中试规模纯化海洋芽孢杆菌源脂肽类化合物

本次研究旨在建立经济可行的海洋芽孢杆菌源脂肽类化合物的中试规模纯化工艺。对包括酸化沉淀、甲醇浸提、溶剂沉淀、盐析、萃取、硅胶柱层析和HZ806大孔树脂吸附工艺在内的可放大的成熟单元工艺进行反复试验,考察脂肽类化合物表面活性对单元工艺的影响。严格遵循以高收率为前提循序渐进逐步减少杂质的原则,组合上述单元工艺对目标产物进行提取和纯化,并最终获得高纯度脂肽样品。新工艺可从1 t海洋芽孢杆菌Bacillus marinus B-9987的发酵液中,以百克量级的规模制备87.51%–100%纯度的脂肽类化合物样品,收率81.73%。本研究首次实现了高纯度的海洋芽孢杆菌源脂肽类化合物的百克量级制备;允许发酵生产阶段使用天然培养基,缓解了脂肽中游发酵生产和下游大规模纯化之间的矛盾;且各单元工艺规避了脂肽类化合物水溶液的乳化起泡和不经济的大体积水溶液蒸发浓缩。新工艺实用可行,经济合理。     

大孔树脂分离纯化辣椒叶总黄酮的工艺研究

目的:筛选适合分离纯化辣椒叶总黄酮的一种大孔树脂,同时用响应面法进行优化得到最佳纯化工艺。方法:采用热回流法提取辣椒叶总黄酮,以吸附率和解吸率为考察指标,考察6种不同型号的大孔树脂(HPD100、HPD450、HPD600、HPD826、D101、AB-8)对辣椒叶总黄酮的吸附能力与解吸能力,确定最佳树脂。通过动态吸附解吸实验考察此树脂对辣椒叶总黄酮的最佳分离纯化工艺。结果:通过对辣椒叶总黄酮吸附分离性能的分析显示HPD600为最佳树脂,最优工艺为:上样浓度为10 mg/mL,上样量为10 mL,洗脱体积为4 BV,洗脱液流速为4 mL/min,洗脱液pH为7,依次用水、10%、30%乙醇冲洗树脂柱,50%乙醇为洗脱液。纯化后的黄酮纯度435.4 mg/g。结论:该方法简便,操作简单,对辣椒叶总黄酮的纯化效果较好。     

小麦紫色素的提取工艺、抗氧化活性及其在毛发染色中的应用

以紫粒小麦麦麸为原料,通过碱水裂解初提、大孔吸附树脂纯化、钠滤膜浓缩和蒸发浓缩等技术手段从中提取天然紫色素。采用单因素实验对其提取条件进行优化,在室温条件（25~30℃）下,确定最佳提取条件为：提取时间60 min,料液比1∶4(g/mL),提取3次。经体外抗氧化实验证明,提取的小麦紫色素具有较高的抗氧化活性。此外,小麦紫色素与不同金属离子作用后可将羊毛染成不同的颜色。上述实验结果表明,利用优化后的工艺提取的小麦紫色素纯度高、抗氧化活性高,具有较大的开发潜力和实用价值。     

发酵液中L-色氨酸分离纯化工艺研究

通过静态吸附实验,考察了温度、pH值对001×7阳离子交换树脂平衡吸附量的影响,并测定了吸附动力学曲线。通过动态实验,测定了动态吸附曲线和洗脱曲线。最后确定了001×7阳离子交换树脂分离纯化L-色氨酸的最佳工艺条件:用001×7阳离子交换树脂吸附L-色氨酸,以浓度为2 mol.L-1氨水进行洗脱,收集的流份经D315阴离子交换树脂脱色,浓缩结晶后得L-色氨酸成品,总提取率为73.0%。     

大孔吸附树脂纯化山茱萸总皂苷的动态吸附条件研究

为了研究大孔吸附树脂纯化山茱萸总皂苷的动态吸附工艺,运用静态吸附与解吸试验对大孔吸附树脂进行筛选,然后通过单因素试验、正交试验和方差分析确定了大孔吸附树脂吸附山茱萸总皂苷的最佳操作条件.结果表明,HPD-300树脂对山茱萸总皂苷的吸附和解吸性能较好.确定的最佳吸附条件为料液浓度3.5 m g.mL-1,上柱速度3.5 BV.h-1,pH值为7.0.HPD-300大孔吸附树脂可较好地纯化山茱萸总皂苷.     

D140大孔吸附树脂银杏黄酮提取纯化性能研究

对国内外部分大孔吸附树脂的银杏黄酮吸附性能进行了比较筛选实验。其中D140型大孔吸附树脂具有较佳的吸附能力,应用该树脂研究了银杏黄酮的树脂法提取纯化工艺,研究结果表明,银杏黄酮提取液的预处理,提取液的pH,提取液过柱流速,洗脱剂种类及用量,洗脱物后处理等因素均对银杏提取物的收率、纯度等产生影响,采用D140树脂提取银杏黄酮的平均收率为3.54％,纯度为24.54％。已用于工业化生产。     

茶氨酸提取纯化工艺研究

系统研究了从茶多酚工业废液中提取纯化茶氨酸的工艺。采用絮凝、吸附、阳离子树脂交换、重结晶工艺来分离纯化茶氨酸。结果表明,絮凝能有效的去除茶多酚工业废液中的蛋白质等杂质,杂质的去除率为50％;吸附能进一步去除色素、多酚类物质及大分子有机物;阳离子交换树脂能较专-吸附氨基酸。茶多酚工业废液经絮凝→吸附→阳离子树脂交换工艺可得纯度50%的茶氨酸,得率为1．8％;通过重结晶可得到纯度90％的茶氨酸,得率为0．8%。     

黄柏中总生物碱的提取及测定方法研究

对黄柏中总生物碱的提取、纯化条件进行了优化研究 ,其提取最佳工艺为醇提取、大孔树脂吸附纯化法。同时采用柱层析—紫外分光光度法对产品进行含量测定 ,并对测定方法进行了系统的方法学考察。该方法重现性好 ,准确度高。     

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...     

葛花不同工艺提取物的异黄酮成分含量比较研究

目的:以不同浓度乙醇和大孔吸附树脂法提取和纯化葛花的提取物,并用HPLC和UV进行异黄酮成分含量比较分析。方法:以不同浓度的乙醇回流提取葛花药材制备提取物,并采用大孔吸附树脂对其进行纯化;然后以HPLC法对提取物中的3种主要黄酮类成分进行定量分析,并以UV法测定其总黄酮含量。结果:葛花不同提取物的黄酮类成分含量差别较大;所建立的HPLC方法可以同时测定葛花提取物中鸢尾苷、6"-O-木糖鸢尾苷和染料木素的含量;UV法与HPLC法联合应用,可对不同葛花提取物中的异黄酮类成分进行评价。结论:不同工艺提取的葛花提取物中异黄酮类成分有较大区别,大孔吸附树脂纯化后可显著提高总黄酮的纯度;HPLC联合UV法可共同用于葛花的质量评价及提取工艺的研究。     

Process development for purifying tacrolimus from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. using adsorption

The immunosuppressant tacrolimus produced by Streptomyces sp. TST10 was purified in a process that included extraction, pre-adsorption using HP20 resin, adsorption using CG161M resin, and crystallization. In this study, the purification process using the adsorption resin CG161M was optimized by correlating tacrolimus yield with analogue load. One-step adsorption and two-step adsorption using CG161M can be applied selectively to the purification process, according to the analogue load of the input solution. We determined a correlation between the analogue load and the first adsorption yield in the two-step adsorption. We also observed yields according to the analogue loads in the one-step adsorption and the second adsorption of the two-step adsorption. As a result, the purification yields can be predicted by input conditions (analogue load). The purification strategy can be modified to achieve specific goals of purity, yield, and cost.     

吸附法提取分离水杉总黄酮的研究

研究了我国重要经济植物水杉总黄酮的提取工艺,实验证明用70％的乙醇渗漉提取水杉总黄酮的工艺是经济实用的。正交实验显示用吸附法分离水杉总黄酮的影响因素依次为吸附剂选择,洗脱剂乙醇浓度,吸附剂用量及洗脱剂乙醇用量。将D140树脂用于水杉总黄酮的分离,具有收率高、操作简单、成本低、环境污染小等优点,适用于工业化生产。     

Process and economic evaluation for monoclonal antibody purification using a membrane-only process

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.     

选择性ADS-21吸附树脂对大豆异黄酮吸附性能的研究

本文筛选了6种不同性能的吸附树脂,对比了这些树脂对大豆异黄酮的吸附规律。结果表明,以大豆胚为原料,用乙醇提取,经选择性吸附树脂——ADS-21树脂吸附,再用70%的乙醇洗脱,可以得到含量达45%以上的大豆异黄酮提取物,表明ADS-21树脂对大豆异黄酮有良好的吸附选择性。     

Application of vortex flow adsorption technology to intein-mediated recovery of recombinant human alpha1-antitrypsin

Vortex flow is a secondary flow pattern that appears above a critical rotation rate in the annular gap between an inner rotating solid cylinder and an outer stationary cylindrical shell. By suspending adsorbent resin in the vortices, a novel unit operation, vortex flow adsorption (VFA), is created. In VFA, the rotation of the inner cylinder facilitates the fluidization of the adsorbent resin. Similar to expanded bed processes, VFA has high fluid voidage so that it can be used to recover biochemical products directly from fermentation broths or cell homogenates without removing cells or cell debris first. In this study, recombinant human alpha1-antitrypsin (alpha1-AT) was expressed in Escherichia coli as a fusion with a modified intein containing a chitin-binding domain. Therefore, the fusion protein can be recovered by chitin resin affinity adsorption. The intein can be induced to undergo in vitro peptide bond cleavage to specifically release alpha1-AT from the bound fusion protein. The capture efficiency of the fusion protein, 26.2%, was obtained in the VFA process. In addition, the specific activity of alpha1-AT was dramatically improved from 0.3 to 205.2 EIC/(mg total protein) after adsorption and cleavage. Therefore, vortex flow adsorption is an integrative technology to combine the primary clarification, concentration, and purification steps in conventional downstream processing into a single unit operation to efficiently recover and purify biochemical products.     

大孔吸附树脂柱层析分离淫羊藿甙的研究

本文通过考察八种大孔吸附树脂对淫羊藿甙的吸附分离性能,筛选出了AB-8树脂作为分离纯化淫羊藿甙的介质。对该树脂的吸附性能研究表明其对浸提物中的淫羊藿甙有良好的吸附选择性,静态饱和吸附容量和动态吸附容量分别为22．97和16．20mg／mL。通过柱层析实验确定了AB-8树脂分离淫羊藿甙的工艺,经一步层析可将淫羊藿甙的纯度从2．78％提高到27％,回收率达97．3％。     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.     

Eremomycin的发酵条件优化及发酵产物的分离纯化

研究确定Nocardia orientalis NRRL18098发酵生产eremomycin的最佳工艺条件,以及对发酵产物进行分离纯化并得到eremomycin的纯品。通过正交设计方法优化发酵培养基的组成。采用树脂吸附、中压液相色谱技术相结合的方法对发酵产物进行分离纯化。在优化条件下,eremomycin的摇瓶发酵单位达115 mg/L,提高了63.5%,并采用树脂吸附和中压液相色谱相结合的方法能有效地将eremomycin从发酵液中分离出来,制备获得eremomycin精制品。     

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019