Facile purification and characterization of recombinant human antithrombin III from transgenic goat milk

BACKGROUND: Antithrombin III (AT III) is a serine protease inhibitor that inhibits thrombin and the activated forms of factors X, VII, IX, XI and XII. Transgenic expression of therapeutic proteins in animal systems has gradually matured from laboratory scale to industrial practice, demanding efficient and scalable purification processes. The purification and characterization of recombinant human antithrombin III (rhAT III) from transgenic goat milk are described here. RESULTS: The rhAT III was purified by isoelectric precipitation, heparin affinity chromatography, and size exclusion chromatography, resulting in a 90.6% yield and > 99% purity. The goat β‐casein secretion peptide introduced to the rhAT III was cut off using enterokinase and removed by size exclusion chromatography using a Superdex 75 column. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT III were measured and found to be the same as those of the plasma‐derived AT III (phAT III). CONCLUSION: A facile process is introduced for the purification of rhAT III from transgenic goat milk. The rhAT III with high purity was achieved after an initial isoelectric precipitation step in which most of the bulk protein impurities are removed, followed by affinity chromatography and size exclusion chromatography. The rhAT III was demonstrated to have the same structure as phAT III. Copyright © 2011 Society of Chemical Industry     

柱层析法纯化基因工程HBsAg中试工艺的研究

由乙肝病毒S基因转化的哺乳动物细胞培养收液，经过Butyl-S-SepharoseFF层析，DEAESepharoseFF层析和Sepharose4FF层析，可获HBsAg纯品。产品检定结果表明，PAGE和SDS-PAGE电泳纯度均合格，小牛血清残余量、细胞DNA残余量和动物免疫效力也均符合规定标准．HBsAg终收率达40％左右．在此工艺中，疏水作用柱层析的纯化效率比较高，可去除绝大部分杂蛋白和细胞DNA。     

Purification of monoclonal antibodies from cell culture supernatants by Gradiflow™ electrophoresis technology

Monoclonal antibodies (mAbs) are used extensively for analytical, diagnostic and therapeutic applications. The purification of mAbs from cell culture supernatants typically consists of protein A, G or L affinity chromatography, often in association with other conventional chromatographic techniques such as ion exchange and gel filtration. We report the application of Gradiflow^? preparative electrophoresis technology, for the separation of mouse and mouse/human chimeric mAbs from cell culture supernatants in their native state. The one‐step purification of murine mAb HuLym3 shows that mAbs can be purified from hybridoma cell culture supernatants to high purity, and is thus an alternative to other purification methods based on conventional and affinity chromatography for the production of mAbs for analytical and diagnostic applications. A mouse/human IgG1 chimeric mAb produced by Chinese hamster ovary cells was also purified from cell culture supernatant, and the purity achieved suggests that Gradiflow^? electrophoresis could replace affinity chromatography in the downstream processing of mAbs for therapeutic use. Gradiflow^? electrophoresis technology is scaleable and thus is applicable to industrial‐scale purification of mAbs. Copyright © 2005 Society of Chemical Industry     

Comparison of affinity membrane adsorbers for the purification of recombinant human erythropoietin (rhEPO)

In this work affinity membrane adsorbers were investigated for the chromatographic purification of recombinant human erythropoietin (rhEPO) produced in mammalian cells. Cibacron Blue (CB), IDA‐Cu⁺², wheat germ agglutinin (WGA), concanavalin A (ConA) and an anti‐EPO monoclonal antibody (MAb) were tested as affinity ligands, attached to microporous Sartobind^® membranes. In experiments carried out with cell culture supernatant, the best results were obtained with Sartobind–CB, Sartobind–WGA and Sartobind–MAb membranes. The thermodynamic parameters were determined by adsorption isotherms of rhEPO onto the membranes. Sartobind–ConA presented the lowest affinity for rhEPO, as evidenced by a lower association constant. For Sartobind–CB, Sartobind–IDA‐Cu⁺² and Sartobind–MAb K_A was in the order of 10⁵ L mol^?1, whereas for Sartobind–WGA it was 10⁶ L mol^?1. Sartobind–CB eluates were also investigated by RP‐HPLC. The purity level achieved in this one‐step purification strategy was 55%, indicating that the Sartobind–CB membrane is a promising affinity membrane for rhEPO purification. Copyright © 2007 Society of Chemical Industry     

重组人白细胞介素-12(CHO细胞)的纯化及其活性

目的建立CHO细胞表达的重组人白细胞介素-12(rhIL-12)的纯化工艺,并检测纯化的rhIL-12的生物活性。方法取高效表达rhIL-12的CHO工程细胞培养上清液,采用层析结合硫酸铵沉淀的方法进行分离纯化,ELISA法测定目的蛋白的含量,并计算回收率;SDS-PAGE、SEC-HPLC和Westernblot对纯化样品进行鉴定;以PBMCIFNγ诱生法检测纯化rhIL-12的生物活性。结果纯化的rhIL-12的总回收率为56.4%;经SEC-HPLC检测,其纯度达99.2%;SDS-PAGE分析显示,两个亚基的相对分子质量分别与理论值(40000和35000)相符;Westernblot分析显示,rhIL-12可与相应抗体发生特异性结合;rhIL-12诱生IFNγ量与rhIL-12浓度呈典型的S型曲线关系,具有剂量依赖性,其效价为8.3×106IU/mg。结论已建立了CHO细胞表达的rhIL-12纯化工艺,该工艺成本低,操作简便,纯化产物回收率和纯度高,适于工业化生产。     

Efficient purification and morphology characterization of paclitaxel from cell cultures of Taxus chinensis

Paclitaxel was purified from cell cultures of Taxus chinensis by a combination of extraction, low‐pressure chromatography, precipitation and HPLC. A crude extract (purity 6.9%) was obtained by methanol extraction of plant cell cultures, followed by liquid–liquid extraction using dichloromethane. The extract was purified to greater than 32% with a 97% step yield by low‐pressure chromatography. After acetone/pentane precipitation, the resulting purity and step yield were 75.8% and 97.4%, respectively. High performance liquid chromatography steps, which were composed of an HPLC step with column‐packed ODS and an HPLC step with column‐packed silica, were applied to give over 99.5% purity with high yield. Amorphous paclitaxel with a fine particle size, which has a solubility advantage compared with the stable crystalline form, was obtained by dissolving in dichloromethane, followed by spray‐drying. Copyright © 2004 Society of Chemical Industry     

Optimal Integration of Directly Combined Hydrophobic Interaction and Ion Exchange Chromatography Purification Processes

Hydrophobic interaction and ion exchange chromatography are basic steps in purification of fermentative biopharmaceuticals. An optimization by statistical design of experiments requires a huge amount of feed. An alternative approach is the combination of model parameter determination using small scale experimental model parameter determination (1‐mL columns) and rigorous process modeling. Applicability for the prediction of the separation of a fermentation mixture of CHO mammalian cell culture is validated and hence IgG is purified from cell culture supernatant. Hydrophobic interaction chromatography directly combined with ion exchange chromatography is optimized. Any direct integration of those two main unit operations in purification processes is a methodological first step towards total process optimization. The potential for cost reduction and overall yield improvement is demonstrated and this leads to the conclusion that single step optimization is a feigned and not a real optimum.     

A methodology for the graphical determination of operating conditions of chromatographic sequences incorporating the trade‐offs between purity and yield

Multiple‐step chromatography sequences are necessary in biopharmaceutical downstream processing to achieve the desired levels of purity for products such as therapeutic proteins. Traditional methods of process design deal with each step individually, but this can result in a sequence that does not achieve best overall performance. This paper proposes a graphical methodology for the identification of operating conditions for a two‐step chromatography sequence. The method uses windows of operation to incorporate the trade‐offs between yield, purity and productivity. A tie‐line procedure is developed that separates the window of operation for the first chromatographic step into two zones. One zone contains those operating conditions that combine to produce a material which can be purified successfully by the second step to produce a product that meets the desired specifications. The second zone consists of operating conditions which will not yield a material that can be adequately purified by a second chromatographic stage to yield a product of the predetermined specifications. The methodology is valuable in that it helps in achieving the rapid design of a two‐step chromatography sequence, and aids in choosing the optimum operating conditions for the first step that are highly dependent upon the operation and specifications of the second chromatographic step. Simulations carried out using a software package based on the general rate model depict the construction and use of the method applied to a sequence of ion exchange and hydrophobic interaction chromatography separating a three‐component protein mixture. Copyright © 2006 Society of Chemical Industry     

人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白重组CHO细胞生产工艺及质控方法的建立

目的 利用国产无血清培养基T22,建立人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)重组CHO细胞大规模生产工艺及质控方法。方法用500 L生物反应器培养重组CHO细胞,通过深层过滤、亲和层析、离子交换层析、疏水层析等方法纯化表达上清中的目的 蛋白,超滤浓缩制成原液,并按照《中国药典》三部(2010版)及现有的质控方法和标准建立相应的质控方法。结果重组CHO细胞在500 L生物反应器中培养15 d左右,细胞密度最高可达8.6×106个/ml,收获时细胞活力为70%左右,rhTNFR:Fc蛋白表达量可达3.3 g/L;纯化的目的 蛋白纯度可达98%以上,总收率超过30%;各项质量指标均符合新药申报标准。结论建立的rhTNFR:Fc重组CHO细胞生产工艺及质控方法稳定可靠,使用国产培养基大大降低了生产成本。     

Development of a pre‐purification process for homoharringtonine

A novel pre‐purification method was developed for producing homoharringtonine from Cephalotaxus koreana, giving high purity and yield. The simple, efficient procedure involved biomass extraction, liquid–liquid extraction, and synthetic adsorbent treatment, followed by low‐pressure chromatography. The use of active clay treatment and silica gel low‐pressure chromatography in the pre‐purification process allowed for the rapid, efficient separation of homoharringtonine from interfering compounds and, compared with alternative processes, increased the yield and purity of crude homoharringtonine for subsequent high‐performance liquid chromatography (HPLC) purification. Homoharringtonine of over 52% purity could be obtained simply with high yield from biomass using this pre‐purification method, while minimizing solvent use and the scale and complexity of HPLC operations for homoharringtonine purification. Copyright © 2005 Society of Chemical Industry     

Multi‐Stage Aqueous Two‐Phase Extraction for the Purification of Monoclonal Antibodies

Using monoclonal antibodies in a cell culture harvest as an example for complex biomolecules, a mini‐plant‐scale aqueous two‐phase purification process was studied. The results of this study indicate that antibodies can be concentrated and purified in a single extraction step employing a small phase ratio. Following the extraction step, a multi‐stage wash‐extraction for further purification was investigated. Starting at a test tube‐scale cross‐current wash, the process was advanced towards a continuous counter‐current mixer‐settler and column wash process. It was shown that a test tube cross‐current extraction operation can predict the multi‐stage purity reasonably well. The hydrodynamic process performance for the multi‐stage wash column was evaluated and related to the separation performance.     

重组人抗HBs-Fab抗体的纯化及结构分析

目的研究重组人抗HBs-Fab抗体的纯化工艺,并对其结构等特性进行分析。方法采用离子交换-分子筛层析法分离纯化由酵母工程菌(GS115/Fab)发酵的重组人抗HBsAg Fab抗体,并经ELISA检测其抗体活性,等电聚焦电泳法检测其等电点(pI),基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)检测其相对分子质量和肽质量图谱。结果纯化的重组Fab抗体的纯度可达90%以上,经Sephacryl-100进一步层析后,纯度达99%以上,总收率可达80%以上。Fab抗体具有较好的抗体活性,其pI值为7·6,为一碱性蛋白,相对分子质量为50494,比其理论值约多2579·35,经Endoglycosidase H内切酶消化后的相对分子质量为49609,证明Fab的一级结构中有糖基化现象,且分布于Fab抗体的H链和L链上。胰酶酶解肽段中有21个与理论肽段相符,另检测到1对二硫键正确。结论已建立了重组人抗HBs-Fab抗体的稳定的纯化工艺,且Fab抗体的一级结构正确。     

疏水作用层析在重组大肠杆菌表达的rProEMAPⅡ/P43蛋白纯化中的作用

目的建立rProEMAPⅡ/P43蛋白的纯化工艺,明确疏水作用层析(HIC)在纯化中的作用。方法采用由疏水作用层析、离子交换层析、亲和肝素层析组成的工艺对rProEMAPⅡ/P43蛋白进行纯化,并与由离子交换层析和亲和肝素层析组成的工艺相比较,分别进行总蛋白含量测定、SDS-PAGE分析目的蛋白含量及纯度检测。结果在pH为7·4~7·5时,采用HIC方法纯化的蛋白较不采用HIC法回收率提高了28·4%,纯化后rProEMAPⅡ/P43蛋白纯度为92%。结论在rProEMAPⅡ/P43蛋白纯化过程中增加疏水作用层析,可以提高rProEMAPⅡ/P43蛋白收率。     

茯苓多糖的纯化及分子量测定

通过水提醇沉的方法得到茯苓粗多糖,精制后采用离子交换柱层析对多糖进行分离纯化,用高效凝胶过滤色谱法(HPGFC)测定多糖分子量.结果表明,茯苓粗多糖用Sevag法脱蛋白效果较好,获得了精制茯苓多糖(TAP);经DEAE-650C层析柱纯化,得到中性多糖(TAP1)和酸性多糖(TAP2),高效凝胶过滤色谱法测定其分子量分...     

重组人肽抗生素hPAB-β的初步纯化

目的　探索重组人肽抗生素hPAB β的纯化工艺 ,为制备高纯度、高活性的重组hPAB β奠定基础。方法　将构建的含pQE32 CP hPAB β重组质粒的基因工程菌扩大培养 ,诱导表达后所得菌体经溶菌酶法裂解 ,鉴定融合蛋白表达形式 ;以含融合蛋白的溶液作为纯化的初始样品 ,采用Ni NTAresin亲和层析柱对融合蛋白进行纯化 ,用CNBr裂解 ,利用SephadexG 5 0、Bio gelP6DG凝胶进行分子筛层析 ,利用Macro PrepHighS进行阳离子交换层析 ,对层析峰行Tris Tricine电泳分析。结果　融合蛋白以包涵体形式存在 ;亲和层析获得了纯度为 82 .6 %的融合蛋白 ,CNBr作用 2 0h可完成融合蛋白的裂解 ;目的肽经纯化后 ,纯度达 95 %以上。结论　已初步建立了重组人肽抗生素hPAB β的纯化工艺 ,并获得了高纯度的肽抗生素hPAB β。     

HCV C区基因片段的克隆、表达及纯化

目的研究 ＨＣＶ Ｃ区片段在大肠杆菌中的表达及纯化工艺。方法采用 ＲＴ－ＰＣＲ技术,从ＨＣＶ感 染者血清中克隆Ｃ区基因片段,插入ｐＥＴ２８ａ（＋）质粒中,并在大肠杆菌ＢＬ２１（ＤＥ３）中表达。采用亲和层析或 Ｓａｐｈａｃｒｙｌ　Ｓ－２００柱层析纯化抗原。结果两种纯化工艺的收率分别为５８ｍｇ／Ｌ和９５ｍｇ／Ｌ发酵液。ＳＤＳ－ＰＡＧＥ显示纯 度分别为９８％和９０％。ＥＬＩＳＡ检测纯化抗原具有较好的抗原性和特异性。结论两种纯化工艺简便,重组蛋白纯 度高,可用于ＥＬＩＳＡ试验。     

水溶性有机试剂PAN-6s的纯化方法

报告了 PAN直接磺化法制备 PAN- 6s的盐析提纯方法 ,筛选出价格低廉 ,本身不含结晶水、并且其溶解度基本不随温度变化而变化的氯化钠为盐析剂 ,避免了水重结晶易使杂质包藏在产品晶体中的缺陷 ,大大地改善了产品的纯度 ,用该法可获纯度达 99.63%的产品 ,而水重结晶法所获产品的纯度仅在 88.83% ,经红外光谱和液相色谱表征 ,结果令人满意     

自制填充介质对人血浆凝血因子Ⅸ的层析分离

目的采用自制的DEAE Bio-SepFF和肝素Bio-Sep FF介质,从人血浆中快速分离纯化凝血因子Ⅸ(FⅨ)。方法低温离心去除冷沉淀后的人血浆,在不同淋洗条件下,经过两步弱阴离子交换和一步亲和层析分离纯化FⅨ,用活性检测试剂盒检测各组分凝血因子的活性,Bradford法测定蛋白浓度,聚丙烯酰胺凝胶电泳分析样品的纯度。结果经过三步层析分离,得到的FⅨ比活达到99.40IU/mg,纯化倍数为3823倍,回收率约为30%,纯度较高。结论采用该方法和介质,可从人血浆中纯化FⅨ,且效果较好。     

葡萄球菌蛋白A的纯化新工艺

目的建立一种高效、简便、实用的分离纯化葡萄球菌蛋白A(SPA)的工艺。方法采用超声波破菌和IgG-Sepharose4B亲和层析分离纯化SPA;以Lowry法检测蛋白含量;双向琼脂免疫扩散法测定效价和特异性;用还原和非还原SDS-PAGE法检测纯度和相对分子质量。结果此法制得的3批SPA的蛋白含量分别为4·7、4·4和5·4mg/ml,收率分别为2·70、2·64和3·78g/100g湿菌。对人IgG的免疫双扩散效价均为1:64;与正常人、豚鼠、家兔和小鼠的血清反应均出现一条沉淀线,而与鸡和羊不出现沉淀线。经非还原SDS-PAGE检测只呈现一条蛋白带,相对分子质量约为160000～180000;经还原SDS-PAGE检测呈现两条蛋白带,相对分子质量分别约为67000和34000。结论已成功建立了分离纯化SPA的新工艺。     

Two-step chromatographic procedure for the preparative separation and purification of epigallocatechin gallate from green tea extracts

The preparative separation and purification of epigallocatechin gallate (EGCG) from green tea extracts was achieved by a two-step chromatographic procedure. In the first step, a preparative β-cyclodextrin (β-CD) bonded silica chromatography operated in the polar organic mode using methanol/acetonitrile/acetic acid as the mobile phase was applied. Due to a high selectivity for EGCG over epicatechin (EC) and caffeine (CA), an efficient separation of EGCG from EC and CA was achieved and the system could be scaled-up with high recovery of EGCG (>90%). The EGCG-rich fraction obtained from the first step was subjected to the second step where a preparative C-18 chromatography operated in the reversed-phase mode using water/methanol/acetic acid as the mobile phase was applied. The resolution of EGCG was significantly enhanced as the sample contained no EC and CA. The system could also be scaled-up and both the purity and recovery of EGCG reached about 90%. In summary, the chromatographic procedure consisting of two such complementary steps gave a satisfactory purification results: the purity and recovery of EGCG were >90% and >80%, respectively. Most significantly, the purification method could be more easily scaled-up, showing good potential for industrial applications.