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Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures
Process Biochemistry 45 (2010) 1368–1374
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures Min-Gyeong Han a , Keum-Young Jeon a , Sungyong Mun b,∗ , Jin-Hyun Kim a,∗∗ a b
Department of Chemical Engineering, Kongju National University, 182 Shinkwan-Dong, Kongju 314-701, South Korea Department of Chemical Engineering, Hanyang University, Haengdang-dong, Seongdong-gu, Seoul 133-791, South Korea
a r t i c l e
i n f o
Article history: Received 22 February 2010 Received in revised form 8 April 2010 Accepted 8 May 2010 Keywords: Paclitaxel Micelle-fractional precipitation hybrid process Pre-purification Surface area per working volume (S/V) Ion exchange resin
a b s t r a c t A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid, has been currently regarded as the most effective anticancer agent. Its chemical structure was elucidated in 1971 by Wani et al. [1]. In 1979, Schiff et al. [2] discovered its unique carcinogenic mechanism, which led the compound to become the focus of attention. Paclitaxel is the most widely used anticarcinogenic material, having relatively low toxicity while strongly inhibiting cancer cell mitosis in a different manner from that of other existing anticarcinogens. The United States Food and Drug Administration has approved the use of paclitaxel for the treatment of refractory ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer. Its application has also been expanded to the treatment of head and neck tumors, rheumatoid arthritis and Alzheimer’s disease [3–5]. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are under way, the demand for paclitaxel is expected to increase [6].
∗ Corresponding author. Tel.: +82 2 2220 0483; fax: +82 2 2298 4101. ∗∗ Corresponding author. Tel.: +82 41 850 8642; fax: +82 41 858 2575. E-mail addresses: [email protected] (S. Mun), [email protected] (J.-H. Kim). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.05.010
For paclitaxel production, three primary methods were reported in the literature. One of the reported methods was a direct extraction from the yew tree [7]. This method is, however, inefficient because it is difficult to ensure a continuous supply of raw materials as well as to establish the corresponding purification process itself. Another method was a semi-synthesis, in which the side chain was chemically synthesized after the precursor had been obtained from yew leaves [8]. This method is also problematic because the precursor is acquired directly from the yew tree, which is usually known as a nurse-tree. The third method reported was to grow yew cells in a bioreactor inoculated with an induced seed or callus culture [9,10]. Since the use of the last method enables stable production in a bioreactor without the influence of external factors such as weather and environment, it allows for the mass production of paclitaxel of consistent quality. To obtain paclitaxel from a plant cell culture, several separation and purification steps are required. Typically, after biomass is collected from the cell culture medium and is extracted with an organic solvent, pre-purification and final purification processes follow. Among these steps, the pre-purification process in particular has a significant impact on the cost of final purification [11–13]. In previous researches, most of efforts were focused on the use of expensive chromatographic methods for pre-purification or high performance liquid chromatography (HPLC) for the final purifica-
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Fig. 1. Principle of the micelle process for pre-purification of paclitaxel.
tion of crude extract without pre-purification. Obviously, such an approach causes economic problems and difficulties in scale-up [14,15]. In general, when paclitaxel is extracted from biomass using an organic solvent, the purity is below 0.5%, and it remains below 10% after simple pre-purification. If the sample proceeds directly to final purification by HPLC, a large amount of organic solvent is consumed, the life of the column packing material (resin) is reduced, and throughput is decreased. Therefore, the cost of final purification, especially when HPLC is used, can be reduced if the purity of the pre-purified sample is increased as much as possible. In 2000, an effective pre-purification process was developed that enabled high purity (>50%) of paclitaxel to be obtained by fractional precipitation [16]. After biomass was collected from the plant cell culture medium and extracted with solvent, adsorbent treatment and hexane precipitation were performed to obtain paclitaxel with 15% purity. Subjecting the paclitaxel thereafter to fractional precipitation enabled a purity of over 50% to be easily achieved. However, the drawback of fractional precipitation is that it is difficult to apply to mass production because it requires a lengthy precipitation (∼3 days). In 2004, the process for pre-purification by micelle formation and precipitation was reported [17]. Although this process enabled paclitaxel of 20–30% purity to be obtained, this level was considered insufficient because it placed too great a burden on the purification process that followed. In 2008, the impact of the surfactant on the micelle process was evaluated [18], leading to the selection of N-cetylpyridinium chloride (CPC), but the purity (20%) achieved remained low. Recently, an improved process for pre-purification of paclitaxel was developed that relies on increasing the surface area per working volume (S/V) of the reaction
solution by the addition of glass beads, thereby making a larger surface area available for precipitation and allowing for a significant reduction in the time required for fractional precipitation [19]. In the present study, we developed a micelle-fractional precipitation hybrid process as an effective method for pre-purification of paclitaxel from plant cell cultures. CPC, a surfactant having both hydrophilic and hydrophobic groups, was used in the micelle process, while fractional precipitation relied on the difference in solubility of paclitaxel in methanol. The effectiveness of this hybrid process was remarkably increased through pretreatment with an adsorbent, and the time required for fractional precipitation was optimized by selecting the most effective material for increasing the S/V in the reactor. In addition to a dramatic reduction of the paclitaxel product cost by resolving the problems inherent in the existing pre-purification process [16–18], this research constitutes the development of a core technology for the commercialized production of similar diterpenoid anticancer agents; as such, it is expected to have a significant ripple effect in this field. 2. Materials and methods 2.1. Plant materials and culture conditions A suspension of cells originating from Taxus chinensis was maintained in darkness at 24.0 ◦ C with shaking at 150 rpm [20]. The cells were cultured in modified Gamborg’s B5 medium supplemented with 30 g/L sucrose, 10 mM naphthalene acetic acid (NAA), 0.2 mM 6-benzylaminopurine (BA), 1 g/L casein hydrolysate, and 1 g/L 2-(N-morpholino) ethanesulfonic acid (MES). Cell cultures were transferred to fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively. Follow-
Fig. 2. Schematic diagram of fractional precipitation for pre-purification of paclitaxel: traditional fractional precipitation (A); improved fractional precipitation by increasing S/V (B).
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M.-G. Han et al. / Process Biochemistry 45 (2010) 1368–1374 2.3. Preparation of crude extract A crude extract was prepared from biomass obtained from T. chinensis cultures using the following steps. (i) Biomass was mixed with methanol and stirred at room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filer paper, where the biomass was preferably added to methanol at a ratio of 100%. Extraction was repeated at least four times. Each methanol extract was collected, pooled and concentrated at a temperature of 40 ◦ C under reduced pressure to reduce the volume of the methanol extract to 30% of original [21]. (ii) Methylene chloride (25% of concentrated liquid) was added and liquid–liquid extraction was performed three times for 30 min [21]. During the application of the liquid–liquid extraction, the polar impurities were dissolved into the methanol layer, which eventually formed the upper phase. After removing the upper phase (i.e., the methanol layer containing the polar impurities), the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) Dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added to the crude extract at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 ◦ C, then filtered. The filtrate was dried at 30 ◦ C under reduced pressure and subjected to the micelle-fractional precipitation hybrid process. 2.4. Micelle-fractional precipitation hybrid process
Fig. 3. Proposed schematic diagram of the pre-purification process.
ing cultivation, biomass was recovered using a decanter (CA150 Clarifying Decanter, Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD-35CDEFP, Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Dried residue was redissolved in methanol for quantitative analysis using an HPLC system (Waters, USA) with a Capcell Pak C18 column (250 mm × 4.6 mm, Shiseido, Japan). Elution was performed in a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm with a UV detector. An authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [9].
2.4.1. Micelle process The principle of the micelle process for the pre-purification of paclitaxel is shown in Fig. 1. The crude extract obtained through the adsorbent treatment was dissolved in 25 mL of methyl t-butyl ether (MtBE). 12.5 mL of 5% (w/v) CPC and 25 mL of hexane were added and then the mixture was agitated at room temperature and set aside. Phase separation resulted in an upper organic solvent (MtBE/hexane) layer and a lower surfactant layer, which corresponded to an aqueous solution containing micelles. The separated lower-phase was then collected. After repeating these procedures four times under the same conditions, the final collected-fractions were pooled and 12.5 mL of MtBE was added to decompose micelles. The paclitaxel molecules released from the MtBE-containing supernatant was then concentrated [18], dried for 24 h in a vacuum oven (UP-2000, EYELA, Japan) and analyzed by HPLC. 2.4.2. Fractional precipitation The dried material obtained from the micelle process was subjected to fractional precipitation utilizing the difference in solubility of paclitaxel in methanol solution as shown in Fig. 2. Ion exchange resin or glass beads were used to increase the S/V of the reacting solution (Fig. 2B). A cation exchange resin (Amberlite 200 or Amberlite
Fig. 4. Effect of adsorbent treatment on the purity of paclitaxel.
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Table 1 Effect of ion exchange resin and glass beads on the purity and yield of paclitaxel at a given fractional precipitation time (9 h). Type
Purity (%)
Yield (%)
Cationic resin Amberlite IR120 Amberlite 200
84 87
82 85
Anionic resin Amberlite IRA400 Amberlite IRA96
84 82
83 80
Nonionic resin Amberlite XAD16 DIAION HP20
– –
– –
83 83
80 63
Glass beads Control
3. Results and discussion 3.1. Effect of adsorbent treatment prior to the hybrid process Fig. 5. Effect of adsorbent treatment on the purity of paclitaxel in the micelle process: without adsorbent treatment (A); with adsorbent treatment (B).
IR120, Rohm and Haas, USA), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96, Rohm and Haas), or a nonionic exchange resin (Amberlite XAD16, Rohm and Haas or DIAION HP20, Mitsubishi, Japan) was used. Ion exchange resins were dried for 1 day at 60 ◦ C prior to use in experiments. Glass beads (0.5–0.75 mm in diameter; Glastechnique Mfg, Germany) were washed with methanol and dried prior to use. Crude extract (pure paclitaxel basis: 0.5%, w/v) obtained from the micelle process was dissolved in methanol, and distilled water was added dropwise until the methanol concentration was 61.5% [16,19]. Ion exchange resin or glass beads were added to the reacting solution and the mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate, which was filtered, vacuum dried for 24 h at 35 ◦ C and analyzed by HPLC. The precipitate was recovered from the resin or beads by washing with methanol for HPLC analysis. The paclitaxel precipitate was visualized during the fractional precipitation process with an SV-35 Video Microscope System (Some Tech, Korea) at high magnification (100×). The size and shape of paclitaxel particles in dynamic images was verified with IT-Plus software (Some Tech, Korea). The diagram of the overall pre-purification process is presented in Fig. 3.
Fig. 6. Effect of adsorbent treatment on the purity and yield of paclitaxel in fractional precipitation: without adsorbent treatment (A); with adsorbent treatment (B).
The purity of the paclitaxel sample was 8% after the application of the liquid–liquid extraction, and it was increased to 9.8% after the treatment with the adsorbent sylopute, as shown in Fig. 4. Although such an increase in purity was slight, the adsorbent treatment was very effective in removing waxy compounds originating from plant cells. This result is consistent with that reported by Pyo et al. [12,21]. Removal of waxy compounds has a considerable influence on the convenience and feasibility of the separation and purification processes that follow pre-purification.
Fig. 7. Chromatograms of fractional precipitation steps analyzed by RP-HPLC: crude extract for fractional precipitation (A); XAD16 washing solution after fractional precipitation (B); filtrate after fractional precipitation (C).
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Fig. 8. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 9 h: control (A); glass beads (B); cation exchange resins Amberlite IR120 (C) and Amberlite 200 (D); anion exchange resins Amberlite IRA400 (E) and Amberlite IRA96 (F); nonionic exchange resins Amberlite XAD16 (G) and DIAION HP20 (H).
3.2. Micelle process The purpose of the micelle process is to remove impurities from paclitaxel via formation of micelles by a surfactant and separation of phases (Fig. 1). When water-insoluble paclitaxel comes into contact with CPC, a cationic surfactant consisting of both hydrophilic and hydrophobic groups, it is surrounded by hydrophobic tails within micelles, enabling hydrophilic heads at micelle surfaces to be dissolved in aqueous solution. As a result, when the organic
solvent layer (MtBE/hexane) is separated from the CPC solution, impurities are effectively removed from the paclitaxel. Results for the micelle process using a sample not treated with adsorbent (paclitaxel purity: 8%) and a sample treated with adsorbent (paclitaxel purity: 9.8%) are compared in Fig. 5. An extract with a purity of 20% (yield > 95%) was obtained when adsorbent treatment was excluded while 45% purity (yield > 95%) was achieved when adsorbent treatment was included. This result is likely due to the removal of waxy substances originating from plant cells by
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the adsorbent; it represents a significant improvement over results obtained with other micelle processes as reported in the existing literature [17,18], demonstrating that adsorbent treatment can considerably increase their effectiveness. 3.3. Fractional precipitation 3.3.1. Effect of adsorbent treatment on fractional precipitation Adsorbent treatment also had a significant effect on fractional precipitation of samples obtained from the micelle process. As shown in Fig. 6, after fractional precipitation for 12 h, paclitaxel of remarkably higher purity (82%) was obtained in high yield (80%) from samples treated with adsorbent while paclitaxel of lower purity (65%) was obtained in very low yield (4%) when the adsorbent treatment was excluded. The removal of waxy plant substances from the sample is considered to be responsible for this result; the higher the purity of sample obtained from the micelle process, the higher the purity of paclitaxel that can be obtained from the subsequent fractional precipitation process. This result is consistent with the recent finding by Jeon and Kim [19] regarding the effect of sample purity on the outcome of fractional precipitation. Thus, adsorbent treatment can improve not only the effectiveness of the subsequent micelle process but the efficiency of fractional precipitation as well. 3.3.2. Effect of increasing S/V of the reacting solution using ion exchange resins Fractional precipitation is a very simple method for the efficient purification of paclitaxel in high yield that relies on solubility differences. The content of methanol solution, precipitation temperature and pure paclitaxel content dissolved in methanol solution, which are the main process variables in fractional precipitation, are 61.5% (v/v), 4 ◦ C, and 0.5% (w/v), respectively [16]. Previously, it was determined that the time required for this process was too long (∼3 days), making separation and purification of large amounts of product difficult. It has also been observed that paclitaxel precipitates as a thin layer on the bottom and walls of the reactor [19]. Since most crystallizations involve formation of a nucleus on a surface (e.g. particulate impurities, reactor wall, and agitator surface) [22], increasing the S/V in the reacting solution, and thus the surface space available for precipitation, reduces the precipitation time and enables high-purity paclitaxel to be obtained in high yield [19]. Using glass beads, Jeon and Kim [19] found the optimal S/V to be 0.428 mm−1 ; this value was selected for the experiments in the present study to determine the optimal material for increasing S/V in an attempt to further improve the fractional precipitation process. In Table 1, the purity and yield of paclitaxel obtained after 9 h of fractional precipitation using cation exchange resin, anion exchange resin, nonionic exchange resin or 7 mm glass beads to increase S/V are compared. In contrast to the control sample, in which S/V was not increased and the resulting purity and yield were 83% and 63%, respectively, the yield, but not the purity, was considerably improved by adding cation exchange resin, anion exchange resin, or glass beads. Fractional precipitation performed with cation exchange resins Amberlite IR120 and Amberlite 200 resulted in 84% purity and 82% yield, and 87% purity and 85% yield, respectively. Using anion exchange resins Amberlite IRA400 and Amberlite IRA96, 84% purity and 83% yield, and 82% purity and 80% yield were achieved, respectively. The addition of glass beads resulted in 83% purity and 80% yield. Therefore, the highest purity (87%) and yield (85%) were obtained for the given fractional precipitation time (9 h) using Amberlite 200. On the other hand, no precipitation occurred when either of the nonionic exchange resins was used. To determine the cause of this result, the nonionic exchange resins were collected by filtration after fractional precipitation and washed with methanol (Fig. 7). Most impuri-
Fig. 9. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm−1 ) on the purity and yield of paclitaxel during fractional precipitation. The methanol composition in water, pure paclitaxel content, crude extract purity, and precipitation temperature were 61.5% (v/v), 0.5% (w/v), 45.0%, and 4 ◦ C, respectively.
ties containing paclitaxel were adsorbed to the resin (Fig. 7B) and did not remain in the filtrate (Fig. 7C), an observation that can be explained by the tendency of a hydrophobic material such as paclitaxel to be strongly adsorbed to the surface of a nonionic exchange resin, which is also hydrophobic. This finding is consistent with the results of a report by Kim and Hong [23], who described the use of nonionic resins (Amberlite XAD2, 4, 7, 8, and 16) to collect small amounts of paclitaxel from plant cell culture medium. In addition, when we observed the morphology of paclitaxel precipitates with an electron microscope (Fig. 8), we found that precipitation occurred in the control and in reactions containing cation or anion exchange resins or glass beads, but not when a nonionic exchange resin was used. 3.3.3. Effect of precipitation time on fractional precipitation Changes in the purity and yield of paclitaxel as a result of varying the fractional precipitation time (6, 9, 12, and 15 h) were determined for reactions in which S/V was increased by adding Amberlite 200, Amberlite IRA400 or glass beads, and for control reactions in which S/V was not increased. As shown in Fig. 9, for a given precipitation time, a generally higher yield was obtained when the S/V of the reacting solution was increased compared to the control. In particular, almost all of the paclitaxel (>98%) could be obtained after 12 h of fractional precipitation using Amberlite 200, enabling a significant reduction in precipitation time when compared to previous methods [16,19]. In addition, no further increase in yield was observed beyond 12 h of fractional precipitation. On the other hand, purity increased over precipitation time but changed little beyond its value at 9 h (∼85%) regardless of the S/V of the reacting solution. In general, purity is low during the early stages of fractional precipitation and increases over time because the precipitation of impurities is relatively high early on but decreases over time. High-purity paclitaxel can be obtained at a relatively early stage of fractional precipitation (6 h), a result that is considered to be due to the charge on the surface of the ion exchange resin.
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4. Conclusions
References
In this research, a micelle-fractional precipitation hybrid process was developed for the effective pre-purification of paclitaxel, an anticancer agent, from plant cell cultures. Treatment with the adsorbent sylopute prior to the hybrid process had a significant effect on the purity and yield obtained in the subsequent prepurification. That is, by effectively removing waxy compounds originating from plant cells through the addition of an adsorbent, the efficiency of the hybrid process could be significantly enhanced. In addition, the S/V of the reacting solution could be increased to the previously determined optimal value of 0.428 mm−1 by adding cation or anion exchange resins or glass beads. Use of the cation exchange resin Amberlite 200 in particular enabled almost all of the paclitaxel (>98%) to be obtained after 12 h of fractional precipitation, a finding which represents a further improvement over the existing methods. Purity was increased to about 85% with time, which trend was continued for 9 h of fractional precipitation. After then, there was little change in purity. On the other hand, the yield could not be enhanced by increasing S/V using nonionic exchange resins because the hydrophobic paclitaxel became strongly adsorbed to their hydrophobic surfaces; as a result, no paclitaxel precipitate was formed when these resins were added to the reacting solution. Since the micelle-fractional precipitation hybrid prepurification process enables not only high-purity paclitaxel to be obtained in high yield but the precipitation time to be reduced as well, it is expected to make a considerable contribution to the final purification process. Implementation of the hybrid prepurification process would enable not only a significant reduction in the consumption of organic solvents and column filler but also a marked increase in throughput in the final purification stage using HPLC. As a result, considerable savings in the cost of paclitaxel production could be achieved.
[1] Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 1971;93:2325–7. [2] Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature 1979;277:665–7. [3] Frye DK, Mahon SM, Palmieri FM. New options for metastatic breast cancer. Clin J Oncol Nurs 2009;13:11–8. [4] Morris PG, Fornier MN. Novel anti-tubulin cytotoxic agents for breast cancer. Expert Rev Anticancer Ther 2009;9:175–85. [5] Hsiao JR, Leu SF, Huang BM. Apoptotic mechanism of paclitaxel-induced cell death in human head and neck tumor cell lines. J Oral Pathol Med 2009;38:188–97. [6] Kim JH. Paclitaxel: recovery and purification in commercialization. Korean J Biotechnol Bioeng 2006;21:1–10. [7] Rao KV, Hanuman JB, Alvarez C, Stoy M, Juchum J, Davies RM, et al. A new large-scale process for taxol and related taxanes from Taxus brevifolia. Pharm Res 1995;12:1003–10. [8] Baloglu E, Kingston DG. A new semisynthesis of paclitaxel from baccatin III. J Nat Prod 1999;62:1068–71. [9] Hyun JE, Kim JH. Microwave-assisted extraction of paclitaxel from plant cell cultures. Korean J Biotechnol Bioeng 2008;23:281–4. [10] Kolewe ME, Gaurav V, Roberts SC. Pharmaceutically active natural product synthesis and supply via plant cell culture technology. Mol Pharm 2008;5:243–56. [11] Kim JH, Choi HK, Hong SS, Lee HS. Development of high performance liquid chromatography for paclitaxel purification from plant cell cultures. J Microbiol Biotechnol 2001;11:204–10. [12] Pyo SH, Song BK, Ju CH, Han BH, Choi HJ. Effects of adsorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree. Process Biochem 2005;40:1113–7. [13] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. A novel pre-purification for paclitaxel from plant cell cultures. Process Biochem 2002;37:679–82. [14] Rao KV. Method for the isolation and purification of taxol and its natural analogues. US Patent 5,670,673; 1997. [15] Castor TP. Method and apparatus for isolating therapeutic compositions from source materials. US Patent 5,750,709; 1998. [16] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis. Biotechnol Lett 2000;22:1753–6. [17] Kim JH. Prepurification of paclitaxel by micelle and precipitation. Process Biochem 2004;39:1567–71. [18] Jeon KY, Kim JH. Effect of surfactant on the micelle process for the prepurification of paclitaxel. Korean J Biotechnol Bioeng 2008;23:557–60. [19] Jeon KY, Kim JH. Improvement of fractional precipitation process for prepurification of paclitaxel. Process Biochem 2009;44:736–41. [20] Choi HK, Adams TL, Stahlhut RW, Kim SI, Yun JH, Song BK, et al. Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture. US Patent 5,871,979; 1999. [21] Pyo SH, Park HB, Song BK, Han BH, Kim JH. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem 2004;39:1985–91. [22] Kim WS, Lee EK. Technological trend of crystallization research for bioproduct separation. Korean J Biotechnol Bioeng 2005;20:164–76. [23] Kim JH, Hong SS. Recovery of paclitaxel from suspension culture medium with hydrophobic resin. Korean J Biotechnol Bioeng 2000;15:366–9.
Acknowledgments This work was supported by the research grant of the Kongju National University in 2009. This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2009-0071452).
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures Min-Gyeong Han a , Keum-Young Jeon a , Sungyong Mun b,∗ , Jin-Hyun Kim a,∗∗ a b
Department of Chemical Engineering, Kongju National University, 182 Shinkwan-Dong, Kongju 314-701, South Korea Department of Chemical Engineering, Hanyang University, Haengdang-dong, Seongdong-gu, Seoul 133-791, South Korea
a r t i c l e
i n f o
Article history: Received 22 February 2010 Received in revised form 8 April 2010 Accepted 8 May 2010 Keywords: Paclitaxel Micelle-fractional precipitation hybrid process Pre-purification Surface area per working volume (S/V) Ion exchange resin
a b s t r a c t A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid, has been currently regarded as the most effective anticancer agent. Its chemical structure was elucidated in 1971 by Wani et al. [1]. In 1979, Schiff et al. [2] discovered its unique carcinogenic mechanism, which led the compound to become the focus of attention. Paclitaxel is the most widely used anticarcinogenic material, having relatively low toxicity while strongly inhibiting cancer cell mitosis in a different manner from that of other existing anticarcinogens. The United States Food and Drug Administration has approved the use of paclitaxel for the treatment of refractory ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer. Its application has also been expanded to the treatment of head and neck tumors, rheumatoid arthritis and Alzheimer’s disease [3–5]. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are under way, the demand for paclitaxel is expected to increase [6].
∗ Corresponding author. Tel.: +82 2 2220 0483; fax: +82 2 2298 4101. ∗∗ Corresponding author. Tel.: +82 41 850 8642; fax: +82 41 858 2575. E-mail addresses: [email protected] (S. Mun), [email protected] (J.-H. Kim). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.05.010
For paclitaxel production, three primary methods were reported in the literature. One of the reported methods was a direct extraction from the yew tree [7]. This method is, however, inefficient because it is difficult to ensure a continuous supply of raw materials as well as to establish the corresponding purification process itself. Another method was a semi-synthesis, in which the side chain was chemically synthesized after the precursor had been obtained from yew leaves [8]. This method is also problematic because the precursor is acquired directly from the yew tree, which is usually known as a nurse-tree. The third method reported was to grow yew cells in a bioreactor inoculated with an induced seed or callus culture [9,10]. Since the use of the last method enables stable production in a bioreactor without the influence of external factors such as weather and environment, it allows for the mass production of paclitaxel of consistent quality. To obtain paclitaxel from a plant cell culture, several separation and purification steps are required. Typically, after biomass is collected from the cell culture medium and is extracted with an organic solvent, pre-purification and final purification processes follow. Among these steps, the pre-purification process in particular has a significant impact on the cost of final purification [11–13]. In previous researches, most of efforts were focused on the use of expensive chromatographic methods for pre-purification or high performance liquid chromatography (HPLC) for the final purifica-
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Fig. 1. Principle of the micelle process for pre-purification of paclitaxel.
tion of crude extract without pre-purification. Obviously, such an approach causes economic problems and difficulties in scale-up [14,15]. In general, when paclitaxel is extracted from biomass using an organic solvent, the purity is below 0.5%, and it remains below 10% after simple pre-purification. If the sample proceeds directly to final purification by HPLC, a large amount of organic solvent is consumed, the life of the column packing material (resin) is reduced, and throughput is decreased. Therefore, the cost of final purification, especially when HPLC is used, can be reduced if the purity of the pre-purified sample is increased as much as possible. In 2000, an effective pre-purification process was developed that enabled high purity (>50%) of paclitaxel to be obtained by fractional precipitation [16]. After biomass was collected from the plant cell culture medium and extracted with solvent, adsorbent treatment and hexane precipitation were performed to obtain paclitaxel with 15% purity. Subjecting the paclitaxel thereafter to fractional precipitation enabled a purity of over 50% to be easily achieved. However, the drawback of fractional precipitation is that it is difficult to apply to mass production because it requires a lengthy precipitation (∼3 days). In 2004, the process for pre-purification by micelle formation and precipitation was reported [17]. Although this process enabled paclitaxel of 20–30% purity to be obtained, this level was considered insufficient because it placed too great a burden on the purification process that followed. In 2008, the impact of the surfactant on the micelle process was evaluated [18], leading to the selection of N-cetylpyridinium chloride (CPC), but the purity (20%) achieved remained low. Recently, an improved process for pre-purification of paclitaxel was developed that relies on increasing the surface area per working volume (S/V) of the reaction
solution by the addition of glass beads, thereby making a larger surface area available for precipitation and allowing for a significant reduction in the time required for fractional precipitation [19]. In the present study, we developed a micelle-fractional precipitation hybrid process as an effective method for pre-purification of paclitaxel from plant cell cultures. CPC, a surfactant having both hydrophilic and hydrophobic groups, was used in the micelle process, while fractional precipitation relied on the difference in solubility of paclitaxel in methanol. The effectiveness of this hybrid process was remarkably increased through pretreatment with an adsorbent, and the time required for fractional precipitation was optimized by selecting the most effective material for increasing the S/V in the reactor. In addition to a dramatic reduction of the paclitaxel product cost by resolving the problems inherent in the existing pre-purification process [16–18], this research constitutes the development of a core technology for the commercialized production of similar diterpenoid anticancer agents; as such, it is expected to have a significant ripple effect in this field. 2. Materials and methods 2.1. Plant materials and culture conditions A suspension of cells originating from Taxus chinensis was maintained in darkness at 24.0 ◦ C with shaking at 150 rpm [20]. The cells were cultured in modified Gamborg’s B5 medium supplemented with 30 g/L sucrose, 10 mM naphthalene acetic acid (NAA), 0.2 mM 6-benzylaminopurine (BA), 1 g/L casein hydrolysate, and 1 g/L 2-(N-morpholino) ethanesulfonic acid (MES). Cell cultures were transferred to fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively. Follow-
Fig. 2. Schematic diagram of fractional precipitation for pre-purification of paclitaxel: traditional fractional precipitation (A); improved fractional precipitation by increasing S/V (B).
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M.-G. Han et al. / Process Biochemistry 45 (2010) 1368–1374 2.3. Preparation of crude extract A crude extract was prepared from biomass obtained from T. chinensis cultures using the following steps. (i) Biomass was mixed with methanol and stirred at room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filer paper, where the biomass was preferably added to methanol at a ratio of 100%. Extraction was repeated at least four times. Each methanol extract was collected, pooled and concentrated at a temperature of 40 ◦ C under reduced pressure to reduce the volume of the methanol extract to 30% of original [21]. (ii) Methylene chloride (25% of concentrated liquid) was added and liquid–liquid extraction was performed three times for 30 min [21]. During the application of the liquid–liquid extraction, the polar impurities were dissolved into the methanol layer, which eventually formed the upper phase. After removing the upper phase (i.e., the methanol layer containing the polar impurities), the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) Dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added to the crude extract at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 ◦ C, then filtered. The filtrate was dried at 30 ◦ C under reduced pressure and subjected to the micelle-fractional precipitation hybrid process. 2.4. Micelle-fractional precipitation hybrid process
Fig. 3. Proposed schematic diagram of the pre-purification process.
ing cultivation, biomass was recovered using a decanter (CA150 Clarifying Decanter, Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD-35CDEFP, Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Dried residue was redissolved in methanol for quantitative analysis using an HPLC system (Waters, USA) with a Capcell Pak C18 column (250 mm × 4.6 mm, Shiseido, Japan). Elution was performed in a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm with a UV detector. An authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [9].
2.4.1. Micelle process The principle of the micelle process for the pre-purification of paclitaxel is shown in Fig. 1. The crude extract obtained through the adsorbent treatment was dissolved in 25 mL of methyl t-butyl ether (MtBE). 12.5 mL of 5% (w/v) CPC and 25 mL of hexane were added and then the mixture was agitated at room temperature and set aside. Phase separation resulted in an upper organic solvent (MtBE/hexane) layer and a lower surfactant layer, which corresponded to an aqueous solution containing micelles. The separated lower-phase was then collected. After repeating these procedures four times under the same conditions, the final collected-fractions were pooled and 12.5 mL of MtBE was added to decompose micelles. The paclitaxel molecules released from the MtBE-containing supernatant was then concentrated [18], dried for 24 h in a vacuum oven (UP-2000, EYELA, Japan) and analyzed by HPLC. 2.4.2. Fractional precipitation The dried material obtained from the micelle process was subjected to fractional precipitation utilizing the difference in solubility of paclitaxel in methanol solution as shown in Fig. 2. Ion exchange resin or glass beads were used to increase the S/V of the reacting solution (Fig. 2B). A cation exchange resin (Amberlite 200 or Amberlite
Fig. 4. Effect of adsorbent treatment on the purity of paclitaxel.
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Table 1 Effect of ion exchange resin and glass beads on the purity and yield of paclitaxel at a given fractional precipitation time (9 h). Type
Purity (%)
Yield (%)
Cationic resin Amberlite IR120 Amberlite 200
84 87
82 85
Anionic resin Amberlite IRA400 Amberlite IRA96
84 82
83 80
Nonionic resin Amberlite XAD16 DIAION HP20
– –
– –
83 83
80 63
Glass beads Control
3. Results and discussion 3.1. Effect of adsorbent treatment prior to the hybrid process Fig. 5. Effect of adsorbent treatment on the purity of paclitaxel in the micelle process: without adsorbent treatment (A); with adsorbent treatment (B).
IR120, Rohm and Haas, USA), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96, Rohm and Haas), or a nonionic exchange resin (Amberlite XAD16, Rohm and Haas or DIAION HP20, Mitsubishi, Japan) was used. Ion exchange resins were dried for 1 day at 60 ◦ C prior to use in experiments. Glass beads (0.5–0.75 mm in diameter; Glastechnique Mfg, Germany) were washed with methanol and dried prior to use. Crude extract (pure paclitaxel basis: 0.5%, w/v) obtained from the micelle process was dissolved in methanol, and distilled water was added dropwise until the methanol concentration was 61.5% [16,19]. Ion exchange resin or glass beads were added to the reacting solution and the mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate, which was filtered, vacuum dried for 24 h at 35 ◦ C and analyzed by HPLC. The precipitate was recovered from the resin or beads by washing with methanol for HPLC analysis. The paclitaxel precipitate was visualized during the fractional precipitation process with an SV-35 Video Microscope System (Some Tech, Korea) at high magnification (100×). The size and shape of paclitaxel particles in dynamic images was verified with IT-Plus software (Some Tech, Korea). The diagram of the overall pre-purification process is presented in Fig. 3.
Fig. 6. Effect of adsorbent treatment on the purity and yield of paclitaxel in fractional precipitation: without adsorbent treatment (A); with adsorbent treatment (B).
The purity of the paclitaxel sample was 8% after the application of the liquid–liquid extraction, and it was increased to 9.8% after the treatment with the adsorbent sylopute, as shown in Fig. 4. Although such an increase in purity was slight, the adsorbent treatment was very effective in removing waxy compounds originating from plant cells. This result is consistent with that reported by Pyo et al. [12,21]. Removal of waxy compounds has a considerable influence on the convenience and feasibility of the separation and purification processes that follow pre-purification.
Fig. 7. Chromatograms of fractional precipitation steps analyzed by RP-HPLC: crude extract for fractional precipitation (A); XAD16 washing solution after fractional precipitation (B); filtrate after fractional precipitation (C).
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Fig. 8. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 9 h: control (A); glass beads (B); cation exchange resins Amberlite IR120 (C) and Amberlite 200 (D); anion exchange resins Amberlite IRA400 (E) and Amberlite IRA96 (F); nonionic exchange resins Amberlite XAD16 (G) and DIAION HP20 (H).
3.2. Micelle process The purpose of the micelle process is to remove impurities from paclitaxel via formation of micelles by a surfactant and separation of phases (Fig. 1). When water-insoluble paclitaxel comes into contact with CPC, a cationic surfactant consisting of both hydrophilic and hydrophobic groups, it is surrounded by hydrophobic tails within micelles, enabling hydrophilic heads at micelle surfaces to be dissolved in aqueous solution. As a result, when the organic
solvent layer (MtBE/hexane) is separated from the CPC solution, impurities are effectively removed from the paclitaxel. Results for the micelle process using a sample not treated with adsorbent (paclitaxel purity: 8%) and a sample treated with adsorbent (paclitaxel purity: 9.8%) are compared in Fig. 5. An extract with a purity of 20% (yield > 95%) was obtained when adsorbent treatment was excluded while 45% purity (yield > 95%) was achieved when adsorbent treatment was included. This result is likely due to the removal of waxy substances originating from plant cells by
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the adsorbent; it represents a significant improvement over results obtained with other micelle processes as reported in the existing literature [17,18], demonstrating that adsorbent treatment can considerably increase their effectiveness. 3.3. Fractional precipitation 3.3.1. Effect of adsorbent treatment on fractional precipitation Adsorbent treatment also had a significant effect on fractional precipitation of samples obtained from the micelle process. As shown in Fig. 6, after fractional precipitation for 12 h, paclitaxel of remarkably higher purity (82%) was obtained in high yield (80%) from samples treated with adsorbent while paclitaxel of lower purity (65%) was obtained in very low yield (4%) when the adsorbent treatment was excluded. The removal of waxy plant substances from the sample is considered to be responsible for this result; the higher the purity of sample obtained from the micelle process, the higher the purity of paclitaxel that can be obtained from the subsequent fractional precipitation process. This result is consistent with the recent finding by Jeon and Kim [19] regarding the effect of sample purity on the outcome of fractional precipitation. Thus, adsorbent treatment can improve not only the effectiveness of the subsequent micelle process but the efficiency of fractional precipitation as well. 3.3.2. Effect of increasing S/V of the reacting solution using ion exchange resins Fractional precipitation is a very simple method for the efficient purification of paclitaxel in high yield that relies on solubility differences. The content of methanol solution, precipitation temperature and pure paclitaxel content dissolved in methanol solution, which are the main process variables in fractional precipitation, are 61.5% (v/v), 4 ◦ C, and 0.5% (w/v), respectively [16]. Previously, it was determined that the time required for this process was too long (∼3 days), making separation and purification of large amounts of product difficult. It has also been observed that paclitaxel precipitates as a thin layer on the bottom and walls of the reactor [19]. Since most crystallizations involve formation of a nucleus on a surface (e.g. particulate impurities, reactor wall, and agitator surface) [22], increasing the S/V in the reacting solution, and thus the surface space available for precipitation, reduces the precipitation time and enables high-purity paclitaxel to be obtained in high yield [19]. Using glass beads, Jeon and Kim [19] found the optimal S/V to be 0.428 mm−1 ; this value was selected for the experiments in the present study to determine the optimal material for increasing S/V in an attempt to further improve the fractional precipitation process. In Table 1, the purity and yield of paclitaxel obtained after 9 h of fractional precipitation using cation exchange resin, anion exchange resin, nonionic exchange resin or 7 mm glass beads to increase S/V are compared. In contrast to the control sample, in which S/V was not increased and the resulting purity and yield were 83% and 63%, respectively, the yield, but not the purity, was considerably improved by adding cation exchange resin, anion exchange resin, or glass beads. Fractional precipitation performed with cation exchange resins Amberlite IR120 and Amberlite 200 resulted in 84% purity and 82% yield, and 87% purity and 85% yield, respectively. Using anion exchange resins Amberlite IRA400 and Amberlite IRA96, 84% purity and 83% yield, and 82% purity and 80% yield were achieved, respectively. The addition of glass beads resulted in 83% purity and 80% yield. Therefore, the highest purity (87%) and yield (85%) were obtained for the given fractional precipitation time (9 h) using Amberlite 200. On the other hand, no precipitation occurred when either of the nonionic exchange resins was used. To determine the cause of this result, the nonionic exchange resins were collected by filtration after fractional precipitation and washed with methanol (Fig. 7). Most impuri-
Fig. 9. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm−1 ) on the purity and yield of paclitaxel during fractional precipitation. The methanol composition in water, pure paclitaxel content, crude extract purity, and precipitation temperature were 61.5% (v/v), 0.5% (w/v), 45.0%, and 4 ◦ C, respectively.
ties containing paclitaxel were adsorbed to the resin (Fig. 7B) and did not remain in the filtrate (Fig. 7C), an observation that can be explained by the tendency of a hydrophobic material such as paclitaxel to be strongly adsorbed to the surface of a nonionic exchange resin, which is also hydrophobic. This finding is consistent with the results of a report by Kim and Hong [23], who described the use of nonionic resins (Amberlite XAD2, 4, 7, 8, and 16) to collect small amounts of paclitaxel from plant cell culture medium. In addition, when we observed the morphology of paclitaxel precipitates with an electron microscope (Fig. 8), we found that precipitation occurred in the control and in reactions containing cation or anion exchange resins or glass beads, but not when a nonionic exchange resin was used. 3.3.3. Effect of precipitation time on fractional precipitation Changes in the purity and yield of paclitaxel as a result of varying the fractional precipitation time (6, 9, 12, and 15 h) were determined for reactions in which S/V was increased by adding Amberlite 200, Amberlite IRA400 or glass beads, and for control reactions in which S/V was not increased. As shown in Fig. 9, for a given precipitation time, a generally higher yield was obtained when the S/V of the reacting solution was increased compared to the control. In particular, almost all of the paclitaxel (>98%) could be obtained after 12 h of fractional precipitation using Amberlite 200, enabling a significant reduction in precipitation time when compared to previous methods [16,19]. In addition, no further increase in yield was observed beyond 12 h of fractional precipitation. On the other hand, purity increased over precipitation time but changed little beyond its value at 9 h (∼85%) regardless of the S/V of the reacting solution. In general, purity is low during the early stages of fractional precipitation and increases over time because the precipitation of impurities is relatively high early on but decreases over time. High-purity paclitaxel can be obtained at a relatively early stage of fractional precipitation (6 h), a result that is considered to be due to the charge on the surface of the ion exchange resin.
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4. Conclusions
References
In this research, a micelle-fractional precipitation hybrid process was developed for the effective pre-purification of paclitaxel, an anticancer agent, from plant cell cultures. Treatment with the adsorbent sylopute prior to the hybrid process had a significant effect on the purity and yield obtained in the subsequent prepurification. That is, by effectively removing waxy compounds originating from plant cells through the addition of an adsorbent, the efficiency of the hybrid process could be significantly enhanced. In addition, the S/V of the reacting solution could be increased to the previously determined optimal value of 0.428 mm−1 by adding cation or anion exchange resins or glass beads. Use of the cation exchange resin Amberlite 200 in particular enabled almost all of the paclitaxel (>98%) to be obtained after 12 h of fractional precipitation, a finding which represents a further improvement over the existing methods. Purity was increased to about 85% with time, which trend was continued for 9 h of fractional precipitation. After then, there was little change in purity. On the other hand, the yield could not be enhanced by increasing S/V using nonionic exchange resins because the hydrophobic paclitaxel became strongly adsorbed to their hydrophobic surfaces; as a result, no paclitaxel precipitate was formed when these resins were added to the reacting solution. Since the micelle-fractional precipitation hybrid prepurification process enables not only high-purity paclitaxel to be obtained in high yield but the precipitation time to be reduced as well, it is expected to make a considerable contribution to the final purification process. Implementation of the hybrid prepurification process would enable not only a significant reduction in the consumption of organic solvents and column filler but also a marked increase in throughput in the final purification stage using HPLC. As a result, considerable savings in the cost of paclitaxel production could be achieved.
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Acknowledgments This work was supported by the research grant of the Kongju National University in 2009. This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant Number: 2009-0071452).