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Evaluation of surface area of mesoporous silica adsorbents for separation and purification of paclitaxel
Microporous and Mesoporous Materials 180 (2013) 109–113
Contents lists available at SciVerse ScienceDirect
Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Short Communication
Evaluation of surface area of mesoporous silica adsorbents for separation and purification of paclitaxel Hyeon-Jeong Oh, Hye Ran Jang, Kyeong Youl Jung ⇑, Jin-Hyun Kim ⇑ Department of Chemical Engineering, Kongju National University, 1223-24 Cheonan-Daero, Seobuk-Gu, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 12 November 2012 Received in revised form 24 April 2013 Accepted 4 June 2013 Available online 15 June 2013 Keywords: Mesoporous silica Adsorbent Evaluation Paclitaxel Purification
a b s t r a c t Several types of mesoporous silica adsorbents with different surface areas at specific pore sizes (2 nm, 4–5 nm, and 29 nm) were prepared by spray pyrolysis and were used for the separation/purification of the anticancer agent paclitaxel. After adsorbent treatment and hexane precipitation, the purity of paclitaxel was found to be increased with increasing surface area of adsorbent. The highest paclitaxel yields were achieved using an absorbent with a surface area of 953 m2/g at a pore size of 2 nm, with a surface area of 575 m2/g at a pore size of 4–5 nm, and with a surface area of 131 m2/g at a pore size of 29 nm. It was clear that the surface area of the adsorbent affected not only the purity but also the yield of paclitaxel. Also, increasing the surface area of the adsorbent resulted in an increase in the adsorption of paclitaxel and impurities (biomass-derived tar and wax components). Removal of these impurities was confirmed by HPLC analysis of the absorbent after treatment and TGA of the organic substances that were adhered to the adsorbent. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid anticancer agent discovered in the bark of the yew tree, is the most widely used treatment for ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer [1]. Its application has also been expanded to the treatment of acute rheumatoid arthritis and Alzheimer’s disease. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are underway, the demand for paclitaxel is expected to increase [2]. The main methods of paclitaxel production include direct extraction from the yew tree, semi-synthesis involving the chemical combination of side chains after obtaining a precursor from the leaves of the yew tree, and plant cell culture from the main bioreactor after inducing callus from the yew tree and performing a seed culture [3–6]. Among these methods, plant cell culture can stably mass produce paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment. To obtain high purity paclitaxel from plant cell cultures, several separation and purification processes are required. These processes generally consist of extraction of paclitaxel from biomass (plant cells containing paclitaxel) using an organic solvent, a pre-purification process, and a final purification process to obtain the purified ⇑ Corresponding authors. Tel.: +82 41 521 9365; fax: +82 41 554 2640 (K.Y. Jung), tel.: +82 41 521 9361; fax: +82 41 554 2640 (J.-H. Kim). E-mail addresses: [email protected] (K.Y. Jung), [email protected] (J.-H. Kim). 1387-1811/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.micromeso.2013.06.009
product. Among these steps, the pre-purification process in particular has a great impact on the total purification cost [7–9]. Existing processes [10–12] either use expensive chromatography methods for pre-purification or directly process the crude extract using high performance liquid chromatography (HPLC) without pre-purification. Obviously, such an approach entails economic problems and difficulties in scale-up and industrial mass production. Crude paclitaxel extracts usually have a low purity of about 10% 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 lifetime of the column packing material (resin) is reduced, and throughput is decreased. Therefore, in order to reduce the cost of final purification using HPLC, it is necessary to increase the purity of the sample as much as possible (>50%) through the pre-purification process. It has been reported that pre-purification with adsorbent treatment and precipitation can provide high purity crude paclitaxel [13]. It is a simple and convenient pretreatment method involving removal of plant-derived tar and wax components by adsorbent treatment followed by precipitation to obtain high purity (>60%) crude paclitaxel. Plant-derived tar and wax components badly affect the separation/purification of paclitaxel, especially because they are the primary contributor to the costly problems with purification described above; therefore, it is necessary to remove these components via a pre-purification process [1]. According to published literature [14], when adsorbent treatment was performed using SiO2, the main ingredient of the synthetic adsorbent sylopute, it proved to be effective in removing plant-derived tar and wax components. Also, pore diameter is a
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major physical property of SiO2; a good adsorbent effect can be achieved at an appropriate pore size (8–15 nm) [15]. In this study, mesoporous silica adsorbents with various surface areas at specific pore diameters were prepared and their effect on the purity and yield of paclitxel was investigated. Ultimately, the physical properties of silica adsorbents (especially surface area) that are the most effective in removing biomass-derived tar and wax components are suggested. 2. Experimental 2.1. Plant materials and culture conditions Plant cell culture was performed using a cell line obtained from the leaves of Taxus chinensis. A suspension of cells was cultured at 24 °C with shaking at 150 rpm in the dark in modified Gamborg’s B5 medium [16] with 30 g/L sucrose, 10 lM naphthalene acetic acid (NAA), 0.2 lM 6-benzylamino purine (BA), 1 g/L casein hydrolysate, and 1 g/L 2-(N-morpholino)ethanesulfonic acid (MES). The culture medium was changed to fresh medium at 2-week intervals, 1–2% maltose was added on days 7 and 21 of culture and 4 lM AgNO3 was also added as an elicitor at the beginning of culture [17]. After plant cell culture, plant cells and cell debris were recovered with a decanter (Westfalia, CA150 Claritying Decanter) and a high speed centrifuge (Alfa Laval, BTPX205GD-35CDEEP). Recovered plant cells and cell debris together were named ‘biomass’. The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Paclitaxel content was analyzed using an HPLC system (Waters, USA) equipped with a Capell 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. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [18].
rene (PS) nanoparticles (29 nm) were used as the pore-directing templates. A typical spray solution was prepared by adding the TEOS aqueous solution to a solution of CTAB or CTAB/P123 mixture. The TEOS concentration was fixed at 0.2 M. Silica particles with the average pore size of 2 nm, 4–5 nm, and 29 nm were synthesized by using CTAB, CTAB/P123, and PS nanoparticles as the templates, respectively. The surface area of silica at a fixed pore size was controlled by varying the content of each template. The molar ratio of CTAB to TEOS was varied from 0.05 to 0.3. At CTAB/TEOS = 0.2, the molar ratio of P123 to TEOS was changed from 0.03 to 0.1. The content of PS nanoparticles was controlled from 1.0 to 4.0 in a weight ratio with respect to SiO2. The spray solutions were atomized by an ultrasonic generator with six vibrators of 1.7 MHz. The generated droplets were carried by air (20 L/min) into a temperature-controlled reactor consisting of two quartz tubes (OD = 55 mm, length = 1200 mm). The first quartz reactor (drying zone) was maintained at 350 °C and the second one (pyrolysis zone) was at 600 °C. The resulting powder was collected by a Teflon bag filter, and followed by a post-thermal treatment at 550 °C (1 °C/min) for 4 h. For all prepared samples, nitrogen adsorption/desorption isotherms were measured at 196 °C using a Micromeritics ASAP 2010 apparatus after pretreatment at 200 °C. The surface area was calculated by the Brunauer– Emmett–Teller method [20] using the adsorption data in the relative pressure (P/P0) range from 0.05 to 0.2. The pore volumes of the samples were determined from the adsorption data at P/P0 = 0.995. The pore diameter range from 17 to 3000 Å was calculated from the desorption branch by the Barrett–Joyner–Halenda method. 2.5. Adsorbent treatment Dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and adsorbent mesoporous silicas (pore size: 2 nm, 4–5 nm, 29 nm) were added at a ratio of 50% (w/w) against dried crude extract. The mixture was agitated for 30 min at 40 °C, and then filtered. The filtrate was dried at 30 °C under vacuum and subjected to a hexane precipitation process. 2.6. Hexane precipitation
2.3. Preparation of sample for adsorbent treatment Biomass recovered from the plant cell culture medium mixed with methanol at a ratio of 1:1 (w/v) was extracted at room temperature for 30 min and filtered. Extraction was performed 4 times by adding another volume of fresh methanol to the biomass using the same method [5]. Extracted filtrate was collected and concentrated (30% of original liquid) using a concentrator (CCA-1100; EYELA, Japan) at 40 °C under vacuum followed by liquid–liquid extraction at room temperature 3 times after adding methylene chloride (25% of concentrated solution) to the concentrated solution. After liquid–liquid extraction, phase separation was carried out to remove polar impurities from the upper methanol layer, while paclitaxel in the lower methylene chloride layer was recovered and concentrated/dried at room temperature under vacuum. The dried sample was used for adsorbent treatments [19]. The purity of the sample used in the adsorbent treatment was 10.6%.
The dried sample obtained from the adsorbent treatment process was dissolved in methylene chloride and dropped into n-hexane (methylene chloride/hexane: 1:10, v/v) to obtain a paclitaxel precipitate [18]. After hexane precipitation, the precipitate was recovered by filtration and dried in a vacuum at 35 °C for 24 h. The yield and purity of the dried precipitate were evaluated by HPLC to determine the effectiveness of the adsorbent treatment. 2.7. TGA analysis The amount of organic impurities adsorbed to the adsorbent was measured via thermogravimetric analysis (TGA) (SDTQ600; Shimadzu, Japan). Different temperatures were applied, and changes in the weight and the amount of organic impurities were measured. The temperature was increased to 700 °C after injecting air.
2.4. Preparation of mesoporous silica adsorbents
3. Results and discussion
A conventional spray pyrolysis process was performed to prepare mesoporous silica particles with a spherical shape. Tetraethylorthosilicate (TEOS, 97%; Aldrich) was used as the precursor of silica. Cetyltrimethylammonium bromide (CTAB; Aldrich) or CTAB/pluronic P123 (EO20PO70EO20; Aldrich) mixture, and polysty-
The effect of adsorbent treatment (removal of biomass-derived tar and wax components) using five different mesoporous silicas with different surface areas was investigated with pore size constant at the 2 nm level (2.3–2.7 nm). The properties of the silica adsorbent used for this study are presented in Table 1 (2S-1–2S-5).
H.-J. Oh et al. / Microporous and Mesoporous Materials 180 (2013) 109–113
The result of hexane precipitation after adsorbent treatment is shown in Fig. 1(a). The purity of paclitaxel increased with increasing surface area of adsorbents in order of 2S-1 (434 m2/g), 2S-2 (703 m2/g), 2S-3 (953 m2/g), 2S-4 (1281 m2/g), and 2S-5 (1670 m2/g). The yield increased with increasing surface area in the order 2S-1 (434 m2/g) < 2S-2 (703 m2/g) < 2S-3 (953 m2/g) and then decreased when the surface area was greater than that of 2S-3 (953 m2/g). The highest paclitaxel yield (70.3%) could be obtained when adsorbent 2S-3 (953 m2/g) was used. In order to determine the tendency of impurities to adhere to the adsorbent, it was recovered by filtration after the adsorbent treatment and washed with methanol, then subjected to HPLC analysis [14]. As shown in Fig. 2(a), differences in the peak pattern in the chromatogram according to changes in surface area were detected. In particular, the peak for paclitaxel as well as those for impurities on the surface of the adsorbent became prominently high using 2S-4 (1281 m2/g). These results suggest that large amounts of paclitaxel as well as impurities including biomass-derived tar and wax components were adsorbed onto the surface of the adsorbent, thereby decreasing paclitaxel yield. In other words, the surface area of adsorbent increased above a certain point, the loss of paclitaxel form adsorbent was increased and the yield of paclitaxel was decreased in the hexane precipitation step. TGA analysis was performed to quantitatively measure the organic matters adhered to the adsorbents [14]. As shown in Fig. 3(a), the amount of organic matters adhered to the adsorbents increased with increasing surface area in order of 2S-1 (10.6%), 2S-2 (15.2%), 2S-3 (15.7%), 2S4 (18.6%), and 2S-5 (23.8%). The total weight of impurities adsorbed to silica decreased sharply at around 350 °C and gradually decreased thereafter until the temperature reached 700 °C. Quantitative analysis revealed that a greater amount of biomass-derived organic matter was adsorbed to/removed from the adsorbent as the surface area of the adsorbent was increased. As a result, the purity of paclitaxel was increased as the surface area was increased. The result is in good agreement with those of existing reports [21–23], which generally show that an adsorbent with a larger surface area (e.g., activated carbon, carbon material) has a high biomass-derived tar removal effect. The effect of adsorbent treatment (removal of biomass-derived tar and wax components) using four types of mesoporous silica with different surface areas was investigated with pore size constant at the 4–5 nm level (4.1–5.6 nm). The properties of the silica adsorbents used for this purpose are presented in Table 1 (5S-1– 5S-4). The hexane precipitation result after adsorbent treatment is illustrated in Fig. 1(b). The purity of paclitaxel increased with increasing surface area of adsorbent. While the yield increased according to the increase in surface area, it decreased when the surface area was increased more than that of 5S-2 (575 m2/g). The highest paclitaxel yield (78.9%) was obtained when adsorbent 5S-2 (575 m2/g) was used. After adsorbent treatment and subTable 1 Physical properties of 2 nm, 4–5 nm, and 29 nm mesoporous silica. Silica
Surface area (m2/g)
Pore volume (cm3/g)
Pore diameter (nm)
2S-1 2S-2 2S-3 2S-4 2S-5 5S-1 5S-2 5S-3 5S-4 29S-1 29S-2 29S-3 29S-4
434 703 953 1281 1670 318 575 779 1003 82 108 131 171
0.25 0.4 0.55 0.67 0.91 0.75 0.66 1.74 1.88 0.35 0.49 0.87 0.81
2.5 2.7 2.7 2.3 2.4 5.6 4.1 4.7 4.9 29 29 29 29
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Fig. 1. Effect of adsorbent on the purity and yield of paclitaxel in hexane precipitation: (a) 2 nm, (b) 4–5 nm, and (c) 29 nm.
sequent filtration, the adsorbent was recovered, washed and analyzed by HPLC to check the trend of substances adhered to the surface [14]. As shown in Fig. 2(b), relatively high paclitaxel peaks were found for adsorbents 5S-3 (779 m2/g) and 5S-4 (1003 m2/g) as compared with 5S-1 (318 m2/g) and 5S-2 (575 m2/g). This result indicates that paclitaxel yield was decreased because more adsorption of paclitaxel occurred along with impurities including biomass-derived tar and wax due to the relatively large surface areas of 5S-3 (779 m2/g) and 5S-4 (1003 m2/g). TGA analysis was performed to quantitatively analyze the amount of organic matter adhered to the adsorbents. As shown in Fig. 3(b), the amount of organic matter on the adsorbent increased with increasing surface area in order of 5S-1 (10.5%), 5S-2 (14.2%), 5S3 (17.9%), and 5S-4 (24.9%). From this result, it is clear that a greater amount of biomass-derived organic matter (tar and wax) was adsorbed/removed using an increased surface area. From the above, it has been found that by increasing the surface area of
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Fig. 2. HPLC chromatograms of washing solution after adsorbent treatment with a pore size of (a) 2 nm, (b) 4–5 nm and (c) 29 nm. The arrow indicates the position of paclitaxel.
the adsorbent, the tendency to adsorb paclitaxel and impurities was increased. The effect of adsorbent treatment (removal of biomass-derived tar and wax compounds) of four types of mesoporous silicas at a constant pore size of 29 nm was investigated. The properties of the silica adsorbents used for the experiment are presented in Table 1 (29S-1–29S-4). The result of hexane precipitation after adsorbent treatment is shown in Fig. 1(c). The paclitaxel yield increased with increasing surface area of the adsorbent, but decreased using 29S-4 (171 m2/g). The highest yield was obtained (74.2%) when 29S-3 (131 m2/g) was used. Meanwhile, the purity of paclitaxel increased slightly as the surface area of the adsorbent was increased. In particular, the change in purity according to changes in the surface area of the adsorbent was relatively insignificant with
Fig. 3. TGA curves of adsorbents with a pore size of (a) 2 nm, (b) 4–5 nm and (c) 29 nm after adsorbent treatment.
a pore size of 29 nm as compared with those obtained using pore sizes of 2 nm and 4–5 nm, suggesting that biomass-derived tar and wax components, which affect the purity of paclitaxel, were sufficiently adsorbed/removed at a larger pore size than the specific level previously described for silica (8–15 nm), thereby being relatively less affected by surface area changes [15]. The components adhered to the adsorbent were analyzed by HPLC after adsorbent treatment, filtration and subsequent recovery of the adsorbent and washing with methanol [14]. As shown in Fig. 2(c), the amount of paclitaxel as well as impurities on the adsorbent was found not to be changed much as the surface area was increased using 29S-1 (82 m2/g), 29S-2 (108 m2/g), and 29S-3 (131 m2/g), while the paclitaxel peak was higher using 29S-4 (171 m2/g). From this result, we found that the paclitaxel yield was decreased because a large amount of paclitaxel was adsorbed and removed by using adsorbent 29S-4 with a relatively large surface area. TGA analysis
H.-J. Oh et al. / Microporous and Mesoporous Materials 180 (2013) 109–113
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4. Conclusions In this study, the effect of adsorbent treatment according to changes in the surface area of mesoporous silica adsorbents at a constant pore size prepared by spray pyrolysis was investigated. The study aimed to determine the most effective physical properties (especially surface area) of the silica adsorbent used in the separation/purification of the biomass-derived anticancer agent paclitaxel. Adsorbent treatment using various mesoporous silicas with different pore sizes from the 2 nm level (2.3–2.7 nm), to the 4–5 nm level (4.1–5.6 nm), and the 29 nm level and subsequent hexane precipitation resulted in an increase in the purity of paclitaxel as the surface area of the adsorbent was increased. Paclitaxel yield was maximal (>70%) using 2S-3 (953 m2/g) at a pore size of 2 nm, 5S-2 (575 m2/g) at a pore size of 4–5 nm, and 29S-3 (131 m2/g) at a pore size of 29 nm. The results indicate that the surface area of the silica adsorbents not only affected the purity of paclitaxel but also the yield. However, the degree to which paclitaxel and impurities (including biomass-derived tar and wax components) were adsorbed increased with increasing surface area of the adsorbent. This effect of removing impurities (including tar and wax components) was clear from the results of HPLC and quantitative analysis using TGA of the organic matter adhered to the adsorbents. On the whole, it has been revealed that a higher surface area is required as the pore size of the adsorbent is decreased to obtain the same paclitaxel purity. Also, a higher surface area is required to achieve maximal paclitaxel yield with decreases in the pore size of the adsorbent. This might be because the surface area could not be utilized to maintain the purity as well as the yield of paclitaxel by decreasing the pore size of the adsorbents. Acknowledgment 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
Fig. 4. Effect of surface area of adsorbent on (a) purity and (b) yield of paclitaxel after hexane precipitation.
was carried out to determine the amount of organic matter on the adsorbents. As shown in Fig. 3(c), a greater amount of organic matter (including tar and wax components) was adsorbed/removed with increases in surface area. The above results indicate that an appropriate surface area was found to be required for the effective purification of paclitaxel using adsorbent silica with a constant pore size. Also, a larger surface area was required to obtain the same purity (Fig. 4(a)), and the maximum yield (Fig. 4(b)), as the pore size of adsorbent was decreased. It is believed that with decreases in the pore size of the adsorbent, the surface area of the adsorbent cannot be utilized effectively for the purification of paclitaxel. However, the larger pores have the better advantages in terms of the utilization of the whole surface area of the adsorbents. The advantage of the large pores in terms of the diffusion of paclitaxel/impurity or the utilization of inner surface area well explains the reason why the 2S-3, 5S-2 and 29S-3 have almost the same maximum yield (>70%) of paclitaxel, even though 29S-3 has about 7.3 times and 4.4 times smaller surface area the 2S-3 and 5S-2 adsorbents, respectively.
[1] J.-H. Kim, Korean J. Biotechnol. Bioeng. 21 (2006) 1–10. [2] J.R. Hsiao, S.F. Leu, B.M. Huang, J. Oral Pathol. Med. 38 (2009) 188–197. [3] K.V. Rao, J.B. Hanuman, C. Alvarez, M. Stoy, J. Juchum, R.M. Davies, R. Baxley, Pharm. Res. 12 (1995) 1003–1010. [4] E. Baloglu, D.G. Kingston, J. Nat. Prod. 62 (1999) 1068–1071. [5] J.E. Hyun, J.-H. Kim, Korean J. Biotechnol. Bioeng. 23 (2008) 281–284. [6] M.E. Kolewe, V. Gaurav, S.C. Roberts, Mol. Pharm. 5 (2008) 243–256. [7] J.-H. Kim, H.-K. Choi, S.-S. Hong, H.-S. Lee, J. Microbiol. Biotechnol. 11 (2001) 204–210. [8] S.-H. Pyo, B.-K. Song, C.-H. Ju, B.-H. Han, H.-J. Choi, Process Biochem. 40 (2005) 1113–1117. [9] J.H. Kim, I.S. Kang, H.K. Choi, S.S. Hong, H.S. Lee, Process Biochem. 37 (2002) 679–682. [10] K.V. Rao, US Patent 5,670,673, 1997. [11] T.P. Castor, US Patent 5,750,709, 1998. [12] H.N. ElSohly, E.M. Croom, M.A. ElSohly, J.D. McChesney, US Patent 5,618,538, 1997. [13] S.-H. Pyo, H.-B. Park, B.-K. Song, B.-H. Han, J.-H. Kim, Process Biochem. 39 (12) (2004) 1985–1991. [14] H.-J. Oh, H.R. Jang, K.Y. Jung, J.-H. Kim, Process Biochem. 47 (2012) 331–334. [15] H.R. Jang, H.-J. Oh, J.-H. Kim, K.Y. Jung, Micropor. Mesopor. Mater. 165 (2013) 219–227. [16] O.L. Gamborg, R.A. Miller, K. Ojima, Exp. Cell Res. 50 (1968) 151–158. [17] H.K. Choi, T.L. Adams, R.W. Stahlhut, S.I. Kim, J.H. Yun, B.K. Song, J.H. Kim, S.S. Hong, H.S. Lee, US Patent 5,871, 979, 1999. [18] S.G. Kim, J.H. Kim, Korean J. Chem. Eng. 26 (2) (2009) 449–452. [19] J.H. Kim, KSBB J. 24 (2009) 212–215. [20] S.J. Gregg, K.S.W. Sing, Adsorption, Surface Area and Porosity, Academic Press, New York, 1982. [21] X. Hu, T. Hanaoka, K. Sakanishi, T. Shinagawa, S. Matsui, M. Tada, T. Iwasaki, J. Jpn. Inst. Energy 86 (2007) 707–711. [22] T. Hanaoka, K. Sakanishi, T. Minowa, J. Jpn. Inst. Energy 83 (2004) 828–831. [23] A.M. Mastral, T. García, M.S. Callén, M.V. Navarro, J. Galbán, Environ. Sci. Technol. 35 (2001) 2395–2400.
Contents lists available at SciVerse ScienceDirect
Microporous and Mesoporous Materials journal homepage: www.elsevier.com/locate/micromeso
Short Communication
Evaluation of surface area of mesoporous silica adsorbents for separation and purification of paclitaxel Hyeon-Jeong Oh, Hye Ran Jang, Kyeong Youl Jung ⇑, Jin-Hyun Kim ⇑ Department of Chemical Engineering, Kongju National University, 1223-24 Cheonan-Daero, Seobuk-Gu, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 12 November 2012 Received in revised form 24 April 2013 Accepted 4 June 2013 Available online 15 June 2013 Keywords: Mesoporous silica Adsorbent Evaluation Paclitaxel Purification
a b s t r a c t Several types of mesoporous silica adsorbents with different surface areas at specific pore sizes (2 nm, 4–5 nm, and 29 nm) were prepared by spray pyrolysis and were used for the separation/purification of the anticancer agent paclitaxel. After adsorbent treatment and hexane precipitation, the purity of paclitaxel was found to be increased with increasing surface area of adsorbent. The highest paclitaxel yields were achieved using an absorbent with a surface area of 953 m2/g at a pore size of 2 nm, with a surface area of 575 m2/g at a pore size of 4–5 nm, and with a surface area of 131 m2/g at a pore size of 29 nm. It was clear that the surface area of the adsorbent affected not only the purity but also the yield of paclitaxel. Also, increasing the surface area of the adsorbent resulted in an increase in the adsorption of paclitaxel and impurities (biomass-derived tar and wax components). Removal of these impurities was confirmed by HPLC analysis of the absorbent after treatment and TGA of the organic substances that were adhered to the adsorbent. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Paclitaxel, a diterpenoid anticancer agent discovered in the bark of the yew tree, is the most widely used treatment for ovarian cancer, breast cancer, Kaposi’s sarcoma, and non-small cell lung cancer [1]. Its application has also been expanded to the treatment of acute rheumatoid arthritis and Alzheimer’s disease. Since clinical trials regarding the combined prescription of paclitaxel with various other treatments are underway, the demand for paclitaxel is expected to increase [2]. The main methods of paclitaxel production include direct extraction from the yew tree, semi-synthesis involving the chemical combination of side chains after obtaining a precursor from the leaves of the yew tree, and plant cell culture from the main bioreactor after inducing callus from the yew tree and performing a seed culture [3–6]. Among these methods, plant cell culture can stably mass produce paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment. To obtain high purity paclitaxel from plant cell cultures, several separation and purification processes are required. These processes generally consist of extraction of paclitaxel from biomass (plant cells containing paclitaxel) using an organic solvent, a pre-purification process, and a final purification process to obtain the purified ⇑ Corresponding authors. Tel.: +82 41 521 9365; fax: +82 41 554 2640 (K.Y. Jung), tel.: +82 41 521 9361; fax: +82 41 554 2640 (J.-H. Kim). E-mail addresses: [email protected] (K.Y. Jung), [email protected] (J.-H. Kim). 1387-1811/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.micromeso.2013.06.009
product. Among these steps, the pre-purification process in particular has a great impact on the total purification cost [7–9]. Existing processes [10–12] either use expensive chromatography methods for pre-purification or directly process the crude extract using high performance liquid chromatography (HPLC) without pre-purification. Obviously, such an approach entails economic problems and difficulties in scale-up and industrial mass production. Crude paclitaxel extracts usually have a low purity of about 10% 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 lifetime of the column packing material (resin) is reduced, and throughput is decreased. Therefore, in order to reduce the cost of final purification using HPLC, it is necessary to increase the purity of the sample as much as possible (>50%) through the pre-purification process. It has been reported that pre-purification with adsorbent treatment and precipitation can provide high purity crude paclitaxel [13]. It is a simple and convenient pretreatment method involving removal of plant-derived tar and wax components by adsorbent treatment followed by precipitation to obtain high purity (>60%) crude paclitaxel. Plant-derived tar and wax components badly affect the separation/purification of paclitaxel, especially because they are the primary contributor to the costly problems with purification described above; therefore, it is necessary to remove these components via a pre-purification process [1]. According to published literature [14], when adsorbent treatment was performed using SiO2, the main ingredient of the synthetic adsorbent sylopute, it proved to be effective in removing plant-derived tar and wax components. Also, pore diameter is a
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major physical property of SiO2; a good adsorbent effect can be achieved at an appropriate pore size (8–15 nm) [15]. In this study, mesoporous silica adsorbents with various surface areas at specific pore diameters were prepared and their effect on the purity and yield of paclitxel was investigated. Ultimately, the physical properties of silica adsorbents (especially surface area) that are the most effective in removing biomass-derived tar and wax components are suggested. 2. Experimental 2.1. Plant materials and culture conditions Plant cell culture was performed using a cell line obtained from the leaves of Taxus chinensis. A suspension of cells was cultured at 24 °C with shaking at 150 rpm in the dark in modified Gamborg’s B5 medium [16] with 30 g/L sucrose, 10 lM naphthalene acetic acid (NAA), 0.2 lM 6-benzylamino purine (BA), 1 g/L casein hydrolysate, and 1 g/L 2-(N-morpholino)ethanesulfonic acid (MES). The culture medium was changed to fresh medium at 2-week intervals, 1–2% maltose was added on days 7 and 21 of culture and 4 lM AgNO3 was also added as an elicitor at the beginning of culture [17]. After plant cell culture, plant cells and cell debris were recovered with a decanter (Westfalia, CA150 Claritying Decanter) and a high speed centrifuge (Alfa Laval, BTPX205GD-35CDEEP). Recovered plant cells and cell debris together were named ‘biomass’. The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel analysis Paclitaxel content was analyzed using an HPLC system (Waters, USA) equipped with a Capell 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. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as a standard [18].
rene (PS) nanoparticles (29 nm) were used as the pore-directing templates. A typical spray solution was prepared by adding the TEOS aqueous solution to a solution of CTAB or CTAB/P123 mixture. The TEOS concentration was fixed at 0.2 M. Silica particles with the average pore size of 2 nm, 4–5 nm, and 29 nm were synthesized by using CTAB, CTAB/P123, and PS nanoparticles as the templates, respectively. The surface area of silica at a fixed pore size was controlled by varying the content of each template. The molar ratio of CTAB to TEOS was varied from 0.05 to 0.3. At CTAB/TEOS = 0.2, the molar ratio of P123 to TEOS was changed from 0.03 to 0.1. The content of PS nanoparticles was controlled from 1.0 to 4.0 in a weight ratio with respect to SiO2. The spray solutions were atomized by an ultrasonic generator with six vibrators of 1.7 MHz. The generated droplets were carried by air (20 L/min) into a temperature-controlled reactor consisting of two quartz tubes (OD = 55 mm, length = 1200 mm). The first quartz reactor (drying zone) was maintained at 350 °C and the second one (pyrolysis zone) was at 600 °C. The resulting powder was collected by a Teflon bag filter, and followed by a post-thermal treatment at 550 °C (1 °C/min) for 4 h. For all prepared samples, nitrogen adsorption/desorption isotherms were measured at 196 °C using a Micromeritics ASAP 2010 apparatus after pretreatment at 200 °C. The surface area was calculated by the Brunauer– Emmett–Teller method [20] using the adsorption data in the relative pressure (P/P0) range from 0.05 to 0.2. The pore volumes of the samples were determined from the adsorption data at P/P0 = 0.995. The pore diameter range from 17 to 3000 Å was calculated from the desorption branch by the Barrett–Joyner–Halenda method. 2.5. Adsorbent treatment Dried crude extract was dissolved in methylene chloride at a ratio of 20% (v/w) and adsorbent mesoporous silicas (pore size: 2 nm, 4–5 nm, 29 nm) were added at a ratio of 50% (w/w) against dried crude extract. The mixture was agitated for 30 min at 40 °C, and then filtered. The filtrate was dried at 30 °C under vacuum and subjected to a hexane precipitation process. 2.6. Hexane precipitation
2.3. Preparation of sample for adsorbent treatment Biomass recovered from the plant cell culture medium mixed with methanol at a ratio of 1:1 (w/v) was extracted at room temperature for 30 min and filtered. Extraction was performed 4 times by adding another volume of fresh methanol to the biomass using the same method [5]. Extracted filtrate was collected and concentrated (30% of original liquid) using a concentrator (CCA-1100; EYELA, Japan) at 40 °C under vacuum followed by liquid–liquid extraction at room temperature 3 times after adding methylene chloride (25% of concentrated solution) to the concentrated solution. After liquid–liquid extraction, phase separation was carried out to remove polar impurities from the upper methanol layer, while paclitaxel in the lower methylene chloride layer was recovered and concentrated/dried at room temperature under vacuum. The dried sample was used for adsorbent treatments [19]. The purity of the sample used in the adsorbent treatment was 10.6%.
The dried sample obtained from the adsorbent treatment process was dissolved in methylene chloride and dropped into n-hexane (methylene chloride/hexane: 1:10, v/v) to obtain a paclitaxel precipitate [18]. After hexane precipitation, the precipitate was recovered by filtration and dried in a vacuum at 35 °C for 24 h. The yield and purity of the dried precipitate were evaluated by HPLC to determine the effectiveness of the adsorbent treatment. 2.7. TGA analysis The amount of organic impurities adsorbed to the adsorbent was measured via thermogravimetric analysis (TGA) (SDTQ600; Shimadzu, Japan). Different temperatures were applied, and changes in the weight and the amount of organic impurities were measured. The temperature was increased to 700 °C after injecting air.
2.4. Preparation of mesoporous silica adsorbents
3. Results and discussion
A conventional spray pyrolysis process was performed to prepare mesoporous silica particles with a spherical shape. Tetraethylorthosilicate (TEOS, 97%; Aldrich) was used as the precursor of silica. Cetyltrimethylammonium bromide (CTAB; Aldrich) or CTAB/pluronic P123 (EO20PO70EO20; Aldrich) mixture, and polysty-
The effect of adsorbent treatment (removal of biomass-derived tar and wax components) using five different mesoporous silicas with different surface areas was investigated with pore size constant at the 2 nm level (2.3–2.7 nm). The properties of the silica adsorbent used for this study are presented in Table 1 (2S-1–2S-5).
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The result of hexane precipitation after adsorbent treatment is shown in Fig. 1(a). The purity of paclitaxel increased with increasing surface area of adsorbents in order of 2S-1 (434 m2/g), 2S-2 (703 m2/g), 2S-3 (953 m2/g), 2S-4 (1281 m2/g), and 2S-5 (1670 m2/g). The yield increased with increasing surface area in the order 2S-1 (434 m2/g) < 2S-2 (703 m2/g) < 2S-3 (953 m2/g) and then decreased when the surface area was greater than that of 2S-3 (953 m2/g). The highest paclitaxel yield (70.3%) could be obtained when adsorbent 2S-3 (953 m2/g) was used. In order to determine the tendency of impurities to adhere to the adsorbent, it was recovered by filtration after the adsorbent treatment and washed with methanol, then subjected to HPLC analysis [14]. As shown in Fig. 2(a), differences in the peak pattern in the chromatogram according to changes in surface area were detected. In particular, the peak for paclitaxel as well as those for impurities on the surface of the adsorbent became prominently high using 2S-4 (1281 m2/g). These results suggest that large amounts of paclitaxel as well as impurities including biomass-derived tar and wax components were adsorbed onto the surface of the adsorbent, thereby decreasing paclitaxel yield. In other words, the surface area of adsorbent increased above a certain point, the loss of paclitaxel form adsorbent was increased and the yield of paclitaxel was decreased in the hexane precipitation step. TGA analysis was performed to quantitatively measure the organic matters adhered to the adsorbents [14]. As shown in Fig. 3(a), the amount of organic matters adhered to the adsorbents increased with increasing surface area in order of 2S-1 (10.6%), 2S-2 (15.2%), 2S-3 (15.7%), 2S4 (18.6%), and 2S-5 (23.8%). The total weight of impurities adsorbed to silica decreased sharply at around 350 °C and gradually decreased thereafter until the temperature reached 700 °C. Quantitative analysis revealed that a greater amount of biomass-derived organic matter was adsorbed to/removed from the adsorbent as the surface area of the adsorbent was increased. As a result, the purity of paclitaxel was increased as the surface area was increased. The result is in good agreement with those of existing reports [21–23], which generally show that an adsorbent with a larger surface area (e.g., activated carbon, carbon material) has a high biomass-derived tar removal effect. The effect of adsorbent treatment (removal of biomass-derived tar and wax components) using four types of mesoporous silica with different surface areas was investigated with pore size constant at the 4–5 nm level (4.1–5.6 nm). The properties of the silica adsorbents used for this purpose are presented in Table 1 (5S-1– 5S-4). The hexane precipitation result after adsorbent treatment is illustrated in Fig. 1(b). The purity of paclitaxel increased with increasing surface area of adsorbent. While the yield increased according to the increase in surface area, it decreased when the surface area was increased more than that of 5S-2 (575 m2/g). The highest paclitaxel yield (78.9%) was obtained when adsorbent 5S-2 (575 m2/g) was used. After adsorbent treatment and subTable 1 Physical properties of 2 nm, 4–5 nm, and 29 nm mesoporous silica. Silica
Surface area (m2/g)
Pore volume (cm3/g)
Pore diameter (nm)
2S-1 2S-2 2S-3 2S-4 2S-5 5S-1 5S-2 5S-3 5S-4 29S-1 29S-2 29S-3 29S-4
434 703 953 1281 1670 318 575 779 1003 82 108 131 171
0.25 0.4 0.55 0.67 0.91 0.75 0.66 1.74 1.88 0.35 0.49 0.87 0.81
2.5 2.7 2.7 2.3 2.4 5.6 4.1 4.7 4.9 29 29 29 29
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Fig. 1. Effect of adsorbent on the purity and yield of paclitaxel in hexane precipitation: (a) 2 nm, (b) 4–5 nm, and (c) 29 nm.
sequent filtration, the adsorbent was recovered, washed and analyzed by HPLC to check the trend of substances adhered to the surface [14]. As shown in Fig. 2(b), relatively high paclitaxel peaks were found for adsorbents 5S-3 (779 m2/g) and 5S-4 (1003 m2/g) as compared with 5S-1 (318 m2/g) and 5S-2 (575 m2/g). This result indicates that paclitaxel yield was decreased because more adsorption of paclitaxel occurred along with impurities including biomass-derived tar and wax due to the relatively large surface areas of 5S-3 (779 m2/g) and 5S-4 (1003 m2/g). TGA analysis was performed to quantitatively analyze the amount of organic matter adhered to the adsorbents. As shown in Fig. 3(b), the amount of organic matter on the adsorbent increased with increasing surface area in order of 5S-1 (10.5%), 5S-2 (14.2%), 5S3 (17.9%), and 5S-4 (24.9%). From this result, it is clear that a greater amount of biomass-derived organic matter (tar and wax) was adsorbed/removed using an increased surface area. From the above, it has been found that by increasing the surface area of
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Fig. 2. HPLC chromatograms of washing solution after adsorbent treatment with a pore size of (a) 2 nm, (b) 4–5 nm and (c) 29 nm. The arrow indicates the position of paclitaxel.
the adsorbent, the tendency to adsorb paclitaxel and impurities was increased. The effect of adsorbent treatment (removal of biomass-derived tar and wax compounds) of four types of mesoporous silicas at a constant pore size of 29 nm was investigated. The properties of the silica adsorbents used for the experiment are presented in Table 1 (29S-1–29S-4). The result of hexane precipitation after adsorbent treatment is shown in Fig. 1(c). The paclitaxel yield increased with increasing surface area of the adsorbent, but decreased using 29S-4 (171 m2/g). The highest yield was obtained (74.2%) when 29S-3 (131 m2/g) was used. Meanwhile, the purity of paclitaxel increased slightly as the surface area of the adsorbent was increased. In particular, the change in purity according to changes in the surface area of the adsorbent was relatively insignificant with
Fig. 3. TGA curves of adsorbents with a pore size of (a) 2 nm, (b) 4–5 nm and (c) 29 nm after adsorbent treatment.
a pore size of 29 nm as compared with those obtained using pore sizes of 2 nm and 4–5 nm, suggesting that biomass-derived tar and wax components, which affect the purity of paclitaxel, were sufficiently adsorbed/removed at a larger pore size than the specific level previously described for silica (8–15 nm), thereby being relatively less affected by surface area changes [15]. The components adhered to the adsorbent were analyzed by HPLC after adsorbent treatment, filtration and subsequent recovery of the adsorbent and washing with methanol [14]. As shown in Fig. 2(c), the amount of paclitaxel as well as impurities on the adsorbent was found not to be changed much as the surface area was increased using 29S-1 (82 m2/g), 29S-2 (108 m2/g), and 29S-3 (131 m2/g), while the paclitaxel peak was higher using 29S-4 (171 m2/g). From this result, we found that the paclitaxel yield was decreased because a large amount of paclitaxel was adsorbed and removed by using adsorbent 29S-4 with a relatively large surface area. TGA analysis
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4. Conclusions In this study, the effect of adsorbent treatment according to changes in the surface area of mesoporous silica adsorbents at a constant pore size prepared by spray pyrolysis was investigated. The study aimed to determine the most effective physical properties (especially surface area) of the silica adsorbent used in the separation/purification of the biomass-derived anticancer agent paclitaxel. Adsorbent treatment using various mesoporous silicas with different pore sizes from the 2 nm level (2.3–2.7 nm), to the 4–5 nm level (4.1–5.6 nm), and the 29 nm level and subsequent hexane precipitation resulted in an increase in the purity of paclitaxel as the surface area of the adsorbent was increased. Paclitaxel yield was maximal (>70%) using 2S-3 (953 m2/g) at a pore size of 2 nm, 5S-2 (575 m2/g) at a pore size of 4–5 nm, and 29S-3 (131 m2/g) at a pore size of 29 nm. The results indicate that the surface area of the silica adsorbents not only affected the purity of paclitaxel but also the yield. However, the degree to which paclitaxel and impurities (including biomass-derived tar and wax components) were adsorbed increased with increasing surface area of the adsorbent. This effect of removing impurities (including tar and wax components) was clear from the results of HPLC and quantitative analysis using TGA of the organic matter adhered to the adsorbents. On the whole, it has been revealed that a higher surface area is required as the pore size of the adsorbent is decreased to obtain the same paclitaxel purity. Also, a higher surface area is required to achieve maximal paclitaxel yield with decreases in the pore size of the adsorbent. This might be because the surface area could not be utilized to maintain the purity as well as the yield of paclitaxel by decreasing the pore size of the adsorbents. Acknowledgment 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
Fig. 4. Effect of surface area of adsorbent on (a) purity and (b) yield of paclitaxel after hexane precipitation.
was carried out to determine the amount of organic matter on the adsorbents. As shown in Fig. 3(c), a greater amount of organic matter (including tar and wax components) was adsorbed/removed with increases in surface area. The above results indicate that an appropriate surface area was found to be required for the effective purification of paclitaxel using adsorbent silica with a constant pore size. Also, a larger surface area was required to obtain the same purity (Fig. 4(a)), and the maximum yield (Fig. 4(b)), as the pore size of adsorbent was decreased. It is believed that with decreases in the pore size of the adsorbent, the surface area of the adsorbent cannot be utilized effectively for the purification of paclitaxel. However, the larger pores have the better advantages in terms of the utilization of the whole surface area of the adsorbents. The advantage of the large pores in terms of the diffusion of paclitaxel/impurity or the utilization of inner surface area well explains the reason why the 2S-3, 5S-2 and 29S-3 have almost the same maximum yield (>70%) of paclitaxel, even though 29S-3 has about 7.3 times and 4.4 times smaller surface area the 2S-3 and 5S-2 adsorbents, respectively.
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