CN108913646A - 一种无血清培养基 - Google Patents

一种无血清培养基 Download PDF

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CN108913646A
CN108913646A CN201810865712.8A CN201810865712A CN108913646A CN 108913646 A CN108913646 A CN 108913646A CN 201810865712 A CN201810865712 A CN 201810865712A CN 108913646 A CN108913646 A CN 108913646A
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growth factor
serum free
free medium
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culture medium
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侯野
张乾顺
邵瑞华
黄广达
王璐
赵英杰
韩丹
张志豪
宋春雨
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Zhuhai Dingan Biological Products Co., Ltd.
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Beijing Ding Biotech Co Ltd
Yishengke (shenzhen) Co Ltd
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Abstract

本发明公开了一种无血清培养基，所述培养基包含以下组分：DMEM/F12；以及在DMEM/F12中添加：0.6‑300ng/mL生长因子、15‑120μg/L激素、3‑150mg/mL蛋白质、0.1‑1.7g/L氨基酸及肽、6.51‑13.1mg/L脂质复合物、22‑220μg/L微量元素、0.5‑5.9mg/L维生素。本发明涉及一种细胞培养基，特别是用于培养悬浮细胞的培养基，其用于重组蛋白的表达，本发明培养基，完全不含有血清成份；生产的抗体纯度大幅度提高；该培养基同时具有高扩增速度及高抗体表达能力；该培养基能长时间保持细胞活性，细胞培养时间大大延长，可以大大延长生产时间，提高抗体产量。

Description

一种无血清培养基
技术领域
本发明涉及一种细胞无血清培养基，属于细胞培养技术领域。
背景技术
无血清培养基是指不需要添加血清就可以维持细胞在体外较长时间生长繁殖的合成培养基，可以应用于培养哺乳动物细胞，例如中国仓鼠卵巢细胞(CHO)、犬肾细胞(MDCK)、猴肾细胞(Vero)、乳仓鼠肾细胞(BHK)等。
目前已广泛采用细胞培养来生产多种生物活性制品，如病毒疫苗、单克隆抗体、非抗体免疫调节剂、多肽生长因子、激素、酶、肿瘤特异性抗原等。这些产品可通过正常的或转化的和经基因工程改造的细胞产生。
以往，用于细胞培养的培养基需要添加血清，作为生产生物活性产品的所有哺乳动物细胞系生长和维持所需的通用营养物质。血清含有激素、生长因子、载体蛋白质、粘附和扩散因子、营养成分、微量元素等。培养基通常含有高达约10％的动物血清，例如胎牛血清(FBS)，也称为胎犊血清(FCS)。
虽然广泛采用了血清，但是其有许多缺点，如产物纯化难度加大、来源限制、批次间生物活性差异(导致过程不稳定)、生物污染(如疯牛病)等。因此，研究含已知成分的无血清培养基成为细胞培养领域中一项重要工作。
目前，市售的培养基培养的细胞扩增倍数低、存活时间短、活性低、产量不高、且抗体纯度低，不能满足临床应用的问题。
发明内容
本发明的目的是针对现有技术的不足，开发出一种高效安全稳定的无血清培养基。
为了实现上述要求和目的，本发明所采用的技术方案如下：
一种无血清培养基，所述培养基包含以下组分：
DMEM/F12；
以及在DMEM/F12中添加：
0.6-300ng/mL生长因子、15-120μg/L激素、3-150mg/mL蛋白质、0.1-1.7g/L氨基酸及肽、6.51-13.1mg/L脂质复合物、22-220μg/L微量元素、0.5-5.9mg/L维生素。
本发明中，在DMEM/F12中添加0.6-300ng/mL生长因子表示的是在1mLDMEM/F12中添加0.6-300ng的生长因子，其他添加组分所表达的含义与此相同，即μg/L、g/L、mg/L中的L均是指作为添加基础的DMEM/F12的量。
优选地，所述的生长因子包括如下组分及含量：
成纤维细胞生长因子0.1-50ng/mL、重组人表皮细胞生长因子(EGF)0.1-50ng/mL、神经生长因子0.1-50ng/mL、转化生长因子0.1-50ng/mL、白血病抑制因子0.1-50ng/mL、胰岛素样生长因子-1 0.1-50ng/mL。
优选地，所述的激素包括如下组分及含量：
氢化可的松12-60μg/L、胰岛素2-20μg/L、孕酮0-20μg/L、地塞米松1-20μg/L。
优选地，所述的蛋白质包括如下组分及含量：
血清白蛋白1-50mg/mL、转铁蛋白1-50mg/mL、胎球蛋白1-50mg/mL。
优选地，所述的氨基酸及肽包括如下组分及含量：
谷氨酰胺0-0.1g/L、还原型谷胱甘肽0.1-1.5g/L、非必需氨基酸0-0.1g/L。
优选地，所述脂类复合物包括如下组分及含量：
亚油酸0.5-1mg/L、胆固醇0.01-0.1mg/L、乙醇胺0.5-1mg/L、β-巯基乙醇0.5-1mg/L、吐温-80 5-10mg/L。
优选地，所述微量元素包括如下组分及含量：
氯化锰1-10μg/L、亚锡酸钠1-10μg/L、柠檬酸铁10-100μg/L、硫酸锌10-100μg/L。
优选地，所述维生素包括如下组分及含量：
生物素0-0.2mg/L、叶酸0.1-3mg/L、核黄素0.1-0.3mg/L、维生素B120.1-0.8mg/L、维生素C 0.1-0.8mg/L、维生素E 0.1-0.8mg/L。
本发明中所用到的DMEM/F12培养基可以商业购买得到，所购买的商业化培养基DMEM/F12可包括如下组分及含量：
无水氯化钙116.6mg/L、L-亮氨酸59.05mg/L、亚油酸0.042mg/L、五水硫酸铜0.0013mg/L、L-赖氨酸盐酸盐91.25mg/L、硫辛酸0.105mg/L、九水硝酸铁0.05mg/L、L-蛋氨酸17.24mg/L、酚红8.1mg/L、七水硫酸亚铁0.417mg/L、L-苯丙氨酸35.48mg/L、1,4-丁二胺二盐酸盐0.081mg/L、氯化钾311.8mg/L、L-丝氨酸26.25mg/L、丙酮酸钠55mg/L、氯化镁28.64mg/L、L-苏氨酸53.45mg/L、维生素H0.0035mg/L、无水硫酸镁48.84mg/L、L-丙氨酸4.45mg/L、D-泛酸钙2.24mg/L、氯化钠6999.5mg/L、L-天门冬酰胺7.5mg/L、氯化胆碱8.98mg/L、无水磷酸二氢钠54.35mg/L、L-天门冬氨酸6.65mg/L、叶酸2.65mg/L、磷酸氢二钠71.02mg/L、L-半胱氨酸盐酸盐17.56mg/L、i-肌醇12.6mg/L、七水硫酸锌0.432mg/L、L-谷氨酸7.35mg/L、烟酰胺2.02mg/L、L-精氨酸盐酸盐147.5mg/L、L-脯氨酸17.25mg/L、盐酸吡哆醛2mg/L、L-胱氨酸盐酸盐31.29mg/L、L-色氨酸9.02mg/L、盐酸吡哆醇0.031mg/L、L-谷氨酰胺365mg/L、L-酪氨酸38.4mg/L、核黄素0.219mg/L、甘氨酸18.75mg/L、L-缬氨酸52.85mg/L、盐酸硫胺2.17mg/L、L-组氨酸盐酸盐31.48mg/L、D-葡萄糖3151mg/L、胸苷0.365mg/L、L-异亮氨酸54.47mg/L、次黄嘌呤2mg/L、维生素B12 0.68mg/L。
本发明与现有技术相比，具有如下的优点和有益效果：
本发明涉及一种细胞培养基，特别是用于培养悬浮细胞的培养基，其用于重组蛋白的表达。本发明一种细胞无血清培养基，完全不含有血清成份；生产的抗体质量大幅度提高；该培养基同时具有高扩增速度及高抗体表达能力；细胞不需要驯化，可以直接从含血清培养直接过渡到无血清培养基培养；该培养基能长时间保持细胞活性，细胞培养时间大大延长，可以大大延长生产时间，提高抗体产量。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定；
图1为分别采用本发明的培养基与DMEM/F12+10％FBS培养基培养CHO细胞生长曲线对比图；
图2为分别采用本发明的培养基与DMEM/F12+10％FBS培养基培养CHO细胞蛋白产量对比图。
具体实施方式
本说明书中公开得任一特征，除非特别叙述，均可被其他等效或具有类似目的的替代特征加以替换。除非特别叙述，每个特征只是一系列等效或者类似特征中的一个例子而已。所述仅仅是为了帮助理解本发明，不应该视为对本发明的具体限制。
下面以附图和具体实施方式对本发明作进一步详细的说明。
实施例1
一种无血清培养基，包括如下浓度的原料：
DMEM/F12；
以及在DMEM/F12中添加：
成纤维细胞生长因子0.1ng/mL、重组人表皮细胞生长因子(EGF)0.1ng/mL、神经生长因子0.1ng/mL、转化生长因子0.1ng/mL、白血病抑制因子0.1ng/mL、胰岛素样生长因子-10.1ng/mL、氢化可的松12μg/L、胰岛素2μg/L、孕酮1μg/L、地塞米松1μg/L、血清白蛋白1mg/mL、转铁蛋白1mg/mL、胎球蛋白1mg/mL、谷氨酰胺0.01g/L、还原型谷胱甘肽0.1g/L、非必需氨基酸0.01g/L、亚油酸0.5mg/L、胆固醇0.01mg/L、乙醇胺0.5mg/L、β-巯基乙醇0.5mg/L、吐温-80 5mg/L、氯化锰1μg/L、亚锡酸钠1μg/L、柠檬酸铁10μg/L、硫酸锌10μg/L、生物素0.01mg/L、叶酸0.1mg/L、核黄素0.1mg/L、维生素B12 0.1mg/L、维生素C 0.1mg/L、维生素E0.1mg/L。
实施例2
一种无血清培养基，包括如下浓度的原料：
DMEM/F12；
以及在DMEM/F12中添加：
成纤维细胞生长因子25ng/mL、重组人表皮细胞生长因子(EGF)25ng/mL、神经生长因子25ng/mL、转化生长因子25ng/mL、白血病抑制因子25ng/mL、胰岛素样生长因子-125ng/mL、氢化可的松30μg/L、胰岛素10μg/L、孕酮10μg/L、地塞米松10μg/L、血清白蛋白25mg/mL、转铁蛋白25mg/mL、胎球蛋白25mg/mL、谷氨酰胺0.05g/L、还原型谷胱甘肽0.75g/L、非必需氨基酸0.05g/L、亚油酸0.75mg/L、胆固醇0.05mg/L、乙醇胺0.75mg/L、β-巯基乙醇0.75mg/L、吐温-80 7.5mg/L、氯化锰5μg/L、亚锡酸钠5μg/L、柠檬酸铁50μg/L、硫酸锌50μg/L、生物素0.1mg/L、叶酸1.5mg/L、核黄素0.15mg/L、维生素B12 0.4mg/L、维生素C 0.4mg/L、维生素E 0.4mg/L。
实施例3
一种无血清培养基，包括如下浓度的原料：
DMEM/F12；
以及在DMEM/F12中添加：
成纤维细胞生长因子50ng/mL、重组人表皮细胞生长因子(EGF)50ng/mL、神经生长因子50ng/mL、转化生长因子50ng/mL、白血病抑制因子50ng/mL、胰岛素样生长因子-150ng/mL、氢化可的松60μg/L、胰岛素20μg/L、孕酮20μg/L、地塞米松20μg/L、血清白蛋白50mg/mL、转铁蛋白50mg/mL、胎球蛋白50mg/mL、谷氨酰胺0.1g/L、还原型谷胱甘肽1.5g/L、非必需氨基酸0.1g/L、亚油酸1mg/L、胆固醇0.1mg/L、乙醇胺1mg/L、β-巯基乙醇1mg/L、吐温-80 10mg/L、氯化锰10μg/L、亚锡酸钠10μg/L、柠檬酸铁100μg/L、硫酸锌100μg/L、生物素0.2mg/L、叶酸3mg/L、核黄素0.3mg/L、维生素B12 0.8mg/L、维生素C 0.8mg/L、维生素E0.8mg/L。
实施例4
实验组：采用本发明实施例1的细胞培养基；
对照组：采用DMEM/F12+10％血清的培养基。
用上述实验组与对比组的培养基培养CHO细胞，并对细胞的生长曲线、蛋白产量和抗体质量进行对比。
图1为分别采用本发明的培养基与DMEM/F12+10％FBS培养基培养CHO细胞生长曲线对比图。如图1所示，实施例1与对照例1均在第四天活细胞密度达到峰值，但实施例1的活细胞密度相比于对照例1的活细胞密度高2×106cells/mL，且实施例1中的细胞活率维持也明显优于对照例1；
图2为分别采用本发明的培养基与DMEM/F12+10％FBS培养基培养CHO细胞蛋白产量对比图。如图2所示，实施例1中的蛋白产量为3.58g/L，对照例1中的蛋白产量为2.81g/L，实施例中的蛋白产量明显高于对照例；
表1为分别采用本发明的培养基与DMEM/F12+10％FBS培养基培养CHO细胞抗体质量对比表；
如表1所示，用体积排阻色谱(SEC)来表征蛋白质量，实施例1的主峰面积明显高于对照例1，且与标准品的主峰面积相似，高聚体(HMW)较标准品略高，但都在可接受范围之内。低聚体(LMW)与标准品基本一致。从蛋白质量来看，实施例1中的蛋白质量较优。
本发明的工艺参数(如温度、时间等)区间上下限取值以及区间值都能实现本法，在此不一一列举实施例。
本发明未详细说明的内容均可采用本领域的常规技术知识。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制。尽管参照实施例对本发明进行了详细说明，本领域的普通技术人员应该理解，对本发明的技术方案进行修改或者等同替换，都不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (8)

1.一种无血清培养基，其特征在于，所述培养基包含以下组分：
DMEM/F12；
以及在DMEM/F12中添加：
0.6-300ng/mL生长因子、15-120μg/L激素、3-150mg/mL蛋白质、0.1-1.7g/L氨基酸及肽、6.51-13.1mg/L脂质复合物、22-220μg/L微量元素、0.5-5.9mg/L维生素。
2.如权利要求1所述的无血清培养基，其特征在于，所述的生长因子包括如下组分：
成纤维细胞生长因子0.1-50ng/mL、重组人表皮细胞生长因子0.1-50ng/mL、神经生长因子0.1-50ng/mL、转化生长因子0.1-50ng/mL、白血病抑制因子0.1-50ng/mL、胰岛素样生长因子-1 0.1-50ng/mL。
3.如权利要求1所述的无血清培养基，其特征在于，所述的激素包括如下组分：
氢化可的松12-60μg/L、胰岛素2-20μg/L、孕酮0-20μg/L、地塞米松1-20μg/L。
4.如权利要求1所述的无血清培养基，其特征在于，所述的蛋白质包括如下组分：
血清白蛋白1-50mg/mL、转铁蛋白1-50mg/mL、胎球蛋白1-50mg/mL。
5.如权利要求1所述的无血清培养基，其特征在于，所述的氨基酸及肽包括如下组分：
谷氨酰胺0-0.1g/L、还原型谷胱甘肽0.1-1.5g/L、非必需氨基酸0-0.1g/L。
6.如权利要求1所述的无血清培养基，其特征在于，所述脂类复合物包括如下组分：
亚油酸0.5-1mg/L、胆固醇0.01-0.1mg/L、乙醇胺0.5-1mg/L、β-巯基乙醇0.5-1mg/L、吐温-80 5-10mg/L。
7.如权利要求1所述的无血清培养基，其特征在于，所述微量元素包括如下组分：
氯化锰1-10μg/L、亚锡酸钠1-10μg/L、柠檬酸铁10-100μg/L、硫酸锌10-100μg/L。
8.如权利要求1所述的无血清培养基，其特征在于，所述维生素包括如下组分：
生物素0-0.2mg/L、叶酸0.1-3mg/L、核黄素0.1-0.3mg/L、维生素B12 0.1-0.8mg/L、维生素C 0.1-0.8mg/L、维生素E 0.1-0.8mg/L。
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