythropoietin research Fisher, J. W.
  4. J. Biol. Chem. v.246 The sialic acids George, W. J.;L. Dean;S. Roseman
  5. Bio/Technology v.9 The oligosaccharides of gylcoproteins: bioprocess factors affecting oligosaccharide structure and their effect on gycoprotein properties Goochee, C F.;M. J. Gramer;D. C. Anderson;J. B. Bahr;J. R. Rasmussen https://doi.org/10.1038/nbt1291-1347
  6. J. Biol. Chem. v.58 Improvement of Interferon-α sialylation in chinese hamster ovary cell culture by feeding of N-acetylmannosamine Gu, X.;D. I. C. Wang
  7. Biotechnol. Bioeng. v.55 Site- and branch-specific sialylation of recombinant human Interferon-α sialylation in chinese hamster ovary cell culture Gu, X.;B. J. Harmon;D. I. C. Wang https://doi.org/10.1002/(SICI)1097-0290(19970720)55:2<390::AID-BIT16>3.0.CO;2-L
  8. Nature v.313 Isolation and characterization of genomic and cDNA clones of human erythropoietin Jacobson, K.;C. Shoemaker;R. Rudersdorf;S. D. Neil;R. J. Kaufman;A. Mufson;J. Seehra;S. S. Jones;R. Hewick;E. F. Fritsh;M. Kawakita;T. Shimizu;T. Miyaki https://doi.org/10.1038/313806a0
  9. Physiol. Rev. v.722 Erythropoietin: Structure, control of production and function Jelkmann, W.
  10. Yakuri Chiryo v.18 Metabolic fate of erythropoietin (TYB-5220) Kazuo, O.;A. Shoichi;S. Takako;O. Tomako;N. Naoki;I. Yoshiko
  11. Blood v.71 Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization Koury, S. T.;M. C. Bondurant;M. J. Koury
  12. Nature v.227 Cleavage of structure proteins during the assembly of the head of bacteriophage T4 Laemmli, U. K. https://doi.org/10.1038/227680a0
  13. Pro. Natl. Acad. Sci. USA v.49 Cloning an expression of human erythropoietin gene Lin, F. K.;S. Suggs;C. H. Lin;J. K. Browne;R. Smalling;J. C. Egrie;K. K. Cjen;G. M. Fox;F. Martin;Z. Stabinsky;S. Bdrawi;P. H. Lin;E. Goldwasser
  14. Blood v.77 The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biological activities of erythropoietin Louise, C. W.;G. Timony;P. Murtha;J. Stoudemire;A. J. Dorner;J. Caro;M. Krieger;R. J. Kaufman
  15. Brit. J. Hematol. v.74 Erythropoietin production in kidney tubular cells Maxwell, A. P.;T. R. J. Lappin;C. F. Johnson;J. M. Bridges;M. G. McGeown https://doi.org/10.1111/j.1365-2141.1990.tb06347.x
  16. Biotechnol. Prog. v.9 Glycosidase activities in chinese hamster ovary cell lysate and cell culture supernatant Michael, J. G.;C. F. Goochee
  17. Bio/Technology v.13 Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase Michael, J. G.;C. F. Goochee;V. Y. Chock;D. T. Brousseau;M .B. Sliwkowski https://doi.org/10.1038/nbt0795-692
  18. Glycoconjugate J. v.13 Biological and physicochemical characterization of recombinant human erythropoietins fractionated by mono Q column chromatography and their modifiction with sialyltransferase Morimoto, K.;E. Tsuda;A. A. Said;E. Uchida;S. Hatakeyama;M. Ueda;T. Hayakawa https://doi.org/10.1007/BF01053197
  19. Blood v.73 Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure Schuster, S. J.;E. V. Badiavas;P. Costa-Giomni;R. Weinmann;A. J. Erslev;J. Caro
  20. Yakuri Chiryo v.18 General pharmacological studies of erythropoietin (TYB-5220) Terumasa, M.;O. Tsuyoshi;H. Yasuko;S. Takaka;H. Hamki
  21. Proc. Natl. Acad. Sci. USA v.76 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Towbin, H. T.;T. Staehelin;J. Gordon https://doi.org/10.1073/pnas.76.9.4350