Purification and Characterization of Paclitaxel from Plant Cell Cultures of Taxus chinensis in Large-Scale Process

식물세포 Taxus chinensis 배양으로부터의 Paclitaxel 대량 정제 및 특성

  • 김진현 ((주)삼양제넥스 생명공학연구소) ;
  • 기은숙 ((주)삼양제넥스 생명공학연구소) ;
  • 민범찬 (삼양사 중앙연구소) ;
  • 최형균 ((주)삼양제넥스 생명공학연구소) ;
  • 홍승서 ((주)삼양제넥스 생명공학연구소) ;
  • 이현수 ((주)삼양제넥스 생명공학연구소)
  • Published : 2000.10.01

Abstract

In developing a HPLC purification process, it was hoped that a single chromatographic system would be sufficient to abtain pure paclitaxel in high yield. However, no such system was found, due in part to the complex taxoid profile of crude paclitaxel and to the rigorous nature of the product specification. A two step HPLC purification was adopted using reverse-phase separation on C(sub)18 as a first step, and normal-phase separation on silica as the final polishing step. Impurity profiles were established and maintained for paclitaxel, which identified and quantified each impurity observed in purified paclitaxel from these two steps, all impurities at or above 0.1% were identified. Results provide information for improving the quality control of paclitaxel production.

일반적으로 paclitaxel과 유사한 구조를 가진 복잡한 유도체의 분리나 최종 제품에서의 엄격한 규정 등으로 인하여 한 단계의 HPLC system으로 정 제하는 것은 상당히 어렵다. 이러한 이유로 두 단계 HPLC system으로 식물세포 배양액으로 부터 paclitaxel의 대량 정제를 수행하였다. 즉, 첫번째 단계에 서 reverse-phase HPLC column, 두번째 단계에서 normal­p phase HPLC column을 사용하여 최종 제품을 얻었다. 또한 최종 정 제 된 paclitaxel 내 에 포함되어 있는 불순물 profile 확 언, 함량 분석, 그리고 이들 물질들에 대한 분리, 정제 및 동 정을 실시하였다 이들 불순물들 중에서 0.1% 이상되는 것은 모두 6종 (side chain derivative, baccatin III, 10-deacetyltaxol, cephalomannine, taxane derivative, 7-epitaxol)이며 NMR과 MS를 사용하여 화학구조를 분석한 결과 이미 알려진 pacli­t taxel 유도체 또는 전구체와 동일한 물칠로 밝혀 졌다. 본 연구 결과는 결국 paclitaxel 생산을 위한 품질관리 (quality con­t tr이)와 허가를 위한 자료로 유용하게 사용되어 진다.

Keywords

References

  1. J. Nat. Prod. v.53 Taxus spp. Needles Contain Amounts of Taxol Comparable to the Bark of Taxus brevifolia: Analysis and Isolation Witherup, K. M.;S. A. Look;M. W. Stasko;T. J. Ghiorzi;G. M. Muschik
  2. J. Liq. Chromatogr. v.14 HPLC Separation of Taxol and Cephalomannine Cardellina Ⅱ, J. H.
  3. J. Liq. Chromatogr. v.15 High-Speed Counter-current Chromatography Separation of Taxol and Related Diterpenoids from Taxus baccata Vanhaelen-Fastre, R.;B. Diallo;M. Jaziri;M. L. Faes;J. Homes;M. Vanhaelen
  4. Phytochem. v.30 Taxanes from Taxus chinensis Zhang, Z.;Z. Jia
  5. J. Liq. Chromatogr. v.16 Determination of Taxol in Taxus Media Needles in the Presence of Interfering Compounds Castor, T. P.;T. A. Tyler
  6. Pharm. Res. v.12 A New Large-Scale Process for Taxol and Related Taxanes from Taxus brevifolia Rao, K. V.;J. B. Hanuman;C. Alvarez;M. Stoy;J. Juchum;R. M. Davies;R. Baxley
  7. U.S. Patent, 5,871,979 Method for Mass Production of Taxol by Semi-Continuous Culture with Taxus chinensis Cell Culture Choi, H. K.;T. L. Adams;R. W. Stahlhut;S. I. Kim;J. H. Yun;B. K. Song;J. H. Kim;S. S. Hong;H. S. Lee
  8. U.S. Patent, 5,900,979 Method for Purifying Taxol from Taxus Biomass Hong, S. S.;B. K. Song;J. H. Kim;C. B. Lim;H. S. Lee;K. W. Kim;I. S. Kang;H. B. Park
  9. J. Nat. Prod. v.55 ¹H- and ¹³C-NMR assignments for taxol. 7-epi-taxol, and cephalomannine Chmurny, G. N.;B. D. Hilton;S. Brobst;S. A. Look;K. M. Witherup;J. A. Beutler
  10. J. of American Chemical Society v.116 First Total Synthesis of Taxol. 1. Functionalization of the B Ring Holton, R. A.;C. Somoza;H. B. Kim;F. Liang;R. J. Biediger;P. D. Boatman;M. Shindo;C. C. Smith;S. Kim;H. Nadizadeh;Y. Suzuki;C. Tao;P. Vu;S. Tang;P. Zhang;K. K. Murthi;L. N. Gentile;J. H. Liu
  11. J. Nat. Prod. v.50 Taxol and derivatives : a biogenetic hypothesis Voegelein, F. G.;D. Guenard;P. Potier
  12. J. Nat. Prod. v.57 Yunnanxane and its homologous esters from cell cultures of Taxus chinensis var. mairei Ma, W.;R. W. Stahlhut;T. L. Adams;G. L. Park;W. A. Evans;S. G. Blumenthal;G. A. Gomez;M. H. Nieder;P. J. Hylands
  13. J. Chem. Soc. Chem. Commun Cephalomannice; a new antitumor alkaloid from Cephalotaxus mannii Powell, R. G.;R. W. Miller;C. S. Smith
  14. J. of American Chemical Society v.116 First Total Synthesis of Taxol. 2. Completion of the C and D Ring Holton, R. A.;H. B. Kim;C. Somoza;F. Liang;R. J. Biediger;R. J. Biediger;P. D. Boatman;M. Shindo;C. C. Smith;S. Kim;H. Nadizadeh;Y. Suzuki;C. Tao;P. Vu;S. Tang;P. Zhang;K. K. Murthi;L. N. Gentile;J. H. Liu
  15. Guidance for industry - Manufacturing, processing, or holding active pharmaceutical ingredients U.S. Department of Health and Human Services, Food and Drug Administration