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Evaluation of the effect of crude extract purity and pure paclitaxel content on the increased surface area fractional precipitation process for the purification of paclitaxel
Process Biochemistry 47 (2012) 2388–2397
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Evaluation of the effect of crude extract purity and pure paclitaxel content on the increased surface area fractional precipitation process for the purification of paclitaxel Ji-Yeon Lee, Jin-Hyun Kim ∗ Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
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Article history: Received 17 July 2012 Received in revised form 24 September 2012 Accepted 26 September 2012 Available online 4 October 2012 Keywords: Paclitaxel Purification Increased surface area fractional precipitation Ion exchange resin Precipitation behavior
a b s t r a c t This study evaluated the effect of crude extract purity and pure paclitaxel content on the behavior in terms of purity, yield, fractional precipitation time, and precipitate shape and size of fractional precipitation in the increased surface area fractional precipitation process for the purification of paclitaxel. With increased pure paclitaxel content and crude extract purity, the purity and yield of paclitaxel were improved and the fractional precipitation time was reduced. Regardless of changes in crude extract purity and pure paclitaxel content, it was possible to obtain a small paclitaxel precipitate size by hindering the growth of precipitate particles using an ion exchange resin to increase the surface area. In addition, according to the type of surface-area increasing substance used, precipitate size and shape differed because of a differing affinity for the paclitaxel particles. The lower the crude extract purity and pure paclitaxel content, the higher the yield and the improvement in purity in the process of increased surface area fractional precipitation, with a greater effect on the decrease in paclitaxel particle size. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid anticancer substance found in the bark of the yew tree, has won FDA approval for the treatment of ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer, and is currently used as the most important anticancer drug [1,2]. In addition, a growing demand for paclitaxel is expected in the future with expanding applications for many diseases including rheumatoid arthritis, Alzheimer’s disease, etc., not to mention ongoing clinical experiments on combined prescription with many other therapies. The main methods of producing paclitaxel include direct extraction from the yew tree, semi-synthesis, plant cell culture, etc. Of these, plant cell culture has the merit of mass-producing a certain quality of paclitaxel because stable production is possible within the bioreactor without being affected by external factors such as climate and environment [2]. To obtain a high purity (>98%) of paclitaxel from a plant cell culture, several separation and purification steps are required. Typically, after biomass is recovered from the cell culture suspension and is extracted with an organic solvent, pre-purification and final purification processes follow. In particular, the pre-purification
∗ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.09.023
process has a significant impact on the cost of final purification [3–5]. Processes reported in the literature [6,7] 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. Usually, the purity of paclitaxel extracted from biomass using an organic solvent is around 0.5%, and it is still very low, under 10%, even after a simple pre-purification process. If the sample proceeds directly to final purification by HPLC, a large amount of organic solvent is consumed, the life of the column packing material (resin) is reduced, and throughput is decreased. Therefore, the cost of final purification, especially when HPLC is used, can be reduced if the purity of the pre-purified sample is increased as much as possible [8]. In 2000, an efficient pre-purification method was developed that enabled a high purity (>50%) of paclitaxel to be obtained by fractional precipitation [8]. After biomass was recovered from the plant cell culture suspension 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 (∼3 days), significantly limiting its application to mass
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Biomass (10 kg) Biomass extraction with methanol (biomass/methanol=1:1 (w/v), four times) → Biomass (discard) Concentration Liquid-liquid extraction with methylene chloride (concentrated methanol/methylene chloride=4:1 (v/v), three times) → Aqueous phase (discard) Dryness (76.7 g) Adsorbent treatment with sylopute (dried crude extract/sylopute=1:0.5 (w/w)) Filtration/Dryness (52.5 g) → Sylopute (discard) Hexane precipitation (methylene chloride/hexane=1:10 (v/v)) Filtration/Dryness (16.2 g) → Filtrate (discard) Sample I (16.2 g, purity: 44.8%) Fractional precipitation (methanol/water=61.5:38.5 (v/v)) Filtration/Dryness (5.8 g) → Filtrate (discard) Sample II (5.8 g, purity: 75.3%) Fig. 1. Preparation of sample for fractional precipitation.
production [3,4,9]. In 2004 and 2008, a pre-purification process involving micelles and precipitation was reported [2,10]. Although these processes enabled paclitaxel of 20–30% purity to be obtained, this level was still considered insufficient because it placed too great a burden on the purification process that followed. In 2009 and 2010, a method was reported that could decrease the time required for fractional precipitation by increasing the surface area per working volume (S/V) using glass beads or ion an exchange resin (Amberlite 200, Amberlite IRA 400) [11,12]. However, the effect of this approach was determined using a limited purity (53.9%) of crude extract and a limited number of surface-areaincreasing substances (glass beads, Amberlite 200, Amberlite IRA 400), so there was difficulty in its extensive use. Therefore, this study was intended to investigate in more detail the behavior in terms of purity, yield, time required, and precipitate shape and size of fractional precipitation according to the purity of the crude extract using diverse types of resins (Amberlite IR 120 H, Amberlite IR 120 Na, Amberlite IRA 400 OH, Amberlite IRA 400 Cl, Amberlite IRA 910). It was also intended to investigate the effect of pure paclitaxel content dissolved in methanol solution in fractional precipitation according to the purity of crude extract in order to evaluate the effect of crude extract purity and pure paclitaxel content in the process of increased surface area fractional precipitation. 2. Materials and methods
Fig. 2. Schematic diagram of increased surface area fractional precipitation for purification of paclitaxel.
Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea.
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. The cells were cultured in modified Gamborg’s B5 medium [13] supplemented with 30 g/L sucrose, 10 mM naphthalene acetic acid, 0.2 mM 6-benzylaminopurine, 1 g/L casein hydrolysate, and 1 g/L 2-(Nmorpholino) ethanesulfonic acid. Cell cultures were transferred to fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of the culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively [14]. Following cultivation, biomass was recovered using a decanter (CA150 Clarifying Decanter; Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD-35CDEFP;
2.2. Paclitaxel analysis Dried residue was redissolved in methanol for quantitative analysis using an HPLC system (SCL-10AVP, Shimadzu, Japan) with a Capcell Pak C18 column (250 mm × 4.6 mm; Shiseido, Japan). Elution was performed in a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm with a UV detector. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [15]. Each sample was analyzed in triplicate.
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Fig. 3. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on the yield and purity of paclitaxel during fractional precipitation. The pure paclitaxel content was 0.4% (A), 0.5% (B) and 0.6% (w/v) (C). The methanol composition in water, purity of crude extract and precipitation temperature were 61.5% (v/v), 44.8% and 4 ◦ C, respectively.
2.3. Preparation of crude extract for fractional precipitation In this study, two samples (Sample I and Sample II) were prepared to investigate the effect of crude extract purity on the process of increased surface area fractional precipitation. Sample I (purity: 44.8%) had a 1:1 (w/v) ratio of biomass to methanol. Extraction was performed four times at room temperature, and liquid–liquid extraction was performed three times by concentrating (30% of the original) the methanol extract using a rotary evaporator (CCA-1100; EYELA, Japan). To the concentrated methanol solution was added methylene chloride (25% of concentrated methanol extract), and it was stirred for 30 min until it stagnated to induce phase separation [16]. By performing liquid–liquid extraction, paclitaxel was recovered from the methylene chloride layer (lower layer) for concentration and drying after vacuum filtration with filter paper (Whatman Grade 4, 20–25 m particle retention, 150 mm diameter). To remove tar and waxy compounds derived from plant cells, the dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 ◦ C, then filtered. The filtrate was dried at 30 ◦ C under vacuum and subjected to a hexane precipitation process. Dried extract was dissolved in methylene chloride and was dropped in hexane to induce precipitation (methylene chloride/hexane = 1:10, v/v). After hexane precipitation, the paclitaxel precipitate was obtained by filtration and it was dried in a vacuum oven (UP-2000; EYELA) at 35 ◦ C for 24 h. The purity of the dried extract was 44.8%. For Sample II (purity: 75.3%), crude extract (pure paclitaxel basis: 0.5%, w/v) obtained through hexane precipitation was dissolved in methanol, and distilled water was added dropwise until the methanol concentration reached 61.5%. The mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate. After precipitation, the precipitate was filtered and vacuum dried for 24 h at 35 ◦ C. The purity of the dried extract was 75.3%.
Fig. 1 shows the process of preparing crude extract for fractional precipitation used in this study. 2.4. Fractional precipitation Fig. 2 shows a schematic diagram of the increased surface area fractional precipitation process using the difference in solubility of paclitaxel in methanol solution. To increase S/V of the reaction solution, the cation exchange resins Amberlte IR 120 H, Amberlite IR 120 Na, and Amberlite 200 (Sigma–Aldrich) and the anion exchange resins Amberlite IRA 400 OH, Amberlite IRA 400 Cl, and Amberlite IRA 910 (Sigma–Aldrich) were used separately. Ion exchange resin was used in the experiment after drying for one day at 35 ◦ C. The effect of pure paclitaxel content dissolved in methanol on increased surface area fractional precipitation was investigated by performing the experiment at constant crude extract purity (44.8%) and varying the pure paclitaxel content (0.4%, 0.5%, 0.6%, w/v). Also, the effect of crude extract purity was investigated by carrying out the experiment with a uniform pure paclitaxel content (0.5%) and varying the crude extract purity (44.8% and 75.3%). With crude extract dissolved in methanol, distilled water was added dropwise with stirring (180 rpm) until the methanol concentration reached 61.5% [8,11]. Ion exchange resins were added to the reacting solution and the mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate. The experiment was conducted with the S/V of the reacting solution fixed at the optimal level of 0.428 mm−1 , as suggested in a previous study [11]. The S/V was calculated for each ion exchange resin as follows: S total surface area of resin (mm2 ) (mm−1 ) = V working volume (mm3 )
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Fig. 4. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–18 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.4% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
The reactor size and experimental volume were 20 mL and 3 mL, respectively. The particle diameter and surface area of the resins were 0.508 mm and 0.811 mm2 , respectively. After fractional precipitation, the precipitate was filtered (Whatman Grade 4, 20–25 m particle retention, 150 mm diameter), vacuum dried for 24 h at 35 ◦ C and analyzed by HPLC. To measure the shape and size of paclitaxel precipitate in the process of fractional precipitation, an SV-35 Video Microscope System (Some Tech, Korea) was used [11]. During fractional precipitation, the paclitaxel precipitate was observed at high magnification (100×). The size and shape of paclitaxel precipitate in dynamic images was verified with IT-Plus software (Some Tech).
3. Results and discussion 3.1. Effects of pure paclitaxel content at constant crude extract purity To investigate the effects in terms of purity, yield, fractional precipitation time, and precipitate shape and size of the pure paclitaxel content dissolved in methanol in increased surface area fractional precipitation using diverse types of ion exchange resins, an
experiment was performed in which the pure paclitaxel content was changed (0.4%, 0.5%, or 0.6%, w/v) while the crude extract purity was held constant (44.8%). As seen in Fig. 3, as the pure paclitaxel content dissolved in methanol was increased, the yield improved and the time required for fractional precipitation was reduced. In the case of increased surface area, a higher paclitaxel yield was obtained, which had the effect of improving the precipitation time. This result is consistent with that of our previous studies [11,12]. Also, when the pure paclitaxel content was lower, the effect on paclitaxel yield of increasing the surface area was greater. That is, the lower the pure paclitaxel content, the greater the difference between the yield obtained when the surface area was increased and not increased; thus, the lower the pure paclitaxel content, the larger the effect on the yield caused by the increased surface area. Such a phenomenon is considered to be due to the fact that as pure paclitaxel content increases, the degree of supersaturation increases and thus it is possible to obtain paclitaxel precipitate in less time [17,18]. The difference in the yield according
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Fig. 5. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–12 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.5% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
to the type of ion exchange resin, which increases the surface area, was insignificant. As seen in Fig. 3, it was found that an increase in surface area generally improved purity. However, as the pure paclitaxel content was increased and precipitation time elapsed, the difference in purity became insignificant. Also, the difference in purity according to the type of ion exchange resin was also insignificant. To investigate the effect of pure paclitaxel content on the shape and size of paclitaxel precipitate, observation was performed using an electron microscope during fractional precipitation (Figs. 4–6). It was found that the paclitaxel precipitate became smaller with increased surface area, regardless of pure paclitaxel content. This phenomenon is considered to have occurred because particle growth is hindered by surface-area increasing substances [17,18]. Also, as the pure paclitaxel content was increased, nuclei were
generated faster due to the effect of high supersaturation [17], resulting in a smaller particle size. The shape of precipitates showed differences according to the type of ion exchange resin used. Ion exchange resins are classified by functional group types and acidic/basic levels. Amberlite IR-120 Na, Amberlite 200, and Amberlite IR-120 H are strongly acidic cation exchange resins, while Amberlite IRA-400 Cl, Amberlite IRA-400 OH, and Amberlite IRA-910 are strongly basic anion exchange resins. It was found that paclitaxel particles precipitated with a zeta potential value measured at −5.96 mV in the fractional precipitation solution were negatively electrically charged (data not shown). Cation exchange resins work by attractive force and the paclitaxel precipitate (nucleus) is generated on the surface of the resin, whereas the repulsive force on an anion exchange resin causes the paclitaxel precipitate to be generated at some distance from the surface of the
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Fig. 6. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–12 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.6% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
resin. Also, compared to precipates that form in the presence of an anion exchange resin, precipitates become smaller in size and the number of nuclei increases in the presence of an cation exchange resin. Such a phenomenon was also found in a previous study using the cation exchange resin Amberlite 200 and the anion exchange resin Amberlite IR 400 [12]. The size of paclitaxel precipitate particles was measured over the course of fractional precipitation and is illustrated in Fig. 7. It was found that the size of the precipitate became smaller when the surface area was increased compared to the control. It was also found that the precipitate size was relatively smaller using a cation exchange resin rather than an anion exchange resin. For all pure paclitaxel contents tested (0.4%, 0.5%, 0.6%, w/v), the smallest paclitaxel precipitate was obtained when the ion exchange resin IR 120 Na was used. The size differed according to the type of ion exchange resin used, a result that is considered to be due
to the differences in affinity for paclitaxel particles among the resins. That is, as the affinity of the S/V-increasing substance for paclitaxel particles increases, it serves as a more effective steric barrier, which inhibits the growth of paclitaxel particles [17–19]. In the case of active pharmaceutical ingredients, their particle sizes are generally manipulated to be smaller during the polishing 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 during formulation [17,20]. Furthermore, a 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 obtained due to the addition of a substance to increase the surface area during the fractional precipitation process is believed to be useful with respect to the usability of the drug.
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Fig. 8. The relationship between particle size, purity, and yield of paclitaxel during fractional precipitation. The pure paclitaxel content was 0.4% (A), 0.5% (B) and 0.6% (w/v) (C). The methanol composition in water, purity of crude extract, and precipitation temperature were 61.5% (v/v), 44.8% and 4 ◦ C, respectively.
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Fig. 9. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on the yield and purity of paclitaxel during fractional precipitation. The crude extract purity was 44.8% (A) and 75.3% (B). The methanol composition in water, pure paclitaxel content, and precipitation temperature were 61.5% (v/v), 0.5% (w/v) and 4 ◦ C, respectively.
Fig. 8 shows the relationship between the size of the paclitaxel precipitate and paclitaxel purity and yield, respectively, according to the pure paclitaxel content. In the case of increased surface area, a precipitate of smaller size was produced compared to when the surface area was not increased. It was found that as the pure paclitaxel content increased, this difference in particle size became gradually smaller. Also, while in the case of increased surface area the yield was increased mainly by nucleus generation, when the surface area was not increased, the yield increased mainly by the growth of generated nuclei. That is, in the case of increased surface area, the yield improved because the generation of small particles (precipitates) was relatively greater. Such a phenomenon is considered to occur because an S/V-increasing substance causes a decrease in the energy needed for generating nuclei, which increases the speed at which nuclei generation occurs and also increases the period of collision between the S/V-increasing substance and the nuclei, resulting in the generation of more nuclei within the same time period [22,23]. 3.2. Effect of changes in crude extract purity at constant pure paclitaxel content The effect of crude extract purity (44.8% and 75.3%) at constant pure paclitaxel content (0.5%, w/v) in increased surface area fractional precipitation using diverse types of ion exchange resins was investigated. As shown in Fig. 9, regardless of the crude extract purity, when the surface area was increased, a higher yield was obtained than when it was not increased, while reducing the time needed for fractional precipitation. Also, as crude extract purity increased, this difference in yield decreased. That is, the effect (in terms of yield) of increased surface area was higher with low crude
extract purity. The purity of paclitaxel was found to be higher with a crude extract purity of 44.8% when the surface area was increased than when it was not increased (Fig. 9). At a crude extract purity of 75.3%, the effect of improving purity by increasing the surface area was insignificant. As a result, as crude extract purity was lower, the effect caused by increasing the surface area was higher. The shape of paclitaxel precipitates obtained from the fractional precipitation process with a 0.5% (w/v) pure paclitaxel content and a 75.3% crude extract purity was observed via an electron microscope (Fig. 10). Comparison of the results with those obtained using a 44.8% crude extract purity (Fig. 5) at the same pure paclitaxel content showed that the shape of the precipitate was different due to the effect of impurities and a difference in affinity of the surface-area increasing substance for the paclitaxel particles. At a crude extract purity of 44.8%, the shape of the precipitate was much different depending on whether a cation or anion exchange resin was used (Fig. 5). At a relatively higher crude extract purity (75.3%), however, it was found that the difference in the shape of the precipitate in the presence of cation or anion exchange resins was insignificant (Fig. 10). Also, as shown in Fig. 11, regardless of the crude extract purity, particle growth of the precipitate was hindered by the addition of a surface-area increasing substance, making the particle size smaller. When the crude extract purity was lower (44.8%), increasing the surface area made more of a difference in particle size than when it was higher (75.3%). As a result, as the purity of the crude extract used in fractional precipitation was relatively lower, the yield was higher and the effect of improving the purity by increasing the surface area during fractional precipitation was greater, with a greater effect on the decrease in paclitaxel particle size at the same pure paclitaxel content (0.5%, w/v).
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Fig. 10. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–9 h: Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.5% (w/v) and 75.3%, respectively. Scale bar indicates 10 m.
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Acknowledgments This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government (MEST) (No. 2011-0010907). References
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Precipitation time (hr) Fig. 11. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on paclitaxel particle size during fractional precipitation. The crude extract purity was 44.8% (A) and 75.3% (B). The methanol composition in water, pure paclitaxel content, and precipitation temperature were 61.5% (v/v), 0.5% (w/v) and 4 ◦ C, respectively.
4. Conclusions This study evaluated the effect of crude extract purity and pure paclitaxel content on the behavior (purity, yield, fractional precipitation time, precipitate shape and size) of the increased surface area fractional precipitation process for the purification of paclitaxel. Diverse types of ion exchange resin were used to increase the surface area, and the effect of crude extract purity and pure paclitaxel content dissolved in methanol solution in fractional precipitation were investigated in detail. As the pure paclitaxel content and crude extract purity increased, paclitaxel purity and yield were improved and the time required for fractional precipitation was reduced as well. Regardless of changes in crude extract purity and pure paclitaxel content, it was possible to obtain small-sized paclitaxel precipitates due to the hindrance of precipitate particle growth by the ion exchange resin. Also, the type of surface-area increasing substance used made a difference in precipitate size and shape due to differences in affinity for paclitaxel particles. As the crude extract purity and pure paclitaxel content were decreased, the greater the yield and effect of improving the purity in the increased surface area fractional precipitation process, with a greater effect on the decrease in paclitaxel particle size.
[1] Kim JH. Paclitaxel: recovery and purification in commercialization step. Korean J Biotechnol Bioeng 2006;21:1–10. [2] Jeon KY, Kim JH. Effect of surfactant on the micelle process for the pre-purification of paclitaxel. Korean J Biotechnol Bioeng 2008;23: 557–60. [3] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. A novel prepurification for paclitaxel from plant cell cultures. Process Biochem 2002;37:679–82. [4] Pyo SH, Park HB, Song BK, Han BH, Kim JH. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem 2004;39: 1985–91. [5] Pyo SH, Song BK, Ju CH, Han BH, Choi HJ. Effects of adsorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree. Process Biochem 2005;40:1113–7. [6] Rao KV. Method for the isolation and purification of taxol and its natural analogues. US Patent 5,670,673; 1997. [7] Castor TP. Method and apparatus for isolating therapeutic compositions from source materials. US Patent 5,750,709; 1998. [8] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis. Biotechnol Lett 2000;22:1753–6. [9] Jeon SI, Mun SY, Kim JH. Optimal temperature control in fractional precipitation for paclitaxel pre-purification. Process Biochem 2006;41:276–80. [10] Kim JH. Prepurification of paclitaxel by micelle and precipitation. Process Biochem 2004;39:1567–71. [11] Jeon KY, Kim JH. Improvement of fractional precipitation process for prepurification of paclitaxel. Process Biochem 2009;44:736–41. [12] Han MG, Jeon KY, Mun SY, Kim JH. Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures. Process Biochem 2010;45:1368–74. [13] Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 1968;50:151–8. [14] Choi HK, Adams TL, Stahlhut RW, Kim SI, Yun JH, Song BK, et al. Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture. US Patent 5,871,979; 1999. [15] Lee JY, Kim JH. 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 2011;80:240–5. [16] Kim JH. Optimization of liquid–liquid extraction conditions for paclitaxel separation from plant cell cultures. Korean J Biotechnol Bioeng 2009;24: 212–5. [17] Cho EB, Cho WK, Cha KH, Park JS. Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additives. Int J Pharm 2010;396:91–8. [18] Dong Y, Ng WK, Shen S, Kim S, Tan RBH. Preparation and characterization of spironolactone nanoparticles by antisolvent precipitation. Int J Pharm 2009;375:84–8. [19] Zhang HX, Wang JX, Zhang ZB, Le Y, Shen ZG, Chen JF. Micronization of atorvastatin calcium by antisolvent precipitation process. Int J Pharm 2009;374:106–13. [20] Yeo SD, Kim MS, Lee J. Recrystallization of sulfathiazole and chlorpropamide using the supercritical fluid antisolvent process. J Supercrit Fluids 2003;25:143–54. [21] Pyo SH, Kim MS, Cho JS, Song BK, Han BH, Choi HJ. Efficient purification and morphology characterization of paclitaxel from cell cultures of Taxus chinensis. J Chem Technol Biotechnol 2005;79:1162–8. [22] Kim JH, Kim KY, Kim DK, Park HS, Lee SC, Lee SI. Effect of struvite crystallization kinetics; seed material, seed particle size, G·td value. J KSEE 2008;30: 207–12. [23] Kim SY, Kim KJ, Ryu SK. In situ analysis of growth mechanism in batch crystallization of phosphoric acid. Theor Appl Chem Eng 2002;8:3445–8.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Evaluation of the effect of crude extract purity and pure paclitaxel content on the increased surface area fractional precipitation process for the purification of paclitaxel 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 17 July 2012 Received in revised form 24 September 2012 Accepted 26 September 2012 Available online 4 October 2012 Keywords: Paclitaxel Purification Increased surface area fractional precipitation Ion exchange resin Precipitation behavior
a b s t r a c t This study evaluated the effect of crude extract purity and pure paclitaxel content on the behavior in terms of purity, yield, fractional precipitation time, and precipitate shape and size of fractional precipitation in the increased surface area fractional precipitation process for the purification of paclitaxel. With increased pure paclitaxel content and crude extract purity, the purity and yield of paclitaxel were improved and the fractional precipitation time was reduced. Regardless of changes in crude extract purity and pure paclitaxel content, it was possible to obtain a small paclitaxel precipitate size by hindering the growth of precipitate particles using an ion exchange resin to increase the surface area. In addition, according to the type of surface-area increasing substance used, precipitate size and shape differed because of a differing affinity for the paclitaxel particles. The lower the crude extract purity and pure paclitaxel content, the higher the yield and the improvement in purity in the process of increased surface area fractional precipitation, with a greater effect on the decrease in paclitaxel particle size. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid anticancer substance found in the bark of the yew tree, has won FDA approval for the treatment of ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer, and is currently used as the most important anticancer drug [1,2]. In addition, a growing demand for paclitaxel is expected in the future with expanding applications for many diseases including rheumatoid arthritis, Alzheimer’s disease, etc., not to mention ongoing clinical experiments on combined prescription with many other therapies. The main methods of producing paclitaxel include direct extraction from the yew tree, semi-synthesis, plant cell culture, etc. Of these, plant cell culture has the merit of mass-producing a certain quality of paclitaxel because stable production is possible within the bioreactor without being affected by external factors such as climate and environment [2]. To obtain a high purity (>98%) of paclitaxel from a plant cell culture, several separation and purification steps are required. Typically, after biomass is recovered from the cell culture suspension and is extracted with an organic solvent, pre-purification and final purification processes follow. In particular, the pre-purification
∗ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.09.023
process has a significant impact on the cost of final purification [3–5]. Processes reported in the literature [6,7] 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. Usually, the purity of paclitaxel extracted from biomass using an organic solvent is around 0.5%, and it is still very low, under 10%, even after a simple pre-purification process. If the sample proceeds directly to final purification by HPLC, a large amount of organic solvent is consumed, the life of the column packing material (resin) is reduced, and throughput is decreased. Therefore, the cost of final purification, especially when HPLC is used, can be reduced if the purity of the pre-purified sample is increased as much as possible [8]. In 2000, an efficient pre-purification method was developed that enabled a high purity (>50%) of paclitaxel to be obtained by fractional precipitation [8]. After biomass was recovered from the plant cell culture suspension 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 (∼3 days), significantly limiting its application to mass
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Biomass (10 kg) Biomass extraction with methanol (biomass/methanol=1:1 (w/v), four times) → Biomass (discard) Concentration Liquid-liquid extraction with methylene chloride (concentrated methanol/methylene chloride=4:1 (v/v), three times) → Aqueous phase (discard) Dryness (76.7 g) Adsorbent treatment with sylopute (dried crude extract/sylopute=1:0.5 (w/w)) Filtration/Dryness (52.5 g) → Sylopute (discard) Hexane precipitation (methylene chloride/hexane=1:10 (v/v)) Filtration/Dryness (16.2 g) → Filtrate (discard) Sample I (16.2 g, purity: 44.8%) Fractional precipitation (methanol/water=61.5:38.5 (v/v)) Filtration/Dryness (5.8 g) → Filtrate (discard) Sample II (5.8 g, purity: 75.3%) Fig. 1. Preparation of sample for fractional precipitation.
production [3,4,9]. In 2004 and 2008, a pre-purification process involving micelles and precipitation was reported [2,10]. Although these processes enabled paclitaxel of 20–30% purity to be obtained, this level was still considered insufficient because it placed too great a burden on the purification process that followed. In 2009 and 2010, a method was reported that could decrease the time required for fractional precipitation by increasing the surface area per working volume (S/V) using glass beads or ion an exchange resin (Amberlite 200, Amberlite IRA 400) [11,12]. However, the effect of this approach was determined using a limited purity (53.9%) of crude extract and a limited number of surface-areaincreasing substances (glass beads, Amberlite 200, Amberlite IRA 400), so there was difficulty in its extensive use. Therefore, this study was intended to investigate in more detail the behavior in terms of purity, yield, time required, and precipitate shape and size of fractional precipitation according to the purity of the crude extract using diverse types of resins (Amberlite IR 120 H, Amberlite IR 120 Na, Amberlite IRA 400 OH, Amberlite IRA 400 Cl, Amberlite IRA 910). It was also intended to investigate the effect of pure paclitaxel content dissolved in methanol solution in fractional precipitation according to the purity of crude extract in order to evaluate the effect of crude extract purity and pure paclitaxel content in the process of increased surface area fractional precipitation. 2. Materials and methods
Fig. 2. Schematic diagram of increased surface area fractional precipitation for purification of paclitaxel.
Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea.
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. The cells were cultured in modified Gamborg’s B5 medium [13] supplemented with 30 g/L sucrose, 10 mM naphthalene acetic acid, 0.2 mM 6-benzylaminopurine, 1 g/L casein hydrolysate, and 1 g/L 2-(Nmorpholino) ethanesulfonic acid. Cell cultures were transferred to fresh medium every 2 weeks. During prolonged culture for production purposes, 4 mM AgNO3 was added at the initiation of the culture as an elicitor, and 1% and 2% (w/v) maltose were added to the medium on days 7 and 21, respectively [14]. Following cultivation, biomass was recovered using a decanter (CA150 Clarifying Decanter; Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD-35CDEFP;
2.2. Paclitaxel analysis Dried residue was redissolved in methanol for quantitative analysis using an HPLC system (SCL-10AVP, Shimadzu, Japan) with a Capcell Pak C18 column (250 mm × 4.6 mm; Shiseido, Japan). Elution was performed in a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm with a UV detector. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [15]. Each sample was analyzed in triplicate.
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Fig. 3. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on the yield and purity of paclitaxel during fractional precipitation. The pure paclitaxel content was 0.4% (A), 0.5% (B) and 0.6% (w/v) (C). The methanol composition in water, purity of crude extract and precipitation temperature were 61.5% (v/v), 44.8% and 4 ◦ C, respectively.
2.3. Preparation of crude extract for fractional precipitation In this study, two samples (Sample I and Sample II) were prepared to investigate the effect of crude extract purity on the process of increased surface area fractional precipitation. Sample I (purity: 44.8%) had a 1:1 (w/v) ratio of biomass to methanol. Extraction was performed four times at room temperature, and liquid–liquid extraction was performed three times by concentrating (30% of the original) the methanol extract using a rotary evaporator (CCA-1100; EYELA, Japan). To the concentrated methanol solution was added methylene chloride (25% of concentrated methanol extract), and it was stirred for 30 min until it stagnated to induce phase separation [16]. By performing liquid–liquid extraction, paclitaxel was recovered from the methylene chloride layer (lower layer) for concentration and drying after vacuum filtration with filter paper (Whatman Grade 4, 20–25 m particle retention, 150 mm diameter). To remove tar and waxy compounds derived from plant cells, the dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and sylopute (Fuji Silysia Chemical Ltd., Japan), an adsorbent, was added at a rate of 50% (w/w). The mixture was agitated for 30 min at 40 ◦ C, then filtered. The filtrate was dried at 30 ◦ C under vacuum and subjected to a hexane precipitation process. Dried extract was dissolved in methylene chloride and was dropped in hexane to induce precipitation (methylene chloride/hexane = 1:10, v/v). After hexane precipitation, the paclitaxel precipitate was obtained by filtration and it was dried in a vacuum oven (UP-2000; EYELA) at 35 ◦ C for 24 h. The purity of the dried extract was 44.8%. For Sample II (purity: 75.3%), crude extract (pure paclitaxel basis: 0.5%, w/v) obtained through hexane precipitation was dissolved in methanol, and distilled water was added dropwise until the methanol concentration reached 61.5%. The mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate. After precipitation, the precipitate was filtered and vacuum dried for 24 h at 35 ◦ C. The purity of the dried extract was 75.3%.
Fig. 1 shows the process of preparing crude extract for fractional precipitation used in this study. 2.4. Fractional precipitation Fig. 2 shows a schematic diagram of the increased surface area fractional precipitation process using the difference in solubility of paclitaxel in methanol solution. To increase S/V of the reaction solution, the cation exchange resins Amberlte IR 120 H, Amberlite IR 120 Na, and Amberlite 200 (Sigma–Aldrich) and the anion exchange resins Amberlite IRA 400 OH, Amberlite IRA 400 Cl, and Amberlite IRA 910 (Sigma–Aldrich) were used separately. Ion exchange resin was used in the experiment after drying for one day at 35 ◦ C. The effect of pure paclitaxel content dissolved in methanol on increased surface area fractional precipitation was investigated by performing the experiment at constant crude extract purity (44.8%) and varying the pure paclitaxel content (0.4%, 0.5%, 0.6%, w/v). Also, the effect of crude extract purity was investigated by carrying out the experiment with a uniform pure paclitaxel content (0.5%) and varying the crude extract purity (44.8% and 75.3%). With crude extract dissolved in methanol, distilled water was added dropwise with stirring (180 rpm) until the methanol concentration reached 61.5% [8,11]. Ion exchange resins were added to the reacting solution and the mixture was kept at 4 ◦ C to obtain a paclitaxel precipitate. The experiment was conducted with the S/V of the reacting solution fixed at the optimal level of 0.428 mm−1 , as suggested in a previous study [11]. The S/V was calculated for each ion exchange resin as follows: S total surface area of resin (mm2 ) (mm−1 ) = V working volume (mm3 )
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Fig. 4. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–18 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.4% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
The reactor size and experimental volume were 20 mL and 3 mL, respectively. The particle diameter and surface area of the resins were 0.508 mm and 0.811 mm2 , respectively. After fractional precipitation, the precipitate was filtered (Whatman Grade 4, 20–25 m particle retention, 150 mm diameter), vacuum dried for 24 h at 35 ◦ C and analyzed by HPLC. To measure the shape and size of paclitaxel precipitate in the process of fractional precipitation, an SV-35 Video Microscope System (Some Tech, Korea) was used [11]. During fractional precipitation, the paclitaxel precipitate was observed at high magnification (100×). The size and shape of paclitaxel precipitate in dynamic images was verified with IT-Plus software (Some Tech).
3. Results and discussion 3.1. Effects of pure paclitaxel content at constant crude extract purity To investigate the effects in terms of purity, yield, fractional precipitation time, and precipitate shape and size of the pure paclitaxel content dissolved in methanol in increased surface area fractional precipitation using diverse types of ion exchange resins, an
experiment was performed in which the pure paclitaxel content was changed (0.4%, 0.5%, or 0.6%, w/v) while the crude extract purity was held constant (44.8%). As seen in Fig. 3, as the pure paclitaxel content dissolved in methanol was increased, the yield improved and the time required for fractional precipitation was reduced. In the case of increased surface area, a higher paclitaxel yield was obtained, which had the effect of improving the precipitation time. This result is consistent with that of our previous studies [11,12]. Also, when the pure paclitaxel content was lower, the effect on paclitaxel yield of increasing the surface area was greater. That is, the lower the pure paclitaxel content, the greater the difference between the yield obtained when the surface area was increased and not increased; thus, the lower the pure paclitaxel content, the larger the effect on the yield caused by the increased surface area. Such a phenomenon is considered to be due to the fact that as pure paclitaxel content increases, the degree of supersaturation increases and thus it is possible to obtain paclitaxel precipitate in less time [17,18]. The difference in the yield according
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Fig. 5. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–12 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.5% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
to the type of ion exchange resin, which increases the surface area, was insignificant. As seen in Fig. 3, it was found that an increase in surface area generally improved purity. However, as the pure paclitaxel content was increased and precipitation time elapsed, the difference in purity became insignificant. Also, the difference in purity according to the type of ion exchange resin was also insignificant. To investigate the effect of pure paclitaxel content on the shape and size of paclitaxel precipitate, observation was performed using an electron microscope during fractional precipitation (Figs. 4–6). It was found that the paclitaxel precipitate became smaller with increased surface area, regardless of pure paclitaxel content. This phenomenon is considered to have occurred because particle growth is hindered by surface-area increasing substances [17,18]. Also, as the pure paclitaxel content was increased, nuclei were
generated faster due to the effect of high supersaturation [17], resulting in a smaller particle size. The shape of precipitates showed differences according to the type of ion exchange resin used. Ion exchange resins are classified by functional group types and acidic/basic levels. Amberlite IR-120 Na, Amberlite 200, and Amberlite IR-120 H are strongly acidic cation exchange resins, while Amberlite IRA-400 Cl, Amberlite IRA-400 OH, and Amberlite IRA-910 are strongly basic anion exchange resins. It was found that paclitaxel particles precipitated with a zeta potential value measured at −5.96 mV in the fractional precipitation solution were negatively electrically charged (data not shown). Cation exchange resins work by attractive force and the paclitaxel precipitate (nucleus) is generated on the surface of the resin, whereas the repulsive force on an anion exchange resin causes the paclitaxel precipitate to be generated at some distance from the surface of the
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Fig. 6. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–12 h. Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.6% (w/v) and 44.8%, respectively. Scale bar indicates 10 m.
resin. Also, compared to precipates that form in the presence of an anion exchange resin, precipitates become smaller in size and the number of nuclei increases in the presence of an cation exchange resin. Such a phenomenon was also found in a previous study using the cation exchange resin Amberlite 200 and the anion exchange resin Amberlite IR 400 [12]. The size of paclitaxel precipitate particles was measured over the course of fractional precipitation and is illustrated in Fig. 7. It was found that the size of the precipitate became smaller when the surface area was increased compared to the control. It was also found that the precipitate size was relatively smaller using a cation exchange resin rather than an anion exchange resin. For all pure paclitaxel contents tested (0.4%, 0.5%, 0.6%, w/v), the smallest paclitaxel precipitate was obtained when the ion exchange resin IR 120 Na was used. The size differed according to the type of ion exchange resin used, a result that is considered to be due
to the differences in affinity for paclitaxel particles among the resins. That is, as the affinity of the S/V-increasing substance for paclitaxel particles increases, it serves as a more effective steric barrier, which inhibits the growth of paclitaxel particles [17–19]. In the case of active pharmaceutical ingredients, their particle sizes are generally manipulated to be smaller during the polishing 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 during formulation [17,20]. Furthermore, a 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 obtained due to the addition of a substance to increase the surface area during the fractional precipitation process is believed to be useful with respect to the usability of the drug.
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Fig. 8. The relationship between particle size, purity, and yield of paclitaxel during fractional precipitation. The pure paclitaxel content was 0.4% (A), 0.5% (B) and 0.6% (w/v) (C). The methanol composition in water, purity of crude extract, and precipitation temperature were 61.5% (v/v), 44.8% and 4 ◦ C, respectively.
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Fig. 9. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on the yield and purity of paclitaxel during fractional precipitation. The crude extract purity was 44.8% (A) and 75.3% (B). The methanol composition in water, pure paclitaxel content, and precipitation temperature were 61.5% (v/v), 0.5% (w/v) and 4 ◦ C, respectively.
Fig. 8 shows the relationship between the size of the paclitaxel precipitate and paclitaxel purity and yield, respectively, according to the pure paclitaxel content. In the case of increased surface area, a precipitate of smaller size was produced compared to when the surface area was not increased. It was found that as the pure paclitaxel content increased, this difference in particle size became gradually smaller. Also, while in the case of increased surface area the yield was increased mainly by nucleus generation, when the surface area was not increased, the yield increased mainly by the growth of generated nuclei. That is, in the case of increased surface area, the yield improved because the generation of small particles (precipitates) was relatively greater. Such a phenomenon is considered to occur because an S/V-increasing substance causes a decrease in the energy needed for generating nuclei, which increases the speed at which nuclei generation occurs and also increases the period of collision between the S/V-increasing substance and the nuclei, resulting in the generation of more nuclei within the same time period [22,23]. 3.2. Effect of changes in crude extract purity at constant pure paclitaxel content The effect of crude extract purity (44.8% and 75.3%) at constant pure paclitaxel content (0.5%, w/v) in increased surface area fractional precipitation using diverse types of ion exchange resins was investigated. As shown in Fig. 9, regardless of the crude extract purity, when the surface area was increased, a higher yield was obtained than when it was not increased, while reducing the time needed for fractional precipitation. Also, as crude extract purity increased, this difference in yield decreased. That is, the effect (in terms of yield) of increased surface area was higher with low crude
extract purity. The purity of paclitaxel was found to be higher with a crude extract purity of 44.8% when the surface area was increased than when it was not increased (Fig. 9). At a crude extract purity of 75.3%, the effect of improving purity by increasing the surface area was insignificant. As a result, as crude extract purity was lower, the effect caused by increasing the surface area was higher. The shape of paclitaxel precipitates obtained from the fractional precipitation process with a 0.5% (w/v) pure paclitaxel content and a 75.3% crude extract purity was observed via an electron microscope (Fig. 10). Comparison of the results with those obtained using a 44.8% crude extract purity (Fig. 5) at the same pure paclitaxel content showed that the shape of the precipitate was different due to the effect of impurities and a difference in affinity of the surface-area increasing substance for the paclitaxel particles. At a crude extract purity of 44.8%, the shape of the precipitate was much different depending on whether a cation or anion exchange resin was used (Fig. 5). At a relatively higher crude extract purity (75.3%), however, it was found that the difference in the shape of the precipitate in the presence of cation or anion exchange resins was insignificant (Fig. 10). Also, as shown in Fig. 11, regardless of the crude extract purity, particle growth of the precipitate was hindered by the addition of a surface-area increasing substance, making the particle size smaller. When the crude extract purity was lower (44.8%), increasing the surface area made more of a difference in particle size than when it was higher (75.3%). As a result, as the purity of the crude extract used in fractional precipitation was relatively lower, the yield was higher and the effect of improving the purity by increasing the surface area during fractional precipitation was greater, with a greater effect on the decrease in paclitaxel particle size at the same pure paclitaxel content (0.5%, w/v).
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Fig. 10. Electron micrograph of paclitaxel precipitate formed by fractional precipitation at 3–9 h: Control (A); cation exchange resins Amberlite IR 120 H (B), Amberlite IR 120 Na (C) and Amberlite 200 (D); and anion exchange resins Amberlite IRA 400 Cl (E), Amberlite IRA 400 OH (F) and Amberlite IRA 910 (G). The pure paclitaxel content and purity of crude extract were 0.5% (w/v) and 75.3%, respectively. Scale bar indicates 10 m.
J.-Y. Lee, J.-H. Kim / Process Biochemistry 47 (2012) 2388–2397
Particle size (radius, µm)
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Control IR 120 H IR 120 Na 200 IRA 400Cl IRA 400OH IRA 910
(A)
50 40 30
Acknowledgments This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government (MEST) (No. 2011-0010907). References
20 10 0 3
6
9
Precipitation time (hr) 60
Particle size (radius, µm)
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(B)
Control IR 120 H IR 120 Na 200 IRA 400Cl IRA 400OH IRA 910
50 40 30 20 10 0 3
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Precipitation time (hr) Fig. 11. Effect of ion exchange resin used to increase S/V (0.428 mm−1 ) on paclitaxel particle size during fractional precipitation. The crude extract purity was 44.8% (A) and 75.3% (B). The methanol composition in water, pure paclitaxel content, and precipitation temperature were 61.5% (v/v), 0.5% (w/v) and 4 ◦ C, respectively.
4. Conclusions This study evaluated the effect of crude extract purity and pure paclitaxel content on the behavior (purity, yield, fractional precipitation time, precipitate shape and size) of the increased surface area fractional precipitation process for the purification of paclitaxel. Diverse types of ion exchange resin were used to increase the surface area, and the effect of crude extract purity and pure paclitaxel content dissolved in methanol solution in fractional precipitation were investigated in detail. As the pure paclitaxel content and crude extract purity increased, paclitaxel purity and yield were improved and the time required for fractional precipitation was reduced as well. Regardless of changes in crude extract purity and pure paclitaxel content, it was possible to obtain small-sized paclitaxel precipitates due to the hindrance of precipitate particle growth by the ion exchange resin. Also, the type of surface-area increasing substance used made a difference in precipitate size and shape due to differences in affinity for paclitaxel particles. As the crude extract purity and pure paclitaxel content were decreased, the greater the yield and effect of improving the purity in the increased surface area fractional precipitation process, with a greater effect on the decrease in paclitaxel particle size.
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