CN107460159A - 无血清、无蛋白补料培养基及其制备方法和运用 - Google Patents
无血清、无蛋白补料培养基及其制备方法和运用 Download PDFInfo
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Abstract
本发明涉及一种无血清、无蛋白高效补料培养基及其制备方法和运用,所述补料培养基包括氨基酸、无机盐、微量元素、维生素、碳水化合物和其它有机物,并且,所述培养基中不含谷氨酰胺。由于该补料培养基无血清、无蛋白、无动物来源成分,可以大大降低病毒污染风险,利于下游纯化,同时因培养基配方成分种类齐全、比例均衡,可以适用于多种CHO细胞株的高密度培养和高蛋白表达。
Description
技术领域
本发明涉及生物技术领域,更具体地,本发明涉及一种无血清、无蛋白高效补料培养基及所述培养基的制备方法和运用。
背景技术
目前CHO细胞作为主要的宿主细胞,广泛用于治疗蛋白药物的生产。一个高产、高效、稳健、低成本、高质量的生产工艺的开发对于治疗蛋白产品工业化生产至关重要。其中关键的上游技术平台包括:高亲和力全人源化抗体筛选平台、高表达的工程细胞株构建和筛选平台,个性化高效基础培养基和补料培养基的开发平台,支持细胞高密度生长和高表达的培养工艺开发平台等,其中低成本、高效的培养基开发对于治疗性蛋白工业化生产至关重要。
治疗性蛋白工业化生产成本主要来自于培养基和填料,因此在工业规模的动物细胞培养中,培养基的设计除了要考虑细胞的生长和抗体表达的基本要求外,过程的可行性和经济性也是重要的考虑因素。含血清培养基因成本、批次间差异、含杂蛋白和可能病毒污染源等问题已经不在工业生产中应用;含蛋白培养基因动物来源因素、成本问题以及监管要求,已被非动物来源培养基(Animal Component Free Medium,ACF)取代。目前商业化的培养基主要采用无蛋白培养基(可能含有水解物)和化学限定培养基(CD)两类。
CHO细胞常用的培养模式有分批培养、补料分批培养和灌流培养,其中补料分批培养过程中采用过程补料可以及时补充消耗的营养物质,避免因营养物质耗尽造成细胞凋亡,并提高抗体的持续表达。补料分批培模式目前已经成为治疗性蛋白生产的主要培养模式。补料培养基中主要补充的是葡萄糖和氨基酸,维生素、无机盐、生长因子等因本身细胞消耗量较少,如何均衡地提供动物细胞所需的培养物质是一个合理补料培养基设计的基础出发点,例如,已显示补充如氨基酸、氯化胆碱或维生素可以增加在无蛋白条件下培养的细胞的存活率和多肽表达产率。
商业补料培养基的价格基本在每升800~2500元,这对于治疗蛋白的工业化生产是一个巨大的成本压力。由于商业补料培养基的成分均属于保密信息,不同细胞株、不同克隆、甚至不同亚克隆之间代谢需求也经常存在显著差异。造成在筛选合适商业补料培养基过程中存在非常大的盲目性,而且在通过调整培养基成分改善蛋白质量指标,无法随意减少某种营养成分来考察影响,会严重影响生物仿制药项目的开发进度。因此本领域迫切需要开发新的适合CHO细胞高密度培养、高蛋白表达的无血清无蛋白补料培养基。
发明内容
本发明所述的补料培养基是一种无血清、无蛋白、无动物来源,可以大大降低病毒污染风险,利于下游纯化,同时因培养基配方成分种类齐全,比例均衡,可以适用于多种CHO细胞株的高密度培养和高蛋白表达。
根据本发明的实施例,提供了一种新型高效的无蛋白培养基,培养基组分包括,氨基酸、无机盐、微量元素、维生素、碳水化合物和其它有机物。所述氨基酸的总浓度为10250-102500mg/L,所述维生素的总浓度为508.7-5087mg/L,总浓度为1900.323-19003.23mg/mL的无机盐和微量元素,以及总浓度为3300.2-33002mg/mL的碳水化合物和其它有机物。
根据本发明的实施例,所述氨基酸中含有950-9500mg/L的脯氨酸,其中脯氨酸包括150-1500mg/L的羟基L-脯氨酸,和800-8000mg/L的L-脯氨酸。
根据本发明的实施例,所述氨基酸可以是选自20种常规氨基酸,如L-苯丙氨酸、L-甲硫氨酸、L-色氨酸、L-组氨酸、L-赖氨酸、L-亮氨酸、L-苏氨酸、L-缬氨酸、L-异亮氨酸、L-天冬酰胺、L-脯氨酸、L-天冬氨酸、L-丝氨酸、L-谷氨酸、L谷氨酰胺、L-精氨酸、L-络氨酸、甘氨酸、L-半胱氨酸、L-丙氨酸。氨基酸作为细胞生长主要的碳源和氮源,即可用于蛋白质、核酸和脂类的生物合成,也能通过几个主要的中间代谢产物进入糖代谢进行供能。通过实验确定每一种氨基酸的合理用量范围,即满足细胞的营养需求,同时避免代谢溢流的出现。
根据本发明的实施例,所述无机盐可以是选自本领域中细胞培养基中常用的无机盐,无机盐:如Na+和K+主要用于维持细胞膜电位;Na+、Cl-和HCO3 -主要用于维持渗透压,HCO3 -和HP04 2-作为pH缓冲体系;P04 3-可作为核酸、磷脂和ATP等的合成原料;Mg2+与ATP合成耦联,与Ca2+一起参与细胞与细胞、细胞与基质的粘附。各个无机盐浓度的选择需要同时考虑对渗透压的影响。
微量元素的选择如铁、锌、铜、锰、铯等,其作为多种酶的辅酶或辅基,参与主要的生命活动,如Fe2+和Cu2+二者均为线粒体中呼吸链的组成部分,参与电子传递,Zn2+作用与胰岛素耦联;Se也可以作为氧化剂,是谷胱甘肽过氧化物酶的辅因子,消除氧化应激反应。微量元素细胞所需量微小,但缺乏会对细胞产生严重影响。
维生素是一类细胞所必需的微量成分,功能多样,通常作为酶的辅酶和辅基,在物质代谢中起重要作用。分为脂溶性维生素和水溶性维生素两大类,如硫胺素(VB1)涉及糖代谢中转酮醇酶和转醛醇酶的羧基转移反应;吡哆醇(VB6)是氨基酸代谢中重要的辅酶,主要参与转氨反应;生物素是活化CO2的载体,参与丙酮酸脱氢酶和丙酮酸羧化酶反应,也参与脂肪酸的合成;钴胺素(VB12)参与分子内部C-C单键的重排,如甲基转移反应;烟酰胺是形成NAD和NADP脱氢酶的辅酶,作为电子载体,参与生物氧化体系;核黄素(VB2)构成黄酶的辅基,作为电子载体,参与生物氧化体系;泛酸钙是辅酶A和磷酸泛酰巯基乙胺的组成成分,主要起传递酰基的作用,在糖代谢、脂质分解代谢、氨基酸代谢以及脂肪酸合成均起作用;叶酸的活性形式是四氢叶酸,是除了CO2外所有一碳单位的重要受体和供体;硫辛酸是酰基载体,在α-酮酸氧化作用和脱羧作用起到酰基转移和电子转移功能。
碳水化合物:作为主要碳源和能源物质,其中最常用的是葡萄糖,可以额外添加补充丙酮酸钠,补充细胞能量的不足。
其它有机物分子:如脂类、抗氧化剂。细胞本身可以合成几乎所有脂类,但是唯一无法合成C9双键脂肪酸,因此需要额外添加C9双键脂肪酸,如亚油酸或者亚麻酸。磷脂主要用于形成生物膜的磷脂双分子层,因此额外添加氯化胆碱、乙醇胺和肌醇。如添加还原性谷胱甘肽作为抗氧化剂,则避免细胞在氧化应激过程中产生的超氧阴离子对细胞本身的严重损伤作用。
本发明的实施例所提供的补料培养基的成分如下:
第一部分:氨基酸
第二部分:无机盐和微量元素
第三部分:维生素
第四部分:其它成分
优选的是,本发明所述补料培养基的成分及配比为:
氨基酸部分
无机盐和微量元素部分
维生素部分
碳水化合物及其它成分
进一步地,本发明提供了一种配制所述补料培养基的方法,包括:
(1)按比例称取补料培养基中各成分,混匀;
(2)将步骤(1)的补料培养基混合物溶解于超纯水中,并加入一定量的氢氧化钠调pH以促进不了培养基混合物的溶解,盐酸调pH至6.9~7.2之间,并用超纯水定容,微孔滤膜过滤,即得所述补料培养基。
将补料培养基混合物溶解于超纯水中时,温度控制在30-40℃条件下进行。
过滤时,微孔滤膜的孔径优选为0.22μm。
根据本发明的再一方面,在于提供本发明所述补料培养基在细胞培养和/或蛋白表达中的运用,比如在CHO细胞培养和/或蛋白表达中的运用。
有益效果
首先,由于补料培养基中不含有谷氨酰胺,因此不存在铵离子累积而对细胞生长引起的毒性,并且同时仍保持高细胞生长和高多肽生产力。
其次,该补料培养基无血清、无蛋白、无动物来源,可以大大降低病毒污染风险,利于下游纯化,同时因培养基配方成分种类齐全,比例均衡,可以适用于多种CHO细胞株的高密度培养和高蛋白表达。将本发明的高效补料培养基加入对数生长中期的CHO细胞培养液后,细胞可达到更高的细胞密度,在高密度的同时,可以长时间保持较高的细胞活力,效果要明显好于其他商业细胞培养基,因此可以很好地运用于重组蛋白和疫苗的生产,降低生产成本,为适应未来工业化培养过程提供了很好的保障。
附图说明
图1是CHO细胞在添加GE Health Hyclone补料培养基培养时活细胞密度-时间曲线图。
图2位CHO细胞在添加GE Health Hyclone补料培养基培养时细胞活率-时间曲线图。
图3是CHO细胞在添加GIBICO补料培养基培养时活细胞密度-时间曲线图。
图4是CHO细胞在添加GIBICO补料培养基培养时细胞活率-时间曲线图。
图5是CHO细胞在添加Irvine补料培养基培养时活细胞密度-时间曲线图。
图6是CHO细胞在添加Irvine补料培养基培养时细胞活率-时间曲线图。
图7是CHO细胞在添加Merk-Sigma补料培养基培养时活细胞密度-时间曲线图。
图8是CHO细胞在添加Merk-Sigma补料培养基培养时细胞活率-时间曲线图。
图9是CHO细胞在添加Kerry水解物补料培养基培养时活细胞密度-时间曲线图。
图10是CHO细胞在添加Kerry水解物补料培养基培养时的细胞活率-时间曲线图。
图11是CHO细胞在添加本发明的实施例的补料培养基培养时的活细胞密度-时间曲线图。
图12是CHO细胞在添加本发明的实施例的补料培养基培养时的细胞活率-时间曲线图。
具体实施方式
下面,结合附图对技术方案的实施作进一步的详细描述。
本领域的技术人员能够理解,尽管以下的说明涉及到有关本发明的实施例的很多技术细节,但这仅为用来说明本发明的原理的示例、而不意味着任何限制。本发明能够适用于不同于以下例举的技术细节之外的场合,只要它们不背离本发明的原理和精神即可。
另外,为了避免使本说明书的描述限于冗繁,在本说明书中的描述中,可能对可在现有技术资料中获得的部分技术细节进行了省略、简化、变通等处理,这对于本领域的技术人员来说是可以理解的,并且这不会影响本说明书的公开充分性。
实施例1
本发明的高效补料培养基的配制方法(1L)
按照本发明的高效补料培养基的成分比例进行配制(参见表1)
加入800ml超纯水,温度35℃左右,按照本发明的高效补料培养基的成分配比称取各个成分,加入一定量的氢氧化钠促进溶解,然后加入浓盐酸,调节pH至6.9~7.2之间,采用0.2μm滤膜过滤,分装于1L玻璃瓶中无菌避光4℃保存。
表1:本发明的高效补料培养基的成分(配方一)及比例
实施例2
按实施例1的配置方式,提供了其它配比的补料培养基。
实施例3
对CHO细胞培养液进行流加培养基(添加补料培养基)
本发明实施例中使用的Hyclone补料培养基购买自GE Healthcare Hyclone,为Cell Boost 2(目录号SH30596)、Cell Boost 4(目录号SH30857)、Cell Boost 5(目录号SH30865)、Cell Boost 6(目录号SH30866);GIBCO补料培养基购买自Thermo FisherInvitrogen,为Feed A+(目录号A25023)、Feed B+(目录号A25030)、Feed C+(目录号A25031)、Feed C(目录号A13275)、TiterEnhancer(目录号A15010);Irvine BalanCD CHOfeed补料培养基购买自Irvine Scientific,为Feed 1(目录号94119)、Feed 2(目录号94121)、Feed 3+(目录号94118);EX-CELL AdvancedTM CHO Feed 1购买自Sigma,目录号为24367C;CellventoFeed-210购买自Merck,目录号为102488;Kerry Sheff-CHO水解物补料购买至西美杰,为ACF(目录号5×00424)、PF ACF(目录号5×00464)、Plus ACF(目录号5×00480)、Plus PF ACF(目录号5×00483)。
试验方式:流加培养(加入补料培养基)
试验程序:在细胞培养至对数生长期中期进行补料,细胞活率降低至50%左右为培养终点。;
培养环境:37℃,5%二氧化碳
摇瓶培养过程:在超净台内向一个2L摇瓶内加入已无菌过滤好的基础培养基,接种密度为5×105细胞/ml,培养体积为800ml,培养至第3天计数:活细胞密度为30.05×105细胞/ml,细胞活率为97.45%。在无菌操作台中均匀分装30mL培养液至125ml摇瓶中,标记为1~20,
按照表2所示进行补料操作。1号仅添加葡萄糖,作为对照实验;2~19号按照商业培养基添加的合适补料策略进行补料操作;20号为本发明是一种新型高效的无蛋白补料培养基,命名为高效补料,补料方式为第3天补加5%,第5天补加5%,第7天补加7.5%,第9天补加7.5%,第11天补加5%,第13天补加3%,随后隔天补加3%。
表2:各个商业补料培养培养结果
注:“对照”代表培养过程中仅补加葡萄糖,不补料;“-”代表无数据
本发明的高效补料培养基加入对数生长期中期的CHO细胞培养液后,细胞可达到更高的细胞密度,在高细胞密度的同时,可以长时间保持较高的细胞活力,为适应未来稳健的工业培养过程提供了很好的保障。
通过实验比较(实验结果参见表2和附图1-12),本发明的高效补料培养基比目前商业上的培养基能达到更高想细胞密度和更长是细胞培养时间,表达的蛋白量也要强于目前商业培养基。尤其是对于本发明的高效补料培养基配方一,比目前商业上的补料培养基能达到更高的细胞密度可至158×105细胞/ml,且培养时间可以大大延长,可至22天。最高活细胞密度比Hyclone的Cell Boost 2高110%,比GIBCO的Feed A+高109%,比Irvine的Feed 3高118%,比Merck的CellventoFeed-210高131%,比Sigma的AdvancedTM Feed 1高140%,比Kerry的ACF高180%。与此同时,表达的某抗体蛋白的表达量也得到明显的提高,培养第14天可至2.32g/L,培养过程表达一直保持积累状态,培养结束后可至3.77g/L。
最后,本领域的技术人员能够理解,对本发明的上述实施例能够做出各种修改、变型、以及替换,其均落入如所附权利要求限定的本发明的保护范围。
Claims (10)
1.一种无血清无蛋白补料培养基,其中所述培养基包含总浓度为10250-102500mg/L的氨基酸,并且所述氨基酸中不含有谷氨酰胺。
2.根据权利要求1所述的补料培养基,其包含浓度为10250-102500mg/L的氨基酸,所述氨基酸中含有浓度为950-9500mg/L的脯氨酸,并且所述氨基酸中不含有谷氨酰胺。
3.根据权利要求2所述的补料培养基,其中,所述脯氨酸包括浓度为150-1500mg/L的羟基L-脯氨酸,以及浓度为800-8000mg/L的L-脯氨酸。
4.根据权利要求1所述的补料培养基,其进一步包括:总浓度为508.7-5087mg/L的维生素;总浓度为1900.323-19003.23mg/mL的无机盐和微量元素;以及总浓度为3300.2-33002mg/mL的碳水化合物和其它有机物。
5.根据权利要求4所述补料培养基,其成分及其含量如下:
氨基酸部分
无机盐和微量元素部分
维生素部分
碳水化合物及其它成分
6.根据权利要求2所述的补料培养基,其成分及其含量如下:
氨基酸部分
无机盐和微量元素部分
维生素部分
碳水化合物及其它成分
7.根据权利要求3所述的补料培养基,其成分及其含量如下:
8.一种制备权利要求1-7中任一项所述的补料培养基的方法,包括如下步骤:
(1)按比例称取补料培养基中各成分,混匀;
(2)将步骤(1)的补料培养基混合物溶解于超纯水中,并加入氢氧化钠调pH促进溶解,以盐酸调pH至6.9~7.2之间,并用超纯水定容,微孔滤膜过滤,即得所述补料培养基。
9.根据权利要求8所述的方法,其中,所述溶解被控制在温度在30-40℃条件下进行,所述微孔滤膜的孔径为0.22μm。
10.权利要求1-7中任一项所述的补料培养基在CHO细胞培养和/或多肽表达中的运用。
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