CN1210866A - 重组因子ⅷ在无蛋白培养基中的制备 - Google Patents
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Abstract
在无任何动物来源蛋白如清蛋白存在时通过在补充以多元醇共聚物,优选在微量金属如铜存在下的无蛋白培养基中培养细胞可连续地从哺乳动物细胞中制备相对大量的重组因子Ⅷ。在优选的实施方案中,培养基包括称为Pluronic F-68的多元醇、硫酸铜、硫酸亚铁/EDTA复合物及微量金属如锰、钼、硅、锂和铬的盐。
Description
领域:本发明一般地涉及重组因子Ⅷ的制备并且特别地涉及在血清或无蛋白培养基中重组因子Ⅷ的制备。
背景技术:血友病A是X-连锁的隐性遗传病,它是由于因子Ⅷ分子有缺陷或不足的而导致的出血倾向。为控制出血,可以用因子Ⅷ治疗血友病。在历史上因子Ⅷ从人类血浆中加以分离。然而,从血浆来源的因子Ⅷ的治疗伴随多种人类病毒,如肝类和人类免疫缺陷病毒的传播。
随着重组DNA技术的问世,人类因子Ⅷ及其基因的结构已被阐明。源自26个外显子的该基因转录产物为约9000个碱基长度的信使RNA分子,其编码一个2351个氨基酸的大型蛋白。对因子Ⅷ的结构研究显示其为含有较多碳水化合物残基的糖蛋白。
编码因子Ⅷ的cDNA己被克隆并在幼年仓鼠肾(BHK-21)和中国仓鼠卵巢(CHO)细胞中稳定表达。已开发出商业方法以制备用于治疗血友病A的重组因子Ⅷ。现在通过遗传工程的哺乳动物细胞制备重组因子Ⅷ,因此,避免了依赖血浆并且使病毒传播的可能风险降为最低。
基因扩增已成为获得高产量治疗蛋白细胞系的首选方法。扩增策略包括将编码所需蛋白的转录与可扩增的标记如二氢叶酸还原酶相连接。然后应用转染技术将载体DNA转入受体细胞。筛选对选择药物如氨甲蝶呤具有增强抗性的细胞种群。通过限制性稀释克隆完成建立稳定的细胞克隆。然后将这些细胞克隆适应于无血清的制备培养基并检测所需蛋白的产量。
对于不稳定的蛋白如因子Ⅷ,在制备和纯化步骤中加入人类清蛋白作为稳定剂。虽然清蛋白通过巴斯德消毒加以病毒灭活,但是如果重组因子Ⅷ可在完全没有人类及动物血液蛋白存在时制备将是理想的。我现在发现通过用一种新的细胞培养基这是可能的。详述如下。
在没有任何人类或动物来源血浆蛋白时从哺乳动物细胞中连续制备相对大量的重组因子Ⅷ(rFⅧ)的方法包括在补充以如Pluronic F-68的多元醇(polyo1)聚合物的无蛋白培养基中培养哺乳动物宿主细胞。优选的培养基包括硫酸铜、硫酸亚铁/EDTA复合物及微量金属盐如锰、钼、硅、锂、及铬。
重组蛋白表达技术的最新进展已使在哺乳动物细胞中大量制备蛋白成为可能。适于因子Ⅷ制备的宿主细胞包括细胞系如幼年仓鼠肾(BHK)细胞、中国仓鼠卵巢(CHO)细胞和人类胚胎肾(HEK)细胞。特别优选的为幼年仓鼠肾细胞,特别是那些以能够按Wood等(1984)所述介导因子Ⅷ表达的基因转染的那些细胞。这些细胞系保藏于美国典型培养物保藏中心(American Type CultureCollection)中并己得到许可号ATCC CRL-8544。
带有因子Ⅷ基因的所需的宿主细胞系一般适于在补充以脂蛋白的无蛋白制备培养基中作为悬浮培养物而培养。选择用于培养宿主细胞系的基本培养基对本发明并非至关重要的并且可以是任何一种本领域众所周知的适于培养哺乳动物细胞的培养基或其组合。商业上可得到的培养基如Dulbecco’sModified Eagle培养基,Ham’s培养基F-2,Eagle’s基本培养基,及RPMI-1640培养基等。在本领域中添加生长因子加重组胰岛素是常见的。
由于因子Ⅷ的不稳定性质,工程宿主细胞产率在无蛋白条件下严重下降。人类血清蛋白常用作重组蛋白制备的无血清培养添加剂。人类血清蛋的具有多种功能,包括:(1)作为脂肪酸、胆固醇和亲脂维生素、类固醇激素及生长因子的载体;(2)作为抵御剪切力损伤的保护剂;(3)作为pH变化的缓冲剂;及(4)作为渗透压调节剂。血清蛋白另一至关重要的作用或许是通过用作蛋白酶的底物而保护不稳定的蛋白如因子Ⅷ免受蛋白水解。
存在于血清制品中的不纯物也可有利于清蛋白的稳定效应。已鉴定出因子如脂蛋白(Chan,1996)作为在无血清条件下用于制备重组因子Ⅷ的人类血清蛋白的替代品。
我们的开发人类血浆来源清蛋白的制备培养基的尝试导致了本公开的发明,即用于重组因子Ⅷ制备的无蛋白基本培养基。优选的培养基由修改的Dulbeceo’s基本培养基(Minimum Essential Medium)和补充以10μg/ml重组胰岛素(Nucellin,Eli Lillg)及FeSO4·EDTA(50μM)的Ham’s F-12培养基所组成(50∶50重量比)。除了因子Ⅷ的制备以外,工程BHK细胞在此无蛋白基本培养基中生长良好。
令人惊奇的是,添加多元醇(polyol)如Pluronic F-68对生长没有影响但增强了BHK细胞因子Ⅷ的特异产率。令人神奇的是,添加硫酸铜进一步增强了因子Ⅷ的产生。包括一组微量金属如锰、钼、硅、锂、及铬也导致因子Ⅷ产量的进一步增加。然后开发出用于在无人类血浆来源蛋白条件下因子Ⅷ制备的连续方法。关于Pluronic多元醇(polyols)使用的进一步信息可见于Papoutsakis(1997)及Schmolka(1977)。
Pluronic F-68,一种聚二醇(polyglycol),(BASF,Wyandot)常用于防止在搅拌的培养物中出现的泡沫形成,并用于保护细胞免受剪切力及少数培养物中水泡的损害。Pluronic F-68为非离子性的嵌段共聚物(blockcopolymer),平均分子量为8400,在两端由聚氧丙烯poly(oxypropylene)中心嵌段(20%重量)和聚氧乙烯poly(oxyethylene)嵌段所组成。对PluronicF-68作用的广泛研究表明Pluronic F-68作为表面活性剂而起作用并通过让细胞避开搅拌或混匀(sparging)过程中生物反应器中形成的水泡而防止对细胞的损害。然而,多位研究者已注意到在剪切力最小培养条件下PluronicF-68对生长的有利影响(Mizrahi,1975;Murhammer和Goochee,1990)。在产品纯化过程中从Pluronic F-68对脂类的共纯化提供奇特的证据,即Pluronic聚体不仅可替代清蛋白作为表面活性剂,而且还可作为脂类的载体而起作用。Pluronic F-68也可能通过直接插入到膜中而可在修复完成之前防止膜损害杀死细胞。Pluronic F-68在作为金属离子缓冲剂而起作用过程中的作用完全未知。
尽管有培养基中的Pluronic F-68可增加容积产率的报导,其作用机制似乎是维持细胞生存力(Schneidet,1989;Qi,1996)。据我们所知,已见到Pluronic F-68增加特定蛋白产品特异性产量尚属首次。由于在我们的系统中具有和不具有Pluronic F-68时生存力和生长速率是相似的,所以在我们的系统中维持细胞生存力不会是Pluronic F-68的作用机制。然而,无论什么机制,Pluronic F-68添加的效应是直接而巨大的。
预计其它多元醇(Polyols)系统将具有类似的效应。这种其它多元醇(Polyols)包括具有约1000至约16,000分子量的非离子聚氧乙烯和聚氧丙烯的嵌段共聚物。
除了常规的悬浮培养技术如摇瓶,旋转瓶及滚动瓶外,本发明方法也可用于灌流及分批生物反应器。培养宿主细胞后,通过常规的方法如超滤或离心可从消耗的培养基中回收因子Ⅷ。如果需要,回收的因子Ⅷ可通过,例如,离子交换或大小排阻层析、免疫亲和或金属螯合层析加以纯化。
如这里所使用的,“无人类或动物蛋白的培养基”为无任何人类来源或动物来源蛋白的细胞培养基。从人类或动物来源分离的蛋白固有导入病毒感染的风险。因此无人类或动物蛋白培养基的目的是排除或至少大大降低病毒传播的风险。
实施例1:以能够指导因子Ⅷ表达的幼年仓鼠肾(BHK-21)细胞获自Genentech,Inc,South San Francisco,加利福利亚,美国。按Wood等(1984)详述制备细胞系并保藏于美国典型培养物保藏中心且得到许可号ATCC CRL-8544该细胞系的克隆变异体也获自Genentech,Inc.,且用于所有实施例中。
用无血清的基本培养基将含有编码因子Ⅷ基因的BHK-21细胞培养成摇瓶中的悬浮培养物,基本培养基中含有如下成分:Ham’s F-12培养基和Dulbecco’s基本培养基(50∶50重量比),Nucellin(重组胰岛素,5-10ug/ml),FeSO4·EDTA(50uM)及MgCl2(15mM)。维持细胞并以48小时的间隔进行传代。以800×g5分钟旋转沉淀细胞,计数并以1×106个细胞/ml的密度重新接种。每瓶中含有50-100ml新鲜培养基。将摇瓶置于旋转器上,于37℃,卵育,并通过在90-1l0r.p.m之间轻旋转维持为悬浮培养物。显示于下表中如F-68(0.1%)的polyol及硫酸铜(50nm)对因子Ⅷ产生的效应在摇瓶中加以检测。因子Ⅷ通过生色分析加以定量。该分析作为称作Coatest Ⅷ:C/4的测试试剂盒在商业上出售并且可从Baxter Health Care Products中获得。通过该方法保持细胞24小时。以Coatest Ⅷ:C/4试剂盒测定的每种培养基中因子Ⅷ的活性显示于表1中。
表1
条件 | 效价(U/ml) | 特异性产率(uU/细胞/天) | 超过基本培养基的增加% |
基本培养基 | 0.15±0.07* | 0.026±0.013 | 0 |
基本+F-68(0.1%**) | 0.24±0.04 | 0.052±0.013 | 200 |
基本+F-68(0.1%)+Cu(50nM**) | 0.42±0.09 | 0.091±0.013 | 350 |
*36个样品的平均值±标准差。按上述监测24天时期内细胞的因子Ⅷ产量。
**效价实验显示0.1%是Pluronic F-68的最佳剂量。增加其浓度至0.3%对因子Ⅷ的产量没有显著影响。剂量反应实验显示50-800nM硫酸铜对因子Ⅷ产量是最适的。
如表1中所示,单独添加Pluronic F-68或优选与硫酸铜共同加入显著地增强了在无蛋白条件下含有编码因子Ⅷ基因的BHK细胞的效价及特异性产率。
实施例2:为了进一步优化无蛋白条件下因子Ⅷ的制备,将微量金属加入到无蛋白制备培养基中。然后按在实施例1中所述通过连续摇瓶培养系统分析16天因子Ⅷ的产量。数据显示于表2中。无硫酸铜时,微量金属对因子Ⅷ的产率没有影响。见表2。
表2
条件 | 效价(U/ml) | 特异性产率(uU/细胞/天) | 超过基本培养基+F-68的增加% |
基本培养基+F-68 | 0.46±0.11 | 0.065±0.013 | 0 |
基本培养基+F-68+Cu | 0.53±0.15 | 0.078±0.026 | 120 |
基本培养基+F-68+Cu+金属* | 0.73±0.16 | 0.104±0.026 | 160 |
*金属包括CuSO4·5H2O(50nM),MnSO4(3nM),Na2SiO3·9H2O(1.5uM),[NH4]Mo7O24·4H2O(3nM),CrK(SO4)2·4H2O(1.5nM),及LiCl(236M)。
实施例3:微量金属及铜对因子Ⅷ产生的影响进一步在灌流发酵罐中加以评估。用表1中所述的基本培养基以2×106个细胞/ml的浓度将两只1.5-升的发酵罐按种以BHK克隆变异体,发酵罐以0.5升/天的速率灌流。将一只发酵罐保留为对照且将另一只发酵罐按表2中所述补充铜和微量金属。将发酵罐以~2-3×106个细胞/ml的平均细胞浓度维持15天。如表3中所示,PluronicF-68,铜及微量金属的添加显著地增强连续灌流条件下无蛋白时,带有编码的因子Ⅷ基因BHK细胞的特异性产率。该制备分法可容易地适应于装配有细胞维持装置如沉淀器(cell retention devices such as settlers)的大型发酵罐(200至500升)。
表3
无数 | 特异性产率(uU/细胞/天) | |
基本培养基 | Cu+金属 | |
1 | 0.02 | 0.04 |
2 | 0.02 | 0.05 |
3 | 0.02 | 0.045 |
4 | 0.018 | 0.05 |
5 | 0.02 | 0.05 |
6 | 0.035 | 0.060 |
7 | 0.025 | 0.055 |
8 | 0.02 | 0.04 |
9 | 0.025 | 0.06 |
10 | 0.02 | 0.065 |
11 | 0.025 | 0.070 |
12 | 0.025 | 0.065 |
13 | 0.02 | 0.060 |
14 | 0.03 | 0.06 |
15 | 0.02 | 0.05 |
提供上述实施例作为说明本发明的手段而不构成对权利要求中单独定义发明的限制。参考文献:Bihoreau,N.,等.,欧洲生物化学杂志.222:41-48(1994)Chan,S.Y.,美国专利.No.5,576,194(1996)Eis-Hubinger,A.M.,等.,Thromb.Haemost.76:1120(1996)Mizrahi,A.,临床微生物学杂志.11-13(19975)Murhammer,D.W.,等.生物技术进度.6:142-148(1990)Papoutsakis,E.T.,生物技术趋势(Tibtech)9:316-324(1991)Qi,Y-M.等.,细胞技术21:95-109(1996)Schmolka,1.R.,美国石油化学家协会杂志.54:110-116Schneidet,Y-J.,免疫学方法杂志.116:65-77(1989)Wood.W.,等.,自然312:330-337(1984)Xu,D.,等.,中国生物技术杂志.11:101-107(1995)Zhang,J.,等.生物技术33:249-258(1994)
Claims (15)
1、一种从带有重组因子Ⅷ基因的哺乳动物细胞中制备重组因子Ⅷ的方法,包括在补充以多元醇的无人类或动物蛋白的培养基中培养所述的哺乳动物宿主细胞。
2、权利要求1的方法,其中的培养基包括铜离子。
3、权利要求1的方法。其中的多元醇为Pluronic F-68并以约0.025至约0.2%重量的浓度范围存在于培养基中。
4、权利要求1的方法,其中的培养基包括约50至约800nM范围的量的硫酸铜。
5、权利要求2的方法,其中的锰离子以约1.5至约4.5nM范围的量存在。
6、权利要求2的方法,其中的包括钼的离子以约1.5至约4.5nM范围的量存在。
7、权利要求2的方法,其中的包括硅的离子以约75至约300nM范围的量存在。
8、权利要求2的方法,其中的铬离子以约1.0至约4.0nM范围的量存在。
9、权利要求2的方法,其中的锂离子以约120至约480nM范围的量存在。
10、权利要求1的方法,其中所述的哺乳动物宿主细胞选自幼年仓鼠肾细胞、人类胚胎肾细胞及中国仓鼠卵巢细胞。
11、根据权利要求1方法制备的重组因子Ⅷ产品,其中的产品无人类或动物蛋白。
12、用于制备重组因子Ⅷ的无人类或动物蛋白的细胞培养基,其中包括含有多元醇的基本培养基。
13、包括铜离子的权利要求12的培养基。
14、权利要求13的培养基,其中包括至少一种选自锰、钼、硅、铬及锂的微量金属。
15、包括胰岛素的权利要求14的培养基。
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