CN116376802A - 一种通用型无血清无蛋白的培养基及其用途 - Google Patents
一种通用型无血清无蛋白的培养基及其用途 Download PDFInfo
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Abstract
本发明涉及现代生物技术领域,具体为一种通用型无血清无蛋白的培养基及其用途。本发明主要用于哺乳细胞在体外悬浮培养的化学成分明确的培养基,通过优化培养基中的营养成分,获得了适合人胚胎肾HEK293细胞和中国仓鼠卵巢细胞(CHO细胞)悬浮培养的无血清培养基,是一款通用型基础培养基。所述培养基具有能使HEK293细胞和CHO细胞具有较高活细胞密度、高活率维持、目的蛋白产物高表达、蛋白产物质量可控等优点。与市场同款竞品相比,该培养基组分种类简单、生产制备方法简便,原材料成本低廉,可适用于HEK293和CHO两大物种细胞系的培养,可提高细胞培养生产工艺的产率,可广泛用于肿瘤和自身免疫性疾病等。
Description
技术领域
本发明涉及现代生物技术领域,具体为一种通用型无血清无蛋白的培养基及其用途。
背景技术
重组蛋白表达平台是科研、生物制药领域中重要的技术工具,其中原核细胞表达平台是最常用的表达系统,经济实惠,可以表达一些无糖基化或者经过简单修饰的重组蛋白,但该平台容易出现载体与菌株不相容、蛋白不表达、包涵体及蛋白纯化等问题。而哺乳动物真核细胞表达平台(如:CHO和HEK293)的出现克服了翻译后修饰蛋白表达的问题,此平台表达的蛋白具备折叠和修饰的功能,高度人源化,可以用于结构、功能分析;同时该平台表达量高,易于大规模生产表达;稳定及瞬时表达平台从细胞转染、筛选、分离到扩大培养,整个操作过程均在无血清培养条件下完成,且蛋白可以实现翻译后的特殊修饰。动物细胞培养技术是目前工业化生产重组蛋白制品的重要技术手段,随着大规模哺乳动物细胞培养生物药物需求的不断增加,基于细胞及产品特性培养基的研制成为了细胞工程领域的重要课题。
HEK293细胞是人类胚胎肾细胞,该细胞系的主要作用是通过细胞的基因改造实现重组蛋白合成。HEK293细胞有很多优点:(1)耐寒、半贴壁、低维护,既可以悬浮培养,也可以单层培养;(2)分裂迅速,倍增时间在20~40小时;(3)容易转染,可用于瞬时和稳定表达;(4)表达蛋白人源化,可完成特殊的翻译后修饰;(5)能为蛋白功能提供所需的辅助因子。目前,HEK293细胞广泛应用于癌症研究、新药开发、抗体蛋白生产、信号转导和蛋白质相互作用研究等领域。因用途不同,HEK293已建立了多种衍生细胞系,如:293F、293H、293T、Expi293、293E等。
中国仓鼠卵巢细胞(CHO细胞)是目前重组蛋白大规模生产中比较广泛的细胞类型,CHO细胞具有高效的基因表达、准确的蛋白后转录修饰和灵活的病原检测等优点,且CHO细胞易于悬浮培养,很少分泌自身蛋白,便于后期蛋白分离纯化。
传统的HEK293和CHO细胞需要在基础培养基中添加5~10%血清中才可以正常生长,但血清外源污染风险大、成本高、批次差异大,且不利于下游分离纯化。优化无血清培养基组分以满足工程细胞维持较高的活细胞密度、细胞活率和延长培养周期、提高蛋白表达量,一种成分明确、货期稳定、质量可控的培养基是企业降低生产成本的一个重要环节。因此,无血清、化学成分明确的培养基的研发是当下哺乳细胞培养的热点。
发明内容
本发明的目的在于提供一种通用型无血清无蛋白的培养基及其用途,以解决现有技术中的问题。
为了解决上述技术问题,本发明提供如下技术方案:本发明所述的一种通用型无血清无蛋白且化学成分明确的的培养基的制备方法。将本发明的较优含量培养基组分拆分为干粉组、碱溶组和其他组,培养基配制方法为:
S1:称取干粉组粉末于80%纯化水中搅拌;
S2:碱溶组溶于5%纯化水中,用5M NaOH调节溶液pH,搅拌;
S3:将溶解后的碱溶组液体加入干粉组溶液中,调节溶液pH;
S4:将其他组粉末加入到混合溶液中,搅拌,用稀盐酸或5M NaOH调节溶液pH;
S5:定容至最终配制体积或重量,搅拌,检测溶液pH、浊度、渗透压;
S6:在超净工作台内,经无菌过滤器无菌过滤,避光保存。
进一步的,S1中,所述搅拌时间为30~40min。
进一步的,S1中,所述干粉组中,各组分及其含量:25~35mg/L丙氨酸、300~400mg/L精氨酸、450~600mg/L天冬酰胺、1400~1500mg/L天冬氨酸、180~220mg/L半胱氨酸、400~500mg/L谷氨酸、30~40mg/L甘氨酸、300~310mg/L组氨酸、500~600mg/L异亮氨酸、420~470mg/L亮氨酸、630~640mg/L赖氨酸、240~260mg/L甲硫氨酸、410~450mg/L苯丙氨酸、250~270mg/L脯氨酸、70~80mg/L羟基脯氨酸、300~400mg/L丝氨酸、400~500mg/L苏氨酸、180~220mg/L色氨酸、530~570mg/L缬氨酸、40~50mg/L泛酸钙、60~70mg/L氯化胆碱、80~90mg/L肌醇、10~20mg/L烟酰胺、3.8~3.9mg/L吡哆醇、8~9mg/L硫胺素、15~20mg/L维生素B12、200~300mg/L丙酮酸钠、2.2~2.7mg/L次黄嘌呤、0.2~0.3mg/L胸苷、5~6mg/L腐胺、50~60mg/L乙醇胺、900~1200mg/L嵌段式聚醚F68、4800~5300mg/L葡萄糖、45~55mg/L硫酸葡聚糖、850~1050mg/L磷酸二氢钠、50~60mg/L硫酸镁、2.1~2.3mg/L氯化钙、400~500mg/L氯化钾、2400~2550mg/L氯化钠、12~20mg/L柠檬酸铵铁、0.6~0.8mg/L硫酸锌、0.003~0.006mg/L硫酸铜、0.01~0.02mg/L亚硒酸钠、0.0002~0.0005mg/L硫酸锰、0.03~0.06mg/L硅酸钠、0.002~0.004mg/L钼酸铵、0.0005~0.001mg/L偏钒酸铵、0.0004~0.0008mg/L硫酸镍、0.0002~0.0006mg/L氯化亚锡、0.002~0.003mg/L醋酸钡、0.001~0.002mg/L氯化镉、0.05~0.1mg/L氯化钴、0.001~0.004mg/L氯化铬、0.001~0.003mg/L氧化锗。
进一步的,S2中,所述NaOH调节pH至11.0~11.2;所述搅拌时间为30~40min。
进一步的,S2中,所述碱溶组中,各组分及其含量:120~160mg/L胱氨酸、200~240mg/L酪氨酸、2.3~2.5mg/L生物素、10~20mg/L叶酸、1.3~1.4mg核黄素。
进一步的,S3中,所述溶液pH调节为6.0~8.0。
进一步的,S4中,所述搅拌时间为5~10min,所述溶液pH调节为6.9~7.20。
进一步的,S4中,所述其他组成分及其含量为2000mg/L碳酸氢钠。
进一步的,S5中,所述搅拌时间为10~15min。
进一步的,S6中,过滤孔径为0.2~0.22μm;所述保存温度为2~8℃。
与现有技术相比,本发明所达到的有益效果是:本发明开发了一款无血清无蛋白化学成分明确的培养基,不仅有利于HEK293和CHO细胞的悬浮培养,而且有利于蛋白产物的高效表达,相较于市场上竞品,大大节省了大规模重组蛋白制备的成本,同时该培养基的广谱性也大大降低了企业物料储备的多样性。本发明包含碳源、氮源、氨基酸、维生素、盐类、脂类、缓冲剂、微量元素和其他营养物来维持HEK293细胞和CHO细胞生长和促进目的产物表达。碳源作为最主要的能源物质,为生物合成提供了所需的能量和产物合成的骨架,不同的碳源形式发挥的功效也不同,常用的有葡萄糖、半乳糖、甘露糖、岩藻糖、谷氨酰胺、丙酮酸钠等。氮源是生物体蛋白合成的主要组织部分,常用的无机氮源包括各种铵盐、硝酸盐等。氨基酸化学成分明确,是培养基中含量最多且非常关键的组分,不同氨基酸含量各异,直接影响了细胞的生长、细胞在培养中后期的维持以及蛋白产物的表达,最终影响产物的功能表现;常用的21种氨基酸分为必需氨基酸和非必需氨基酸,不同细胞类型对氨基酸的需求量也不尽相同,同一细胞在细胞培养周期内对氨基酸的需求也相差较大,细胞代谢研究至关重要。维生素为机体提供了大量的辅酶因子,在新陈代谢过程中发挥重要的作用;维生素不稳定,对强光、热敏感,且易被氧化,故培养基制备和储存的条件需要密切关注。脂类物质是细胞膜的重要组成部分,在细胞中既提供了能量,同时可作为血清替代物,常用的有脂肪酸、磷脂和胆固醇等。微量元素在培养基中含量较低,但有利于维持体内酶活性,可作为血清替代物,可提高培养基的稳定性,不同种类细胞对微量元素的需求不同。盐类物质和缓冲剂可以调节细胞膜的通透性,维持细胞内外正常的渗透压和酸碱平衡,促进细胞生长。其他营养物包括一些能源物质、激素和血清替代物,在维持细胞生长和表达过程中起重要的作用。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1是HEK293细胞传代过程中的活细胞密度和细胞活率;
图2是CHO细胞传代过程中的活细胞密度和细胞活率;
图3是HEK293细胞瞬时表达培养的活细胞密度和细胞活率;
图4是HEK293细胞瞬时表达培养的产物浓度;
图5是CHO重组细胞株A流加培养过程中的活细胞密度和细胞活率;
图6是CHO重组细胞株B流加培养过程中的活细胞密度和细胞活率;
图7是CHO重组细胞株流加培养过程中的产物浓度;
图8是CHO重组细胞株流加培养过程中的蛋白纯度;
图9是CHO重组细胞株流加培养过程中的蛋白电荷变异体分布;
图10是CHO重组细胞株流加培养过程中的蛋白糖型分布。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明中使用的原料及其来源如下表所示。
其中,细胞传代培养基:OPM-CD 293CD05(OPM,适用于HEK293细胞)和CD CHO(Gibco,适用于CHO细胞);HEK293瞬转培养基:OPM-CD 293CD05;CHO Fed-Batch流加培养基:基础培养基Dynamis、补料培养基EfficientFeed C+。
实施例1
本发明所述的一种通用型无血清无蛋白且化学成分明确的的培养基的制备方法。
(1)称取干粉组粉末于80%纯化水中搅拌30min;其中,所述干粉组中各组分及其含量如下表1所示:
表1
(2)碱溶组溶于5%纯化水中,用5M NaOH调节溶液pH至11.1,搅拌30min;所述碱溶组中各组分及其含量如下表2所示:
表2
(3)将溶解后的碱溶组液体加入干粉组溶液中,调节溶液pH至7.0;
(4)将其他组粉末加入到混合溶液中,搅拌5min,用稀盐酸或5M NaOH调节溶液pH至7.0;所述其他组中,各组分及其含量为2000mg/L碳酸氢钠(NaHCO3);
(6)定容至最终配制体积或重量,搅拌10min,检测溶液pH、浊度、渗透压;
(7)在超净工作台内,经0.22μm无菌过滤器无菌过滤,5℃避光保存。
实施例2
将各细胞按0.5×106细胞/毫升的密度稀释至含本发明培养基的摇瓶中,置于36.5℃、6%CO2、130r/min的二氧化碳摇床中培养,每3天传一代,监测每一代活细胞密度和细胞活率。如图1~2所示,与对照组相比,实验组活细胞密度和细胞活率更高,表现更优,各细胞株传代培养稳健,实验表明本发明培养基同时适用于HEK293细胞和CHO细胞悬浮细胞生长,生长稳定且状态良好。
其中,细胞复苏及传代培养方法如下:
(1)提前开启水浴锅,温度调节到37度;
(2)将细胞冻存管从-80℃或液氮中取出,迅速置于水浴锅中,轻柔晃动至完全融化
(3)将融化后的细胞液迅速转移至含9毫升新鲜培养基的离心管中,充分混匀;
(4)将混匀后的细胞液离心,1000转、5分钟,离心后弃上清,加入新鲜培养液混匀;
(5)将混匀后的细胞液加入含有新鲜培养基的摇瓶中,置于36.5℃、130转、6%CO2的摇床中进行培养。
(6)将细胞按0.5×106细胞/毫升的活细胞密度扩增至含新鲜培养基的摇瓶中,置于36.5℃、130转、6%CO2的摇床中进行培养,细胞每2~4天扩增一代。
实施例3
首先将HEK293细胞在本发明培养基中进行细胞适应培养2~3代,待细胞倍增时间稳定、状态良好时进行瞬时转染。如图3所示,与对照组相比,本发明在HEK293瞬时表达的细胞生长良好,活细胞密度和细胞活率较高,后期维持较好,细胞活率在90%以上;如图4所示,与对照组相比,实验组蛋白表达量更高,不同目的蛋白的瞬时表达水平各异,蛋白表达量在50~1000毫克/升。
HEK293瞬转操作方法如下:
(1)细胞准备:HEK293细胞按照0.5×106细胞/毫升的密度进行传代培养,瞬转当天活细胞密度达到3~4×106细胞/毫升,活率>95%;
(2)转染试剂准备:用培养基稀释质粒(1~3毫克/升),稀释PEI(2~6毫克/升),将稀释后的质粒和PEI混合,室温孵育20分钟;
(3)转染:将转染试剂缓慢加入准备好的细胞液中,置于36.5度、6%CO2、130转/分钟的二氧化碳摇床中培养;
(4)细胞培养:20小时后加入初始细胞体积5%的补料培养基(293ProFeed,OPM)
(5)每天取样监测活细胞密度、细胞活率和生化指标(葡萄糖、乳酸、蛋白含量),当细胞液中葡萄糖浓度低于3克/升时,补加葡萄糖至4~6克/升;当细胞活率<60%或培养第7天时离心取细胞上清进行蛋白纯化(分泌蛋白)。
实施例4
选取两种CHO重组细胞株A和B(表达重组单克隆抗体细胞株)中进行流加培养,如图5~6所示,与对照组相比,在不同细胞株中,本发明培养基更有利于细胞生长和活率维持;如图7~8所示,与对照组相比,本发明培养基的蛋白表达量较高,蛋白纯度更高;如图9~10所示,对照组与实验组的蛋白电荷变异体和蛋白糖型无明显差异,均符合行业要求。
CHO细胞Fed-Batch流加培养操作方法如下:
以本发明培养基为基础培养基,接种密度1.0×106细胞/毫升,前期培养温度36.5℃,培养第5天将培养温度降温至33.0℃,在培养周期第3、5、7、9、11天时分别添加初始接种体积5%的补料培养基EfficientFeed C+,每天监测活细胞密度、细胞活率、蛋白含量和生化指标(葡萄糖、乳酸、氨),当细胞液中葡萄糖浓度低于3克/升时,添加葡萄糖浓缩液至细胞液中使葡萄糖浓度提高到4~6克/升;培养至14天结束培养,离心取细胞上清进行蛋白纯化,检测纯化后蛋白纯度、蛋白电荷变异体和蛋白糖型分布。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种通用型无血清无蛋白的培养基的制备方法,包括以下步骤:
S1:称取干粉组粉末于纯化水中搅拌得到干粉组液体;
S2:碱溶组溶于纯化水中得到碱溶组液体,调节溶液pH,搅拌;
S3:将溶解后的碱溶组液体加入干粉组溶液中得到混合溶液,调节溶液pH;
S4:将碳酸氢钠加入到混合溶液中,搅拌,调节溶液pH;
S5:定容至最终配制体积或重量,搅拌,检测溶液pH、浊度、渗透压后过滤得到所述培养基。
2.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S1中,搅拌时间为30~40min。
3.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S1中,所述干粉组中,各组分及其含量:25~35mg/L丙氨酸、300~400mg/L精氨酸、450~600mg/L天冬酰胺、1400~1500mg/L天冬氨酸、180~220mg/L半胱氨酸、400~500mg/L谷氨酸、30~40mg/L甘氨酸、300~310mg/L组氨酸、500~600mg/L异亮氨酸、420~470mg/L亮氨酸、630~640mg/L赖氨酸、240~260mg/L甲硫氨酸、410~450mg/L苯丙氨酸、250~270mg/L脯氨酸、70~80mg/L羟基脯氨酸、300~400mg/L丝氨酸、400~500mg/L苏氨酸、180~220mg/L色氨酸、530~570mg/L缬氨酸、40~50mg/L泛酸钙、60~70mg/L氯化胆碱、80~90mg/L肌醇、10~20mg/L烟酰胺、3.8~3.9mg/L吡哆醇、8~9mg/L硫胺素、15~20mg/L维生素B12、200~300mg/L丙酮酸钠、2.2~2.7mg/L次黄嘌呤、0.2~0.3mg/L胸苷、5~6mg/L腐胺、50~60mg/L乙醇胺、900~1200mg/L嵌段式聚醚F68、4800~5300mg/L葡萄糖、45~55mg/L硫酸葡聚糖、850~1050mg/L磷酸二氢钠、50~60mg/L硫酸镁、2.1~2.3mg/L氯化钙、400~500mg/L氯化钾、2400~2550mg/L氯化钠、12~20mg/L柠檬酸铵铁、0.6~0.8mg/L硫酸锌、0.003~0.006mg/L硫酸铜、0.01~0.02mg/L亚硒酸钠、0.0002~0.0005mg/L硫酸锰、0.03~0.06mg/L硅酸钠、0.002~0.004mg/L钼酸铵、0.0005~0.001mg/L偏钒酸铵、0.0004~0.0008mg/L硫酸镍、0.0002~0.0006mg/L氯化亚锡、0.002~0.003mg/L醋酸钡、0.001~0.002mg/L氯化镉、0.05~0.1mg/L氯化钴、0.001~0.004mg/L氯化铬、0.001~0.003mg/L氧化锗。
4.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S2中,溶液pH调节至11.0~11.2;搅拌时间为30~40min。
5.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S2中,所述碱溶组中,各组分及其含量:120~160mg/L胱氨酸、200~240mg/L酪氨酸、2.3~2.5mg/L生物素、10~20mg/L叶酸、1.3~1.4mg核黄素。
6.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S3中,溶液pH调节为6.0~8.0。
7.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S4中,搅拌时间为5~10min,所述溶液pH调节为6.9~7.20。
8.根据权利要求1所述的一种通用型无血清无蛋白的培养基的制备方法,其特征在于:S4中,碳酸氢钠含量为2000mg/L。
9.根据权利要求1~8中任一项所述的一种通用型无血清无蛋白的培养基的制备方法制备得到的通用型无血清无蛋白的培养基。
10.根据权利要去9所述的一种通用型无血清无蛋白的培养基的应用,其特征在于:所述培养基适用于HEK293和CHO细胞的培养。
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