CN113913368A - 适合cho细胞大规模悬浮扩增培养的无血清培养基及其制备和应用 - Google Patents
适合cho细胞大规模悬浮扩增培养的无血清培养基及其制备和应用 Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种无血清培养基及其制备方法和在CHO细胞大规模悬浮扩增培养中的应用。该无血清培养基pH为6.9~7.0,包括氨基酸组合物、无机盐组合物、维生素组合物、微量元素组合物、缓冲体系组合物、补充因子组合物和水;以无血清培养基为基准,氨基酸组合物包括谷丙氨酸二肽GlutaMAX‑I 1~2mg/L,缓冲体系组合物包括HEPES 3~8g/L、碳酸氢钠1~5g/L、磷酸二氢钠100~300mg/L、磷酸氢二钠100~300mg/L、氯化钠3~10g/L、氯化钾200~500mg/L,补充因子组合物包括大豆提取物10~30g/L、酵母提取物10~30g/L、Long‑R3‑IGF‑I 80~100μg/L。制备方法包括:将各组分按配方称量混合,调节pH至6.9~7.0后用孔径为0.22μm的滤膜过滤。
Description
技术领域
本发明涉及细胞生物学技术领域,具体涉及一种适合CHO细胞大规模悬浮扩增培养的无血清培养基及其制备和应用。
背景技术
利用动物细胞工程发酵表达蛋白药物和抗体是现代医药发展的重要方向,世界上有近半的重组药物是哺乳动物细胞表达获得,而其中70%是由中国仓鼠卵巢细胞(ChineseHamsters Ovary,CHO)表达获得,CHO细胞作为重组药物表达的工程细胞具有重要价值。
体外培养重组CHO细胞过程中往往需要在培养基中加入动物血清,动物血清含有激素、生长因子、载体蛋白、粘附扩散因子、微量元素等营养成分,通常以高达10%的含量加入到培养基中维持细胞生长,如胎牛血清(Fetal Bovine Serum,FBS)。
但动物血清成分复杂、价格昂贵、批次间生物活性不稳定,且极易感染病毒或支原体,这往往导致后期生产的目标蛋白在分离纯化上难度增加,同时对重组蛋白产品的品质造成影响,这令含动物血清的培养基很难进行工业化运用,而无血清培养基的出现解决了这一问题。
无血清培养基是指不需要添加血清就可以支持哺乳动物细胞在体外生长繁殖、维持较高的细胞密度和产物表达量的合成培养基,如犬肾细胞(MDCK)、猴肾细胞(Vero)、中国仓鼠卵巢细胞(CHO)等。无血清培养基在制药工程中的广泛使用提高了细胞培养过程中的可重复性,也避免了由不同血清批次之间差异带来的质控不稳定的影响,同时也减少了血清中携带的病毒、支原体等微生物的污染风险,使通过动物细胞培养生产的生物活性制品易于规模化稳定培养及纯化利用。
目前市场上的CHO无血清培养基,无论进口还是国产,价格均相对昂贵,规模化使用成本较高,这使得无血清培养基很难在重组蛋白药物生产中得到大规模应用,限制了制药工程的发展。在无血清培养基的开发上,张立等开发了一种无血清培养基(CN1696283A),主要对中国仓鼠卵巢细胞CHO-GS培养进行优化,以DMEM/F12为基础培养基,添加有胰岛素(2-20mg/L),L-谷氨酰胺(0-500mg/L)等物质,然而胰岛素、L-谷氨酰胺容易降解,且L-谷氨酰胺降解产物具有细胞毒性,不利于细胞生长。寇庚等开发了一种无血清培养基(CN101117624A),以DMEM/F12为基础培养基,添加一定量的维生素及微量元素,然而该培养基未添加细胞生长所需的生长因子及氮源,以及未添加保护细胞免受剪切伤害的表面活性剂,因此不适合大规模悬浮培养。陈亮等开发的无血清培养基(CN104293729A),主要成分包括了氨基酸、无机盐、缓冲体系,维生素,微量元素等。然而该培养基不含有细胞生长所必须的氮源L-谷氨酰胺,且含有1.0-2.0mg/L的酚红,酚红具有固醇类激素的作用,在细胞培养中可引起固醇类反应,同时,酚红对环境尤其是水体可能有危害,因此不利于大规模培养。宋国芳等开发的CHO细胞无血清培养基(CN107460160A),以CHO-S-SFM II为基础培养基,同时添加了1mg/ml谷氨酰胺、1mg/ml蛋氨酸、1mg/ml脯氨酸、1mg/ml色氨酸、10mg/ml胰岛素5种添加物,然而CHO-S-SFM II本身为无血清培养基,价格高昂,不利于大规模培养,同时,胰岛素、L-谷氨酰胺也存在容易降解的问题,L-谷氨酰胺降解产物具有细胞毒性,不利于细胞生长。王晓柯开发的CHO细胞无血清培养基(CN109337861A),主要成分包括氨基酸,无机盐成分,维生素、微量元素、酵母水解物及白蛋白等成分,其只采用一种缓冲体系,pH只适合7.0-7.4的区间,且放大培养时摇床需要用到CO2维持pH,而细胞高密度培养后期易产生大量代谢废物,培养溶液变酸,低缓冲能力培养基不利于高密度细胞培养。
综上所述,虽然市场上开发了很多无血清培养基,但各种培养基或者只适合一种细胞生长,或者添加产物不稳定,容易降解后并产生细胞毒性。同时,多数无血清培养基还含有白蛋白等大分子蛋白类添加剂,严重影响了后期表达制品的分离纯化,提高了生产成本。因此,发明一款适合大规模CHO细胞生长的无血清培养基是目前市场亟需解决的问题,对满足临床用重组蛋白的生产具有重要意义。
发明内容
针对本领域存在的不足之处,本发明提供了一种适合CHO细胞大规模悬浮扩增培养的无血清培养基,通过添加大豆提取物和酵母提取物提供无血清培养基中细胞所需的短肽类氨基酸,同时用可在溶液中稳定存在的类胰岛素生长因子Long-R3-IGF-I和谷丙氨酸二肽GlutaMAX-I代替生长激素和氮源,优化了无血清培养基的组成和pH,使得本发明无血清培养基更加适合CHO细胞的大规模悬浮扩增培养,具有体外长时间维持较高细胞密度的作用,具有较高的市场应用价值。
一种适合CHO细胞大规模悬浮扩增培养的无血清培养基,包括氨基酸组合物、无机盐组合物、维生素组合物、微量元素组合物、缓冲体系组合物、补充因子组合物和水;
以所述无血清培养基为基准,所述氨基酸组合物包括谷丙氨酸二肽GlutaMAX-I 1~2mg/L,所述缓冲体系组合物包括HEPES(4-羟乙基哌嗪乙磺酸)3~8g/L、碳酸氢钠1~5g/L、磷酸二氢钠100~300mg/L、磷酸氢二钠100~300mg/L、氯化钠3~10g/L、氯化钾200~500mg/L,所述补充因子组合物包括大豆提取物10~30g/L、酵母提取物10~30g/L、Long-R3-IGF-I 80~100μg/L;
所述无血清培养基的pH为6.9~7.0。
本发明在无血清培养基中添加特定浓度的酵母提取物和大豆提取物,可为无血清培养基提供短肽类氨基酸,该类氨基酸所含的部分肽段可作为外部分子信号对细胞代谢、生物合成、细胞生长及产物表达等生命获得产生特殊的调控作用,同时避免了无血清培养基中添加动物组分后,动物组分对实验研究的影响。
本发明还优化了缓冲体系组合物,其中HEPES可在pH=7.2~7.4范围内提供优良的缓冲能力,配合磷酸缓冲体系及碳酸缓冲体系并通过浓度调整可保证细胞在高密度时的合适pH值以及无血清培养基在pH=6.9~7.0范围内的精确控制。
类胰岛素生长因子Long-R3-IGF-I在细胞分化、增殖中具有重要促进作用,富集效率高,可提高无血清培养基的稳定性,延长无血清培养基的半衰期。
作为优选,以所述无血清培养基为基准,所述氨基酸组合物还包括至少一种以下浓度的组分:丙氨酸5~10mg/L、精氨酸1~1.5g/L、天冬酰胺200~500mg/L、天冬氨酸30~50mg/L、半胱氨酸80~110mg/L、胱氨酸80~110mg/L、谷氨酸50~90mg/L、甘氨酸40~70mg/L、组氨酸90~110mg/L、异亮氨酸100~110mg/L、亮氨酸100~120mg/L、赖氨酸200~300mg/L、蛋氨酸30~40mg/L、苯丙氨酸60~90mg/L、缬氨酸95~130mg/L、脯氨酸200~500mg/L、丝氨酸50~90mg/L、苏氨酸80~120mg/L、色氨酸15~25mg/L、酪氨酸170~250mg/L。
作为优选,以所述无血清培养基为基准,所述无机盐组合物包括至少一种以下浓度的组分:四水钼酸铵0.005~0.009mg/L、九水硝酸铁0.1~0.8mg/L、七水硫酸亚铁1~3mg/L、偏钒酸铵0.0005~0.0009mg/L、九水硅酸钠0.01~0.04mg/L、七水硫酸锌1~4mg/L、二水氯化钙105~130mg/L、无水硫酸铜0.01~0.05mg/L、硫酸锰0.001~0.005mg/L、氯化镍0.0001~0.0004mg/L、硒酸钠0.003~0.007mg/L、六水氯化镁80~110mg/L、二水氯化锡0.0001~0.0005mg/L。
作为优选,以所述无血清培养基为基准,所述维生素组合物包括至少一种以下浓度的组分:维生素H 0.03~0.08mg/L、氯化胆碱70~130mg/L、叶酸7~13mg/L、肌醇50~120mg/L、烟酰胺2~5mg/L、盐酸吡哆醛3~10mg/L、盐酸吡哆醇1~7mg/L、核黄素1~6mg/L、盐酸硫胺3~8mg/L、维生素B12 10~20mg/L。
作为优选,以所述无血清培养基为基准,所述微量元素组合物包括至少一种以下浓度的组分:腐胺0.5~1.5mg/L、丙酮酸钠1~3g/L、泛酸钙3~5mg/L、α-硫辛酸0.1~0.3mg/L、柠檬酸铁50~80mg/L、胸苷0.2~0.6mg/L、硒酸10~30μg/L、亚油酸1~5mg/L、乙醇胺5~20mg/L、氢化可的松3~10μg/L、甲状腺激素100~150ng/L、泛酸钙5~10mg/L。
作为优选,以所述无血清培养基为基准,所述补充因子组合物还包括至少一种以下浓度的组分:聚醚F-68 1~3g/L、葡萄糖3~30g/L、次黄嘌呤10~20mg/L、胸腺嘧啶3~10mg/L。
本发明还提供了所述的无血清培养基的制备方法,包括:将各组分按配方称量混合,调节pH至6.9~7.0后用孔径为0.22μm的滤膜过滤即得所述的无血清培养基。可采用浓度为0.1mol/L的盐酸、氢氧化钠等本领域常用的酸碱调节pH。
本发明还提供了所述的无血清培养基在CHO细胞大规模悬浮扩增培养中的应用。
本发明与现有技术相比,主要优点包括:
1、本发明的无血清培养基中以葡萄糖为主要碳源,GlutaMAX-I为主要氮源,GlutaMAX-I二肽是L-谷氨酰胺的衍生物,将其不稳定的α-氨基用L-丙氨酸来保护,提高了培养基的长期稳定性及由L-谷氨酰胺降解带来的细胞毒性。
2、本发明的无血清培养基添加了大豆提取物和酵母提取物,优化了无血清培养基的组成,提供了无血清培养基中细胞所需的短肽类氨基酸,使得本发明的无血清培养基更加适合CHO细胞大规模悬浮扩增培养,具有体外长时间维持较高细胞密度的作用。
附图说明
图1为CHO细胞在实施例1的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b);
图2为CHO细胞在实施例1的无血清培养基中培养和在对比例1的不含大豆提取物、酵母提取物的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b);
图3为CHO细胞在实施例1的无血清培养基中培养和在对比例2的不含HEPES的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b);
图4为CHO细胞在实施例1的无血清培养基中培养和在对比例3的不含Long-R3-IGF-I的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b);
图5为CHO细胞在实施例1的无血清培养基中培养和在对比例4的不含GlutaMAX-I的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b);
图6为CHO细胞在实施例1的无血清培养基中培养和在对比例5的使用胰岛素样生长因子IGF及L-谷氨酰胺等量替代Long-R3-IGF-I及GlutaMAX-I配制的无血清培养基中培养的生长曲线图(a)及细胞状态照片(b)。
具体实施方式
下面结合附图及具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的操作方法,通常按照常规条件,或按照制造厂商所建议的条件。如无特殊说明,所用组分均可商业购买获得且均为细胞培养级,并按照相关要求储存。大豆提取物,Gibco,货号:212489。酵母提取物,Gibco,货号:210941。Long-R3-IGF-I,Merck,货号:85580C。GlutaMAX-I,Gibco,货号:A1286001。
实施例1
以配制1L无血清培养基为例。
氨基酸组合物在1L无血清培养基中的具体含量如下:
无机盐组合物在1L无血清培养基中的具体含量如下:
维生素组合物在1L无血清培养基中的具体含量如下:
微量元素组合物在1L无血清培养基中的具体含量如下:
缓冲体系组合物在1L无血清培养基中的具体含量如下:
补充因子组合物在1L无血清培养基中的具体含量如下:
以上组分充分溶解于无菌去离子水中,并调节pH至6.9~7.0,定容至1000mL后用孔径为0.22μm的滤膜过滤,4℃保存待用。
本发明中,CHO细胞无血清培养基通过以下方法进行CHO细胞培养并计数:
常规方法复苏冻存的CHO细胞至T25方瓶,并采用5mL上述无血清培养基。待细胞密度长成0.8×106cells/mL后,扩繁至2 T25方瓶,每瓶5mL无血清培养基。2 T25方瓶细胞密度分别达到1×106cells/mL左右,转入100mL三角瓶,同时添加10ml新无血清培养基,保持摇瓶接种密度0.5×106cells/mL,接种体积20mL。将三角摇瓶置于恒温摇床中,37℃、60rpm振荡培养,每天取样计数,台盼蓝染色计算细胞活率,按规定流加,待细胞活率低于80%时,培养结束。
由图1可以看出,CHO细胞在实施例1的无血清培养基悬浮放大培养过程中,细胞生长状态良好。6-7天扩增后,细胞在悬浮培养过程中能达到7.8×106cells/mL的较高细胞密度,且维持较高细胞活力,连续培养表现稳定。
对比例1
无血清培养基与实施例1相比,区别仅在于无大豆提取物(H粉)、酵母提取物(Y粉),其余均相同。
对比例1和实施例1的两种无血清培养基培养的CHO细胞密度见图2。
由图2可以看出,实施例1的无血清培养基添加H粉和Y粉后,CHO细胞在无血清培养基悬浮放大培养过程中,细胞生长状态良好,细胞单个分散透亮,均匀悬浮,无结团现象。6-7天扩增后,细胞在悬浮培养过程中能达到更高的细胞密度,且维持较高细胞活力,连续培养表现稳定,而未添加H粉、Y粉的无血清培养基则在第5天,密度4.0×106cells/mL左右达到最大值,之后出现死亡及密度下降。
对比例2
无血清培养基与实施例1相比,区别仅在于无HEPES,其余均相同。
对比例2和实施例1的两种无血清培养基培养的CHO细胞密度见图3。
由图3可以看出,不添加HEPES后,CHO细胞悬浮放大3天以后密度不再上升,之后维持2-3天后密度开始下降,不太适合连续培养。
对比例3
无血清培养基与实施例1相比,区别仅在于无Long-R3-IGF-I,其余均相同。
对比例3和实施例1的两种无血清培养基培养的CHO细胞密度见图4。
由图4可以看出,不添加Long-R3-IGF-I的无血清培养基与添加了Long-R3-IGF-I的无血清培养基相比,CHO细胞在第5天只能达到细胞密度3.0×106cells/mL,不适合工业化高密度培养。
对比例4
无血清培养基与实施例1相比,区别仅在于无GlutaMAX-I,其余均相同。
对比例4和实施例1的两种无血清培养基培养的CHO细胞密度见图5。
由图5可以看出,不添加GlutaMAX-I的无血清培养基在第5天左右细胞密度达到最大值,且该密度不能长时间维持,在高密度后的第2天密度开始下降,说明不添加GlutaMAX-I不能维持细胞较高密度稳定培养。
对比例5
无血清培养基与实施例1相比,区别仅在于使用胰岛素样生长因子IGF及L-谷氨酰胺替代Long-R3-IGF-I及GlutaMAX-I,其余均相同。
对比例5和实施例1的两种无血清培养基均在4℃保存30天后再进行CHO细胞培养,培养的CHO细胞密度见图6。
由图6可以看出,在4℃保存30天后进行试验,CHO细胞在实施例1的无血清培养基中细胞生长状态良好,细胞密度一直缓慢上升,并在第8天左右达到密度最大值7.8×106cells/mL左右,细胞活力较高,连续培养表现稳定,而含有IGF及L-谷氨酰胺培的无血清培养基在相同培养条件下,在5-7天出现密度最大值4.7×106cells/mL左右,之后密度、活率开始下降,这可能是因为IGF及L-谷氨酰胺在培养基中易降解失活,同时产生毒性降解产物所致,因此,对比例5的无血清培养基不是CHO细胞扩增放大的最佳培养基。
此外应理解,在阅读了本发明的上述描述内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (8)
1.一种适合CHO细胞大规模悬浮扩增培养的无血清培养基,其特征在于,包括氨基酸组合物、无机盐组合物、维生素组合物、微量元素组合物、缓冲体系组合物、补充因子组合物和水;
以所述无血清培养基为基准,所述氨基酸组合物包括谷丙氨酸二肽GlutaMAX-I 1~2mg/L,所述缓冲体系组合物包括HEPES 3~8g/L、碳酸氢钠1~5g/L、磷酸二氢钠100~300mg/L、磷酸氢二钠100~300mg/L、氯化钠3~10g/L、氯化钾200~500mg/L,所述补充因子组合物包括大豆提取物10~30g/L、酵母提取物10~30g/L、Long-R3-IGF-I 80~100μg/L;
所述无血清培养基的pH为6.9~7.0。
2.根据权利要求1所述的无血清培养基,其特征在于,以所述无血清培养基为基准,所述氨基酸组合物还包括至少一种以下浓度的组分:丙氨酸5~10mg/L、精氨酸1~1.5g/L、天冬酰胺200~500mg/L、天冬氨酸30~50mg/L、半胱氨酸80~110mg/L、胱氨酸80~110mg/L、谷氨酸50~90mg/L、甘氨酸40~70mg/L、组氨酸90~110mg/L、异亮氨酸100~110mg/L、亮氨酸100~120mg/L、赖氨酸200~300mg/L、蛋氨酸30~40mg/L、苯丙氨酸60~90mg/L、缬氨酸95~130mg/L、脯氨酸200~500mg/L、丝氨酸50~90mg/L、苏氨酸80~120mg/L、色氨酸15~25mg/L、酪氨酸170~250mg/L。
3.根据权利要求1所述的无血清培养基,其特征在于,以所述无血清培养基为基准,所述无机盐组合物包括至少一种以下浓度的组分:四水钼酸铵0.005~0.009mg/L、九水硝酸铁0.1~0.8mg/L、七水硫酸亚铁1~3mg/L、偏钒酸铵0.0005~0.0009mg/L、九水硅酸钠0.01~0.04mg/L、七水硫酸锌1~4mg/L、二水氯化钙105~130mg/L、无水硫酸铜0.01~0.05mg/L、硫酸锰0.001~0.005mg/L、氯化镍0.0001~0.0004mg/L、硒酸钠0.003~0.007mg/L、六水氯化镁80~110mg/L、二水氯化锡0.0001~0.0005mg/L。
4.根据权利要求1所述的无血清培养基,其特征在于,以所述无血清培养基为基准,所述维生素组合物包括至少一种以下浓度的组分:维生素H 0.03~0.08mg/L、氯化胆碱70~130mg/L、叶酸7~13mg/L、肌醇50~120mg/L、烟酰胺2~5mg/L、盐酸吡哆醛3~10mg/L、盐酸吡哆醇1~7mg/L、核黄素1~6mg/L、盐酸硫胺3~8mg/L、维生素B12 10~20mg/L。
5.根据权利要求1所述的无血清培养基,其特征在于,以所述无血清培养基为基准,所述微量元素组合物包括至少一种以下浓度的组分:腐胺0.5~1.5mg/L、丙酮酸钠1~3g/L、泛酸钙3~5mg/L、α-硫辛酸0.1~0.3mg/L、柠檬酸铁50~80mg/L、胸苷0.2~0.6mg/L、硒酸10~30μg/L、亚油酸1~5mg/L、乙醇胺5~20mg/L、氢化可的松3~10μg/L、甲状腺激素100~150ng/L、泛酸钙5~10mg/L。
6.根据权利要求1所述的无血清培养基,其特征在于,以所述无血清培养基为基准,所述补充因子组合物还包括至少一种以下浓度的组分:聚醚F-68 1~3g/L、葡萄糖3~30g/L、次黄嘌呤10~20mg/L、胸腺嘧啶3~10mg/L。
7.根据权利要求1~6任一权利要求所述的无血清培养基的制备方法,其特征在于,包括:将各组分按配方称量混合,调节pH至6.9~7.0后用孔径为0.22μm的滤膜过滤即得所述的无血清培养基。
8.根据权利要求1~6任一权利要求所述的无血清培养基在CHO细胞大规模悬浮扩增培养中的应用。
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