CN1105776C - 在高细胞密度发酵中控制磷酸金属盐沉淀的方法 - Google Patents
在高细胞密度发酵中控制磷酸金属盐沉淀的方法 Download PDFInfo
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Abstract
本发明是利用一类磷酸盐玻璃作为高细胞密度发酵培养基的磷源,从而减少磷酸金属盐的沉淀反应。此发明展示了一个实用的方法,可用来有效控制在营养型培养基中或产生高细胞密度的发酵罐中磷酸金属盐沉淀的程度。它适用于各种高细胞密度细菌发酵。
Description
发明背景
本发明是关于生产重组蛋白的细菌发酵方法,其中监测和调节选料中某些培养基营养成分,来控制磷酸金属盐在培养基中的沉淀。具体地,本发明是关于高细胞密度发酵中的一种方法,其中在培养基中使用多聚磷酸盐和/或偏磷酸盐,以消除养份的沉淀并提高细胞密度。
随着分子生物学的进步和重组DNA的开发,使得我们在一些宿主细胞系统中生产大量的外源蛋白质成为可能。通过将编码有意义的蛋白质的DNA转入到宿主细胞中,然后让这些细胞在一定条件下生长,使重组蛋白在这些宿主细胞中产生这样使新的重组蛋白得以表达。一些细菌宿主细胞系统能用于产生大量的重组蛋白,而这些蛋白在自然资源中只能极有限地获得。
原核生物大肠杆菌是一种研究极广泛的细菌,它通常被选作高表达宿主细胞,部分原因是大肠杆菌细胞能产生极大量的重组蛋白。其核宿主细胞和酵母无法象大肠杆菌一样产生大量重组蛋白,而且高密度细胞发酵能使大肠杆菌产生更大量的重组蛋白质。(Yee and Blanch,生物技术和生物工程(Biotechnology and Bioengineering)41:221-230,1993)。
象大肠杆菌等,生产重组蛋白的宿主细胞体系的发酵通常是在有限的物理容器中进行(如发酵罐,反应器等)。搅拌釜式发酵罐仍是最常见几何形状的发酵罐,尽管大量其它物理形状的发酵容器也在发展。发酵操作模式大体分为如下三种形式:(1),不连续发酵(批量式发酵),(2),连续式发酵,(3),各种半连续发酵,如碱间歇式补料。
根据操作模式和选用的宿主系统,必须设计一种精确均衡的批量式和/或补料式培养基,以使细胞生长并表达重组蛋白。指定的培养基里若仅含有细胞生长所必需的养份被称为“最低限度”培养基。如大肠杆菌系统,最低限度培养基必须包含碳源,氮源,磷源,镁源和一些微量的铁和钙。(Gunsalus and Stanter细菌(The Bacteria)Vol 1 Chapter1.Academic Press Inc.N.Y.1960);大部分最低限度培养基都以葡萄糖作碳源,氨作氮源,正磷酸盐(如PO4)作磷源。理想的培养基包含有供细胞生长消耗的各种准确养分,既无养分的过分积累而抑制细胞生长,也不会因缺少任何一种养分而使细胞饥饿。(Thompson et al,生物技术和生物工程(Biotechnology and Bioengineering),27:818-824,1985)大肠杆菌均衡的最低限度培养基已在理论上被计算出来,用于低细胞密度摇瓶发酵(细胞密度最大为1.5克细胞干重/升)。Neidhordt et al细菌学杂志(Journal of Bacteriology),119:736-747,1974)。
除培养基的化学组成外,一些其它环境参数如pH值,时间,培养温度,溶解氧分压等都必须给予考虑。例如,大肠杆菌生长的最适pH是7.0,但在发酵中,由于氨的消耗或一些微生物合成的代谢产物如乙酸、乳酸等,都可能改变发酵pH值。pH值的改变不适细胞的最佳生长,因而保持培养基的pH值是很关键的,可通过加酸或加碱来达到目的。发酵中pH值及其它参数可通过手工或自动化仪器监测。
高细胞密度发酵(细胞密度>20克细胞干重/升)就必须使用高浓度培养基。而在高细胞密度发酵过程中发现,往往由于含磷酸盐溶液与含养分溶液的混合而在培养基中出现沉淀。这些沉淀物涉及正磷酸盐沉淀,它包括NH4MgPO4,Mg3(PO4)和金属离子磷酸盐类(Me)n(PO4)m(Me包括Fe,Ca,Zn,Cu,Co)。这些化合物在水中溶解度很低。(Dean,John A朗氏化学手册(Lange′s Handbook of Chemistry),12th edition,McGraw-Hill,New York,Pages 7-12 1979)。沉淀多少随pH值,葡萄糖浓度及培养基浓度的不同而变化。
沉淀的形成可能对补充培养基和发酵罐带来一些问题。如在培养基里导致供料的不均匀(这里因为沉淀物在容器或管线中沉积下来)而细胞因得不到关键可溶性养分而饥饿。沉淀物也会磨损进料泵和管道,以及堵塞管道等。
在发酵罐中,如果培养基相对细胞生长不均衡,就可能产生沉淀。在罐中的沉淀可能会堵塞住空气喷嘴,导致培养基的不均匀混合(沉淀物在发酵罐中轻微的沉积),降低细胞对溶解性养分的利用率。细胞可利用的养分浓度将取决于因沉淀形成而损失的养分的损失率与细胞对养分吸收率之比。这些影响都可以通过自动加酸加碱控制pH值来解决,但这些条件都降低了发酵过程的再现性。甚至沉淀的存在还能影响到蛋白质的纯化操作,要将沉淀物从产品中分离出去就不得不增加额外的纯化步骤。
在商业化生产中,要使发酵更加实用,就必须解决沉淀问题。一种推荐方法可以防止最低限度培养基中的养分沉淀,这就是加入EDTA或柠檬酸盐以螯合培养基中的金属离子(Pirt,S.J.微生物和细胞培养原理(Principles of Microbe and cell Cultivation),Page 134,1975)。但是加入螯合剂的方法并不是很理想的因为这些试剂并不能被代谢掉,因而造成大量积累从而增加了细胞环境的渗透压。高渗透压对细胞的代谢有重要影响(Gouesbet等细菌学杂志(Journal of Bacteriology),175:214-221,1993)。高浓度的螯合剂也可能破坏细胞膜(Ryan等,生物技术和生物工程(Biotechnology and Bioengineering)37:430-444(1991))。
在一份申请号为290,212 A5的德国专利(申请日期为1988,7,28)中,Riesenberg等人描述了一种制备最低限度葡萄糖培养基的方法,这种培养基用于培养大量大肠杆菌以得到重组DNA产品。据Riesenberg讲,设计这种培养基可克服工作中常遇到的严重沉淀问题。但这种以正磷酸盐为磷源的该专利申请描述的培养基并不能完全消除养分的沉淀,它仅能减轻培养基沉淀的形成,使培养基呈现较低的湍动性。
除上述讨论外,文献中尚无报道关于制备能有效消除养分沉淀的高细胞密度发酵的培养基。因此现在仍需要一个方法来减少那些在各成分混合(对于大肠杆菌在pH中性下混合)时并无沉淀出现的批量式和/或补料式的培养基,并且保证在随后发酵过程中也无沉淀问题。本发明就提供了这样一个方法,使用磷酸钠玻璃作为高浓度营养培养基中的磷源。不同于上述引用的参考文献的方法,本发明方法有三大优点:(1)该方法能制备高浓度,完全均衡,无沉淀的批量式和/或补料式培养基,(2)能被大肠杆菌完全代谢利用,和(3)能增大细胞密度和生长速度。该发明方法适用于各种细菌发酵,尤其是高细胞密度的发酵。
发明概述
本发明是关于生产重组蛋白的细菌发酵方法,其中,监测和调节某些培养基中的营养成分,从而控制磷酸金属盐在发酵过程的沉淀。具体地,本发明是关于高细胞密度发酵中的一种方法,该方法采用磷酸钠玻璃作为高浓度营养液的磷源。令人惊异地该方法不仅能消除培养基中的沉淀形成,而且能在标准条件下促进细胞密度的增加。
附图简述
图1示出C组(·-·)和D组(o-o)的细胞密度变化曲线图形。该图表明在培养基中用磷酸盐玻璃作为磷源,其细胞密度的增加大大高于以正磷酸盐为磷源的情况。细胞密度是用一台Perkin-Elmer M35分光光度计来测定的,将不同时间的OD600光密度值对时间作图。
发明详述
通过此方法监测和调节培养基中某些养分,从而控制在发酵过程中磷酸金属盐的沉淀。该方法将在下面讨论中将详细描述并通过实例加以例证说明。实例表明改变磷源在高密度发酵中可用于消除养分的沉淀。令人惊异的结果是用磷酸钠玻璃在批量式和/或补料式高浓度培养基中作为磷源,可消除培养基中的磷酸金属盐沉淀,同时它又能提高发酵中的细胞密度。
用于生产重组蛋白的发酵过程都会使用下列操作方式中的一种:(1)不连续(批量式)操作,(2)连续操作,(3)半连续(补料式)操作。批量式方法具有下列特征,在方法开始时,将微生物接入无菌的培养基(批量培养基)中,培养一特定的反应时间。在培养期间,培养基中的细胞浓度,底物浓度(碳源,营养盐类,维生素等)以及产物浓度都一直在变化中。要防止在反应混合液中局部成分不匀或温度不匀,就须将培养基混合充分。其反应应是非静态的,细胞一直生长到其生长必需的底物被消耗完为止(通常是碳源耗尽)。
连续操作具有如下特征;新鲜的培养基(给料培养基)不断地加入到发酵罐中,而消耗过的培养基和细胞以同样的速率不断地从罐中流出。在连续操作中,细胞的生长速率取决于培养基加入的速率,细胞得率取决于生长限制性底物(即碳源)的浓度。所有反应变量和控制参数在时间上是恒定的,因此,可根据恒定的生产量和输出来确立在发酵罐中的稳定状态。
半连续操作可看作是将批量式和连续式结合在一起的一种操作模式。在该模式中,发酵开始时同批量式操作一样,当生长限制性底物被消耗完后,就以一个特别方式(补料)连续地补加入含有葡萄糖和矿物质的补料培养基。换句话说,该操作即使用了批量培养基又使用了补料培养基,从而使细胞充分生长并产生足够多的蛋白质产物,在培养过程中既不加入细胞也不带出细胞,因而就微生物而论,发酵罐仍是分批操作的。本发明适用于上面提到的所有发酵操作方式,而最优选则是补料式方法。
在上述每个方法中,细胞的生长和产物的积累都可间接地通过择优地选择代谢产物的形成和其量的相关关系来检测,如培养基的pH值,光密度值,颜色,酸值滴定等变化。例如光密度值可显示不溶性的细胞颗粒的聚集,和可用与显示器或记录仪相连的微型光密度计来连续测定,或通过非连续性测定样品在600nm波长的光密度值(OD600)来确定细胞的干重。
高细胞密度发酵是通常指那些细胞回收大于20克细胞干重/升(OD600>30)的发酵,所有的高细胞密度发酵都使用高浓度营养型培养基,它们通过“补料方式”逐步加入发酵罐中。高浓度营养培养基符合高细胞密度方法的要求,可减少发酵中在反应罐中养分的稀释。补料过程是必须的,因为它可使操作者能有效控制碳源的供给,这是很重要的,因为如果细胞在高浓度碳源中就能产生高密度细胞,而细胞也会产生许多抑制性副产物,如乙酸等,这样细胞的生长就停止(Majewski andDomach,生物技术和生物工程(Biotechnology and Bioengineering),35:732-738,1990)。
通过发酵产生重组蛋白的标准条件是:pH值在5.0~8.0之间,大肠杆菌的培养温度在20℃~50℃。在本发明里,以大肠杆菌为宿主系统是最优选的;最佳pH值为7.0,最佳培养温度为37℃。
在通常的发酵过程中,标准的营养成分包括有能源,碳源,氮源,磷源,镁盐和微量的Fe,Ca,另外培养基里可能还含有一些生长因子(如维生素和氨基酸),无机盐和其它产物形成所必需的前体。微生物的基本组成可用作计算适于细胞生长所需各养分比例的参考。各成分的浓度取决于是低密度发酵还是高细胞密度发酵。例如在低细胞密度的批量式发酵中,葡萄糖的浓度在1~5克/升之间,而在高细胞密度发酵中,葡萄糖的浓度在45~75克/升之间。
本发明可使用广泛围的各种磷酸盐玻璃作为培养基磷源。磷酸盐玻璃是具有特别用途的线性多聚磷酸盐。关于线性多聚磷酸盐,包括磷酸盐玻璃的一般性讨论可参考Corbridge,D.E.C.的文章“磷:它的化学、生化与技
术概观(Phosporus:An Outline of its Chemistry,Biochemistryand Technology)”第四版,第三章,210-301页,1990。磷酸盐玻璃可由广泛成分来制备,它主要由阳离子和分开的多聚磷酸盐链的混合物组成。这种由Na+阳离子构成的玻璃经过全面分析,它是由一系列在常温下稳定的,从P2O5到5Na2O100P2O5的化学成分构成。磷酸盐玻璃是由正磷酸根阴离子,即NaH2PO4加热到650℃然后急冷,凝结而成的。这种典型玻璃是从650℃及55乇水蒸汽中急冷后的熔体而得,是平均链长
n=60的PO4四方体。
各种玻璃态的多聚磷酸钠可市售购得。它们可制成各种不同平均链长(从
n=5到
n=200)的产品。
本发明方法中,通常采用的多聚磷酸盐是磷酸钠玻璃。具体地,磷酸钠玻璃的链长范围是从
n=2到大约
n=100,更优选是链长在
n=4到
n=20。这些磷酸盐玻璃之所以用于本发明是因为(1)它们的溶解度性质可使高浓度培养基制备混合时不产生沉淀,(2)象大肠杆菌的宿主细胞系统都有一定代谢途径来代谢磷酸盐玻璃,和(3)由于没有沉淀,所有的养分都可全部被利用,这就能使细胞密度及生长速率得到提高。在优选的实施中,平均链长
n=11的玻璃化多聚磷酸盐用作磷源。
本发明适用于各种重组蛋白的生产方法。较为典型的有下列蛋白质:细胞分裂素,包括各种造血因子如G-CSF因子,SCF,EPO,GM-CSF,CSF-1等因子,中介白细胞素如IL-1到IL-12,IGFs,M-CSF,TNF,或LIF。以及其它一些有疗效的蛋白质如干扰素(α、β、γ或杂型干扰素),和一些有用的生长因子或激素,如人或动物生长激素(如猪,鸡生长激素),FGF,KGF,EGF及PDGF。蛋白酶抑制剂,具有代表性的有金属蛋白酶抑制剂,如TIMP-1,TIMP-2等。神经生长因子如BDNF,NT-3等。还包括一些具有原蛋白部分或全部一级结构的肽链,这些肽链保留着至少一种以上的原蛋白质的生物性质。
通常本发明使用的KGF因子拥有人KGF因子的序列式相似序列。已发表的申请号为WO 90108771的PCT专利申请描述了下列KGF纯化方法:从人胚胎成纤维细胞培养液中纯化KGF分离部分肽段的氨基酸序列,基因克隆,和在大肠杆菌中表达,得到具有生物活性的KGF重组蛋白。
在方法优选实施中以大肠杆菌作为宿主细胞较好,因为大肠杆菌的遗传性质已得到充分研究,它可以形成高细胞密度并在常规条件下很好生长(如pH值,温度等)。
本发明的优选实施还包括一些辅助条件:复合氮源,如酵母膏和大豆粉、酪蛋白水解物,肉汁,血液或棉籽的化学消化液。本技术领域的人员都会理解,本发明涵盖了可以包括各种附加因素的组合的控制磷酸金属盐沉淀的方法。
实例1
均衡的最低限度营养型补料培养基使用各自不同的磷源制备,这些典型的培养基可被用于高细胞密度发酵生产重组蛋白。一种“标准”的批量式最低限度培养基(将它命名为培养基A),它用正磷酸盐为磷源,与另一种用HexophosTM玻璃态多聚磷酸钠为磷源的培养基(命名为培养基B)作比较实验。其中HexophosTM多聚磷酸钠链长
n=11,由FMC公司提供。
培养基A的成分为:45克/升葡萄糖,3克/升酵母膏,1克/升(NH4)2SO4,4克/升K2HPO4,4.56克/升KH2PO4,0.71克/升MgSO4·7H2O,0.74克/升KCl,4.0毫升/升微量金属离子溶液A(27克/升FeCl3·6H2O,2.0克/升ZnCl2·4H2O,2.0克/升CoCl2·6H2O,2.0克/升MnMoO4·2H2O,1.0克/升CaCl2.2H2O,1.9克/升CuSO4·5H2O,0.5克/升H3BO3,1.6克/升MnCl2·4H2O,73.5克/升柠檬酸钠·H2O)和4毫升/升维生素溶液A(0.06克/升生物素,0.04克/升叶酸,1.4克/升维生素B6盐酸盐,0.42克/升核黄素,5.4克/升泛酸,和6.1克/升烟酸)。培养基B与培养基A仅一种成份不同,这就是用3.33克/升HexaphosTM作为磷源代替K2HPO4和KH2PO4。
在每种培养基灭菌时,葡萄糖和MgSO4是在一起灭菌的,微量金属离子溶液A及培养基其它成分(如正磷酸盐)是分开灭菌的。这样做是为了防止产生不必要的副反应。在培养基B中,HexaphosTM也是分开灭菌的。当所有灭过菌的培养基成分混和在一起时,培养基A溶液里有雾状出现,这种混浊是因为有沉淀反应产生。但培养基B在混和后仍保持透明。
沉淀的重量是通过用0.2μm孔径的尼龙膜(Nalgene,215-4020)过滤器(Nalgene,300-4100)过滤10毫升培养基样品来确定的。第二过滤器用来计算因进入膜上的可溶性固体而导致重量的总增加。此过滤器在空气中干燥几个小时,然后在Labware 9000中微波干燥至恒重。Labware 9000可被用来确定过滤器在过滤前后的干重变化,这些结果都如下表1所示。
表1
培养基 沉淀重量
A 0.2克/升
B 未检测到
实例2
此例将通过在大肠杆菌中生产重组蛋白的高细胞密度补料式发酵,以比较在批量式培养基和高浓度营养型补料式培养基中使用磷酸盐玻璃或正磷酸盐作为不同磷源而产生的不同结果。它将确定这些磷源对培养基中磷酸金属盐沉淀的影响以及细胞密度的变化。一种以正磷酸盐为磷源的“标准”补料式最低限度培养基(编名为C组)同以HexaphosTM为磷源的D组相对照。
在C组里,批量式发酵培养基成分见如下;5克/升葡萄糖,1.68克/升(NH4)2SO4,0.05克/升K2SO4,0.36克/升NaH2PO4·H2O,0.136克/升MgSO4·7H2O,0.008克/升KCl,0.78毫升/升微量元素溶液A(27克/升FeCl3·6H2O,2.0克/升ZnCl2·4H2O,2.0克/升CoCl2·6H2O,2.0克/升MnMoO4·2H2O,1.0克/升CaCl2·2H2O,1.9克/升CuSO4·5H2O,0.5克/升H3BO3,1.6克/升MnCl2·4H2O,73.5克/升柠檬酸钠·H2O)。在D组里,除了用0.28克/升HexaphosTM取代NaH2PO4·H2O外,其它成分与C组一样。
在C组里补料式培养基成分为;651.5克/升葡萄糖,6.52克/升K2SO4,46.91克/升NaH2PO4·H2O,17.69克/升MgSO4·7H2O,1.08克/升KCl,以及102毫升/升的微量元素溶液A。在D组里的补料式培养基除用37.2克/升HexaphosTM取代NaH2PO4·H2O外,其它成分与C组中的一样。每组中的补料式培养基都通过加入36毫升/升的40%(v/v)NaOH溶液来将其pH值调至pH7.0。
就象实例1中一样,用正磷酸盐为磷源的均衡的最低限度补料培养基在各灭菌成分混和时,有沉淀产生,而用HexaphosTM作磷源的培养基则没有沉淀产生。沉淀的多少可通过上述例1的方法进行测定,其结果如下表2所示。
表2
组 沉淀重量
C 0.33克/升
D 未检测到
同时,在C组和D组中的细胞密度变化也可监测得到。在每组中,细胞在批量式培养基中开始分批发酵生长。当葡萄糖被耗尽时,开始连续地加入补料式培养基,在补料发酵过程中,补料速度可根据细胞密度大小间隔2小时而增加。在C组中,细胞的生长速率开始为0.08h-1,当48小时后,细胞密度达到OD=65,开始补料时,其生长率下降到0.04h-1,其OD值也同时下降。在D组里,经43小时发酵,细胞密度达到OD=92,其生长率维持在0.09h-1水平上,然后OD值下降(如图1所示)。最终,D组的细胞密度可达61克细胞干重/升,而C组中,细胞密度仅有43克细胞干重/升。结果还表明,D组中可生产180毫克/升的KGF,而C组中仅能生产120毫克/升的KGF。
实例3
除HexaphosTM外,其它几种多聚磷酸盐(其链长范围在
n=2到
n=19之间)也被用作高细胞密度发酵培养基中的磷源。在大肠杆菌发酵中,通过将高浓度的葡萄糖/MgSO4贮液(888克/升葡萄糖)与不同的磷源化合物及水混和,精确配制成几种培养基,它们的葡萄糖/磷,葡萄糖/Mg的比例都符合高细胞密度发酵所必需条件。每种培养基的pH值都用40%NaOH或30%HCl调到pH7.0。
每天通过对各种培养基的透光率的自测分析来确定沉淀的形成,一直持续五天,其结果如下表3所示。结果显示,当用HexaphosTM作为磷源,可允许使用浓度高达800克/升的葡萄糖溶液而无沉淀产生,当使用玻璃HTM,一种玻璃态多聚磷酸钠,其链长为
n=19和SodaphosTM,一种链长
n=4的玻璃态多聚磷酸钠时,培养基中葡萄糖浓度可达600克/升,而另一方面,当用正磷酸盐作磷源时,其葡萄糖浓度只能达到462克/升。
表3
磷源 链长 沉淀形成时间(无)
800克/升 600克/升 400克/升 200克/升 100克/升
葡萄糖 葡萄糖 葡萄糖 葡萄糖 葡萄糖Glass HTM 19 2 无 无 无 无HexophosTM 11 无 无 无 无 无SodaphosTM 4 2 无 无 无 无Tripoly- 1 1 1 1 无 无Phosphate正磷酸盐 N/A N/A 0* 无 无 无*得到最大葡萄糖浓度是462克/升。
结果表明,各种玻璃状多聚磷酸钠都能有效消除高浓度培养基中的养分沉淀,这些培养基都可用于高细胞密度发酵生产重组蛋白。
上述结果也证实了本发明是控制在高细胞密度细菌发酵过程中培养基中金属磷酸盐沉淀反应的实用方法,并能促进细胞的生长及蛋白的产生。
Claims (7)
1、在生产重组蛋白质的细菌发酵中,减少沉淀的方法,该方法包括在生产蛋白的培养基中使用磷酸盐玻璃作为磷源;其中所述发酵方法为细胞密度>20克细胞干重/升的高细胞密度发酵。
2、权利要求1的方法,其中所述磷酸盐玻璃是选自链长范围约为n=2到n=100的一组磷酸钠玻璃。
3、权利要求2的方法,其中所述磷酸钠玻璃的链长约为n=4到n=20之间。
4、权利要求3的方法,其中,磷酸钠玻璃的链长为n=11。
5、权利要求4的方法,其中,高细胞密度发酵是批量式,连续式或补料式发酵中的任意一种。
6、在用大肠杆菌生产重组蛋白的细胞密度>20克细胞干重/升的高细胞密度的发酵中减少沉淀的方法,该方法包括使用链长约为n=4到n=20的磷酸盐玻璃作为生产蛋白质的培养基中的磷源。
7、权利要求6的方法,其中磷酸盐玻璃的链长约为n=11。
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US7381545B2 (en) | 2008-06-03 |
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US7118884B1 (en) | 2006-10-10 |
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