- Email: [email protected]
pentane precipitation process for the purification of paclitaxel from plant cell cultures
Separation and Purification Technology 89 (2012) 112–116
Contents lists available at SciVerse ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Evaluation of a high surface area acetone/pentane precipitation process for the purification of paclitaxel from plant cell cultures Hyun A Sim, Ji-Yeon Lee, Jin-Hyun Kim ⇑ Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 9 January 2012 Accepted 9 January 2012 Available online 16 January 2012 Keywords: Paclitaxel Acetone/pentane precipitation Purification Surface area per working volume (S/V) Precipitate size
a b s t r a c t In this study, we evaluated the efficiency and the behavior as well as shapes and sizes of paclitaxel precipitate as the surface area per working volume (S/V) of the reacting solution is increased in an acetone/ pentane precipitation process for the purification of paclitaxel. The purity of paclitaxel after 24 h of precipitation was 54.0% when there was no surface area increase, while it was 54.2% and 77.7% when the surface area was increased by the use of glass beads and anion exchange resin (Amberlite IRA-400OH). The purity was significantly improved when Amberlite IRA-400OH is used as an agent to increase surface area. The yield of paclitaxel improved when glass beads were used but decreased when Amberlite IRA400OH was used. Compared with the case where no surface area increasing agent was employed, the addition of glass beads or Amberlite IRA-400OH as a surface area increasing agent resulted in a considerable decrease in the size of the paclitaxel precipitate. When Amberlite IRA-400OH was added, the zeta potential value in the precipitation solution was higher (zeta potential value: 21.36 mV) than when no surface area increasing agent was employed (zeta potential value: 0.52 mV), indicating that the precipitated particles are much more stabilized with the addition of Amberlite IRA-400OH. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is one of the most effective anticancer drugs for treating ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer [1–3]. Its application has also been expanded to the treatment of head and neck tumors, acute rheumatoid arthritis, and Alzheimer’s disease [4,5]. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are underway, the demand for paclitaxel is expected to increase [6]. The main paclitaxel production methods are direct extraction from the yew tree, semi-synthesis, and plant cell culture [7–9]. Among these methods, plant cell culture enables stable mass production of paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment. To obtain high purity (>98%) paclitaxel from plant cell cultures, several separation and purification steps are required. Typically, paclitaxel is first extracted from biomass (plant cells containing paclitaxel) with an organic solvent, then pre-purified, and finally purified to obtain the product. Among these steps, the pre-purification process in particular has a significant impact on the total purification cost [10–12]. In previous studies, most efforts were focused on the use of expensive chromatographic methods for
⇑ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2012.01.017
pre-purification or high performance liquid chromatography (HPLC) for the final purification of a crude extract without prepurification. This approach thus suffers from economic problems and difficulties in scale-up and mass production [13,14]. Therefore, the purity of the pre-purified sample needs to be increased as much as possible in order to reduce the cost of final purification, especially by HPLC. Precipitation is a very simple method for the efficient purification of paclitaxel with high purity and yield. Notably, the precipitation process is dependent on solubility differences. The methanol/ water fractional precipitation process reported in 2000 [15] and the acetone/pentane precipitation process reported in 2005 [16] are typical precipitation methods. Observation of the precipitation trend of paclitaxel in the fractional precipitation process has revealed that a thin layer of paclitaxel precipitates is formed on the bottom and walls of the reactor. In reality, most of the crystallization occurs around the nucleus that is created with the help of the surface area (e.g. particulate impurities, the reactor wall, and agitator surface) [17]. Utilizing this phenomenon, a new method for improving the precipitation efficiency has been developed in which the internal surface of the precipitator is increased by adding an surface area increasing agent in the methanol/water fractional precipitation process [18,19]. The aim of this study is to propose an optimal acetone/pentane precipitation strategy by evaluating the efficiency and the behavior as well as shapes and sizes of the precipitate as the surface area per working volume (S/V) of the
H.A Sim et al. / Separation and Purification Technology 89 (2012) 112–116
reacting solution in the reactor is increased in the acetone/pentane precipitation process. Also, the effects of the use of different solvent systems in the precipitation process for paclitaxel purification have been studied in comparison with the methanol/water fractional precipitation process.
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 °C with shaking at 150 rpm [20]. The cells were cultured in modified Gamborg’s B5 medium [21] 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 a fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of the culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively. Following 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. Analysis of paclitaxel Dried residue was redissolved in methanol for a 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 lL, 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 [22].
2.3. Preparation of crude extract for acetone/pentane precipitation 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 filter 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 the original [18,23]. (ii) Methylene chloride (25% of concentrated liquid) was added and liquid–liquid extraction was performed three times for 30 min, during which polar impurities were dissolved into the methanol layer, eventually forming the upper phase. After removal of the upper phase, the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) The dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/ w) and sylopute (Fuji Silysia Chemical Ltd., Japan), used as an adsorbent, was added at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 °C, and then filtered. The filtrate was dried at 30 °C under reduced pressure and subjected to a hexane precipitation process. (iv) The filtrate obtained in the adsorbent treatment step was added to n-hexane to obtain a crude paclitaxel (filtrate/ hexane = 1/10, v/v) [18,23].
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2.4. Acetone/pentane precipitation Acetone/pentane precipitation with increased surface area is shown in Fig. 1. The crude paclitaxel (purity 38.5%) obtained from the hexane precipitation is dissolved in acetone (0.02 g crude paclitaxel/1.196 mL acetone) and drops of n-pentane are added at a ratio of 1/7 (acetone/pentane, v/v) under agitation (335 rpm) [16]. Turning off the agitating operation, glass beads (0.5–0.7 mm in diameter: Glastechnique Mfg, Germany) or anion exchange resin (Amberlite IRA-400OH or Amberlite IRA-910, Rohm and Haas, USA) and cation exchange resin (Amberlite 200 or Amberlite IRA-120, Rohm and Haas, USA) were added. Ion exchange resins were dried for 1 day at 35 °C prior to use in experiments. Glass beads were washed with methanol and dried prior to use. The experiment was conducted with the surface area per working volume (S/V) of the reacting solution fixed at the optimal level of 0.428 mm 1, as suggested in a previous study [18]. In order to obtain the paclitaxel precipitate after the addition of the surface area increasing agent, the preparation was stored for 24 h at 6.5 °C. After the precipitation period, the paclitaxel precipitate was filtered and vacuum dried (635 mm Hg, 30 °C). The morphology and the size of the paclitaxel precipitate in the acetone/pentane precipitation process were observed using an electron microscope (SV-35 Video Microscope system, Some Tech., Korea) [18]. The paclitaxel precipitate from the acetone/pentane precipitation process was then observed at high magnification (100). Video observation of the paclitaxel precipitate was performed using an IT-Plus system (Some Tech., Korea). Also, the difference in the zeta potential (ELS-Z, Photal, Japan) between the precipitation solution with and without addition of a surface area increasing agent was examined. 3. Results and discussion 3.1. Effect of increasing S/V of the reacting solution Successful formation of paclitaxel precipitate could be achieved when glass beads or an anion exchange resin, Amberlite IRA400OH, was used as a surface area increasing agent in the high surface area acetone/pentane precipitation process. The yield increased slowly with precipitation time (12–24 h), and the yield at 24 h of precipitation was 94.4% in the control without an increase of the internal surface area of the precipitator, 98.7% when glass beads were added to increase the surface, area and 60.4% when an ion exchange resin, Amberlite IRA-400OH, was added (Fig. 2). Compared with the results of the control, the addition of glass beads to increase the surface area improved the yield whereas the addition of Amberlite IRA-400OH resulted in a decreased yield. The results of the analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation indicated that the yield of paclitaxel decreased. This is
Fig. 1. Schematic diagram of high surface area acetone/pentane precipitation for purification of paclitaxel from crude extracts.
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H.A Sim et al. / Separation and Purification Technology 89 (2012) 112–116
Fig. 2. Effect of increased surface area per working volume (S/V: 0.428 mm yield of paclitaxel from acetone/pentane precipitation.
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presumably because some paclitaxel is adsorbed to Amberlite IRA400OH. On the other hand, almost no formation of paclitaxel precipitate was observed when the anion exchange resin, Amberlite IRA-910, and the cation exchange resin, Amberlite 200, IRA-120, were used as surface area increasing agents. The results of analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation revealed that most of the paclitaxel was adsorbed to the ion exchange resin when Amberlite IRA-910 or Amberlite 200, IRA-120 was used (Fig. 3(B–D)). The purity after a precipitation period of 24 h was 54.0% in the control without an increase of the internal surface area in the reactor, 54.2% when glass beads were added to increase the surface
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area, and 77.7% when the anion exchange resin, Amberlite IRA400OH, was added (Fig. 4). The purity increased slowly with the precipitation time (12–24 h). Compared with the results of the control, the addition of glass beads for increased surface area showed almost no effect in terms of improving purity whereas the addition of anion exchange resin, Amberlite IRA-400OH, improved the purity considerably. The results of the analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation indicated that almost no adsorption of paclitaxel or impurities occurred when glass beads were added (Fig. 3(E)). However, when Amberlite IRA-400OH was added (Fig. 3(A)), the purity of the precipitate was relatively higher, presumably because a considerable portion of the impurities as well as paclitaxel was adsorbed to the ion exchange resin. These results are in contrast with the results of the methanol/water fractional precipitation process [19], where increasing the surface area per working volume (S/V) of reacting solution (S/V: 0.428 mm 1) by adding a surface area increasing agent (Amberlite IRA-400OH) resulted in almost no improvement of paclitaxel purity while it improved the yield. This is presumably due to the difference in the type and the properties of the solvent used in the precipitation of paclitaxel [18,19]. 3.2. Change in shape and size of the paclitaxel precipitate over time The morphology and sizes of the paclitaxel precipitate in the acetone/pentane precipitation process with increased surface area were observed using an electron microscope. It could be seen that the precipitate branched out from around the nucleus, forming a general shape of a disk. The size of the paclitaxel precipitate grew steadily through the precipitation period (Fig. 5). Also, the number of branches from paclitaxel particles increased with time, forming
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Time (min) Fig. 3. Chromatograms of methanol washing solution after acetone/pentane precipitation analyzed by RP-HPLC: Amberlite IRA-400OH (A); IRA-910 (B); IRA-120 (C); Amberlite 200 (D); and glass beads (E), respectively. The arrow indicates the peak of paclitaxel.
H.A Sim et al. / Separation and Purification Technology 89 (2012) 112–116
Fig. 4. Effect of increased surface area per working volume (S/V: 0.428 mm purity of paclitaxel from acetone/pentane precipitation.
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denser structures as the amount of precipitation thus increased. Compared with the results of the control, the size of the paclitaxel precipitate decreased considerably within the same precipitation time frame when the surface area increasing agent, glass beads, or the ion exchange resin, Amberlite IRA-400OH, were added (Fig. 6). The size of the paclitaxel precipitate at the same precipitation time was in the order of control > glass beads > Amberlite IRA400OH, showing that the smallest paclitaxel precipitate could be obtained when Amberlite IRA-400OH is added. This is presumably because the growth of crystals was impeded by the addition of glass beads or Amberlite IRA-400OH as compared with the results of the control. Also, particle size differed depending on the type of surface area increasing agent used. This is likely due to the difference in the affinity between the surface area increasing agent and paclitaxel particles. In other words, Amberlite IRA-400OH acts as an effective steric barrier, inhibiting the growth of paclitaxel particles, since its affinity to the paclitaxel particles is higher than that of the surface area increasing agent, glass beads. This phenomenon has also been observed with active pharmaceutical ingredients,
Fig. 6. Effect of increased surface area per working volume (S/V: 0.428 mm 1) on the size of the paclitaxel precipitate through precipitation time in acetone/pentane precipitation.
megestrol acetate [24], atorvastatin calcium [25], and spironolactone [26]. The particle sizes of these active pharmaceutical ingredients can be effectively controlled by adding high molecular substances in anti-solvent precipitation processes. From the results of this study, it was found that the size of the paclitaxel precipitate is much smaller in the acetone/pentane precipitation process than in the methanol/water fractional precipitation process [18] regardless of whether a surface area increasing agent is employed. From measurement of the zeta potential values of the solution with or without the addition of a surface area increasing agent, the value was 0.52 mV when no surface area increasing agent was added (control), 0.15 mV when glass beads were added as a surface area increasing agent, and 21.36 mV when Amberlite IRA-400OH was added as a surface area increasing agent. These results indicate that the addition of Amberlite IRA-400 contributes to stabilization of the precipitation solution, as a high zeta potential value can be achieved. More specifically, the addition of Amberlite
Fig. 5. Electron micrograph of the paclitaxel precipitate from high surface area acetone/pentane precipitation. Scale bar indicates 10 lm.
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H.A Sim et al. / Separation and Purification Technology 89 (2012) 112–116
IRA-400OH produces crystals of small sizes, which remain stabilized in the solution without coagulating [24,25]. In the case of active pharmaceutical ingredients (API), their particle sizes are generally manipulated to be smaller during the crystallization process in order to enhance their usability. This is because a better dissolution rate, uniformity of drug dispersion, and oral bioavailability can be achieved with smaller particle size [24,27]. Furthermore, smaller particle size facilitates the removal of residual water and solvent during the drying process after purification [16]. From this point of view, paclitaxel with reduced particle size due to the addition of a surface area increasing agent during the acetone/pentane process is believed to be useful in respect of the usability of the drug. 4. Conclusions This goal of this study was to identify an optimal acetone/pentane precipitation strategy by evaluating the efficiency and the state as well as the shape and the size of paclitaxel precipitates as the surface area per working volume (S/V) of the reacting solution is increased (S/V: 0.428 mm 1) in the acetone/pentane precipitation process for separation/purification of the anticancer agent paclitaxel from plant cell cultures. The yield after 24 h of precipitation was 94.4% in the control where the internal surface area of the reactor was not increased, 98.7% when glass beads were added to increase the surface area, and 60.4% when an anion exchange resin, Amberlite IRA-400OH, was added. The yield increased slowly with precipitation time (12–24 h). The use of glass beads as a surface area increasing agent improved the yield compared with the results of the control while the use of IRA-400OH resulted in decreased yield. Purity increased slowly with precipitation time. After 24 h of precipitation, the purity was 54.0% in the control where the internal surface area of the reactor was not increased, 54.2% when glass beads were added to increase the surface area, and 77.7% when an anion exchange resin, Amberlite IRA-400OH, was added. Compared with the results of the control, almost no improvement in purity was achieved with the use of glass beads as a surface area increasing agent while the purity was considerably enhanced when Amberlite IRA-400OH was employed. The size of the precipitate increased with precipitation time. At the same precipitation time, the size of the paclitaxel precipitate decreased considerably when the surface area increasing agent, glass beads, or the ion exchange resin, Amberlite IRA-400OH, was added. The size of the precipitate was in the order of control > glass beads > Amberlite IRA-400OH, indicating that the smallest paclitaxel precipitate could be obtained when the anion exchange resin, Amberlite IRA-400OH, was added. Also, it was found that the addition of Amberlite IRA-400OH as a surface area increasing agent resulted in a decrease of the zeta potential value, stabilizing small crystals in the solution. From these results, it was found that the precipitation behavior varies considerably depending on the use of the solvent system (methane/water or acetone/pentane). Acknowledgment This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0010907).
References [1] P.G. Morris, M.N. Fornier, Novel anti-tubulin cytotoxic agents for breast cancer, Expert Rev. Anticancer Ther. 9 (2009) 175–185. [2] E.B. Baskan, S. Tunali, S.B. Adim, M. Kiyici, R. Ali, Treatment of advanced classic Kaposi’s sarcoma with weekly low-dose paclitaxel therapy, Int. J. Dermatol. 45 (2006) 1441–1443. [3] D.K. Frye, S.M. Mahon, F.M. Palmieri, New options for metastatic breast cancer, Clin. J. Oncol. Nurs. 13 (2009) 11–18. [4] T.M. Mekhail, M. Markman, Paclitaxel in cancer therapy, Expert Opin. Pharmacother. 3 (2002) 755–766. [5] J.R. Hsiao, S.F. Leu, B.M. Huang, Apoptotic mechanism of paclitaxel-induced cell death in human head and neck tumor cell lines, J. Oral Pathol. Med. 38 (2009) 188–197. [6] J.H. Kim, Paclitaxel: recovery and purification in commercialization step, Korean J. Biotechnol. Bioeng. 21 (2006) 1–10. [7] K.V. Rao, J.B. Hanuman, C. Alvarez, M. Stoy, J. Juchum, R.M. Davies, R. Baxley, A new large-scale process for taxol and related taxanes from Taxus brevifolia, Pharm. Res. 12 (1995) 1003–1010. [8] E. Baloglu, D.G. Kingston, A new semisynthesis of paclitaxel from baccatin III, J. Nat. Prod. 62 (1999) 1068–1071. [9] H.K. Choi, T.L. Adams, R.W. Stahlhut, S.I. Kim, J.H. Yun, B.K. Song, J.H. Kim, S.S. Hong, H.S. Lee, Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture, US Patent No. 5871,979 (1999). [10] J.H. Kim, H.K. Choi, S.S. Hong, H.S. Lee, Development of high performance liquid chromatography for paclitaxel purification from plant cell cultures, J. Microbiol. Biotechnol. 11 (2001) 204–210. [11] S.H. Pyo, B.K. Song, C.H. Ju, B.H. Han, H.J. Choi, Effects of adsorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree, Process Biochem. 40 (2005) 1113–1117. [12] J.H. Kim, I.S. Kang, H.K. Choi, S.S. Hong, H.S. Lee, A novel pre-purification for paclitaxel from plant cell cultures, Process Biochem. 37 (2002) 679–682. [13] K.V. Rao, Method for the isolation and purification of taxol and its natural analogues, US Patent No. 5670,673 (1997). [14] T.P. Castor, Method and apparatus for isolating therapeutic compositions from source materials, US Patent No. 5750,709 (1998). [15] J.H. Kim, I.S. Kang, H.K. Choi, S.S. Hong, H.S. Lee, Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis, Biotechnol. Lett. 22 (2000) 1753–1756. [16] S.H. Pyo, M.S. Kim, J.S. Cho, B.K. Song, B.H. Han, H.J. Choi, Efficient purification and morphology characterization of paclitaxel from cell cultures of Taxus chinensis, J. Chem. Technol. Biotechnol. 79 (2005) 1162–1168. [17] W.S. Kim, E.K. Lee, Technological trend of crystallization research for bioproduct separation, Korean J. Biotechnol. Bioeng. 20 (2005) 164–176. [18] K.Y. Jeon, J.H. Kim, Improvement of fractional precipitation process for prepurification of paclitaxel, Process Biochem. 44 (2009) 736–741. [19] M.G. Han, K.Y. Jeon, S. Mun, J.H. Kim, Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures, Process Biochem. 45 (2010) 1368–1374. [20] H.K. Choi, J.S. Son, G.H. Na, S.S. Hong, Y.S. Park, J.Y. Song, Mass production of paclitaxel by plant cell culture, Korean J. Plant Biotechnol. 29 (2002) 59–62. [21] O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158. [22] J.Y. Lee, J.H. Kim, Development and optimization of a novel simultaneous microwave-assisted extraction and adsorbent treatment process for separation and recovery of paclitaxel from plant cell cultures, Sep. Purif. Technol. 80 (2011) 240–245. [23] S.H. Pyo, H.B. Park, B.K. Song, B.H. Han, J.H. Kim, A large-scale purification of paclitaxel from cell cultures of Taxus chinensis, Process Biochem. 39 (2004) 1985–1991. [24] E.B. Cho, W.K. Cho, K.H. Cha, J.S. Park, Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additives, Int. J. Pharm. 396 (2010) 91–98. [25] H.X. Zhang, J.X. Wang, Z.B. Zhang, Y. Le, Z.G. Shen, J.F. Chen, Micronization of atorvastatin calcium by antisolvent precipitation process, Int. J. Pharm. 374 (2009) 106–113. [26] Y. Dong, W.K. Ng, S. Shen, S. Kim, R.B.H. Tan, Preparation and characterization of spironolactone nanoparticles by antisolvent precipitation, Int. J. Pharm. 375 (2009) 84–88. [27] S.D. Yeo, M.S. Kim, J.C. Lee, Recrystallization of sulfathiazole and chlorpropamide using the supercritical fluid antisolvent process, J. Supercrit. Fluids 25 (2003) 143–154.
Contents lists available at SciVerse ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Evaluation of a high surface area acetone/pentane precipitation process for the purification of paclitaxel from plant cell cultures Hyun A Sim, Ji-Yeon Lee, Jin-Hyun Kim ⇑ Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 9 January 2012 Accepted 9 January 2012 Available online 16 January 2012 Keywords: Paclitaxel Acetone/pentane precipitation Purification Surface area per working volume (S/V) Precipitate size
a b s t r a c t In this study, we evaluated the efficiency and the behavior as well as shapes and sizes of paclitaxel precipitate as the surface area per working volume (S/V) of the reacting solution is increased in an acetone/ pentane precipitation process for the purification of paclitaxel. The purity of paclitaxel after 24 h of precipitation was 54.0% when there was no surface area increase, while it was 54.2% and 77.7% when the surface area was increased by the use of glass beads and anion exchange resin (Amberlite IRA-400OH). The purity was significantly improved when Amberlite IRA-400OH is used as an agent to increase surface area. The yield of paclitaxel improved when glass beads were used but decreased when Amberlite IRA400OH was used. Compared with the case where no surface area increasing agent was employed, the addition of glass beads or Amberlite IRA-400OH as a surface area increasing agent resulted in a considerable decrease in the size of the paclitaxel precipitate. When Amberlite IRA-400OH was added, the zeta potential value in the precipitation solution was higher (zeta potential value: 21.36 mV) than when no surface area increasing agent was employed (zeta potential value: 0.52 mV), indicating that the precipitated particles are much more stabilized with the addition of Amberlite IRA-400OH. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is one of the most effective anticancer drugs for treating ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer [1–3]. Its application has also been expanded to the treatment of head and neck tumors, acute rheumatoid arthritis, and Alzheimer’s disease [4,5]. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are underway, the demand for paclitaxel is expected to increase [6]. The main paclitaxel production methods are direct extraction from the yew tree, semi-synthesis, and plant cell culture [7–9]. Among these methods, plant cell culture enables stable mass production of paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment. To obtain high purity (>98%) paclitaxel from plant cell cultures, several separation and purification steps are required. Typically, paclitaxel is first extracted from biomass (plant cells containing paclitaxel) with an organic solvent, then pre-purified, and finally purified to obtain the product. Among these steps, the pre-purification process in particular has a significant impact on the total purification cost [10–12]. In previous studies, most efforts were focused on the use of expensive chromatographic methods for
⇑ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim). 1383-5866/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2012.01.017
pre-purification or high performance liquid chromatography (HPLC) for the final purification of a crude extract without prepurification. This approach thus suffers from economic problems and difficulties in scale-up and mass production [13,14]. Therefore, the purity of the pre-purified sample needs to be increased as much as possible in order to reduce the cost of final purification, especially by HPLC. Precipitation is a very simple method for the efficient purification of paclitaxel with high purity and yield. Notably, the precipitation process is dependent on solubility differences. The methanol/ water fractional precipitation process reported in 2000 [15] and the acetone/pentane precipitation process reported in 2005 [16] are typical precipitation methods. Observation of the precipitation trend of paclitaxel in the fractional precipitation process has revealed that a thin layer of paclitaxel precipitates is formed on the bottom and walls of the reactor. In reality, most of the crystallization occurs around the nucleus that is created with the help of the surface area (e.g. particulate impurities, the reactor wall, and agitator surface) [17]. Utilizing this phenomenon, a new method for improving the precipitation efficiency has been developed in which the internal surface of the precipitator is increased by adding an surface area increasing agent in the methanol/water fractional precipitation process [18,19]. The aim of this study is to propose an optimal acetone/pentane precipitation strategy by evaluating the efficiency and the behavior as well as shapes and sizes of the precipitate as the surface area per working volume (S/V) of the
H.A Sim et al. / Separation and Purification Technology 89 (2012) 112–116
reacting solution in the reactor is increased in the acetone/pentane precipitation process. Also, the effects of the use of different solvent systems in the precipitation process for paclitaxel purification have been studied in comparison with the methanol/water fractional precipitation process.
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 °C with shaking at 150 rpm [20]. The cells were cultured in modified Gamborg’s B5 medium [21] 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 a fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of the culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively. Following 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. Analysis of paclitaxel Dried residue was redissolved in methanol for a 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 lL, 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 [22].
2.3. Preparation of crude extract for acetone/pentane precipitation 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 filter 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 the original [18,23]. (ii) Methylene chloride (25% of concentrated liquid) was added and liquid–liquid extraction was performed three times for 30 min, during which polar impurities were dissolved into the methanol layer, eventually forming the upper phase. After removal of the upper phase, the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) The dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/ w) and sylopute (Fuji Silysia Chemical Ltd., Japan), used as an adsorbent, was added at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 °C, and then filtered. The filtrate was dried at 30 °C under reduced pressure and subjected to a hexane precipitation process. (iv) The filtrate obtained in the adsorbent treatment step was added to n-hexane to obtain a crude paclitaxel (filtrate/ hexane = 1/10, v/v) [18,23].
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2.4. Acetone/pentane precipitation Acetone/pentane precipitation with increased surface area is shown in Fig. 1. The crude paclitaxel (purity 38.5%) obtained from the hexane precipitation is dissolved in acetone (0.02 g crude paclitaxel/1.196 mL acetone) and drops of n-pentane are added at a ratio of 1/7 (acetone/pentane, v/v) under agitation (335 rpm) [16]. Turning off the agitating operation, glass beads (0.5–0.7 mm in diameter: Glastechnique Mfg, Germany) or anion exchange resin (Amberlite IRA-400OH or Amberlite IRA-910, Rohm and Haas, USA) and cation exchange resin (Amberlite 200 or Amberlite IRA-120, Rohm and Haas, USA) were added. Ion exchange resins were dried for 1 day at 35 °C prior to use in experiments. Glass beads were washed with methanol and dried prior to use. The experiment was conducted with the surface area per working volume (S/V) of the reacting solution fixed at the optimal level of 0.428 mm 1, as suggested in a previous study [18]. In order to obtain the paclitaxel precipitate after the addition of the surface area increasing agent, the preparation was stored for 24 h at 6.5 °C. After the precipitation period, the paclitaxel precipitate was filtered and vacuum dried (635 mm Hg, 30 °C). The morphology and the size of the paclitaxel precipitate in the acetone/pentane precipitation process were observed using an electron microscope (SV-35 Video Microscope system, Some Tech., Korea) [18]. The paclitaxel precipitate from the acetone/pentane precipitation process was then observed at high magnification (100). Video observation of the paclitaxel precipitate was performed using an IT-Plus system (Some Tech., Korea). Also, the difference in the zeta potential (ELS-Z, Photal, Japan) between the precipitation solution with and without addition of a surface area increasing agent was examined. 3. Results and discussion 3.1. Effect of increasing S/V of the reacting solution Successful formation of paclitaxel precipitate could be achieved when glass beads or an anion exchange resin, Amberlite IRA400OH, was used as a surface area increasing agent in the high surface area acetone/pentane precipitation process. The yield increased slowly with precipitation time (12–24 h), and the yield at 24 h of precipitation was 94.4% in the control without an increase of the internal surface area of the precipitator, 98.7% when glass beads were added to increase the surface, area and 60.4% when an ion exchange resin, Amberlite IRA-400OH, was added (Fig. 2). Compared with the results of the control, the addition of glass beads to increase the surface area improved the yield whereas the addition of Amberlite IRA-400OH resulted in a decreased yield. The results of the analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation indicated that the yield of paclitaxel decreased. This is
Fig. 1. Schematic diagram of high surface area acetone/pentane precipitation for purification of paclitaxel from crude extracts.
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Fig. 2. Effect of increased surface area per working volume (S/V: 0.428 mm yield of paclitaxel from acetone/pentane precipitation.
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presumably because some paclitaxel is adsorbed to Amberlite IRA400OH. On the other hand, almost no formation of paclitaxel precipitate was observed when the anion exchange resin, Amberlite IRA-910, and the cation exchange resin, Amberlite 200, IRA-120, were used as surface area increasing agents. The results of analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation revealed that most of the paclitaxel was adsorbed to the ion exchange resin when Amberlite IRA-910 or Amberlite 200, IRA-120 was used (Fig. 3(B–D)). The purity after a precipitation period of 24 h was 54.0% in the control without an increase of the internal surface area in the reactor, 54.2% when glass beads were added to increase the surface
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area, and 77.7% when the anion exchange resin, Amberlite IRA400OH, was added (Fig. 4). The purity increased slowly with the precipitation time (12–24 h). Compared with the results of the control, the addition of glass beads for increased surface area showed almost no effect in terms of improving purity whereas the addition of anion exchange resin, Amberlite IRA-400OH, improved the purity considerably. The results of the analysis using HPLC (Fig. 3(A)) after washing the ion exchange resin with methanol after precipitation indicated that almost no adsorption of paclitaxel or impurities occurred when glass beads were added (Fig. 3(E)). However, when Amberlite IRA-400OH was added (Fig. 3(A)), the purity of the precipitate was relatively higher, presumably because a considerable portion of the impurities as well as paclitaxel was adsorbed to the ion exchange resin. These results are in contrast with the results of the methanol/water fractional precipitation process [19], where increasing the surface area per working volume (S/V) of reacting solution (S/V: 0.428 mm 1) by adding a surface area increasing agent (Amberlite IRA-400OH) resulted in almost no improvement of paclitaxel purity while it improved the yield. This is presumably due to the difference in the type and the properties of the solvent used in the precipitation of paclitaxel [18,19]. 3.2. Change in shape and size of the paclitaxel precipitate over time The morphology and sizes of the paclitaxel precipitate in the acetone/pentane precipitation process with increased surface area were observed using an electron microscope. It could be seen that the precipitate branched out from around the nucleus, forming a general shape of a disk. The size of the paclitaxel precipitate grew steadily through the precipitation period (Fig. 5). Also, the number of branches from paclitaxel particles increased with time, forming
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Fig. 4. Effect of increased surface area per working volume (S/V: 0.428 mm purity of paclitaxel from acetone/pentane precipitation.
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denser structures as the amount of precipitation thus increased. Compared with the results of the control, the size of the paclitaxel precipitate decreased considerably within the same precipitation time frame when the surface area increasing agent, glass beads, or the ion exchange resin, Amberlite IRA-400OH, were added (Fig. 6). The size of the paclitaxel precipitate at the same precipitation time was in the order of control > glass beads > Amberlite IRA400OH, showing that the smallest paclitaxel precipitate could be obtained when Amberlite IRA-400OH is added. This is presumably because the growth of crystals was impeded by the addition of glass beads or Amberlite IRA-400OH as compared with the results of the control. Also, particle size differed depending on the type of surface area increasing agent used. This is likely due to the difference in the affinity between the surface area increasing agent and paclitaxel particles. In other words, Amberlite IRA-400OH acts as an effective steric barrier, inhibiting the growth of paclitaxel particles, since its affinity to the paclitaxel particles is higher than that of the surface area increasing agent, glass beads. This phenomenon has also been observed with active pharmaceutical ingredients,
Fig. 6. Effect of increased surface area per working volume (S/V: 0.428 mm 1) on the size of the paclitaxel precipitate through precipitation time in acetone/pentane precipitation.
megestrol acetate [24], atorvastatin calcium [25], and spironolactone [26]. The particle sizes of these active pharmaceutical ingredients can be effectively controlled by adding high molecular substances in anti-solvent precipitation processes. From the results of this study, it was found that the size of the paclitaxel precipitate is much smaller in the acetone/pentane precipitation process than in the methanol/water fractional precipitation process [18] regardless of whether a surface area increasing agent is employed. From measurement of the zeta potential values of the solution with or without the addition of a surface area increasing agent, the value was 0.52 mV when no surface area increasing agent was added (control), 0.15 mV when glass beads were added as a surface area increasing agent, and 21.36 mV when Amberlite IRA-400OH was added as a surface area increasing agent. These results indicate that the addition of Amberlite IRA-400 contributes to stabilization of the precipitation solution, as a high zeta potential value can be achieved. More specifically, the addition of Amberlite
Fig. 5. Electron micrograph of the paclitaxel precipitate from high surface area acetone/pentane precipitation. Scale bar indicates 10 lm.
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IRA-400OH produces crystals of small sizes, which remain stabilized in the solution without coagulating [24,25]. In the case of active pharmaceutical ingredients (API), their particle sizes are generally manipulated to be smaller during the crystallization process in order to enhance their usability. This is because a better dissolution rate, uniformity of drug dispersion, and oral bioavailability can be achieved with smaller particle size [24,27]. Furthermore, smaller particle size facilitates the removal of residual water and solvent during the drying process after purification [16]. From this point of view, paclitaxel with reduced particle size due to the addition of a surface area increasing agent during the acetone/pentane process is believed to be useful in respect of the usability of the drug. 4. Conclusions This goal of this study was to identify an optimal acetone/pentane precipitation strategy by evaluating the efficiency and the state as well as the shape and the size of paclitaxel precipitates as the surface area per working volume (S/V) of the reacting solution is increased (S/V: 0.428 mm 1) in the acetone/pentane precipitation process for separation/purification of the anticancer agent paclitaxel from plant cell cultures. The yield after 24 h of precipitation was 94.4% in the control where the internal surface area of the reactor was not increased, 98.7% when glass beads were added to increase the surface area, and 60.4% when an anion exchange resin, Amberlite IRA-400OH, was added. The yield increased slowly with precipitation time (12–24 h). The use of glass beads as a surface area increasing agent improved the yield compared with the results of the control while the use of IRA-400OH resulted in decreased yield. Purity increased slowly with precipitation time. After 24 h of precipitation, the purity was 54.0% in the control where the internal surface area of the reactor was not increased, 54.2% when glass beads were added to increase the surface area, and 77.7% when an anion exchange resin, Amberlite IRA-400OH, was added. Compared with the results of the control, almost no improvement in purity was achieved with the use of glass beads as a surface area increasing agent while the purity was considerably enhanced when Amberlite IRA-400OH was employed. The size of the precipitate increased with precipitation time. At the same precipitation time, the size of the paclitaxel precipitate decreased considerably when the surface area increasing agent, glass beads, or the ion exchange resin, Amberlite IRA-400OH, was added. The size of the precipitate was in the order of control > glass beads > Amberlite IRA-400OH, indicating that the smallest paclitaxel precipitate could be obtained when the anion exchange resin, Amberlite IRA-400OH, was added. Also, it was found that the addition of Amberlite IRA-400OH as a surface area increasing agent resulted in a decrease of the zeta potential value, stabilizing small crystals in the solution. From these results, it was found that the precipitation behavior varies considerably depending on the use of the solvent system (methane/water or acetone/pentane). Acknowledgment This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0010907).
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