基于正交实验法优化从栀子黄色素中制备藏红花酸的工艺

基于正交实验法,优化从栀子黄色素中提取制备藏红花酸的碱水解工艺,以期可以简单高效地获得高纯度藏红花酸。建立高效液相色谱法(HPLC)测定藏红花酸含量,以藏红花酸的含量和得率为考察指标,采用正交实验法考察碱水解工艺中的料液比、NaOH浓度、水解温度和水解时间对产品中藏红花酸含量和得率的影响。确定栀子黄色素碱水解的最佳条件:料液比1∶6 g/mL、NaOH浓度3 mol/L、水解温度55℃、水解时间60 min。在该条件下制备获得的藏红花酸得率可达15.33%±1.25%;含量可达到97.24%±0.78%。优化后方法步骤简单易行,绿色无污染,一步制得高纯度藏红花酸,适用于工业化生产。     

同柱串联凝胶过凝从重组人肿瘤坏死因子中脱除PEG和盐

A serial gel filtration operation with the combination of G25 and G100 in one column was developed to remove salt and PEG from upper phase rich in recombinant human tumor necrosis factor alpha after simultaneous cell disruption and two-phase aqueous extraction. Buffer exchanging and primary purification were also achieved at same time. The purification factor of 4.4 was obtained with an activity recovery of 97% by one single step of serial operation.     

UGT75L6 and UGT94E5 mediate sequential glucosylation of crocetin to crocin in Gardenia jasminoides

Crocin is an apocarotenoid glycosyl ester accumulating in fruits of Gardenia jasminoides and used as a food coloring and nutraceutical. For the first time, the two glucosyltransferases UGT75L6 and UGT94E5 that sequentially mediate the final glucosylation steps in crocin biosynthesis in G. jasminoides have been identified and functionally characterized. UGT75L6 preferentially glucosylates the carboxyl group of crocetin yielding crocetin glucosyl esters, while UGT94E5 glucosylates the 6' hydroxyl group of the glucose moiety of crocetin glucosyl esters. The expression pattern of neither UGT75L6 nor UGT94E5 correlated with the pattern of crocin accumulation in G. jasminoides.     

The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration

Crocetin, a unique carotenoid with a short carbon chain length, is an active compound of saffron and Gardenia jasminoides Ellis used as traditional herbal medicine. The present study was undertaken to investigate the pharmacokinetic profiles of crocetin in healthy adult subjects. The study was conducted as an open-label, single dose escalation with 10 Filipino volunteers (5 men and 5 women). The subjects received a single dose of crocetin at three doses (7.5, 15 and 22.5 mg) in one week interval. Blood samples were collected from the brachial vein before and at 1, 2, 4, 6, 8, 10 and 24 h after administration. Plasma concentrations of crocetin were determined by high-performance liquid chromatography (HPLC). Crocetin was rapidly absorbed and detected within an hour of administration with a mean time to reach maximum concentration (T_max) of crocetin ranging from 4.0 to 4.8 h. The mean values of C_max and AUC_0-24 h ranged from 100.9 to 279.7 ng/ml and 556.5 to 1720.8 ng^.h/ml respectively. C_max and AUC values increased with dose proportional manner. Crocetin was eliminated from human plasma with a mean elimination half life (T_1/2) of 6.1 to 7.5 h.In summary, there were no serious adverse events up to 22.5 mg dose of crocetin while crocetin was found to be absorbed more quickly than the other carotenoids such as β-carotene, lutein and lycopene.     

Purification of monomeric mAb from associated aggregates using selective desorption chromatography in hydroxyapatite systems

Selective desorption on ceramic hydroxyapatite (CHT) was implemented for the purification of monomeric monoclonal antibody (mAb) from associated aggregates and other post-protein A step impurities. A robotic liquid handling system was employed to carry out a parallel batch screen of selective desorbents on a post-protein A step mAb mixture. The effect of different phosphate concentrations was also investigated. Selective batch separations were achieved between monomeric mAb and associated aggregates/impurities. The batch screen results also established optimal mobile phase conditions for each selective desorbent. These initial batch results were then used to guide column separations, and baseline separation of monomeric mAb from associated aggregates and impurities was achieved, validating the screening results. Selective desorption also resulted in improved separations on CHT, with 100% yield of pure monomeric mAb as compared to 61% and 79%, respectively, for conventional linear and step gradient operations. This proof of concept study demonstrates selective desorption on CHT as an effective separation technique for the purification of monomeric mAb from associated aggregates and other post-protein A step impurities in a single process step.     

大孔树脂——溶剂萃取法精制高色价栀子黄色素的集成技术研究

以栀子为原料提取栀子黄色素,采用大孔吸附树脂--有机溶剂萃取相结合的集成技术,从栀子中分离纯化得到高色价的栀子黄色素.先采用大孔吸附树脂对栀子黄色素进行初步精制,以306型大孔吸附树脂为研究对象,探讨了大孔树脂对栀子黄色素的静态吸附率、吸附流速和洗脱剂浓度对吸附的影响,从而得到较为合适的工艺:吸附流速2.O mT/mi...     

Liquid–liquid extraction of a recombinant protein, cytochrome b5, from an impure extract using aqueous two-phase systems

The recovery and purification of a recombinant protein, cytochrome b₅, from an impure extract of Escherichia coli disrupted cells was carried out in one step using a liquid–liquid extraction process of aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts. With this separation process it was possible in one single step to remove the cell debris, that precipitate at interface of the system, and to obtain relatively high recovery yields, nearly 67%, of the target protein in the salt-rich phase, with purification factors up to 6.     

栀子黄色素制备藏红花酸研究

以栀子黄色素为原料,对其水解反应制备藏红花酸及其纯化条件进行了研究,结果表明,栀子黄色素经碱水解法制备藏红花酸的最佳条件为:KOH溶液10%、温度为60℃、反应时间120 min,水解所得藏红花酸通过甲醇除杂,重结晶后,所得结晶其mp.、uv与文献报道一致,其质谱的诱导碰撞解离技术获得碎片裂解信息均表明所得到的结晶为藏红花酸,经HPLC检测纯度达到98.43%,得率为8.42%。     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

Purification of native dehydrin from Glycine Max cv., Pisum sativum,and Rosmarinum officinalis by affinity chromatography

An improved method for the purification of dehydrin from soy (glycine max) is described. Acidic extraction of soy whey was followed by a three step chromatographic process: capture on copper charged Chelating Sepharose Big Beads, intermediate hydrophobic interaction chromatography on Source 15 PHE, and a polishing step on blue Sepharose. The 32-kDa native soy dehydrin was purified to a purity of greater than 98.5% with an overall recovery of 63%. When compared to a previously published purification procedure, recovery, time requirements, and sample preparation steps were improved. The developed method is readily scaleable. Preliminary results show that the process can be used for dehydrins from rosemary (Rosmarinum officinalis) and pea (Pisum sativum).     

A群脑膜炎球菌荚膜多糖纯化工艺的优化

目的对A群脑膜炎球菌荚膜多糖纯化工艺的关键步骤进行分步研究,优化每一步工艺参数。方法优化十六烷基三甲基溴化铵的加入浓度、复合多糖的解离浓度和解离时间、不同厂家的苯酚、超滤和透析等工艺过程对荚膜多糖的影响。结果十六烷基三甲基溴化铵质量体积终浓度0.10%(w/v)沉淀效果更好,纯化获得的荚膜多糖产量更高相对分子质量更大。复合多糖解离浓度越高,纯化获得的荚膜多糖相对分子质量越小。延长复合多糖解离时间有利于提高荚膜多糖产量。不同厂家的苯酚、超滤和透析等工艺对荚膜多糖的产量和分子大小没有影响。结论现行A群脑膜炎球菌荚膜多糖纯化工艺复杂,优化后的工艺提高了荚膜多糖产量,缩短了工艺用时,增加了工艺稳定性。     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

Optimization of micellar extraction for the pre-purification of paclitaxel from<Emphasis Type="Italic">Taxus chinensis</Emphasis>

Currently, there are many reports in the literature regarding technological methods for paclitaxel purification. However, there have been few reports on the purification of paclitaxel using a micellar system. This method is based on the transfer of paclitaxel within the crude extract to an aqueous surfactant solution as a micelle, allowing the use of organic solvents to be used for the removal of lipids and non-polar impurities. In this study, we optimized the important process parameters of micellar extraction to obtain a high purity and yield of paclitaxel in a pre-purification step. The optimal surfactant (N-cetylpyridinium chloride, CPC) concentration, initial crude paclitaxel concentration, organic solvents (methylt-butyl ether/hexane) ratio, extraction temperature, and extraction time were 7.5% (w/v), 16.4 mg/mL, 1.5/1 (v/v), 25°C, and 30 min, respectively. The crude extracts from the liquid-liquid extracts were efficiently pre-purified by micellar extraction, increasing in purity from 6% to over 21%, with a yield of 92%. Overall, the use of micellar extraction in the pre-purification process allowed for rapid and efficient separation of paclitaxel from interfering compounds, and dramatically increased the yield and purity of the crucle paclitael for subsequent purification steps.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography

In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.     

Downstream processing of extracellular phytase from Aspergillus niger: Chromatography process vs. aqueous two phase extraction for its simultaneous partitioning and purification

The application of single step aqueous two-phase extraction (ATPE) for the downstream processing of phytase from Aspergillus niger NCIM 563, produced under solid state fermentation, has been studied and compared with the traditional multi-step procedure involving salt precipitation and column chromatography. High phytase recovery (98.5%) within a short time (3 h) and improved thermostability was attained by ATPE in comparison to 20% recovery in 96 h by chromatography process. The ATPE method, therefore, seems to be an interesting alternative for simultaneous partitioning and purification of phytase. The influence of system parameters; such as, phase forming salts, polymer molecular weight and system pH on the partitioning behavior of phytase was evaluated. The ATPE system consisting of combination of polyethylene glycol (PEG) 6000 and 8000 (10.5%) and sodium citrate (20.5%) resulted in one-sided partitioning of phytase in bottom phase with a purification factor of 2.5. This is the first report on phytase purification using liquid–liquid extraction and the results are likely to be beneficial in the poultry feed industry.     

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads? was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni²⁺ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.     

Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling

The partitioning behaviour of endo-polygalacturonase (endo-PG) and total protein from a clarified Kluyveromyces marxianus fermentation broth in polyethylene glycol (PEG)-ammonium sulfate and PEG-potassium phosphate (pH=7) aqueous two-phase systems was experimentally investigated. Both the enzyme and total protein partitioned in the bottom phase for these two kinds of systems. The enzyme partitioning coefficient can be lower than 0.01 in PEG8000-(NH₄)₂SO₄ ATPS with a large phase volume ratio and a moderate tie-line length, which implies the possibility of concentration operation using aqueous two phase partitioning. An ion-exchange separation of high purification efficiency was applied to analyze the clarified and dialyzed fermentation broth. A total purification factor of only 2.3 was obtained, which indicated the high enzyme protein content in the total protein of the fermentation broth. Consequently, the main purpose for separating endo-PG is concentration rather than purification. A separation scheme using an aqueous two-phase extraction process with polymer recycling and a dialysis was proposed to recover endo-PG from the fermentation supernatant of K. marxianus for commercial purpose. A high enzyme recovery up to 95% and a concentration factor of 5 to 8 with a purification factor of about 1.25 were obtained using the single aqueous two-phase extraction process. More than 95% polymer recycled will not affect the enzyme recovery and purification factor. Dialysis was used mainly to remove salts in the bottom phase. The dialysis step has no enzyme loss and can further remove small bulk proteins. The total purification factor for the scheme is about 1.7.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.