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Effect of reactor type on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process
Separation and Purification Technology 99 (2012) 14–19
Contents lists available at SciVerse ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Effect of reactor type on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process Insoo Kang, 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 6 March 2012 Received in revised form 13 August 2012 Accepted 21 August 2012 Available online 30 August 2012 Keywords: Paclitaxel Purification Increased surface area fractional precipitation Reactor type Evaluation
a b s t r a c t In this study, we evaluated the effect of reactor type with increased surface area on the fractional precipitation process for the efficient purification of paclitaxel. The three types of reactors (spiral, round, and twisted types) all exhibited higher yields than in cases where the surface area had not been increased at the same precipitation time. It was possible to obtain a high paclitaxel yield of 70% or above after 4 h of precipitation. In particular, in the case of the spiral-type reactor, high yields of 64% and 72%, respectively, were obtained 2 h and 4 h after precipitation, thus exhibiting the highest precipitation efficiency. In addition, it was also possible to dramatically reduce the time necessary for fractional precipitation. On the other hand, the reactor type hardly affected the purity. The size of the paclitaxel precipitate increased with precipitation time. With the spiral-, round-, and twisted-type reactors, it was possible to obtain paclitaxel precipitates with smaller sizes than cases where the surface area had not been increased. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is a diterpenoid anticancer agent discovered in the bark of the yew tree. Its chemical structure was described in 1971 [1]. The anticancer mechanism of paclitaxel, which differs from those of other anticancer drugs, was revealed in 1979 to be the restriction of cancer cell division in the mitotic phase with relatively low toxicity and high activity [2]. It was approved by the Food and Drug Administration (FDA) as a treatment for ovarian cancer in 1992, breast cancer in 1994, Kaposi’s sarcoma in 1997, and non-small cell lung cancer (NSCLC) in 1999. Paclitaxel is currently the most widely used anticancer drug. Its application to the treatment of diseases including rheumatic arthritis and Alzheimer’s disease continues to be expanded, and clinical tests on combined prescription with many other treatment methods are under way, and thus demand for paclitaxel is expected to increase continuously in the future [3,4]. There are three main production methods for paclitaxel. The first method is direct extraction from yew trees [5], where both continuous supply of raw materials and extraction/purification are difficult. Furthermore, this approach requires the harvest of yew trees, which are environmentally protected in some areas. Second, precursors (baccatin III, 10-deacetylbaccatin III, 10-deacetylpaclitaxel, etc.) can be obtained from yew tree leaves and side
⇑ 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. http://dx.doi.org/10.1016/j.seppur.2012.08.025
chains are chemically bonded via the semi-synthesis method [6]. Because this method likewise must obtain precursors from yew trees, it has problems identical to those of direct extraction. Third, calluses are induced from yew trees, and plant cells are cultured in a bioreactor through seed culture [7]. Among these methods, the third has the advantage that stable production in the bioreactor is possible without being affected by external factors such as the climate and the environment, and thus it is possible to mass produce paclitaxel of consistent quality. Other methods include the total synthesis method [8] and production through microbial fermentation [9], but there remain many difficulties with commercialization due to low paclitaxel yields. To obtain the high purity (>98%) of paclitaxel from plant cell cultures, several separation and purification steps are required. Typically, after biomass (plant cells containing paclitaxel) is collected from the cell culture broth and is extracted with an organic solvent, pre-purification and final purification processes follow. In these processes, the pre-purification process has a particularly strong impact on the final purification costs [10–12]. Processes reported in the literature [13–15] either use highly expensive chromatography methods for pre-purification or directly process the crude extract using high performance liquid chromatography (HPLC) without pre-purification; the latter approach entails serious problems with cost as well as with scaling-up and industrial mass production. 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
I. Kang, J.-H. Kim / Separation and Purification Technology 99 (2012) 14–19
solvent is consumed, the lifetime 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 [16]. In 2000, an effective pre-purification process was first developed that enabled high purity (>50%) of paclitaxel to be obtained by fractional precipitation [16]. After biomass was collected from the culture broth 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, this method requires a very long precipitation time (up to 3 days), significantly limiting its application to mass production [10,12,17]. Improved fractional precipitation processes were reported in 2009 and 2010 [18,19] in which efficiency was enhanced by increasing the surface area per working volume (S/V) using glass beads or ion exchange resin (Amberlite 200, Amberlite IRA 400). However, fractional precipitation has only been performed using spherical surface area-increasing materials (glass beads, ion exchange resin), and the evaluation of fractional precipitation efficiency using diverse methods to increase the surface area is considerably inadequate. In this study, therefore, we evaluated the effect of reactor type, which is prepared by different method to increase the surface area, on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process. The results are expected to contribute considerably to the improvement of precipitation efficiency by evaluating the effects of the reactor type on fractional precipitation. 2. Materials and methods 2.1. Plant materials and culture conditions A suspension of cells originating from Taxus chinensis was maintained in darkness at 24.8 °C under shaking at 150 rpm. The cells were cultured in a modified Gamborg‘s B5 medium [20] 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) ethanesulphonic 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 culture as an elicitor and maltose (1 and 2%, w/v) was added to the medium at days 7 and 21, respectively [21]. Following cultivation, biomass was recovered using a decanter (Westfalia, CA150 Clarifying Decanter) and a high-speed centrifuge (Alfa-Laval, BTPX 205GD-35CDEFP). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Dried residue was redissolved in methanol for a quantitative analysis using an HPLC system (SCL-10AVP; Shimadzu, Japan) with a Capcell Pak C18 column (250 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 [22]. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard. 2.3. Preparation of crude extracts for fractional precipitation A crude extract was prepared from biomass obtained from T. chinensis cultures using the following steps. (i) Biomass was mixed
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with methanol and agitated 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. (ii) Methylene chloride (25% of concentrated methanol extract) was added and liquid–liquid extraction was performed three times for 30 min. During the extraction, polar impurities were dissolved in the methanol layer, which eventually formed the upper phase. After this layer was removed, the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) The dried crude extract obtained from the liquid–liquid extraction step was dissolved in methylene chloride at a ratio of 20% (v/ w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added at a ratio of 50% (w/w) of dried crude extract. The mixture was agitated for 30 min at 40 °C, and then filtered. (iv) The filtrate obtained in the adsorbent treatment step was added to nhexane to obtain a precipitate (filtrate/hexane = 1/10, v/v). The crude paclitaxel precipitate was filtered and dried under a vacuum at 635 mmHg and 30 °C. The dried material (purity: 42.9%) obtained from the hexane precipitation was subjected to fractional precipitation [22]. 2.4. Reactor type for fractional precipitation Three types of aluminum reactors prepared for fractional precipitation are shown in Fig. 1. In order to investigate the effects of the reactor type on the fractional precipitation process, three different types of reactors (spiral, round, and twisted types) were prepared. The experiment was conducted with the S/V of the reacting solution fixed at the optimal level of 0.428 mm1, as suggested in a previous study [18]. The reactor size and working volume were 40 and 2.84 mL, respectively. The S/V was calculated as follows:
S=V ¼ ½Total surface area inside the reactor=working volume ð1Þ 2.5. Fractional precipitation The crude extract obtained from hexane precipitation was subjected to fractional precipitation utilizing the difference in the solubility of paclitaxel in methanol solutions [10,12]. Three different types of reactors (Fig. 1) were used to increase the S/V. Fig. 2 shows the fractional precipitation process using the spiral-type reactor, among the three different types of reactors. The crude extract (purity: 42.9%) obtained through hexane precipitation was dissolved in methanol and the solution was diluted with distilled water to 61.5% (v/v) methanol. After completing the addition of distilled water, agitation was stopped, surface area-increasing materials prepared to increase the S/V were added. The experiments were conducted with the S/V fixed at 0.428 mm1, a value optimized through earlier study [18]. The solution was maintained at 4 °C to obtain a paclitaxel precipitate. The resultant precipitate was filtered and dried at 35 °C for 24 h under a vacuum, and analyzed using HPLC. 2.6. Analysis of the shapes and sizes of the paclitaxel precipitate The paclitaxel precipitate was visualized during the fractional precipitation process with an SV-35 Video Microscope System (Some Tech, Korea) at high magnification (100 times). The sizes and shapes of paclitaxel particles in dynamic images were verified with IT-Plus software (Some Tech, Korea).
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Fig. 1. Three different types of reactors for the increased surface area fractional precipitation: spiral type (A), round type (B), and twisted type (C).
Fig. 2. Schematic diagram of increased surface area fractional precipitation for purification of paclitaxel from crude extracts.
3. Results and discussion 3.1. Effect of the reactor type on the purity and yield of paclitaxel Fractional precipitation is a very simple method for the efficient purification of paclitaxel in high yield that relies on solubility differences. It has also been observed that paclitaxel precipitates as a thin layer on the bottom and walls of the reactor. In most cases of crystallization, the nuclei are generated with the assistance of external surfaces (external impurity particles, reactor walls, agitators, etc.) [23]. Based on this reason, a recently reported fractional precipitation process [18,19], increasing the S/V by using spherical glass beads or ion exchange resin, and thus the surface space available for precipitation, reduces the precipitation time. For this experiments, in addition to spherical glass beads, three different types of reactors (spiral, round, and twisted types) were produced to evaluate the effect of reactor type on the purity and yield of paclitaxel according to changes in the fractional precipitation time (2 h, 4 h, 8 h, 12 h, and 16 h). As can be seen in Fig. 3, the purity of paclitaxel increased with precipitation time. However, cases where surface area-increasing materials had been added exhibited nearly no difference in purity from the control without surface area-increasing materials, thus showing that the reactor type (surface area-increasing method) had a negligible effect on the purity. In the case of the yield, overall, the yield tended to increase with precipitation time. Up to 4 h after precipitation, the yield increased dramatically, and the rate of increase later gradually slowed. The yield of the control, where the surface area had not been increased, and in the case where glass beads had been added gradually increased 4 h after precipitation, reaching approximately 70% after a precipitation time of 16 h and 12 h, respectively. On the other hand, in cases where the surface area in the reactors had been
Fig. 3. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm1) on the purity (A) and yield (B) of paclitaxel from 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), 42.9%, and 4 °C, respectively.
increased with the spiral, round, and twisted configurations, it was possible to obtain a paclitaxel yield of 70% or more after 4 h of precipitation, thus dramatically reducing the time required for precipitation. In addition, after 4 h of precipitation, the yield showed little change. The driving force that induces precipitation tends to change the supersaturated and unstable state of the solution, or the solution in a state of supersaturation, into a stable state following a metastable state through crystal growth. Consequently, in a saturated solution or an unsaturated solution, neither nucle-
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Fig. 4. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 2 h, 16 h: control (A); spiral type (B); round type (C); twisted type (D); and glass beads (E). Scale bar indicates 10 lm.
ation nor nucleus growth occurs [18,24]. In particular, when the reactor surface area was increased with the spiral type, it was possible to obtain the greatest paclitaxel yield after 2 h of precipitation, thus showing that this type of reactor surpassed others in terms of precipitation efficiency. This appears to be because the surface area-increasing materials of the spiral type effectively increased the surface area within the reactor in comparison with other surface area-increasing materials. Such results constitute a reduction in the fractional precipitation time in comparison with
the results of already reported fractional precipitation using spherical glass beads [18]. 3.2. Changes in the shapes and sizes of the paclitaxel precipitate The shapes and sizes of paclitaxel precipitates according to the reactor type in the fractional precipitation process were investigated using electron microscopy. While it is important to obtain a high yield of high-purity products in precipitation, the shapes
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Fig. 5. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm1) on the particle size (radius) 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), 42.9%, and 4 °C, respectively.
in the magma for the growth of precipitates. When glass beads were placed in the reactor, up to the precipitation time of 4 h, paclitaxel precipitates grew with precipitation time, and exhibited nearly no change afterwards. In comparison with the control, and the case where glass beads had been added inside the reactor, in the cases of the spiral-, round-, and twisted-type reactors, the particle sizes increased gradually and paclitaxel precipitate sizes decreased far more. This is because, unlike the case where glass beads had been added, where precipitates were thinly spread on the floor of the reactor, surface area-increasing materials (spiral, round, and twisted types), which had effectively established independent space inside the reactor, acted as an effective steric barriers [26–28]. In addition, precipitate sizes differ slightly per reactor type, likely because when surface area-increasing materials are added into the reactor, they each form different types of space in the reactor. In the case of active pharmaceutical ingredients (APIs), 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 [26,29]. Furthermore, smaller particle size facilitates the removal of residual water and solvent during the drying process after purification [21]. From this point of view, paclitaxel with reduced particle size due to the addition of a surface area-increasing material during the fractional precipitation process is believed to be useful in respect of the usability of the drug. 3.3. Relationship between the precipitate size and the paclitaxel yield
Fig. 6. The relation between particle size (radius) 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), 42.9%, and 4 °C, respectively.
and sizes of the precipitates are also important for the follow-up processes [25]. As can be seen in Fig. 4, the paclitaxel precipitates increased in the number of branches around the nuclei and grew with precipitation time (2 h and 16 h). In particular, the generated precipitates were confirmed to be laminar or discoid rather than spherical in shape, and this seems to be due to invariants, which maintain geometric similarity. With invariant crystals, the sides of normally growing crystals differ in the translation velocity, and the sides with low translation velocity according to the overlapping principle dominate the growth process. Consequently, sides with high translation velocity disappear, to near-extinction, and only those with the least translation velocity survive. Fig. 5 shows the sizes of paclitaxel precipitates according to the precipitation time. In the case of the control, the size of the paclitaxel precipitate increased with precipitation time. According to previous report [18], the growth of precipitates finally stops only when precipitates collide. In the case of the control, paclitaxel particles appear to continue growing because there is adequate space
The crystallization process can be divided into two main stages, namely nucleation and crystal growth. These two processes proceed in parallel rather than in series and depend on supersaturation. Generally, when the temperature is decreased, supersaturation increases; this in turn increases the driving force of nucleation, thus increasing the nucleation speed geometrically. In other words, stable particles created through nucleation spontaneously grow under supersaturation concentration into physically observable sizes. The saturated zone is divided into an unstable zone and a metastable zone; while new crystals are dramatically generated with the result that crystal growth does not occur in the unstable zone, existing crystals can grow without allowing the generation of new crystals in the metastable zone [24]. Fig. 6 shows the relationship between the sizes of the precipitated paclitaxel particles and the yield. Under the same conditions, as the particle size increased, so did the yield. In the cases of the spiral-, round-, and twisted-type reactors, although the particle sizes were considerably smaller than those of the control, the yield was actually higher. In the cases of the spiral-, round-, and twisted-type reactors, the generation of small but numerous particles (nucleation) led to an increase in the yield. In contrast, in the case of the control, growth of the generated particles (crystal growth) rather than nucleation appears to have led to an increase in the yield. In other words, this is because when surface area-increasing materials were placed in the reactor, the S/V was increased in comparison with the case of the control, and consequently, during the same precipitation time, a large number of nuclei were generated and grew. In addition, in the case of the control, particles whose nuclei were generated and began to grow interfered with the crystal growth of nuclei generated relatively late, thus resulting in a relatively uneven crystal size distribution (CSD). 4. Conclusions In this study, we evaluated the effects of the type of reactor with increased surface area on the fractional precipitation process,
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which is employed for efficient purification of paclitaxel, an anticancer agent, from plant cell culture. After conducting fractional precipitation while maintaining the S/V identical at 0.428 mm1 and only changing the reactor type (spiral, round, and twisted types), precipitation efficiency (purity and yield) and the shapes and sizes of the precipitates were assessed. The three types of reactors all exhibited higher yields than the control, where the surface area had not been increased, at the same precipitation time, and it was possible to obtain a high paclitaxel yield of 70% or above after 4 h of precipitation. In particular, the spiral-type reactor obtained a high yield of 64% and 72%, respectively, 2 h and 4 h after precipitation, exhibiting the highest precipitation efficiency. In addition, it was also possible to dramatically reduce the time necessary for fractional precipitation. On the other hand, the reactor type had little effect on the purity. With the spiral-, round-, and twisted-type reactors, it was possible to obtain paclitaxel precipitates with smaller sizes than the case of the control. This appears to be because surface area-increasing materials inhibited the growth of paclitaxel particles in the reactors. In the cases of the spiral-, round-, and twisted-type reactors, while the generation of small but numerous particles (nucleation) led to an increase in the yield, in the case of control, growth of the generated nuclei (crystal growth) rather than nucleation led to an increase in the yield. 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] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, J. Am. Chem. Soc. 93 (1971) 2325–2327. [2] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 665–667. [3] T.M. Mekhail, M. Markman, Paclitaxel in cancer therapy, Expert Opin. Pharmacother. 3 (2002) 755–766. [4] 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. [5] 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. [6] E. Baloglu, D.G. Kingston, A new semisynthesis of paclitaxel from baccatin III, J. Nat. Prod. 62 (1999) 1068–1071. [7] H.K. Choi, S.J. 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.
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[8] K.C. Nicolaou, Z.J.J. Liu, P.G. Nantermet, R.K. Guy, C.F. Claiborne, J. Renaud, E.A. Clouladouros, K. Paulvannan, E.J. Sorensen, Total synthesis of taxol, Nature 367 (1994) 630–634. [9] A. Stierle, G. Strobel, D. Stierle, P. Grothaus, G. Bibnami, The search for a taxolproducing microorganism among the endophytic fungi of the pacific yew, Taxus brevifolia, J. Nat. Prod. 58 (1995) 1315–1324. [10] 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. [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. 5,670,673, 1997. [14] T.P. Castor, Method and apparatus for isolating therapeutic compositions from source materials, US Patent No. 5,750,709, 1998. [15] H.N. ElSohly, E.M. Croom, M.A. ElSohly, J.D. McChesney, Methods and compositions for isolating taxanes, US Patent No. 5,618,538, 1997. [16] 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. [17] S.I. Jeon, S. Mun, J.H. Kim, Optimal temperature control in fractional precipitation for paclitaxel pre-purification, Process Biochem. 41 (2006) 276–280. [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] O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158. [21] 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. [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] W.S. Kim, E.K. Lee, Technological trend of crystallization research for bioproduct separation, Korean J. Biotechnol. Bioeng. 20 (2005) 164–176. [24] W.S. Kim, Principles and applications of crystallization technology, KIC News 10 (2007) 9–24. [25] J.W. Yoon, J.H. Kim, Establishment of a solvent map for formation of crystalline and amorphous paclitaxel by solvent evaporation process, Korean J. Chem. Eng. 28 (2011) 1918–1923. [26] 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. [27] 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. [28] 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. [29] 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
Effect of reactor type on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process Insoo Kang, 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 6 March 2012 Received in revised form 13 August 2012 Accepted 21 August 2012 Available online 30 August 2012 Keywords: Paclitaxel Purification Increased surface area fractional precipitation Reactor type Evaluation
a b s t r a c t In this study, we evaluated the effect of reactor type with increased surface area on the fractional precipitation process for the efficient purification of paclitaxel. The three types of reactors (spiral, round, and twisted types) all exhibited higher yields than in cases where the surface area had not been increased at the same precipitation time. It was possible to obtain a high paclitaxel yield of 70% or above after 4 h of precipitation. In particular, in the case of the spiral-type reactor, high yields of 64% and 72%, respectively, were obtained 2 h and 4 h after precipitation, thus exhibiting the highest precipitation efficiency. In addition, it was also possible to dramatically reduce the time necessary for fractional precipitation. On the other hand, the reactor type hardly affected the purity. The size of the paclitaxel precipitate increased with precipitation time. With the spiral-, round-, and twisted-type reactors, it was possible to obtain paclitaxel precipitates with smaller sizes than cases where the surface area had not been increased. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Paclitaxel is a diterpenoid anticancer agent discovered in the bark of the yew tree. Its chemical structure was described in 1971 [1]. The anticancer mechanism of paclitaxel, which differs from those of other anticancer drugs, was revealed in 1979 to be the restriction of cancer cell division in the mitotic phase with relatively low toxicity and high activity [2]. It was approved by the Food and Drug Administration (FDA) as a treatment for ovarian cancer in 1992, breast cancer in 1994, Kaposi’s sarcoma in 1997, and non-small cell lung cancer (NSCLC) in 1999. Paclitaxel is currently the most widely used anticancer drug. Its application to the treatment of diseases including rheumatic arthritis and Alzheimer’s disease continues to be expanded, and clinical tests on combined prescription with many other treatment methods are under way, and thus demand for paclitaxel is expected to increase continuously in the future [3,4]. There are three main production methods for paclitaxel. The first method is direct extraction from yew trees [5], where both continuous supply of raw materials and extraction/purification are difficult. Furthermore, this approach requires the harvest of yew trees, which are environmentally protected in some areas. Second, precursors (baccatin III, 10-deacetylbaccatin III, 10-deacetylpaclitaxel, etc.) can be obtained from yew tree leaves and side
⇑ 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. http://dx.doi.org/10.1016/j.seppur.2012.08.025
chains are chemically bonded via the semi-synthesis method [6]. Because this method likewise must obtain precursors from yew trees, it has problems identical to those of direct extraction. Third, calluses are induced from yew trees, and plant cells are cultured in a bioreactor through seed culture [7]. Among these methods, the third has the advantage that stable production in the bioreactor is possible without being affected by external factors such as the climate and the environment, and thus it is possible to mass produce paclitaxel of consistent quality. Other methods include the total synthesis method [8] and production through microbial fermentation [9], but there remain many difficulties with commercialization due to low paclitaxel yields. To obtain the high purity (>98%) of paclitaxel from plant cell cultures, several separation and purification steps are required. Typically, after biomass (plant cells containing paclitaxel) is collected from the cell culture broth and is extracted with an organic solvent, pre-purification and final purification processes follow. In these processes, the pre-purification process has a particularly strong impact on the final purification costs [10–12]. Processes reported in the literature [13–15] either use highly expensive chromatography methods for pre-purification or directly process the crude extract using high performance liquid chromatography (HPLC) without pre-purification; the latter approach entails serious problems with cost as well as with scaling-up and industrial mass production. 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
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solvent is consumed, the lifetime 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 [16]. In 2000, an effective pre-purification process was first developed that enabled high purity (>50%) of paclitaxel to be obtained by fractional precipitation [16]. After biomass was collected from the culture broth 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, this method requires a very long precipitation time (up to 3 days), significantly limiting its application to mass production [10,12,17]. Improved fractional precipitation processes were reported in 2009 and 2010 [18,19] in which efficiency was enhanced by increasing the surface area per working volume (S/V) using glass beads or ion exchange resin (Amberlite 200, Amberlite IRA 400). However, fractional precipitation has only been performed using spherical surface area-increasing materials (glass beads, ion exchange resin), and the evaluation of fractional precipitation efficiency using diverse methods to increase the surface area is considerably inadequate. In this study, therefore, we evaluated the effect of reactor type, which is prepared by different method to increase the surface area, on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process. The results are expected to contribute considerably to the improvement of precipitation efficiency by evaluating the effects of the reactor type on fractional precipitation. 2. Materials and methods 2.1. Plant materials and culture conditions A suspension of cells originating from Taxus chinensis was maintained in darkness at 24.8 °C under shaking at 150 rpm. The cells were cultured in a modified Gamborg‘s B5 medium [20] 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) ethanesulphonic 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 culture as an elicitor and maltose (1 and 2%, w/v) was added to the medium at days 7 and 21, respectively [21]. Following cultivation, biomass was recovered using a decanter (Westfalia, CA150 Clarifying Decanter) and a high-speed centrifuge (Alfa-Laval, BTPX 205GD-35CDEFP). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Dried residue was redissolved in methanol for a quantitative analysis using an HPLC system (SCL-10AVP; Shimadzu, Japan) with a Capcell Pak C18 column (250 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 [22]. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard. 2.3. Preparation of crude extracts for fractional precipitation A crude extract was prepared from biomass obtained from T. chinensis cultures using the following steps. (i) Biomass was mixed
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with methanol and agitated 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. (ii) Methylene chloride (25% of concentrated methanol extract) was added and liquid–liquid extraction was performed three times for 30 min. During the extraction, polar impurities were dissolved in the methanol layer, which eventually formed the upper phase. After this layer was removed, the methylene chloride layer containing paclitaxel was collected and concentrated/dried under reduced pressure. (iii) The dried crude extract obtained from the liquid–liquid extraction step was dissolved in methylene chloride at a ratio of 20% (v/ w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added at a ratio of 50% (w/w) of dried crude extract. The mixture was agitated for 30 min at 40 °C, and then filtered. (iv) The filtrate obtained in the adsorbent treatment step was added to nhexane to obtain a precipitate (filtrate/hexane = 1/10, v/v). The crude paclitaxel precipitate was filtered and dried under a vacuum at 635 mmHg and 30 °C. The dried material (purity: 42.9%) obtained from the hexane precipitation was subjected to fractional precipitation [22]. 2.4. Reactor type for fractional precipitation Three types of aluminum reactors prepared for fractional precipitation are shown in Fig. 1. In order to investigate the effects of the reactor type on the fractional precipitation process, three different types of reactors (spiral, round, and twisted types) were prepared. The experiment was conducted with the S/V of the reacting solution fixed at the optimal level of 0.428 mm1, as suggested in a previous study [18]. The reactor size and working volume were 40 and 2.84 mL, respectively. The S/V was calculated as follows:
S=V ¼ ½Total surface area inside the reactor=working volume ð1Þ 2.5. Fractional precipitation The crude extract obtained from hexane precipitation was subjected to fractional precipitation utilizing the difference in the solubility of paclitaxel in methanol solutions [10,12]. Three different types of reactors (Fig. 1) were used to increase the S/V. Fig. 2 shows the fractional precipitation process using the spiral-type reactor, among the three different types of reactors. The crude extract (purity: 42.9%) obtained through hexane precipitation was dissolved in methanol and the solution was diluted with distilled water to 61.5% (v/v) methanol. After completing the addition of distilled water, agitation was stopped, surface area-increasing materials prepared to increase the S/V were added. The experiments were conducted with the S/V fixed at 0.428 mm1, a value optimized through earlier study [18]. The solution was maintained at 4 °C to obtain a paclitaxel precipitate. The resultant precipitate was filtered and dried at 35 °C for 24 h under a vacuum, and analyzed using HPLC. 2.6. Analysis of the shapes and sizes of the paclitaxel precipitate The paclitaxel precipitate was visualized during the fractional precipitation process with an SV-35 Video Microscope System (Some Tech, Korea) at high magnification (100 times). The sizes and shapes of paclitaxel particles in dynamic images were verified with IT-Plus software (Some Tech, Korea).
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Fig. 1. Three different types of reactors for the increased surface area fractional precipitation: spiral type (A), round type (B), and twisted type (C).
Fig. 2. Schematic diagram of increased surface area fractional precipitation for purification of paclitaxel from crude extracts.
3. Results and discussion 3.1. Effect of the reactor type on the purity and yield of paclitaxel Fractional precipitation is a very simple method for the efficient purification of paclitaxel in high yield that relies on solubility differences. It has also been observed that paclitaxel precipitates as a thin layer on the bottom and walls of the reactor. In most cases of crystallization, the nuclei are generated with the assistance of external surfaces (external impurity particles, reactor walls, agitators, etc.) [23]. Based on this reason, a recently reported fractional precipitation process [18,19], increasing the S/V by using spherical glass beads or ion exchange resin, and thus the surface space available for precipitation, reduces the precipitation time. For this experiments, in addition to spherical glass beads, three different types of reactors (spiral, round, and twisted types) were produced to evaluate the effect of reactor type on the purity and yield of paclitaxel according to changes in the fractional precipitation time (2 h, 4 h, 8 h, 12 h, and 16 h). As can be seen in Fig. 3, the purity of paclitaxel increased with precipitation time. However, cases where surface area-increasing materials had been added exhibited nearly no difference in purity from the control without surface area-increasing materials, thus showing that the reactor type (surface area-increasing method) had a negligible effect on the purity. In the case of the yield, overall, the yield tended to increase with precipitation time. Up to 4 h after precipitation, the yield increased dramatically, and the rate of increase later gradually slowed. The yield of the control, where the surface area had not been increased, and in the case where glass beads had been added gradually increased 4 h after precipitation, reaching approximately 70% after a precipitation time of 16 h and 12 h, respectively. On the other hand, in cases where the surface area in the reactors had been
Fig. 3. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm1) on the purity (A) and yield (B) of paclitaxel from 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), 42.9%, and 4 °C, respectively.
increased with the spiral, round, and twisted configurations, it was possible to obtain a paclitaxel yield of 70% or more after 4 h of precipitation, thus dramatically reducing the time required for precipitation. In addition, after 4 h of precipitation, the yield showed little change. The driving force that induces precipitation tends to change the supersaturated and unstable state of the solution, or the solution in a state of supersaturation, into a stable state following a metastable state through crystal growth. Consequently, in a saturated solution or an unsaturated solution, neither nucle-
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Fig. 4. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 2 h, 16 h: control (A); spiral type (B); round type (C); twisted type (D); and glass beads (E). Scale bar indicates 10 lm.
ation nor nucleus growth occurs [18,24]. In particular, when the reactor surface area was increased with the spiral type, it was possible to obtain the greatest paclitaxel yield after 2 h of precipitation, thus showing that this type of reactor surpassed others in terms of precipitation efficiency. This appears to be because the surface area-increasing materials of the spiral type effectively increased the surface area within the reactor in comparison with other surface area-increasing materials. Such results constitute a reduction in the fractional precipitation time in comparison with
the results of already reported fractional precipitation using spherical glass beads [18]. 3.2. Changes in the shapes and sizes of the paclitaxel precipitate The shapes and sizes of paclitaxel precipitates according to the reactor type in the fractional precipitation process were investigated using electron microscopy. While it is important to obtain a high yield of high-purity products in precipitation, the shapes
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Fig. 5. Effect of the material used to increase surface area per working volume (S/V: 0.428 mm1) on the particle size (radius) 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), 42.9%, and 4 °C, respectively.
in the magma for the growth of precipitates. When glass beads were placed in the reactor, up to the precipitation time of 4 h, paclitaxel precipitates grew with precipitation time, and exhibited nearly no change afterwards. In comparison with the control, and the case where glass beads had been added inside the reactor, in the cases of the spiral-, round-, and twisted-type reactors, the particle sizes increased gradually and paclitaxel precipitate sizes decreased far more. This is because, unlike the case where glass beads had been added, where precipitates were thinly spread on the floor of the reactor, surface area-increasing materials (spiral, round, and twisted types), which had effectively established independent space inside the reactor, acted as an effective steric barriers [26–28]. In addition, precipitate sizes differ slightly per reactor type, likely because when surface area-increasing materials are added into the reactor, they each form different types of space in the reactor. In the case of active pharmaceutical ingredients (APIs), 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 [26,29]. Furthermore, smaller particle size facilitates the removal of residual water and solvent during the drying process after purification [21]. From this point of view, paclitaxel with reduced particle size due to the addition of a surface area-increasing material during the fractional precipitation process is believed to be useful in respect of the usability of the drug. 3.3. Relationship between the precipitate size and the paclitaxel yield
Fig. 6. The relation between particle size (radius) 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), 42.9%, and 4 °C, respectively.
and sizes of the precipitates are also important for the follow-up processes [25]. As can be seen in Fig. 4, the paclitaxel precipitates increased in the number of branches around the nuclei and grew with precipitation time (2 h and 16 h). In particular, the generated precipitates were confirmed to be laminar or discoid rather than spherical in shape, and this seems to be due to invariants, which maintain geometric similarity. With invariant crystals, the sides of normally growing crystals differ in the translation velocity, and the sides with low translation velocity according to the overlapping principle dominate the growth process. Consequently, sides with high translation velocity disappear, to near-extinction, and only those with the least translation velocity survive. Fig. 5 shows the sizes of paclitaxel precipitates according to the precipitation time. In the case of the control, the size of the paclitaxel precipitate increased with precipitation time. According to previous report [18], the growth of precipitates finally stops only when precipitates collide. In the case of the control, paclitaxel particles appear to continue growing because there is adequate space
The crystallization process can be divided into two main stages, namely nucleation and crystal growth. These two processes proceed in parallel rather than in series and depend on supersaturation. Generally, when the temperature is decreased, supersaturation increases; this in turn increases the driving force of nucleation, thus increasing the nucleation speed geometrically. In other words, stable particles created through nucleation spontaneously grow under supersaturation concentration into physically observable sizes. The saturated zone is divided into an unstable zone and a metastable zone; while new crystals are dramatically generated with the result that crystal growth does not occur in the unstable zone, existing crystals can grow without allowing the generation of new crystals in the metastable zone [24]. Fig. 6 shows the relationship between the sizes of the precipitated paclitaxel particles and the yield. Under the same conditions, as the particle size increased, so did the yield. In the cases of the spiral-, round-, and twisted-type reactors, although the particle sizes were considerably smaller than those of the control, the yield was actually higher. In the cases of the spiral-, round-, and twisted-type reactors, the generation of small but numerous particles (nucleation) led to an increase in the yield. In contrast, in the case of the control, growth of the generated particles (crystal growth) rather than nucleation appears to have led to an increase in the yield. In other words, this is because when surface area-increasing materials were placed in the reactor, the S/V was increased in comparison with the case of the control, and consequently, during the same precipitation time, a large number of nuclei were generated and grew. In addition, in the case of the control, particles whose nuclei were generated and began to grow interfered with the crystal growth of nuclei generated relatively late, thus resulting in a relatively uneven crystal size distribution (CSD). 4. Conclusions In this study, we evaluated the effects of the type of reactor with increased surface area on the fractional precipitation process,
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which is employed for efficient purification of paclitaxel, an anticancer agent, from plant cell culture. After conducting fractional precipitation while maintaining the S/V identical at 0.428 mm1 and only changing the reactor type (spiral, round, and twisted types), precipitation efficiency (purity and yield) and the shapes and sizes of the precipitates were assessed. The three types of reactors all exhibited higher yields than the control, where the surface area had not been increased, at the same precipitation time, and it was possible to obtain a high paclitaxel yield of 70% or above after 4 h of precipitation. In particular, the spiral-type reactor obtained a high yield of 64% and 72%, respectively, 2 h and 4 h after precipitation, exhibiting the highest precipitation efficiency. In addition, it was also possible to dramatically reduce the time necessary for fractional precipitation. On the other hand, the reactor type had little effect on the purity. With the spiral-, round-, and twisted-type reactors, it was possible to obtain paclitaxel precipitates with smaller sizes than the case of the control. This appears to be because surface area-increasing materials inhibited the growth of paclitaxel particles in the reactors. In the cases of the spiral-, round-, and twisted-type reactors, while the generation of small but numerous particles (nucleation) led to an increase in the yield, in the case of control, growth of the generated nuclei (crystal growth) rather than nucleation led to an increase in the yield. 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] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, J. Am. Chem. Soc. 93 (1971) 2325–2327. [2] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 665–667. [3] T.M. Mekhail, M. Markman, Paclitaxel in cancer therapy, Expert Opin. Pharmacother. 3 (2002) 755–766. [4] 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. [5] 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. [6] E. Baloglu, D.G. Kingston, A new semisynthesis of paclitaxel from baccatin III, J. Nat. Prod. 62 (1999) 1068–1071. [7] H.K. Choi, S.J. 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.
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