摘要
人血清白蛋白为人体血浆中含量最丰富的蛋白,具有很多重要的生理功能。临床上可作为很多疾病的药品治疗剂,还可作为生物产品的保护剂和赋形剂及细胞培养的添加成分,得到了广泛应用。近年来大规模生产重组人血清白蛋白作为血源白蛋白替代品。针对从高密度发酵液中分离纯化重组人血清白蛋白中存在的固液分离困难、工艺复杂、杂质难去除等问题,本论文从以下方面做了探索性研究。
首先,考察了不同乙醇/无机盐双水相体系对重组人血清白蛋白的萃取能力,其中乙醇/磷酸氢二钾双水相体系的回收率最高,且在4-30℃的温度和1-30g/L的重组人血清白蛋白浓度均能保持较高萃取收率。该双水相体系能够直接从发酵液中萃取重组人血清白蛋白,当操作条件为乙醇20%(w/w)/磷酸氢二钾18%(w/w)时,重组人血清白蛋白回收率达到108.13%。综合考虑回收率和相比因素,选择乙醇18%(w/w)/磷酸氢二钾20%(w/w)体系放大实验,0.01-73L规模的平均回收率为100.4%,菌体的平均去除率达到99%。同时该双水相体系能够有效去除发酵液中的部分杂蛋白,还可以去除87.2%的多糖,并使上相的蛋白酶A活性降低了24.4%,部分抑制了蛋白酶B的活性,提高了重组人血清白蛋白的稳定性。双水相萃取将固液分离、萃取、浓缩、初级纯化整合为一步进行。通过研究成相物质的回收,上相中的乙醇和下相中的磷酸氢二钾回收率均在80%以上。
其次,根据双水相上相的特点,建立了与之相适应的纯化方法：包括减压蒸馏去除乙醇、Phenyl SepharoseTM疏水层析纯化、DEAE阴离子交换层析去除多聚体等步骤。其中向平衡液中添加含1.2mol/L的磷酸氢二钾,为疏水层析的最优条件,此时收率为74.94%,还原SDS-PAGE电泳结果表明纯化的重组人血清白蛋白达到了电泳纯。而且纯化的重组人血清白蛋白能够长期保存,其中蛋白酶A活性降至上清液的9.18%,蛋白酶B可能被去除。
最后,对上述方法分离纯化得到的重组人血清白蛋白纯度、杂质含量、二级结构及分子量进行了分析。高效液相色谱检测其纯度达到了99.77%,色素比值A350/A280为0.028。同时,其多糖含量为2.98μg/mg,蛋白酶A活性降为上清液的2.93%。而且,纯化的重组人血清白蛋白分子量及二级结构与血源白蛋白具有一致性。
双水相萃取耦合柱层析技术提供了一种简便的、分离纯化得到高纯度重组人血清白蛋白的方法,具有分离时间短、收率高、杂质去除效果好的优点,具有产业化的前景。
Human serum albumin (HSA) is the most abundant protein component contained in plasma and has many functions. HSA has wide applications, such as pharmaceuticals under various clinical conditions, an excipient or stabilizer in other biotechnology based products, a component in cell culture media, and so on. Large scale production of recombination human serum albumin (rHSA) which is as a substitute for the HSA originating in plasma (pHSA) has developed. For the problem in separation and purification of rHSA from high density fermentation broths such as complex process, difficulties in solid-liquid separation and removal of impurities,some technical problems were explored from the following aspects.
Firstly, the extraction ability of different ethanol/salt aqueous two-phase systems (ATPS) was investigated. The ethanol/dipotassium hydrogen phosphate system showed the highest recovery for rHSA. When the concentration of rHSA changed from lg/L to 30g/L, and temperature was from 4℃to 30℃, aqueous two-phase extraction (ATPE) of rHSA exhibited high extraction efficiency. And ATPE could recover rHSA directly from a crude culture medium. The recovery (Y) of rHSA reached up to 108.13%when the system was composed of 20%(w/w) ethanol and18%(w/w) dipotassium hydrogen phosphate. Take suitable volume ratio of the top to bottom phase (R) into consideration, extrations which ATPS was composed of 18%(w/w) ethanol and 20%(w/w) dipotassium hydrogen phosphate were scaled up from 0.01 to 73L successfully.The average recovery of rHSA at different scales was 100.4%,and average removal ratios of cells was up to 99%. Simultaneously, partial protein impurities and 87.2%polysaccharides were eliminated, proteinase A (PrA) activity was decreased by 24.4%, proteinase B(PrB) activity was partially inhibited, and thus the stability of rHSA was improved. ATPE has integrated solid-liquid separation, extraction, concentration and primary purification into a single step. Moreover, recycle of phase components were reaserched. More than 80% of ethanol in the top phase and 80% dipotassium hydrogen phosphate in the bottom phase can be recovered.
Secondly, according to the characteristics of the top phase, the downstream purification process of rHSA has been established. It included vacuum distillation for removing ethanol, purification by Phenyl SepharoseTM hydrophobic interaction chromatography(HIC), DEAE anion exchange chromatography for removal of polymers,. When the equilibrium solution contained 1.2mol/L K2HPO4, HIC condition was optimum.The recovery of HIC attained 74.9%, and reduced SDS-PAGE analysis showed that rHSA was electrophoretic pure. Moreover, purified rHSA remained stable for a long time because PrA activity was decreased to 9.18% compared with the supernatant, and PrB might be removed.
Finally, purity, impurities content, secondary structure and molecular characteristics of purified rHSA has been analyzed. The HPLC purity and A350/A280 of rHSA attained 99.77% and 0.028 respectively. Meanwhile, the content of polysaccharides was reduced to 2.98ug/mg, and PrA activities was 2.93% compared with the supernatant. Furthermore, purified rHSA was essentially identical with pHSA in molecular weight and secondary structure.
Integration process of hydrophilic organic solvents/inorganic salts ATPE and chromatography technology has provided a simple process for the separation and purification of rHSA to a high purity level, which has advantages of short time, high yield and removal rate of impurity, and produces large-scale industrial prospect.
引文
[1]赵颖怡,汪嵘,韦宇拓,等.人血清白蛋白的研究进展[J].广西农业生物科学,2002,21(2)：126-128.
[2]满初日嘎,张英霞.重组人血清白蛋白的研究进展[J].生物医学工程学杂志,2009,26(4)：900-903.
[3]郭美锦,储炬,杭海峰,等.重组人血清白蛋白表达研究进展[J].生物工程进展,2000,20(5)：39-42.
[4]李梅彦.重组白蛋白(rHSA)纯化工艺优化研究[D].天津：天津大学化工学院,2006.
[5]Takao Ohmura, Akinori Sumi, Wataru Ohtani, et al. Recombinant human serum albumin,process for producing the same and pharmaceutical preparation containing the same:United States,5986062 [P].1999,11,16.
[6]刘彦丽.巴斯德毕赤酵母发酵生产重组人血清白蛋白的研究[D].北京：北京化工大学化学工程学院,2003.
[7]Tuyoshi Takahashi, Kazuo Ikegaya, Shinobu Mochizuki, et al. Process for producing albumin preparation:United States,5616691[P].1997,04,01.
[8]刘永东,王云山,苏志国.重组人血清白蛋白生产工艺研究进展[J].微生物学通报.2003,30(5)：128-132.
[9]刘茉娥,陈欢林.新型分离技术基础：第2版[M].浙江：浙江大学出版社,1999.
[10]孙彦.生物分离工程[M].北京：化学工业出版社,1998.
[11]王志华,马会民,马泉莉,等.双水相萃取体系的研究[J].应用化学,2001,18(3)：173-175.
[12]Ariel Louwrier. Model phase separations of proteins using aqueous/ethanolcomponents [J].Biotechnology Techniques,1998,12(5):363-365.
[13]田明玉.双水相萃取白蛋白和酶的初步研究[D].大连：大连理工大学,2009.
[14]Curry S, Mandelkow H, Brick P, et al. Crystal structure of human serum albumin [J].Nature Structural & Molecular Biology,1998,5:827-836.
[15]Ueno Y, Mio M, Sato C, et al. Single particle conformations of human serum albumin by electron microscopy [J]. Electron Microscopy,2007,56 (3):103-110.
[16]Kaoru Kobayashi, Shinobu Kuwae, Tomoshi Ohya, et al. High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast Pichia pastoris, based on optimal methanol feeding strategy[J]. Journal of Bioscience and Bioengineering,2000,90(3):280-288.
[17]Peters T, Putnam F W. All about albumin[J]. Academic Press Inc,1996,5:251-284.
[18]Shinya E, Dervillez X, Edwards-Levy F, et al.In vivo delivery of therapeutic proteins by genetically modified cells:comparison of organoids and human serum albumin alginate-coated beads[J].Biomed Pharmacother,1999,53 (10):471-481.
[19]殷艺伟,陈建华.重组人血请白蛋白的药学应用研究进展[J].药学进展,2006,30(2)：70-73.
[20]Kindberg G M, Tolleshaug H, Skotland T.Uptake and degradation of radioactively labeled albumin microspheres as markers for Kupffer cell phagocytosis [J].Cell Tissue Res,2000,300 (3):397-400.
[21]Wunderlich G, Pinkert J, Andreeff M, et al.Preparat ion and biodistribution of thenium-188 labeled albumin microspheres B20:a promising new agent for radiotherapy [J].Appl Radiat Isot,2000,52 (1):63-68.
[22]黄宇彬,小松晃之,土田英俊.一种全合成型人工红血球的研究进展[J].应用化学,2004,21(11)：1081-1086.
[23]Syed S, Schuyler P D, Kulcz yxky M, et al.Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin [J].Blood,1997,89(9):3243-3252.
[24]朱振齐,刘进,马大龙,等.人重组GM-CSF/HAS-D3嵌合蛋白(GM-HSA)在大肠杆菌中的表达及其产物稳定性的研究[J].生物化学杂志,1996,12(3)：284-288.
[25]Bickel U, Yoshikawa T, Pardridge W M. Delivery of peptides and proteins through the blood-brain barrier[J].Adv Drug Deliv Rev,2001,46(13):247-279.
[26]Bekeredjian R, Grabum P A, Shohet R V.Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine [J]. J Am Colf Cardiol,2005,45(3):329-335.
[27]Petersen C E, Ha C E, Harohalli K, et al.A dynamic model for bilirubin binding to human serum albumin [J].J Biol Chem,2000,275(28):20985-20995.
[28]徐宝全.国内人血白蛋白市场供需状况及应对策略研究[D].沈阳：沈阳药科大学,2009.
[29]孙战胜.重组人血清白蛋白在巴斯德毕赤酵母中的表达[D].北京：北京化工大学,2004.
[30]P.Matejtschuk, C.H.Dash, E.W.Gascoigne. Production of human albumin solution:a continually developing colloid[J].British Journal of Anaesthesia,2000,85(6):887-895.
[31]DamJ.Plasma fractionation based on chromatography and precipition by polythylene glycol and caprylic[J].A report from the first Plasma Products Biotechnology meeting,1999,3:27-30.
[32]王清和,樊绍文,刘文方,等.利凡诺法制备人白蛋白的新工艺：中国,88107797[P].1988,11,17.
[33]陈继廷,杨鹏云,吕秀华.辛酸钠含量对人白蛋白稳定性的影响[J].中国输血杂志,1995,8(1)：42.
[34]徐金凤,史稚飞.利凡诺一氢氧化钠去除血浆白蛋白热原质[J].广东医学,1995,16(5)：344.
[35]张雪萍,郑宇红.人血白蛋白中辛酸钠含量与热稳定性试验相关性研究[J].中国现代应用药学杂志,1999,16(3)：29-30.
[36]刘力,刘文芳.人血白蛋白分离工艺的历史沿革及发展[J].中国输血杂志,2008,21(4)：323-326.
[37]浦奕奕.低温乙醇法在人血白蛋白工业化生产中的应用[J].考试周刊,2008,3：236-237.
[38]Kim I S, Eo H G, Chang C E, et al. Partitioning and inactivation of viruses by cold ethanol fractionation and pasteurization during manufacture of albumin from human plasma [J].Journal of Microbiology and Biotechnology,2000,10(6):858-864.
[39]刘隽湘.输血疗法与血液制剂[M].北京：人民卫生出版社,1996.
[40]杨成民.基础输血学[M].北京：中国科学技术出版社,2001.
[41]Titchener-Hooker NJ, Me Intosh RV. A study of effect of low-frequency conditioning on the size distribution properties and centrifugal recovery of human albumin precipitate [J]. Bioprocess Eng,1993,8:215-222.
[42]刘欣晏.人血白蛋白连续流压滤工艺研究[D].山东：山东大学,2008.
[43]刘晓宇,周昌华.低温乙醇法工艺中影响白蛋白收率的若干因素探讨[J].中国输血杂志,2001,6：355-357.
[44]路秀岭,苏志国.人血清白蛋白纯化技术研究进展[J].生物工程学报,2002,18(6)：761-766.
[45]Joseph D, Willie M,Michael E.Purification of plasma albumin by ion exchange chromatography:United States,4228154 [P].1980,10,14.
[46]SU Z G, JIANG W, FENG P S.Purification of serum albumin with dye-ligand adsorption chromatography[J].Chinese Journal of Biotechnology,1991,7(2):161-168.
[47]杨金菊,王继武,李永红.人血清白蛋白的仿生亲和配基研究[J].中国药学杂志,2003,38(10)：802-804.
[48]Gebauer K H, Thoemmes J, Kula M R. Plasma protein fractionation with advanced membrane adsorbents [J].Biotechnology and Bioengineering,1997,54(2):181-189.
[49]Janson J C, Ryden L.Protein purification:principles,high resolution methods,and applications[M].New York:John Wiley& Sons Inc,Publication,1997.
[50]钦晓峰,蔡刚明,周杏琴,等HiTrap Q柱FPLC快速纯化人血清白蛋白(HSA)[J]生命科学仪器,2004,2(6)：28-30.
[51]K.Tanaka, E.M.Shigueoka, E.Sawatani, et al.Purification of human albumin by the combination of the method of Cohn with liquid chromatography[J].Medical and Biological Research,1998,31:1383-1388.
[52]杨海荣,罗坚,董爱华.疏水层析结合冷乙醇沉淀纯化人血清白蛋白[J].生物工程学报,2004,20(6)：943-947.
[53]Yap HB, Young IF, MicucciV, et al. Development of a process for the preparation of human serum albumin using chromatographic methods [J].Biotechnol of Blood Proteins,1993,227:143-149.
[54]Kiyoshi N, Naoki M, Yoshio Y et al.Comparison of plasma and recombinant human serum albumin by microanalysis techniques for proteins [J].Perspect,Protein Eng Collect Pap,Int Symp,3ndMeeting,1994,317-323.
[55]Ohtani W, Atsushi M, Yoshitaka et al. Structure of recombinant human serum albumin from Pichia pastoris[J]. Yakugaku Zassh,1997,117(4):220-232.
[56]Matsushita Sadaharu, Isima Yu, Chuang V T.Functional analysis of recombinant human serum albumin domains for Pharmaceutical applications[J].Pharm Ras,2004,21(10): 1924-1932.
[57]Schindel F, Andresen C, Bosse D.Comparison of recombinant human albumin with human serum albumin:safety,tolerability and pharmacodynamics[J]. Clinical Pharmacology,2003,43:1032-1034.
[58]Watanabe H, Yamasaki K, Kragh U.In Vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time[J]. Pharmaceutical Research,2001,18(12):1775-1781.
[59]Kaoru Kobayashi.Summary of recombinant human serum albumin development [J].The International Association for Biologicals,2006,34:55-59.
[60]Andrew.R.Goodey, Darrell.Sleep, Hendrik.vanurk. High purity albumin production process:Britain,WO97/31947 [P].1997,04,09.
[61]Noda M, Sumi A, Ohmura T et al. Process for purifying recombinant human serum albumin:United States,6617133 B1[P].2003,09,09.
[62]Hendrik.vanurk,David.J.Mead,Philip.H.Morton. Process:Britain,WO00/44772[P]. 2000,03,08.
[63]陈光明,周长林,魏元刚.发酵液中重组人血清白蛋白的纯化[J].药物生物技术,2003,10(1)：25-28.
[64]袁中一,邱荣德,吴祥甫,等.人血清白蛋白在毕赤酵母中的表达与纯化：中国,981110844.X[P].1998,05,15.
[65]野内俊伸,沟上宽,田岛义高,等.人血清白蛋白多聚体的单体化方法：中国,200410100061.1[P].2001,10,24.
[66]贝洛.麦考恩,李梅彦,张炜.白蛋白纯化：中国,03123502.6[P].2009,02,25.
[67]李梅彦,贾茜,张炜,等.一种纯化rHSA的方法：中国,200510068187.X[P].2006,11,01.
[68]Tomoshi Ohya, Toyoo Ohda, Shinobu Kuwae, et al. Method for sterilizing recombinant human serum albumin pharmaceutical preparation:United States,5691451[P].1997,11,25.
[69]Wataru Ohtani, Naoto Furuhata, Akinori Sumi, et al. Method for decoloring human serum albumin:United States,5710253 [P].1998,01,20.
[70]Cornelis J, Marcellinus P.Process for the purification of serum albumin:United States,20030204060A1[P].2003,10,30.
[71]Frerick C, Kreis P.Simulation and optimization of the downstream process for purification of human serum albumin by using ion exchange membrane adsorbers[J].Desalination,2006,200:468-469.
[72]M.Belew, Li Mei Yan, Zhang Wei, et al. Purification of recombinant human serum albumin (rHSA) produced by genetically modified Pichia Pastoris[J]. Separation Science and Technology,2008,43(11):3134-3153.
[73]朱家文,武斌,陈葵.离子交换层析分离纯化重组人血清白蛋白[J].华东理工大学学报,2002,28(4)：341-345.
[74]苑隆国.重组人血清白蛋白的纯化工艺研究[D].沈阳：沈阳药科大学,2001.
[75]邱荣德,李士云,陈俊刚.重组人血清白蛋白在Pichia pastor is中的表达与纯化[J].生物化学与生物物理学报,2000,32(1)：59-62.
[76]Ohtani W, Ohda T, Sumi A et al. Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection [J]. Analytical Chemistry,70(2):425-429.
[77]Hiroto Kondol, Hideko Yomo, Susumu Furukubo, etal.Advanced Method for Measuring Proteinase A in Beer and Application to Brewing [J].Journal of The Institute of Brewing,1999,105(5):293-300.
[78]李梅彦,张炜,葛志强,等.引起重组白蛋白电泳检测假性结果的研究[J].生物技术通讯,2006,17(6)：908-910.
[79]Harris P, Wright G, Andrew A T, etal.The application of aqueous two-phase partitioning to the purification of protein from transgenic sheep milk[J].Prog Biotechnol, 1994,9:381-384.
[80]焦庆才,刘茜,陈耀祖.双水相萃取法从发酵液中分离提取α-淀粉酶和蛋白酶的研究[J].高等学校化学学报,1998,19(3)：391-394.
[81]Morre DM, Morre DJ.Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells [J]. Chromatogr.B,2000, 743:377-387.
[82]Trindade IP, Diogo MM, D.Prazeres MF, et al. Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography[J]. Chromatogr.A,2005,1082:176-184.
[83]Datar.R, Rosen C G.Extraction of human growth hormone (hGH) from Escherichia coli cell by aqueous two-phase polymer extraction [J]. J of Biotech,1986,3:207-219.
[84]朱慎林,朴香兰,沈刚.第一届全国化学工程与生物化工年会论文集[C].南京：[出版者不祥],2004.
[85]潘杰,秦德华.全发酵液萃取丙酰螺旋霉素的研究[J].中国医药工业杂志,1998,29(12)：534-537.
[86]Mishima K, Krita H, Miyake A, et al.日本溶剂萃取论文报告会Ⅱ[C].福冈：[出版者不祥],1995.
[87]Krone K H, Hustedt H, Kula M R. Process Biochemistry,1984,119:170-176.
[88]江波.双水相萃取分离发酵液中2,3-丁二醇[D].大连：大连理工大学,2008.
[89]谭天伟,沈忠耀.双水相萃取分离技术的评价和展望[J].微生物学通报,1996,23(6)：368-370.
[90]刘琳,董悦生,修志龙.微波辅助双水相提取盾叶薯蓣中的皂苷成分[J].过程工程学报,2009,9(6)：1147-1152.
[91]王辉.微生物转化虎杖中白藜芦醇苷及其产物的分离纯化[D].大连：大连理工大学,2008.
[92]E.Kiss J, Szamos B,Tames R B.Interfacial behavior of proteins in three-phase partitioning using salt-containing water/tert-butanol systems[J].Colloids and Surfaces A: Physicochemical and Engineering Aspects,1998,142:295-302.
[93]修志龙,李志刚,江波,等.一种从发酵液中分离1,3-丙二醇的双水相萃取方法：中国,101012151B[P].2010,07,28.
[94]Zhigang Li, Bo Jiang, Daijia Zhang, Zhilong Xiu.Aqueous two-phase extraction of 1,3-propanediol from glycerol-based fermentation broths[J].Separation and Purification Technology,2009,66:472-478.
[95]霍清,林强,赵玉娥.利用双水相乙醇-磷酸氢二钾体系萃取甘草有效成分的研究[J].精细化工,2002,19(2)：65-67.
[96]林金清,董军芳,李夏兰.乙醇/硫酸铵双水相体系萃取甘草酸钾的研究[J].精细化工,2004,21(3)：165-173.
[97]王志辉,朱建航,郑楠,等.葛根素在乙醇/硫酸铵两水相体系中的分配特性[J].时珍国医国药,2008,19(1)：13-15.
[98]张华.双水相/三液相萃取分离天然产物有效成分[D].大连：大连理工大学,2009.
[99]刑云,雷军,李全民.硫酸铵存在下碘化物-乙基紫-异丙醇体系萃取分离镉[J].分析试验室,2001,20(5)：62-64.
[100]马万山,钟黎,高素英,等.硫酸铵—溴化钾—乙醇体系萃取分离金[J].分析化学,2004,32(1)：56-59.
[101]高云涛,王伟.丙醇-硫酸铵双水相体系中萃取Au(Ⅲ)-氯化物-罗丹明B[J].化学研究与应用,2002,12(3)：320-321.
[102]董军芳,林金清.乙醇-水-硫酸铵三元体系的溶解度和液相平衡[J].福建化工,2002,4：49-52.
[103]栾雨时,包永明.生物工程实验技术手册[M].北京：化学工业出版社,2005.
[104]Dubois K.A, Gilles J.K. Hamilton,et al.Colorimetric method for determination of sugars and related substances[J]..Anal.Chem,1956,2:350-356.
[105]食物中磷的测定方法.中华人民共和国国家标准,1990,GB12393-90.
[106]于海英,程秀民,王晓坤.圆二色光谱及其在药物研究方面的应用[J].光谱实验室,2007,24(5)：877-885.
[107]吴明和.圆二色光谱在蛋白质结构研究中的应用[J].氨基酸和生物资源,2010,32(4)：77-80.
