CN116790477A - 一种支持cho细胞高密度培养的无血清培养基及其制备方法与应用 - Google Patents
一种支持cho细胞高密度培养的无血清培养基及其制备方法与应用 Download PDFInfo
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- CN116790477A CN116790477A CN202310699847.2A CN202310699847A CN116790477A CN 116790477 A CN116790477 A CN 116790477A CN 202310699847 A CN202310699847 A CN 202310699847A CN 116790477 A CN116790477 A CN 116790477A
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Abstract
本发明公开了一种支持CHO细胞高密度培养的无血清培养基及其制备方法与应用。该无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,能够支持CHO细胞在悬浮状态下快速增殖,缩短了细胞增殖所需时间,细胞高密度维持时间长,可实现CHO悬浮细胞的高密度培养。
Description
技术领域
本发明属于细胞培养领域,具体涉及一种支持CHO细胞高密度培养的无血清培养基及其制备方法与应用。
背景技术
细胞培养作为生物制药工程技术的核心环节之一,覆盖抗体药物、蛋白药物、疫苗等方面,而细胞培养基作为细胞培养的基础,为细胞体外培养提供营养物质,促进细胞增殖和蛋白表达。中国仓鼠卵巢(Chinese Hamster Ovary,CHO)细胞在生物制药领域具有重要的地位,由于其具有翻译后修饰功能,表达的蛋白质活性比较高,因此,被广泛的应用于重组蛋白的表达。传统上,CHO细胞的培养需要在基础培养基(如DMEM/F12)中添加10%胎牛血清,以满足CHO细胞对生长的营养需求,从而实现细胞高密度培养。但胎牛血清的添加会带来一系列的问题:批次间差异大、价格昂贵、存在潜在的致病因子、下游纯化工艺复杂等。
为了避免添加胎牛血清所造成的一系列问题,无血清培养基登上舞台。无血清培养基通过添加细胞生长所需的碳源、氮源、维生素、无机盐、微量元素等成分,为细胞生长提供最佳的营养环境,可以使细胞在不添加胎牛血清的条件下快速增殖,满足生物制药对细胞的要求。并且由于无血清培养基中,成分明确,为下游的纯化提供了便利,降低了生产成本。但目前无血清培养基配方中常添加胰岛素或IGF-1、转铁蛋白来实现与含血清培养基同等的培养性能,但胰岛素或IGF-1、转铁蛋白为蛋白类物质,成本较高。我国国内无血清培养基研究起步晚,产品质量参差不齐,而进口的各大品牌无血清培养基价格较高,限制了其大规模应用。而且,最关键的是现有CHO细胞无血清培养基细胞生长密度无法达到特别高的水平。因此,开发一款性能佳、稳定性好、生长倍数高的CHO无血清培养基是市场的迫切需求。
发明内容
本发明的第一个目的在于提供一种支持CHO细胞高密度培养的无血清培养基。该培养基无血清、无蛋白、无动物源成分,不存在潜在致病因子风险,批间一致性好,利于下游纯化等工艺。
本发明的第二个目的在于提供一种上述无血清培养基的制备方法。
本发明的第三个目的在于提供上述无血清培养基的应用。
为达到第一个目的,本发明提供了一种支持CHO细胞高密度培养的无血清培养基,所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中,
所述氨基酸的组成为:L-酪氨酸二钠二水合物100~460mg/L、L-色氨酸50~150mg/L、L-赖氨酸盐酸盐350~650mg/L、L-组氨酸盐酸盐一水合物100~300mg/L、甘氨酸5~50mg/L、L-异亮氨酸260~550mg/L、L-丙氨酸10~100mg/L、L-精氨酸盐酸盐320~760mg/L、L-天冬酰胺一水合物360~820mg/L、L-亮氨酸45~225mg/L、L-甲硫氨酸100~300mg/L、L-苯丙氨酸90~400mg/L、L-脯氨酸100~500mg/L、L-丝氨酸50~250mg/L、L-苏氨酸165~500mg/L、L-缬氨酸280~550mg/L、L-谷氨酸200~400mg/L、L-胱氨酸10~85mg/L、L-半胱氨酸250~450mg/L、L-天冬氨酸265~515mg/L;
所述维生素的组成为:肌醇50~170mg/L、氯化胆碱100~300mg/L、尼克酰胺1.6~8.9mg/L、核黄素0.1~0.5mg/L、D-泛酸钙3.5~8.8mg/L、盐酸吡哆辛2.0~5.15mg/L、维生素B12 0.5~5.5mg/L、抗坏血酸10~100mg/L、盐酸硫胺2~10mg/L、生物素0.01~0.1mg/L、叶酸0.5~5.5mg/L;
所述无机盐的组成为:氯化钠3000~5500mg/L、氯化钾300~1000mg/L、无水磷酸氢二钠140~320mg/L、硫酸镁100~260mg/L、碳酸氢钠1500~3700mg/L、无水氯化钙75~235mg/L;
所述微量元素的组成为:七水硫酸亚铁2~6mg/L、五水硫酸铜0.01~0.15mg/L、七水硫酸锌0.05~0.5mg/L、六水氯化铝0.0001~0.002mg/L、硝酸银0.01~0.1mg/L、溴化钾0.01~0.1mg/L、六水氯化钴0.0001~0.001mg/L、六水氯化铬0.000001~0.00001mg/L、乙酸钡0.0001~0.001mg/L、硫酸锰一水合物0.000001~0.00001mg/L、四水钼酸铵0.00001~0.0001mg/L、偏钒酸铵0.0005~0.01mg/L、偏硅酸钠0.005~0.05mg/L、亚硒酸钠0.002~0.02mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖3000~8000mg/L、丙酮酸钠105~550mg/L、脱氧-D-核糖1.0~5.0mg/L、腺嘌呤1.0~5.0mg/L、胸腺嘧啶0.1~2.0mg/L、脱氧尿苷20~125mg/L、1,4-丁二胺双盐酸盐0.15~3mg/L、牛磺酸85~165mg/L、对氨基苯甲酸0.001~0.0035mg/L、还原型谷胱甘肽0.001~0.025mg/L、乙醇胺盐酸盐0.2~2.5mg/L、硫辛酸1.0~2.65mg/L、金精三羧酸8.44~84.4mg/L、花生四烯酸0.005~0.025mg/L、胆固醇0.1~2.5mg/L、DL-α-生育酚醋酸酯0.5~1.5mg/L、亚油酸0.01~0.1mg/L、亚麻酸0.01~0.1mg/L、硬脂酸0.01~0.1mg/L、酵母浸粉500~5000mg/L、吐温80 1~10mg/L、硫酸葡聚糖15~60mg/L、Pluronic F-68 800~1500mg/L。
在本发明的培养基中,使用金精三羧酸(ATA)可同时替代胰岛素/IGF-1和转铁蛋白,大大降低了配方成本和复杂性。各组分协同作用对CHO细胞生长具有显著促进作用,能够支持CHO悬浮细胞的高密度快速生长,特别是培养基中加入了脱氧尿苷。
进一步的,所述支持CHO细胞高密度培养的无血清培养基中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物150~300mg/L、L-色氨酸65~100mg/L、L-赖氨酸盐酸盐400~550mg/L、L-组氨酸盐酸盐一水合物150~265mg/L、甘氨酸8~40mg/L、L-异亮氨酸300~500mg/L、L-丙氨酸25~85mg/L、L-精氨酸盐酸盐400~600mg/L、L-天冬酰胺一水合物430~695mg/L、L-亮氨酸80~170mg/L、L-甲硫氨酸135~230mg/L、L-苯丙氨酸140~320mg/L、L-脯氨酸176~360mg/L、L-丝氨酸75~200mg/L、L-苏氨酸180~420mg/L、L-缬氨酸310~415mg/L、L-谷氨酸238~340mg/L、L-胱氨酸20~65mg/L、L-半胱氨酸295~380mg/L、L-天冬氨酸300~445mg/L;
所述维生素的组成为:肌醇65~120mg/L、氯化胆碱160~270mg/L、尼克酰胺2.5~8.0mg/L、核黄素0.2~0.4mg/L、D-泛酸钙5.0~8.0mg/L、盐酸吡哆辛3.0~5.0mg/L、维生素B12 1.5~4.3mg/L、抗坏血酸25~90mg/L、盐酸硫胺3.0~8.5mg/L、生物素0.05~0.09mg/L、叶酸2.5~4.5mg/L;
所述无机盐的组成为:氯化钠3500~5000mg/L、氯化钾400~850mg/L、无水磷酸氢二钠200~300mg/L、硫酸镁120~240mg/L、碳酸氢钠2000~3300mg/L、无水氯化钙100~200mg/L;
所述微量元素的组成为:七水硫酸亚铁2~4mg/L、五水硫酸铜0.02~0.1mg/L、七水硫酸锌0.1~0.35mg/L、六水氯化铝0.0005~0.001mg/L、硝酸银0.02~0.075mg/L、溴化钾0.015~0.06mg/L、六水氯化钴0.0003~0.00085mg/L、六水氯化铬0.000002~0.000008mg/L、乙酸钡0.0002~0.00075mg/L、硫酸锰一水合物0.0000025~0.0000085mg/L、四水钼酸铵0.00002~0.00007mg/L、偏钒酸铵0.00085~0.005mg/L、偏硅酸钠0.015~0.04mg/L、亚硒酸钠0.005~0.015mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖4000~6500mg/L、丙酮酸钠150~450mg/L、脱氧-D-核糖2.5~4.0mg/L、腺嘌呤2.0~4.5mg/L、胸腺嘧啶0.5~1.5mg/L、脱氧尿苷25~85mg/L、1,4-丁二胺双盐酸盐0.3~2.0mg/L、牛磺酸90~130mg/L、对氨基苯甲酸0.0015~0.003mg/L、还原型谷胱甘肽0.007~0.02mg/L、乙醇胺盐酸盐0.5~2.0mg/L、硫辛酸1.5~2.5mg/L、金精三羧酸25.3~63.4mg/L、花生四烯酸0.01~0.02mg/L、胆固醇0.5~2.0mg/L、DL-α-生育酚醋酸酯0.75~1.25mg/L、亚油酸0.025~0.085mg/L、亚麻酸0.025~0.085mg/L、硬脂酸0.025~0.08mg/L、酵母浸粉1000~4000mg/L、吐温80 2~6mg/L、硫酸葡聚糖20~50mg/L、Pluronic F-68 900~1200mg/L。
进一步的,所述支持CHO细胞高密度培养的无血清培养基中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物255.5mg/L、L-色氨酸79.5mg/L、L-赖氨酸盐酸盐456mg/L、L-组氨酸盐酸盐一水合物205mg/L、甘氨酸18.6mg/L、L-异亮氨酸380.8mg/L、L-丙氨酸55.3mg/L、L-精氨酸盐酸盐490.4mg/L、L-天冬酰胺一水合物515mg/L、L-亮氨酸109.5mg/L、L-甲硫氨酸163.1mg/L、L-苯丙氨酸197.4mg/L、L-脯氨酸268.9mg/L、L-丝氨酸112mg/L、L-苏氨酸248mg/L、L-缬氨酸360.5mg/L、L-谷氨酸279.7mg/L、L-胱氨酸34.8mg/L、L-半胱氨酸312mg/L、L-天冬氨酸419mg/L;
所述维生素的组成为:肌醇84mg/L、氯化胆碱216mg/L、尼克酰胺6.4mg/L、核黄素0.35mg/L、D-泛酸钙7.1mg/L、盐酸吡哆辛4.7mg/L、维生素B12 2.25mg/L、抗坏血酸75mg/L、盐酸硫胺6.55mg/L、生物素0.08mg/L、叶酸3.25mg/L;
所述无机盐的组成为:氯化钠4000mg/L、氯化钾521.8mg/L、无水磷酸氢二钠240.33mg/L、硫酸镁180mg/L、碳酸氢钠2600mg/L、无水氯化钙179.5mg/L;
所述微量元素的组成为:七水硫酸亚铁3.5mg/L、五水硫酸铜0.035mg/L、七水硫酸锌0.24mg/L、六水氯化铝0.00065mg/L、硝酸银0.035mg/L、溴化钾0.025mg/L、六水氯化钴0.00065mg/L、六水氯化铬0.0000039mg/L、乙酸钡0.00035mg/L、硫酸锰一水合物0.000006mg/L、四水钼酸铵0.000045mg/L、偏钒酸铵0.0025mg/L、偏硅酸钠0.022mg/L、亚硒酸钠0.0077mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖5000mg/L、丙酮酸钠320mg/L、脱氧-D-核糖3.5mg/L、腺嘌呤3.5mg/L、胸腺嘧啶1.12mg/L、脱氧尿苷37.5mg/L、1,4-丁二胺双盐酸盐1.55mg/L、牛磺酸95mg/L、对氨基苯甲酸0.0025mg/L、还原型谷胱甘肽0.015mg/L、乙醇胺盐酸盐1.25mg/L、硫辛酸2.0mg/L、金精三羧酸42.2mg/L、花生四烯酸0.015mg/L、胆固醇1.75mg/L、DL-α-生育酚醋酸酯0.85mg/L、亚油酸0.045mg/L、亚麻酸0.045mg/L、硬脂酸0.065mg/L、酵母浸粉1500mg/L、吐温80 3.5mg/L、硫酸葡聚糖25mg/L、Pluronic F-681000mg/L。
进一步的,所述支持CHO细胞高密度培养的无血清培养基中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物200mg/L、L-色氨酸70mg/L、L-赖氨酸盐酸盐410mg/L、L-组氨酸盐酸盐一水合物180mg/L、甘氨酸10mg/L、L-异亮氨酸300mg/L、L-丙氨酸30mg/L、L-精氨酸盐酸盐410mg/L、L-天冬酰胺一水合物445mg/L、L-亮氨酸85mg/L、L-甲硫氨酸140mg/L、L-苯丙氨酸140mg/L、L-脯氨酸180mg/L、L-丝氨酸80mg/L、L-苏氨酸180mg/L、L-缬氨酸310mg/L、L-谷氨酸240mg/L、L-胱氨酸20mg/L、L-半胱氨酸300mg/L、L-天冬氨酸305mg/L;
所述维生素的组成为:肌醇70mg/L、氯化胆碱180mg/L、尼克酰胺4mg/L、核黄素0.2mg/L、D-泛酸钙5.0mg/L、盐酸吡哆辛3.5mg/L、维生素B122.0mg/L、抗坏血酸30mg/L、盐酸硫胺4.0mg/L、生物素0.06mg/L、叶酸3.0mg/L;
所述无机盐的组成为:氯化钠3800mg/L、氯化钾450mg/L、无水磷酸氢二钠200mg/L、硫酸镁150mg/L、碳酸氢钠2200mg/L、无水氯化钙115mg/L;
所述微量元素的组成为:七水硫酸亚铁2mg/L、五水硫酸铜0.025mg/L、七水硫酸锌0.15mg/L、六水氯化铝0.0005mg/L、硝酸银0.02mg/L、溴化钾0.015mg/L、六水氯化钴0.0004mg/L、六水氯化铬0.000003mg/L、乙酸钡0.0002mg/L、硫酸锰一水合物0.000004mg/L、四水钼酸铵0.00003mg/L、偏钒酸铵0.001mg/L、偏硅酸钠0.015mg/L、亚硒酸钠0.006mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖4000mg/L、丙酮酸钠200mg/L、脱氧-D-核糖3.0mg/L、腺嘌呤3.0mg/L、胸腺嘧啶0.75mg/L、脱氧尿苷25mg/L、1,4-丁二胺双盐酸盐0.5mg/L、牛磺酸90mg/L、对氨基苯甲酸0.0015mg/L、还原型谷胱甘肽0.01mg/L、乙醇胺盐酸盐0.85mg/L、硫辛酸1.5mg/L、金精三羧酸33.8mg/L、花生四烯酸0.01mg/L、胆固醇1.0mg/L、DL-α-生育酚醋酸酯0.75mg/L、亚油酸0.03mg/L、亚麻酸0.03mg/L、硬脂酸0.04mg/L、酵母浸粉1000mg/L、吐温80 2.5mg/L、硫酸葡聚糖20mg/L、Pluronic F-68 900mg/L。
进一步的,所述支持CHO细胞高密度培养的无血清培养基中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物276mg/L、L-色氨酸100mg/L、L-赖氨酸盐酸盐525mg/L、L-组氨酸盐酸盐一水合物255mg/L、甘氨酸30mg/L、L-异亮氨酸450mg/L、L-丙氨酸75mg/L、L-精氨酸盐酸盐575mg/L、L-天冬酰胺一水合物685mg/L、L-亮氨酸160mg/L、L-甲硫氨酸215mg/L、L-苯丙氨酸300mg/L、L-脯氨酸300mg/L、L-丝氨酸200mg/L、L-苏氨酸400mg/L、L-缬氨酸400mg/L、L-谷氨酸310mg/L、L-胱氨酸60mg/L、L-半胱氨酸360mg/L、L-天冬氨酸430mg/L;
所述维生素的组成为:肌醇105mg/L、氯化胆碱245mg/L、尼克酰胺7.5mg/L、核黄素0.4mg/L、D-泛酸钙8.0mg/L、盐酸吡哆辛5.0mg/L、维生素B12 4.0mg/L、抗坏血酸90mg/L、盐酸硫胺8.0mg/L、生物素0.09mg/L、叶酸4.0mg/L;
所述无机盐的组成为:氯化钠4500mg/L、氯化钾760mg/L、无水磷酸氢二钠285mg/L、硫酸镁220mg/L、碳酸氢钠3000mg/L、无水氯化钙200mg/L;
所述微量元素的组成为:七水硫酸亚铁4mg/L、五水硫酸铜0.085mg/L、七水硫酸锌0.3mg/L、六水氯化铝0.0008mg/L、硝酸银0.05mg/L、溴化钾0.05mg/L、六水氯化钴0.0008mg/L、六水氯化铬0.0000065mg/L、乙酸钡0.0006mg/L、硫酸锰一水合物0.0000075mg/L、四水钼酸铵0.00006mg/L、偏钒酸铵0.004mg/L、偏硅酸钠0.035mg/L、亚硒酸钠0.01mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖6000mg/L、丙酮酸钠400mg/L、脱氧-D-核糖4.0mg/L、腺嘌呤4.0mg/L、胸腺嘧啶1.25mg/L、脱氧尿苷70mg/L、1,4-丁二胺双盐酸盐2.0mg/L、牛磺酸110mg/L、对氨基苯甲酸0.003mg/L、还原型谷胱甘肽0.02mg/L、乙醇胺盐酸盐1.7mg/L、硫辛酸2.5mg/L、金精三羧酸63.4mg/L、花生四烯酸0.02mg/L、胆固醇2.0mg/L、DL-α-生育酚醋酸酯1.0mg/L、亚油酸0.065mg/L、亚麻酸0.065mg/L、硬脂酸0.07mg/L、酵母浸粉3000mg/L、吐温80 5mg/L、硫酸葡聚糖40mg/L、Pluronic F-68 1100mg/L。
为达到第二个目的,本发明提供了上述支持CHO细胞高密度培养的无血清培养基的制备方法,该制备方法包括:取配方量的组分溶解混合,调节pH至7.0~7.4,调节渗透压至280~320mOsm/kg,过滤除菌即得。
本发明还要求保护上述支持CHO细胞高密度培养的无血清培养基在培养CHO细胞中的应用。
进一步,培养CHO细胞的条件为:37℃,5%CO2,85r/min恒温摇床培养。
本发明的有益效果如下:
本发明的无血清培养基培养,能够支持CHO细胞在悬浮状态下快速增殖,缩短了细胞增殖所需时间,细胞高密度维持时间长,可实现CHO悬浮细胞的高密度培养,批次培养最大细胞密度可达1.4×107个/ml,活率维持98%以上,大大提高了生产效率。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1为倒置显微镜下观察的CHO-K1细胞在实施例3中制备的无血清培养基中悬浮生长的细胞图;
图2为CHO-K1细胞在实施例1、2、3、4、5和对比例1、2和3中制备的无血清培养基中悬浮生长的细胞密度及细胞活率变化对比图;
图3为CHO-K1细胞在实施例3中制备的无血清培养基中连续传代培养图,细胞生长稳定;
图4为CHO-K1细胞在实施例3中制备的无血清培养基及市售1、2培养基中培养对比图。
具体实施方式
为了更清楚地说明本发明,下面结合优选实施例和附图对本发明做进一步的说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
本发明的所有原料均为细胞培养级或药用级原料,并按相关要求储存。下述实施例中的试验方法,如无特殊说明,均为常规方法。
实施例1:制备CHO无血清培养基
采用万分之一天平按以下成分及浓度进行精确称量:
所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物100mg/L、L-色氨酸50mg/L、L-赖氨酸盐酸盐350mg/L、L-组氨酸盐酸盐一水合物100mg/L、甘氨酸5mg/L、L-异亮氨酸260mg/L、L-丙氨酸10mg/L、L-精氨酸盐酸盐320mg/L、L-天冬酰胺一水合物360mg/L、L-亮氨酸45mg/L、L-甲硫氨酸100mg/L、L-苯丙氨酸90mg/L、L-脯氨酸100mg/L、L-丝氨酸50mg/L、L-苏氨酸165mg/L、L-缬氨酸280mg/L、L-谷氨酸200mg/L、L-胱氨酸10mg/L、L-半胱氨酸250mg/L、L-天冬氨酸265mg/L;
所述维生素的组成为:肌醇50mg/L、氯化胆碱100mg/L、尼克酰胺1.6mg/L、核黄素0.1mg/L、D-泛酸钙3.5mg/L、盐酸吡哆辛2.0mg/L、维生素B12 0.5mg/L、抗坏血酸10mg/L、盐酸硫胺2.0mg/L、生物素0.01mg/L、叶酸0.5mg/L;
所述无机盐的组成为:氯化钠3000mg/L、氯化钾300mg/L、无水磷酸氢二钠140mg/L、硫酸镁100mg/L、碳酸氢钠1500mg/L、无水氯化钙75mg/L;
所述微量元素的组成为:七水硫酸亚铁2mg/L、五水硫酸铜0.01mg/L、七水硫酸锌0.05mg/L、六水氯化铝0.0001mg/L、硝酸银0.01mg/L、溴化钾0.01mg/L、六水氯化钴0.0001mg/L、六水氯化铬0.000001mg/L、乙酸钡0.0001mg/L、硫酸锰一水合物0.000001mg/L、四水钼酸铵0.00001mg/L、偏钒酸铵0.0005mg/L、偏硅酸钠0.005mg/L、亚硒酸钠0.002mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖3000mg/L、丙酮酸钠105mg/L、脱氧-D-核糖1.0mg/L、腺嘌呤1.0mg/L、胸腺嘧啶0.1mg/L、脱氧尿苷20mg/L、1,4-丁二胺双盐酸盐0.15mg/L、牛磺酸85mg/L、对氨基苯甲酸0.001mg/L、还原型谷胱甘肽0.005mg/L、乙醇胺盐酸盐0.2mg/L、硫辛酸1.0mg/L、金精三羧酸8.44mg/L、花生四烯酸0.005mg/L、胆固醇0.1mg/L、DL-α-生育酚醋酸酯0.5mg/L、亚油酸0.01mg/L、亚麻酸0.01mg/L、硬脂酸0.01mg/L、酵母浸粉500mg/L、吐温80 1mg/L、硫酸葡聚糖15mg/L、Pluronic F-68 800mg/L。
将上述成分按配制1L培养基所需量,逐一称量加入到900ml注射水中进行溶解,若某物质在水中难溶,则按其溶解特性溶解后,再加入到注射水中。全部称量添加完毕后,在磁力搅拌器上进行搅拌半小时;之后调节pH至7.0~7.4,定容至1L,测定渗透压为280~320mOsm/kg,0.22μm滤芯过滤除菌后,4℃避光保存备用。
实施例2:制备CHO无血清培养基
采用万分之一天平按以下成分及浓度进行精确称量:
所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物460mg/L、L-色氨酸150mg/L、L-赖氨酸盐酸盐650mg/L、L-组氨酸盐酸盐一水合物300mg/L、甘氨酸50mg/L、L-异亮氨酸550mg/L、L-丙氨酸100mg/L、L-精氨酸盐酸盐760mg/L、L-天冬酰胺一水合物820mg/L、L-亮氨酸225mg/L、L-甲硫氨酸300mg/L、L-苯丙氨酸400mg/L、L-脯氨酸500mg/L、L-丝氨酸250mg/L、L-苏氨酸500mg/L、L-缬氨酸550mg/L、L-谷氨酸400mg/L、L-胱氨酸85mg/L、L-半胱氨酸450mg/L、L-天冬氨酸515mg/L;
所述维生素的组成为:肌醇170mg/L、氯化胆碱300mg/L、尼克酰胺8.9mg/L、核黄素0.5mg/L、D-泛酸钙8.8mg/L、盐酸吡哆辛5.15mg/L、维生素B12 5.5mg/L、抗坏血酸100mg/L、盐酸硫胺10mg/L、生物素0.1mg/L、叶酸5.5mg/L;
所述无机盐的组成为:氯化钠5500mg/L、氯化钾1000mg/L、无水磷酸氢二钠320mg/L、硫酸镁260mg/L、碳酸氢钠3700mg/L、无水氯化钙235mg/L;
所述微量元素的组成为:七水硫酸亚铁6mg/L、五水硫酸铜0.15mg/L、七水硫酸锌0.5mg/L、六水氯化铝0.002mg/L、硝酸银0.1mg/L、溴化钾0.1mg/L、六水氯化钴0.001mg/L、六水氯化铬0.00001mg/L、乙酸钡0.001mg/L、硫酸锰一水合物0.00001mg/L、四水钼酸铵0.0001mg/L、偏钒酸铵0.01mg/L、偏硅酸钠0.05mg/L、亚硒酸钠0.02mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖8000mg/L、丙酮酸钠550mg/L、脱氧-D-核糖5.0mg/L、腺嘌呤5.0mg/L、胸腺嘧啶2.0mg/L、脱氧尿苷125mg/L、1,4-丁二胺双盐酸盐3.0mg/L、牛磺酸165mg/L、对氨基苯甲酸0.0035mg/L、还原型谷胱甘肽0.025mg/L、乙醇胺盐酸盐2.5mg/L、硫辛酸2.65mg/L、金精三羧酸84.4mg/L、花生四烯酸0.025mg/L、胆固醇2.5mg/L、DL-α-生育酚醋酸酯1.5mg/L、亚油酸0.1mg/L、亚麻酸0.1mg/L、硬脂酸0.1mg/L、酵母浸粉5000mg/L、吐温80 10mg/L、硫酸葡聚糖60mg/L、Pluronic F-68 1500mg/L。
将上述成分按配制1L培养基所需量,逐一称量加入到900ml注射水中进行溶解,若某物质在水中难溶,则按其溶解特性溶解后,再加入到注射水中。全部称量添加完毕后,在磁力搅拌器上进行搅拌半小时;之后调节pH至7.0~7.4,定容至1L,测定渗透压为280~320mOsm/kg,0.22μm滤芯过滤除菌后,4℃避光保存备用。
实施例3:制备CHO无血清培养基
采用万分之一天平按以下成分及浓度进行精确称量:
所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物255.5mg/L、L-色氨酸79.5mg/L、L-赖氨酸盐酸盐456mg/L、L-组氨酸盐酸盐一水合物205mg/L、甘氨酸18.6mg/L、L-异亮氨酸380.8mg/L、L-丙氨酸55.3mg/L、L-精氨酸盐酸盐490.4mg/L、L-天冬酰胺一水合物515mg/L、L-亮氨酸109.5mg/L、L-甲硫氨酸163.1mg/L、L-苯丙氨酸197.4mg/L、L-脯氨酸268.9mg/L、L-丝氨酸112mg/L、L-苏氨酸248mg/L、L-缬氨酸360.5mg/L、L-谷氨酸279.7mg/L、L-胱氨酸34.8mg/L、L-半胱氨酸312mg/L、L-天冬氨酸419mg/L;
所述维生素的组成为:肌醇84mg/L、氯化胆碱216mg/L、尼克酰胺6.4mg/L、核黄素0.35mg/L、D-泛酸钙7.1mg/L、盐酸吡哆辛4.7mg/L、维生素B12 2.25mg/L、抗坏血酸75mg/L、盐酸硫胺6.55mg/L、生物素0.08mg/L、叶酸3.25mg/L;
所述无机盐的组成为:氯化钠4000mg/L、氯化钾521.8mg/L、无水磷酸氢二钠240.33mg/L、硫酸镁180mg/L、碳酸氢钠2600mg/L、无水氯化钙179.5mg/L;
所述微量元素的组成为:七水硫酸亚铁3.5mg/L、五水硫酸铜0.035mg/L、七水硫酸锌0.24mg/L、六水氯化铝0.00065mg/L、硝酸银0.035mg/L、溴化钾0.025mg/L、六水氯化钴0.00065mg/L、六水氯化铬0.0000039mg/L、乙酸钡0.00035mg/L、硫酸锰一水合物0.000006mg/L、四水钼酸铵0.000045mg/L、偏钒酸铵0.0025mg/L、偏硅酸钠0.022mg/L、亚硒酸钠0.0077mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖5000mg/L、丙酮酸钠320mg/L、脱氧-D-核糖3.5mg/L、腺嘌呤3.5mg/L、胸腺嘧啶1.12mg/L、脱氧尿苷37.5mg/L、1,4-丁二胺双盐酸盐1.55mg/L、牛磺酸95mg/L、对氨基苯甲酸0.0025mg/L、还原型谷胱甘肽0.015mg/L、乙醇胺盐酸盐1.25mg/L、硫辛酸2.0mg/L、金精三羧酸42.2mg/L、花生四烯酸0.015mg/L、胆固醇1.75mg/L、DL-α-生育酚醋酸酯0.85mg/L、亚油酸0.045mg/L、亚麻酸0.045mg/L、硬脂酸0.065mg/L、酵母浸粉1500mg/L、吐温80 3.5mg/L、硫酸葡聚糖25mg/L、Pluronic F-681000mg/L。
将上述成分按配制1L培养基所需量,逐一称量加入到900ml注射水中进行溶解,若某物质在水中难溶,则按其溶解特性溶解后,再加入到注射水中。全部称量添加完毕后,在磁力搅拌器上进行搅拌半小时;之后调节pH至7.0~7.4,定容至1L,测定渗透压为280~320mOsm/kg,0.22μm滤芯过滤除菌后,4℃避光保存备用。
实施例4制备CHO无血清培养基
采用万分之一天平按以下成分及浓度进行精确称量:
所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物200mg/L、L-色氨酸70mg/L、L-赖氨酸盐酸盐410mg/L、L-组氨酸盐酸盐一水合物180mg/L、甘氨酸10mg/L、L-异亮氨酸300mg/L、L-丙氨酸30mg/L、L-精氨酸盐酸盐410mg/L、L-天冬酰胺一水合物445mg/L、L-亮氨酸85mg/L、L-甲硫氨酸140mg/L、L-苯丙氨酸140mg/L、L-脯氨酸180mg/L、L-丝氨酸80mg/L、L-苏氨酸180mg/L、L-缬氨酸310mg/L、L-谷氨酸240mg/L、L-胱氨酸20mg/L、L-半胱氨酸300mg/L、L-天冬氨酸305mg/L;
所述维生素的组成为:肌醇70mg/L、氯化胆碱180mg/L、尼克酰胺4mg/L、核黄素0.2mg/L、D-泛酸钙5.0mg/L、盐酸吡哆辛3.5mg/L、维生素B122.0mg/L、抗坏血酸30mg/L、盐酸硫胺4.0mg/L、生物素0.06mg/L、叶酸3.0mg/L;
所述无机盐的组成为:氯化钠3800mg/L、氯化钾450mg/L、无水磷酸氢二钠200mg/L、硫酸镁150mg/L、碳酸氢钠2200mg/L、无水氯化钙115mg/L;
所述微量元素的组成为:七水硫酸亚铁2mg/L、五水硫酸铜0.025mg/L、七水硫酸锌0.15mg/L、六水氯化铝0.0005mg/L、硝酸银0.02mg/L、溴化钾0.015mg/L、六水氯化钴0.0004mg/L、六水氯化铬0.000003mg/L、乙酸钡0.0002mg/L、硫酸锰一水合物0.000004mg/L、四水钼酸铵0.00003mg/L、偏钒酸铵0.001mg/L、偏硅酸钠0.015mg/L、亚硒酸钠0.006mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖4000mg/L、丙酮酸钠200mg/L、脱氧-D-核糖3.0mg/L、腺嘌呤3.0mg/L、胸腺嘧啶0.75mg/L、脱氧尿苷25mg/L、1,4-丁二胺双盐酸盐0.5mg/L、牛磺酸90mg/L、对氨基苯甲酸0.0015mg/L、还原型谷胱甘肽0.01mg/L、乙醇胺盐酸盐0.85mg/L、硫辛酸1.5mg/L、金精三羧酸33.8mg/L、花生四烯酸0.01mg/L、胆固醇1.0mg/L、DL-α-生育酚醋酸酯0.75mg/L、亚油酸0.03mg/L、亚麻酸0.03mg/L、硬脂酸0.04mg/L、酵母浸粉1000mg/L、吐温80 2.5mg/L、硫酸葡聚糖20mg/L、Pluronic F-68900mg/L。
将上述成分按配制1L培养基所需量,逐一称量加入到900ml注射水中进行溶解,若某物质在水中难溶,则按其溶解特性溶解后,再加入到注射水中。全部称量添加完毕后,在磁力搅拌器上进行搅拌半小时;之后调节pH至7.0~7.4,定容至1L,测定渗透压为280~320mOsm/kg,0.22μm滤芯过滤除菌后,4℃避光保存备用。
实施例5制备CHO无血清培养基
采用万分之一天平按以下成分及浓度进行精确称量:
所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中:
所述氨基酸的组成为:L-酪氨酸二钠二水合物276mg/L、L-色氨酸100mg/L、L-赖氨酸盐酸盐525mg/L、L-组氨酸盐酸盐一水合物255mg/L、甘氨酸30mg/L、L-异亮氨酸450mg/L、L-丙氨酸75mg/L、L-精氨酸盐酸盐575mg/L、L-天冬酰胺一水合物685mg/L、L-亮氨酸160mg/L、L-甲硫氨酸215mg/L、L-苯丙氨酸300mg/L、L-脯氨酸300mg/L、L-丝氨酸200mg/L、L-苏氨酸400mg/L、L-缬氨酸400mg/L、L-谷氨酸310mg/L、L-胱氨酸60mg/L、L-半胱氨酸360mg/L、L-天冬氨酸430mg/L;
所述维生素的组成为:肌醇105mg/L、氯化胆碱245mg/L、尼克酰胺7.5mg/L、核黄素0.4mg/L、D-泛酸钙8.0mg/L、盐酸吡哆辛5.0mg/L、维生素B12 4.0mg/L、抗坏血酸90mg/L、盐酸硫胺8.0mg/L、生物素0.09mg/L、叶酸4.0mg/L;
所述无机盐的组成为:氯化钠4500mg/L、氯化钾760mg/L、无水磷酸氢二钠285mg/L、硫酸镁220mg/L、碳酸氢钠3000mg/L、无水氯化钙200mg/L;
所述微量元素的组成为:七水硫酸亚铁4mg/L、五水硫酸铜0.085mg/L、七水硫酸锌0.3mg/L、六水氯化铝0.0008mg/L、硝酸银0.05mg/L、溴化钾0.05mg/L、六水氯化钴0.0008mg/L、六水氯化铬0.0000065mg/L、乙酸钡0.0006mg/L、硫酸锰一水合物0.0000075mg/L、四水钼酸铵0.00006mg/L、偏钒酸铵0.004mg/L、偏硅酸钠0.035mg/L、亚硒酸钠0.01mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖6000mg/L、丙酮酸钠400mg/L、脱氧-D-核糖4.0mg/L、腺嘌呤4.0mg/L、胸腺嘧啶1.25mg/L、脱氧尿苷70mg/L、1,4-丁二胺双盐酸盐2.0mg/L、牛磺酸110mg/L、对氨基苯甲酸0.003mg/L、还原型谷胱甘肽0.02mg/L、乙醇胺盐酸盐1.7mg/L、硫辛酸2.5mg/L、金精三羧酸63.4mg/L、花生四烯酸0.02mg/L、胆固醇2.0mg/L、DL-α-生育酚醋酸酯1.0mg/L、亚油酸0.065mg/L、亚麻酸0.065mg/L、硬脂酸0.07mg/L、酵母浸粉3000mg/L、吐温80 5mg/L、硫酸葡聚糖40mg/L、Pluronic F-68 1100mg/L。
将上述成分按配制1L培养基所需量,逐一称量加入到900ml注射水中进行溶解,若某物质在水中难溶,则按其溶解特性溶解后,再加入到注射水中。全部称量添加完毕后,在磁力搅拌器上进行搅拌半小时;之后调节pH至7.0~7.4,定容至1L,测定渗透压为280~320mOsm/kg,0.22μm滤芯过滤除菌后,4℃避光保存备用。
对比例1:制备CHO无血清培养基
本实施例是将实施例3配方中脱氧尿苷去除,其余成分及实施方式均不变。
对比例2:制备CHO无血清培养基
本实施例是将实施例3配方中金精三羧酸(ATA)去除,其余成分及实施方式均不变。
对比例3:制备CHO无血清培养基
本实施例是将实施例3配方中金精三羧酸(ATA)去除,添加8mg/L胰岛素、10mg/L转铁蛋白,其余成分及实施方式均不变。
试验例
将在市售培养基中稳定培养的CHO-K1悬浮细胞以4.0×105个/ml的密度接种入实施例1、2、3、4、5及对比例1、2、3的培养基中进行培养。采用125ml透气盖摇瓶,加液量30ml,培养条件为37℃,5%CO2,85r/min恒温摇床培养。每天取样,倒置显微镜下观察细胞状态、拍照,并采用AO/PI染色进行细胞数量和细胞活率测定。另外,CHO-K1细胞在实施例3培养基中连续传代培养,在培养第3天的对数生长期,以4.0×105个/ml的密度接种传代,测定细胞在培养基中的生长稳定性。实验结果见附图。
由培养结果可以看出,CHO-K1细胞在本发明实施例1、2、3、4、5的无血清培养基中生长迅速,细胞分散良好,圆润透亮,无细胞团,见图1。
从图2可以看出,培养4天,细胞密度达到1.4×107个/ml以上,细胞活率98%以上,高密度维持1~2天后细胞逐渐衰退。与对比例1对比,添加了脱氧尿苷的培养基中细胞生长更快,最大细胞密度更高;可以得出:脱氧尿苷对细胞生长具有显著的促进作用,可以提高细胞生长速度及最大细胞密度;与对比例2和对比例3对比,在不含胰岛素和转铁蛋白且没有ATA的培养基中,细胞无法正常生长,添加ATA或添加胰岛素、转铁蛋白后,细胞生长恢复正常,且细胞在含有ATA的培养基中最大细胞密度稍大于含胰岛素、转铁蛋白的培养基,因此可以得出:本发明采用的ATA单独添加使用即可同时替代胰岛素和转铁蛋白,大大降低了培养基的成本,并简化了配方组成。各实施例的无血清培养基培养CHO效果与含胰岛素和转铁蛋白的培养基效果相当,甚至优于其效果。同时CHO细胞在本发明的培养基中生长迅速,可在4天达到最大细胞密度,应用于工业生产可以缩短生产周期,极大地降低生产成本。
从图3可以看出,CHO-K1细胞在本发明实施例的无血清培养基中连续传代培养,各代次之间细胞的生长速度表现出良好的一致性,说明本发明的培养基培养细胞性能稳定。
从图4可以看出,与市售1、市售2无血清培养基相比,本发明实施例的无血清培养基培养CHO-K1细胞,生长速度明显更快,最大细胞密度明显更高,达到最大细胞密度所需时间更短,并且在高细胞密度下维持细胞活力时间更长,说明本发明的无血清培养基综合性能显著占优,可以极大程度提高生产效率。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化或变动,这里无法对所有的实施方式予以穷举,凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (8)
1.一种支持CHO细胞高密度培养的无血清培养基,其特征在于,所述无血清培养基由氨基酸、维生素、无机盐、微量元素、碳水化合物及其他成分组成,其中,
所述氨基酸的组成为:L-酪氨酸二钠二水合物100~460mg/L、L-色氨酸50~150mg/L、L-赖氨酸盐酸盐350~650mg/L、L-组氨酸盐酸盐一水合物100~300mg/L、甘氨酸5~50mg/L、L-异亮氨酸260~550mg/L、L-丙氨酸10~100mg/L、L-精氨酸盐酸盐320~760mg/L、L-天冬酰胺一水合物360~820mg/L、L-亮氨酸45~225mg/L、L-甲硫氨酸100~300mg/L、L-苯丙氨酸90~400mg/L、L-脯氨酸100~500mg/L、L-丝氨酸50~250mg/L、L-苏氨酸165~500mg/L、L-缬氨酸280~550mg/L、L-谷氨酸200~400mg/L、L-胱氨酸10~85mg/L、L-半胱氨酸250~450mg/L、L-天冬氨酸265~515mg/L;
所述维生素的组成为:肌醇50~170mg/L、氯化胆碱100~300mg/L、尼克酰胺1.6~8.9mg/L、核黄素0.1~0.5mg/L、D-泛酸钙3.5~8.8mg/L、盐酸吡哆辛2.0~5.15mg/L、维生素B12 0.5~5.5mg/L、抗坏血酸10~100mg/L、盐酸硫胺2~10mg/L、生物素0.01~0.1mg/L、叶酸0.5~5.5mg/L;
所述无机盐的组成为:氯化钠3000~5500mg/L、氯化钾300~1000mg/L、无水磷酸氢二钠140~320mg/L、硫酸镁100~260mg/L、碳酸氢钠1500~3700mg/L、无水氯化钙75~235mg/L;
所述微量元素的组成为:七水硫酸亚铁2~6mg/L、五水硫酸铜0.01~0.15mg/L、七水硫酸锌0.05~0.5mg/L、六水氯化铝0.0001~0.002mg/L、硝酸银0.01~0.1mg/L、溴化钾0.01~0.1mg/L、六水氯化钴0.0001~0.001mg/L、六水氯化铬0.000001~0.00001mg/L、乙酸钡0.0001~0.001mg/L、硫酸锰一水合物0.000001~0.00001mg/L、四水钼酸铵0.00001~0.0001mg/L、偏钒酸铵0.0005~0.01mg/L、偏硅酸钠0.005~0.05mg/L、亚硒酸钠0.002~0.02mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖3000~8000mg/L、丙酮酸钠105~550mg/L、脱氧-D-核糖1.0~5.0mg/L、腺嘌呤1.0~5.0mg/L、胸腺嘧啶0.1~2.0mg/L、脱氧尿苷20~125mg/L、1,4-丁二胺双盐酸盐0.15~3mg/L、牛磺酸85~165mg/L、对氨基苯甲酸0.001~0.0035mg/L、还原型谷胱甘肽0.005~0.025mg/L、乙醇胺盐酸盐0.2~2.5mg/L、硫辛酸1.0~2.65mg/L、金精三羧酸8.44~84.4mg/L、花生四烯酸0.005~0.025mg/L、胆固醇0.1~2.5mg/L、DL-α-生育酚醋酸酯0.5~1.5mg/L、亚油酸0.01~0.1mg/L、亚麻酸0.01~0.1mg/L、硬脂酸0.01~0.1mg/L、酵母浸粉500~5000mg/L、吐温80 1~10mg/L、硫酸葡聚糖15~60mg/L、Pluronic F-68 800~1500mg/L。
2.根据权利要求1所述的无血清培养基,其特征在于,
所述氨基酸的组成为:L-酪氨酸二钠二水合物150~300mg/L、L-色氨酸65~100mg/L、L-赖氨酸盐酸盐400~550mg/L、L-组氨酸盐酸盐一水合物150~265mg/L、甘氨酸8~40mg/L、L-异亮氨酸300~500mg/L、L-丙氨酸25~85mg/L、L-精氨酸盐酸盐400~600mg/L、L-天冬酰胺一水合物430~695mg/L、L-亮氨酸80~170mg/L、L-甲硫氨酸135~230mg/L、L-苯丙氨酸140~320mg/L、L-脯氨酸176~360mg/L、L-丝氨酸75~200mg/L、L-苏氨酸180~420mg/L、L-缬氨酸310~415mg/L、L-谷氨酸238~340mg/L、L-胱氨酸20~65mg/L、L-半胱氨酸295~380mg/L、L-天冬氨酸300~445mg/L;
所述维生素的组成为:肌醇65~120mg/L、氯化胆碱160~270mg/L、尼克酰胺2.5~8.0mg/L、核黄素0.2~0.4mg/L、D-泛酸钙5.0~8.0mg/L、盐酸吡哆辛3.0~5.0mg/L、维生素B12 1.5~4.3mg/L、抗坏血酸25~90mg/L、盐酸硫胺3.0~8.5mg/L、生物素0.05~0.09mg/L、叶酸2.5~4.5mg/L;
所述无机盐的组成为:氯化钠3500~5000mg/L、氯化钾400~850mg/L、无水磷酸氢二钠200~300mg/L、硫酸镁120~240mg/L、碳酸氢钠2000~3300mg/L、无水氯化钙100~200mg/L;
所述微量元素的组成为:七水硫酸亚铁2~4mg/L、五水硫酸铜0.02~0.1mg/L、七水硫酸锌0.1~0.35mg/L、六水氯化铝0.0005~0.001mg/L、硝酸银0.02~0.075mg/L、溴化钾0.015~0.06mg/L、六水氯化钴0.0003~0.00085mg/L、六水氯化铬0.000002~0.000008mg/L、乙酸钡0.0001~0.00075mg/L、硫酸锰一水合物0.0000025~0.0000085mg/L、四水钼酸铵0.00002~0.00007mg/L、偏钒酸铵0.00085~0.005mg/L、偏硅酸钠0.015~0.04mg/L、亚硒酸钠0.005~0.015mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖4000~6500mg/L、丙酮酸钠150~450mg/L、脱氧-D-核糖2.5~4.0mg/L、腺嘌呤2.0~4.5mg/L、胸腺嘧啶0.5~1.5mg/L、脱氧尿苷25~85mg/L、1,4-丁二胺双盐酸盐0.3~2.0mg/L、牛磺酸90~130mg/L、对氨基苯甲酸0.0015~0.003mg/L、还原型谷胱甘肽0.007~0.02mg/L、乙醇胺盐酸盐0.5~2.0mg/L、硫辛酸1.5~2.5mg/L、金精三羧酸25.3~63.4mg/L、花生四烯酸0.01~0.02mg/L、胆固醇0.5~2.0mg/L、DL-α-生育酚醋酸酯0.75~1.25mg/L、亚油酸0.025~0.085mg/L、亚麻酸0.025~0.085mg/L、硬脂酸0.025~0.08mg/L、酵母浸粉1000~4000mg/L、吐温80 2~6mg/L、硫酸葡聚糖20~50mg/L、Pluronic F-68 900~1200mg/L。
3.根据权利要求1所述的无血清培养基,其特征在于,
所述氨基酸的组成为:L-酪氨酸二钠二水合物255.5mg/L、L-色氨酸79.5mg/L、L-赖氨酸盐酸盐456mg/L、L-组氨酸盐酸盐一水合物205mg/L、甘氨酸18.6mg/L、L-异亮氨酸380.8mg/L、L-丙氨酸55.3mg/L、L-精氨酸盐酸盐490.4mg/L、L-天冬酰胺一水合物515mg/L、L-亮氨酸109.5mg/L、L-甲硫氨酸163.1mg/L、L-苯丙氨酸197.4mg/L、L-脯氨酸268.9mg/L、L-丝氨酸112mg/L、L-苏氨酸248mg/L、L-缬氨酸360.5mg/L、L-谷氨酸279.7mg/L、L-胱氨酸34.8mg/L、L-半胱氨酸312mg/L、L-天冬氨酸419mg/L;
所述维生素的组成为:肌醇84mg/L、氯化胆碱216mg/L、尼克酰胺6.4mg/L、核黄素0.35mg/L、D-泛酸钙7.1mg/L、盐酸吡哆辛4.7mg/L、维生素B122.25mg/L、抗坏血酸75mg/L、盐酸硫胺6.55mg/L、生物素0.08mg/L、叶酸3.25mg/L;
所述无机盐的组成为:氯化钠4000mg/L、氯化钾521.8mg/L、无水磷酸氢二钠240.33mg/L、硫酸镁180mg/L、碳酸氢钠2600mg/L、无水氯化钙179.5mg/L;
所述微量元素的组成为:七水硫酸亚铁3.5mg/L、五水硫酸铜0.035mg/L、七水硫酸锌0.24mg/L、六水氯化铝0.00065mg/L、硝酸银0.035mg/L、溴化钾0.025mg/L、六水氯化钴0.00065mg/L、六水氯化铬0.0000039mg/L、乙酸钡0.00035mg/L、硫酸锰一水合物0.000006mg/L、四水钼酸铵0.000045mg/L、偏钒酸铵0.0025mg/L、偏硅酸钠0.022mg/L、亚硒酸钠0.0077mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖5000mg/L、丙酮酸钠320mg/L、脱氧-D-核糖3.5mg/L、腺嘌呤3.5mg/L、胸腺嘧啶1.12mg/L、脱氧尿苷37.5mg/L、1,4-丁二胺双盐酸盐1.55mg/L、牛磺酸95mg/L、对氨基苯甲酸0.0025mg/L、还原型谷胱甘肽0.015mg/L、乙醇胺盐酸盐1.25mg/L、硫辛酸2.0mg/L、金精三羧酸42.2mg/L、花生四烯酸0.015mg/L、胆固醇1.75mg/L、DL-α-生育酚醋酸酯0.85mg/L、亚油酸0.045mg/L、亚麻酸0.045mg/L、硬脂酸0.065mg/L、酵母浸粉1500mg/L、吐温80 3.5mg/L、硫酸葡聚糖25mg/L、Pluronic F-681000mg/L。
4.根据权利要求1所述的无血清培养基,其特征在于,
所述氨基酸的组成为:L-酪氨酸二钠二水合物200mg/L、L-色氨酸70mg/L、L-赖氨酸盐酸盐410mg/L、L-组氨酸盐酸盐一水合物180mg/L、甘氨酸10mg/L、L-异亮氨酸300mg/L、L-丙氨酸30mg/L、L-精氨酸盐酸盐410mg/L、L-天冬酰胺一水合物445mg/L、L-亮氨酸85mg/L、L-甲硫氨酸140mg/L、L-苯丙氨酸140mg/L、L-脯氨酸180mg/L、L-丝氨酸80mg/L、L-苏氨酸180mg/L、L-缬氨酸310mg/L、L-谷氨酸240mg/L、L-胱氨酸20mg/L、L-半胱氨酸300mg/L、L-天冬氨酸305mg/L;
所述维生素的组成为:肌醇70mg/L、氯化胆碱180mg/L、尼克酰胺4mg/L、核黄素0.2mg/L、D-泛酸钙5.0mg/L、盐酸吡哆辛3.5mg/L、维生素B12 2.0mg/L、抗坏血酸30mg/L、盐酸硫胺4.0mg/L、生物素0.06mg/L、叶酸3.0mg/L;
所述无机盐的组成为:氯化钠3800mg/L、氯化钾450mg/L、无水磷酸氢二钠200mg/L、硫酸镁150mg/L、碳酸氢钠2200mg/L、无水氯化钙115mg/L;
所述微量元素的组成为:七水硫酸亚铁2mg/L、五水硫酸铜0.025mg/L、七水硫酸锌0.15mg/L、六水氯化铝0.0005mg/L、硝酸银0.02mg/L、溴化钾0.015mg/L、六水氯化钴0.0004mg/L、六水氯化铬0.000003mg/L、乙酸钡0.0002mg/L、硫酸锰一水合物0.000004mg/L、四水钼酸铵0.00003mg/L、偏钒酸铵0.001mg/L、偏硅酸钠0.015mg/L、亚硒酸钠0.006mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖4000mg/L、丙酮酸钠200mg/L、脱氧-D-核糖3.0mg/L、腺嘌呤3.0mg/L、胸腺嘧啶0.75mg/L、脱氧尿苷25mg/L、1,4-丁二胺双盐酸盐0.5mg/L、牛磺酸90mg/L、对氨基苯甲酸0.0015mg/L、还原型谷胱甘肽0.01mg/L、乙醇胺盐酸盐0.85mg/L、硫辛酸1.5mg/L、金精三羧酸33.8mg/L、花生四烯酸0.01mg/L、胆固醇1.0mg/L、DL-α-生育酚醋酸酯0.75mg/L、亚油酸0.03mg/L、亚麻酸0.03mg/L、硬脂酸0.04mg/L、酵母浸粉1000mg/L、吐温80 2.5mg/L、硫酸葡聚糖20mg/L、Pluronic F-68900mg/L。
5.根据权利要求1所述的无血清培养基,其特征在于,
所述氨基酸的组成为:L-酪氨酸二钠二水合物276mg/L、L-色氨酸100mg/L、L-赖氨酸盐酸盐525mg/L、L-组氨酸盐酸盐一水合物255mg/L、甘氨酸30mg/L、L-异亮氨酸450mg/L、L-丙氨酸75mg/L、L-精氨酸盐酸盐575mg/L、L-天冬酰胺一水合物685mg/L、L-亮氨酸160mg/L、L-甲硫氨酸215mg/L、L-苯丙氨酸300mg/L、L-脯氨酸300mg/L、L-丝氨酸200mg/L、L-苏氨酸400mg/L、L-缬氨酸400mg/L、L-谷氨酸310mg/L、L-胱氨酸60mg/L、L-半胱氨酸360mg/L、L-天冬氨酸430mg/L;
所述维生素的组成为:肌醇105mg/L、氯化胆碱245mg/L、尼克酰胺7.5mg/L、核黄素0.4mg/L、D-泛酸钙8.0mg/L、盐酸吡哆辛5.0mg/L、维生素B12 4.0mg/L、抗坏血酸90mg/L、盐酸硫胺8.0mg/L、生物素0.09mg/L、叶酸4.0mg/L;
所述无机盐的组成为:氯化钠4500mg/L、氯化钾760mg/L、无水磷酸氢二钠285mg/L、硫酸镁220mg/L、碳酸氢钠3000mg/L、无水氯化钙200mg/L;
所述微量元素的组成为:七水硫酸亚铁4mg/L、五水硫酸铜0.085mg/L、七水硫酸锌0.3mg/L、六水氯化铝0.0008mg/L、硝酸银0.05mg/L、溴化钾0.05mg/L、六水氯化钴0.0008mg/L、六水氯化铬0.0000065mg/L、乙酸钡0.0006mg/L、硫酸锰一水合物0.0000075mg/L、四水钼酸铵0.00006mg/L、偏钒酸铵0.004mg/L、偏硅酸钠0.035mg/L、亚硒酸钠0.01mg/L;
所述碳水化合物及其他成分的组成为:葡萄糖6000mg/L、丙酮酸钠400mg/L、脱氧-D-核糖4.0mg/L、腺嘌呤4.0mg/L、胸腺嘧啶1.25mg/L、脱氧尿苷70mg/L、1,4-丁二胺双盐酸盐2.0mg/L、牛磺酸110mg/L、对氨基苯甲酸0.003mg/L、还原型谷胱甘肽0.02mg/L、乙醇胺盐酸盐1.7mg/L、硫辛酸2.5mg/L、金精三羧酸63.4mg/L、花生四烯酸0.02mg/L、胆固醇2.0mg/L、DL-α-生育酚醋酸酯1.0mg/L、亚油酸0.065mg/L、亚麻酸0.065mg/L、硬脂酸0.07mg/L、酵母浸粉3000mg/L、吐温80 5mg/L、硫酸葡聚糖40mg/L、Pluronic F-68 1100mg/L。
6.权利要求1-5任一所述的支持CHO细胞高密度培养的无血清培养基的制备方法,其特征在于,该制备方法包括:取配方量的组分溶解混合,调节pH至7.0~7.4,调节渗透压至280~320mOsm/kg,过滤除菌即得。
7.权利要求1-5任一所述的支持CHO细胞高密度培养的无血清培养基在培养CHO细胞中的应用。
8.根据权利要求7所述的应用,其特征在于,培养CHO细胞的条件为:37℃,5%CO2,85r/min恒温摇床培养。
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