Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.     

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

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.     

The Wittig-cyclization procedure: acid promoted intramolecular formation of 3-C-branched-chain 3,6-anhydro furano sugars via 2'-oxopropylene derivatives

Some olefinic Wittig products, 3-deoxy-5,6-O-isopropylidene-3-C-(2'-oxopropylene)-1,2-O-alkylidene hexofuranose derivatives were converted to the branched-chain 3,6-anhydro-3-C-(2'-oxopropyl) derivatives on treatment with ion exchange resin Amberlite 120 (H(+)) in methanol-water at room temperature. Hydrolysis of 5,6-isopropylidene groups and intramolecular ring-closures took place in one pot reactions.     

Improvement of a crystallization process for the purification of vancomycin

We evaluated the effects of the sample feeding and mixing methods on the efficiency of vancomycin crystallization and developed a method to dramatically shorten the time required for crystal formation by increasing the available surface area inside the reactor. The highest purity (97%) and yield (99%) of vancomycin could be obtained with an initial one-step feeding and mixing method, resulting in the formation and growth of spherical fan-shaped crystals. The yield of vancomycin increased when the surface area per working volume (S/V) was increased to 0.428 mm⁻¹ using glass beads (0.5 mm), the cation exchange resin Amberlite 200, or the anion exchange resin Amberlite IRA-400. In particular, most of the vancomycin (>99%) could be recovered after 6 h of crystallization using Amberlite 200, resulting in a dramatic reduction in the time required for crystallization. On the other hand, increasing S/V had almost no effect on vancomycin purity and crystal size.     

Improvement of fractional precipitation process for pre-purification of paclitaxel

Fractional precipitation is a simple, efficient method for pre-purifying paclitaxel extracted from plant cell cultures. However, the fractional precipitation process has been inherently problematic due to the lengthy precipitation time (～3 days) that is required. An improved fractional precipitation process could significantly reduce the precipitation time by increasing the purity of crude extract and the surface area available for precipitation. Glass beads (7 mm) were used to increase the surface area, and the optimal surface area per working volume (i.e. volume of reaction solution) (S/V) for achieving the highest purity and yield of paclitaxel possible was found to be 0.428 mm^?1. The content of paclitaxel dissolved in methanol that can be processed during fractional precipitation was evaluated, and it was established that up to 0.9% (w/v) pure paclitaxel content could be processed. This improved pre-purification process serves to minimize solvent usage and the size and complexity of the high performance liquid chromatography operation required for paclitaxel purification.     

Potential biofuel additive from renewable sources--Kinetic study of formation of butyl acetate by heterogeneously catalyzed transesterification of ethyl acetate with butanol

Butyl acetate holds great potential as a sustainable biofuel additive. Heterogeneously catalyzed transesterification of biobutanol and bioethylacetate can produce butyl acetate. This route is eco-friendly and offers several advantages over the commonly used Fischer Esterification. The Amberlite IR 120- and Amberlyst 15-catalyzed transesterification is studied in a batch reactor over a range of catalyst loading (6–12 wt.%), alcohol to ester feed ratio (1:3 to 3:1), and temperature (303.15–333.15 K). A butanol mole fraction of 0.2 in the feed is found to be optimum. Amberlite IR 120 promotes faster kinetics under these conditions. The transesterifications studied are slightly exothermic. The moles of solvent sorbed per gram of catalyst decreases (ethanol > butanol > ethyl acetate > butyl acetate) with decrease in solubility parameter. The dual site models, the Langmuir Hinshelwood and Popken models, are the most successful in correlating the kinetics over Amberlite IR 120 and Amberlyst 15, respectively.     

Quantitative Aspects of Growth of the Methane Oxidizing Bacterium Methylococcus capsulatus on Methane in Shake Flask and Continuous Chemostat Culture

S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed.     

Toward the Synthesis of Pyroglutamate Lauroyl Ester: Biocatalysis Versus Chemical Catalysis

The synthesis of dodecyl pyroglutamate (or pyroglutamate lauroyl ester) was achieved in a two-step process involving a pyroglutamic acid alkyl ester intermediate. The reaction was carried out either by lipase or by chemical catalysis using ion exchange resin. Among the various tested lipases, the one from Candida antarctica B gave the best results allowing 73% formation of the desired ester after 6 h. Comparing the efficiency of this latter lipase with the one of Amberlyst IR120H resin in catalyzing this reaction, the biocatalyst gave a molar yield of pyroglutamate lauroyl ester of 79% compared to 69% when using the ion exchange resin starting with 1.04 mmol substrate in each case.     

Biosorption of lead and nickel by biomass of marine algae

Screening tests of different marine algae biomas types revealed a high passive biosorptive uptake of lead up to 270 mg Pb/g of biomass in some brown marine algae. Members of the order Fucales perfomed particularly well in this descending sequence: Fucus > Ascophyllum > Sargassum. Although decreasing the swelling of wetted biomass particles, their reinforcement by crosslinking may significantly affect the biosorption performance. Lead uptakes up to 370 mg Pb/g were observed in crosslinked Fucus vesiculosus and Ascophyllum nodosum. At low equilibrium residual concentrations of lead in solution, however, ion exchange resin Amberlite IR-120 had a higher lead uptake than the biosorbent materials. An order-of-magnitude lower uptake of nickel was observed in all of the sorbent materials examined. (c) 1994 John Wiley & Sons, Inc.     

Effect of a Pharmaceutical Cationic Exchange Resin on the Properties of Controlled Release Diphenhydramine Hydrochloride Matrices Using Methocel K4M or Ethocel 7cP as Matrix Formers

This work was aimed at evaluating the effect of a pharmaceutical cationic exchange resin (Amberlite IRP-69) on the properties of controlled release matrices using Methocel K4M (HPMC) or Ethocel 7cP (EC) as matrix formers. Diphenhydramine hydrochloride (DPH), which was cationic and water soluble, was chosen as a model drug. HPMC- and EC-based matrices with varying amounts (0–40%w/w) of resin incorporation were prepared by a direct compression. Matrix properties including diameter, thickness, hardness, friability, surface morphology and drug release were evaluated. The obtained matrices were comparable in diameter and thickness regardless of the amount of resin incorporation. Increasing the incorporated resin decreased the hardness of HPMC- and EC-based matrices, correlating with the degree of rupturing on the matrix surfaces. The friability of HPMC-based matrices increased with increasing the incorporated resin, corresponding to their decreased hardness. In contrast, the EC-based matrices showed no significant change in friability in spite of decreasing hardness. The incorporated resin differently influenced DPH release from HPMC- and EC-based matrices in deionized water. The resin further retarded DPH release from HPMC-based matrices due to the gelling property of HPMC and the ion exchange property of the resin. In contrast, the release from EC-based matrices initially increased because of the disintegrating property of the resin, but thereafter declined due to the complex formation between released drug and dispersed resin via the ion exchange process. The release in ionic solutions was also described. In conclusion, the incorporated resin could alter the release and physical properties of matrices.     

The amino acid composition of crystalline pepsin

The amino acid composition of twice recrystallized pepsin (Worthington Biochemical Corporation) has been determined chromatographically on columns of Amberlite IR 120 resin. The results of the analyses obtained on four different preparations indicate a close agreement in their amino acid composition. Pepsin is unique in that it has a great predominance of acidic amino acids over basic ones. Moreover, all the preparations contain a small and constant amount of hydroxyproline, corresponding to about 0.1 residue per molecule.     

Improved fractional precipitation method for purification of paclitaxel

This study investigated changing the methanol/water ratio during fractional precipitation of paclitaxel, and adding all the distilled water at room temperature, followed mixing for an additional 10 min. When the methanol/water ratio was 50:50, 40:60, and 30:70 (v/v), the paclitaxel yield was 42.0%, 84.3%, and 92.0%, respectively. When using a methanol/water ratio of 50:50 (v/v), a similar high purity and yield of paclitaxel to the case of storing at a low temperature was achieved when adding all the distilled water at room temperature, followed by additional mixing for 10 min and further mixing at room temperature during fractional precipitation. Thus, additional mixing after adding all the distilled water is confirmed as important during fractional precipitation. Furthermore, the present results show that a high yield of high-purity paclitaxel is possible with additional mixing at room temperature after adding all the distilled water, which is significantly more economical than the existing method of storing at a low temperature for a long time after adding all the distilled water during fractional precipitation.     

Integration of ion exchange resin materials for a downstream-processing approach of an imine reductase-catalyzed reaction

In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of d -glucose. Seven commercially available cation- and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding ~1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Glycosylation in room temperature ionic liquid using unprotected and unactivated donors

Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated donors. Modest yields of simple benzyl glycosides and disaccharides of glucose, mannose and N-acetylgalactosamine were obtained in 1-ethyl-3-methylimidazolium benzoate with Amberlite IR-120 (H(+)) resin or p-toluenesulfonic acid as promoters.     

Evaluation of feeding and mixing conditions for fractional precipitation of paclitaxel from plant cell cultures

Fractional precipitation based on the difference in solubility of crude paclitaxel dissolved in methanol, to which distilled water must be added, is the most effective method for the pre-purification of paclitaxel. In this study, the effect of the distilled water feeding and mixing method on the efficiency of fractional precipitation and the formation of paclitaxel precipitate were evaluated. When the distilled water was added all at once, the highest purity (∼57.0%) and yield (∼81.0%) were obtained, and a spherical precipitate was formed by the clustering of crystal branches. On the other hand, when the distilled water was added intermittently in several aliquots, the purity and yield tended to decrease with increasing number of additions, and the precipitate took the form of a cross or pentagon with less clustering of branches. Not mixing after the addition of distilled water resulted in high purity (∼57.0%) and the formation of a spherical precipitate that showed increased branching around the nucleus over time. In contrast, when the sample was mixed intermittently after adding distilled water, paclitaxel was obtained in high yield (∼99.7%). Continuously mixing the sample after adding distilled water, however, caused the precipitate crystals to be broken into smaller pieces.     

Enhancing protein capacity of rigid macroporous polymeric adsorbent.

A macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin was synthesized and modified with diethylamine to yield an anion-exchange resin suitable for protein adsorption. Efforts were made to enhance protein ion exchange capacity of the resin by investigating the copolymer composition. Different synthesis recipes were attempted, and the resultant resins were characterized by measuring the specific surface area and the adsorption ability using bovine serum albumin (BSA) as a model protein. The intraparticle pore size distribution measured by mercury porosimetry showed that the pores in the range of 40-120 nm took 75% of the total pore volume, indicating that the ion exchanger was favorable for protein adsorption. BSA capacity obtained with an appropriate recipe was as high as 78.6 mg/g wet resin or 50 mg/mL packed volume, which was higher than the capacities of some commercially available ion exchangers. Moreover, by using a pore diffusion model, the effective pore diffusivity of BSA was found to be 5.5 x 10(-12) m(2)/s, similar to those in the commercial ion exchangers.     

Isolation and Identification of Phenolic Acids in Rice Vinegar

The fractions obtained from the ether soluble part of rice vinegar were separated by means of cellulose column and of Amberlite XE-64 ion exchange resin. The following compounds were identified: p-hydroxyphenylethanol, p-hydroxyphenylacetic acid, p-hydroxyphenyllactic acid, p-hydroxybenzoic acid and phenyllactic acid.     

Analysis of tyrosine and deuterium labelled tyrosine in tissues and body fluids

A gas chromatographic mass spectrometric method has been developed to determine tyrosine and deuterium labelled tyrosine in biological samples using alpha-methyltyrosine (alpha-Me Ty) or m-hydroxyphenylalanine as internal standards. With the latter standard both labelled and unlabelled tyrosine as well as alpha-Me Ty can be determined simultaneously, in for example human blood, following administration of alpha-Me Ty to inhibit catecholamine synthesis. After isolation of the amino acids on an ion exchange column (Amberlite IR 120) the butyl ester pentafluoropropionyl derivatives were prepared. In human plasma the precision of the method was determined at a level of 80 nmol tyrosine ml-1 and found to be +/- 5% (coefficient of variation, n = 10).     

Isolation of Alkaline Proteinase from Aspergillus sojae in Homogeneous Form

The alkaline proteinase of Aspergillus sojae was isolated in gram quantities as a homogeneous form. The purification procedures were, (1) batchwise-treatment with ion exchange resin Duolite CS 101, (2) fractional precipitation with ammonium sulfate, (3) precipitation with acetone, (4) column chromatography on DEAE-cellulose, and (5) gel filtration with Sephadex G-100. The recovery of the activity was about 12%. The purified enzyme preparation was found to be homogeneous by several criteria such as ultracentrifugation, paper and moving-boundary electrophoreses, etc. Any kinds of carbohydrate and phosphorus were not detected in this preparation, suggesting that this enzyme is a simple protein.