실리콘 큐브 이원상 주목세포 현탁배양을 통한 패클리탁셀의 생산성 향상

김창헌, 정충식, 정인식

경희대학교 유전공학과, 수원, 449-701

Improved Paclitaxel Production in a Two-Phase Suspension Culture of

Taxus cuspidata Using Silicone Cubes

Chang-Heon Kim, Chung-Sik Chung and In-Sik Chung

Department of Genetic Engineering, Kyung Hee University, Suwon, 449-701

INTRODUCTION

Paclitaxel is a diterpenoid mainly extracted from bark of Pacific yew trees [1]. It was approved by the US FDA for the treatment of advanced ovarian and breast cancers in 1993 and is being used in clinical trials for the treatment of other types of cancer. However, demand of the paclitaxel has not been satisfied and further medical application has been hindered since its current supply source relies on the bark of pacific yew trees only. Several solutions to the paclitaxel supply problem are under investigation [2]. Among them, cell culture of Taxus spp. is potential alternative source for the production of paclitaxel [3-4]. Despite many works for paclitaxel production using Taxus spp. cell culture [5-6], the low productivity and slow growth are still remained as the major barriers for commercial production .

In this work, we investigated two-phase culture of Taxus cuspidata using silicone cubes as a second phase for paclitaxel production.

MATERIALS AND METHODS

Plant material, culture medium and conditions

Suspension culture of T. cuspidata was established from young stem segments of yew trees growing at Suwon, Korea, as previously described [7]. Cell suspension was maintained at 110 rpm and 26℃ under dark condition using modified Gamborg's B5 medium supplemented with 1.0 g/L casein hydrolyzates, 4 mg/L 2,4-D and 30 g/L sucrose. The pH of medium was adjusted to 5.6 before autoclaving. Cultures were maintained by transferring 10 mL suspensions into 40 mL fresh medium in 200 mL Erlenmeyer flasks every 20 day.

Adsorbent preparation

Silicone sheet (2 mm thick, 1 m2) was obtained from local agency in Korea (Korea Ace, Seoul, Korea) and cut into 2 mm cubes. The silicone cubes were washed with methanol for 24 hours. The methanol was removed and the cubes were washed repeatedly three times with deionized water. Subsequently, they were filtered under vacuum and dried before use.

Adsorption and desorption of taxanes in silicone cubes

Silicone cubes were added to 20 mL of fresh medium containing 2.5 mg/L of authentic taxanes in 100 mL Erlenmeyer flasks and incubated for 7 days at 110 rpm, 26℃. After 7 days, the silicone cubes and medium were separated and extracted for taxanes analysis. The adsorbed taxanes in silicone cubes were recovered by 24 hrs of methanol extraction.

Two-phase culture

Two-phase culture was carried out using silicone cubes in 200 mL Erlenmeyer flask containing 40 mL production medium (modified B5 medium containing 80 g/L sucrose, 6 mg/L NAA, without nitrogen source and phosphate source). The culture was operated for 35 days after seeding with 10 mL of 20-day-old cultures. The other conditions were the same as those of subculture. After 35 days, the cultures were separated into silicone phase and cellular (cells and medium) phase. Each phase was extracted and analyzed for taxanes.

Taxane extraction and analysis

The taxanes in fresh medium were extracted by solid phase extraction with Supelclean LC-18 SPE tubes (SUPELCO Inc., Bellefonte, PA, USA). The taxanes in the silicone cube were extracted in methanol for 24 hours. Taxanes in the extracts were analyzed by HPLC, as described previously [7]. Taxane analysis was carried out in the intracellular, medium and silicon cube phase of the culture.

RESULTS AND DISCUSSION

Effective in situ removal of paclitaxel is important in improving paclitaxel production as well as separating paclitaxel for downstream processing operations. An ideal material for in situ removal should bind the desired substances in a specific manner, if possible [8].

Table 1. Adsorption of various taxanes using silicone cube.

Taxane	Adsorbed amount (%)	Adsorption capacity (mg/g adsorbent)
10-Deacetyl baccatin Ⅲ	8.44 %	0.004
Baccatin Ⅲ	4.44 %	0.002
Paclitaxel	71.8 %	0.036

An experiment was made to see if silicon cubes could paclitaxel, 10-deacetyl baccatin Ⅲ(DAB) and baccatin Ⅲ(BAC) in the fresh medium. Silicone cubes adsorbed 71.8%, 8.4 %, 4.4 % of paclitaxel, DAB and BAC, respectively in the medium (Table 1). Most of the paclitaxel absorbed in silicone cubes was recovered by single treatment of methanol. DAB and BAC are well known for intermediate metabolites in paclitaxel production. Interestingly, silicone cube has lower adsorption capacity to DAB and BAC than paclitaxel. It means that silicone cube could enhance paclitaxel production not preventing biosynthesis of paclitaxel from intermediate metabolites. Our results indicate that the silicone cube can be used as a second phase for in situ removal of paclitaxel in suspension culture of T. cuspidata.

The effect of silicone cube addition on taxane production was investigated using suspension culture of T. cuspidata cell culture. The suspension culture was performed in shake flasks for 35 days after 10 % of silicone cubes were added to the production medium. The final cell concentration for two-phase culture using silicone cube was 4.17 g/L DCW, whereas that for the control culture was 5.3 g/L DCW.  As shown in Figure 1, the addition of silicone cube enhanced paclitaxel production. The paclitaxel content in silicone phase was 3.57 mg/L and it was 45 times higher than that of the control. In the medium phase, paclitaxel and BAC were not detected, but DAB was accumulated to the similar level as the control (data not shown). For two-phase culture, the % composition of paclitaxel in the detected taxanes was 84.2%, whereas that of DAB and BAC was 6.0% and 9.8%, respectively. For the control culture, the % composition of paclitaxel, DAB and BAC was 5%, 59% and 36%, respectively. This indicates that the % ratio (= % composition) in produced taxanes for two-phase and control culture was changed markedly. Our findings suggests that silicone cube could affect the production of paclitaxel by selective adsorption.  Since silicone cube did not adsorb the precursors of paclitaxel such as DAB and BAC, the production of paclitaxel would not be restricted. The marked change in % composition for two-phase culture also suggests that biosynthetic pathway of paclitaxel could be altered by silicone cube.

Fig. 1. Effect of the silicone cube addition on the production of various taxane compounds. □, 10-Deacetyl baccatin Ⅲ; ▥, baccatin Ⅲ; ▦, paclitaxel.	Fig. 2. Effect of silicone cube content on paclitaxel production in two-phase culture. □, concentration; □, specific productivity.

The effect of silicone cube content on paclitaxel production was investigated (Fig. 2). Addition of 10 % (w/v) silicone cube was the optimum for paclitaxel production. At this point the specific productivity of paclitaxel was 0.022 mg/g day-1. Increased silicone cube content higher than 10% reduced paclitaxel production. It might be speculated that addition of higher concentrations of silicone cube might reduce paclitaxel production due to its removal of nutrient or precursor of paclitaxel biosynthesis in culture medium.

To find out optimal addition time of silicone cube to suspension culture of T. cuspidata, equally inoculated 18 culture flasks were prepared and silicone cubes were added at every 7 days to each three flasks. The suspension culture was incubated for 35 days. Figure 3 shows the effect of addition time of silicone cube on paclitaxel accumulation. The maximum paclitaxel production was 3.95 mg/L when 10% of silicone cubes were added to the culture at 7 days after inoculation. This result shows that the addition time of silicone cube can be an important parameter for enhanced paclitaxel production in two-phase culture of T. cuspidata.

Fig. 3. Effect of addition time of silicone cube on cell growth and paclitaxel production in two-phase culture. Initial cell concentration: 1 g dry cells /L

It is necessary to investigate further operating parameters which may affect silicone cube addition for improved paclitaxel production in T. cuspidata cell culture. However, our preliminary data suggest that the use of a second phase such as silicone cube may be useful to produce paclitaxel from T. cuspidata cell culture.

REFERENCES

[1] Slichenmyer, W.J. and Von Hoff, D.D. : Anti-cancer drugs, 2, 519(1991).

[2] Stull, D.P., Scales, T.A., Daughenbaugh, R., Jans, N.A. and Bailey D.T. :

Applied Biochemistry and Biotechnology 54, 133(1995).

[3] Jaziri, M., Zhiri, A., Guo, Y.W., Dupont, J.P., Shimomura, K., Hamada, H.,

Vanhaelen, M. and Homes J. : Plant Cell, Tissue and Organ Culture 46,

59(1996).

[4] Pestchanker, L.J., Roberts S.C. and Shuler, M.L. : Enzyme & Microbial

Technology, 19, 256(1996).

[5] Ciddi, V., Srinivasan, V. and Shuler, M.L. : Biotechnol. Lett. 17, 1343

(1995).

[6] Moon, W.J., Yoo, B.S., Kim, D.I. and Byun, S.Y. : Biotechnology

Techniques 12, 79(1998).

[7] Park, J.H., Kim, C.H., Lee, S.W. and Chung, I.S. : Acta Horticulturae. 390,

127(1995)

[8] Beiderbeck, R. and Knoop, B. : "Two-phase culture" In: Vasil, I.K. (ed)

Cell Culture and Somatic Cell Genetics of Plants vol 4. Academic Press,

San Diego, (1987)