CN111676184A - 一种由补料培养基共混的基础培养基及其制备方法和应用 - Google Patents
一种由补料培养基共混的基础培养基及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及无血清细胞培养基技术领域,具体公开了一种由补料培养基共混的基础培养基,包括补料培养基A、补料培养基B和添加剂,所述补料培养基B的体积用量是补料培养基A的5‑15%;所述补料培养基A包含有总浓度为12900‑124000mg/L的氨基酸,总浓度为1210‑12560mg/mL的无机盐和微量元素,总浓度为780‑10260mg/L的维生素,以及总浓度为1300‑8020mg/mL的其它成分;所述补料培养基B包含有总浓度为18000‑180000mg/L的L‑色氨酸、L‑酪氨酸、L‑半胱氨酸;所述添加剂包括总浓度0.103‑1.251g/L的丙酮酸钠、乙醇胺、谷胱甘肽、精胺、柠檬酸铁铵、亚硒酸钠。本发明基础培养基能够支撑工业界和实验室主流细胞株的生长代谢,具有广谱的适用性,原料价格低廉,工艺操作简单,适合大批量工业生产。
Description
技术领域
本发明涉及无血清细胞培养基技术领域,具体涉及一种由补料培养基共混的基础培养基及该基础培养基的制备方法,以及该基础培养基在细胞培养中的应用。
背景技术
我国医药发展历史源远流长,先人们用中草药缓解病痛,积累了丰富的药理学知识;近代以来,随着对疾病机理的深入研究,小分子化药由于其针对性强,疗效好,作用快等优势,在医药市场占据主导地位;然而,在面对肿瘤,免疫系统疾病,以及病毒感染等挑战时,小分子化药还是显得力不从心。随着免疫学、分子生物学的发展,单克隆抗体药物的出现为解决这些疑难杂症提供了新的途径。
单克隆抗体药物是基于抗原和抗体特异性结合的原理发展而来。单抗类药物能中和多种毒素,使其丧失生物学活性;激活巨噬细胞,吞食入侵的抗原;启动抗体和补体依赖细胞毒作用,杀死肿瘤细胞,病原体。可以说单抗药物的发展,给人类战胜疾病带来了新的曙光。
动物细胞是生产单克隆抗体最主要的载体,但是动物细胞比较脆弱,对外界环境敏感,且营养需求高,因此开发适合其生长代谢的培养基和培养工艺十分重要。细胞培养基的组分丰富,且组分含量的不同会对细胞密度,抗体蛋白的表达量和质量造成影响。当前国内的细胞培养基被国外生物公司所垄断,使得抗体药物的研发和生产成本居高不下,故抗体药物细胞培养基的国产化具有很重要的现实意义。
单抗药物生产所用的细胞培养基分为基础培养基和补料培养基。基础培养基的开发和优化通常会采用DOE实验、培养基成分消耗分析、代谢组学、高通量筛选系统等技术手段。这些方法耗时长,且工作量大。因此本领域迫切需要开发新的具有广谱适用性的基础培养基。
发明内容
针对现有技术的不足,本发明提供了一种由补料培养基共混的基础培养基及该基础培养基的制备方法,以及该基础培养基在细胞培养中的应用,很好的支持当前工业界和实验室常用细胞株的生长代谢,具有广谱的应用价值。
本发明是通过如下的技术方案实现的:
一种由补料培养基共混的基础培养基,包括补料培养基A、补料培养基B和添加剂,所述补料培养基B的体积用量是补料培养基A的5-15%;所述补料培养基A包含有总浓度为12900-124000mg/L的氨基酸,总浓度为1210-12560mg/mL的无机盐和微量元素,总浓度为780-10260mg/L的维生素,以及总浓度为1300-8020mg/mL的其它成分;所述补料培养基B包含有总浓度为18000-180000mg/L的氨基酸,所述补料培养基B中的氨基酸由L-色氨酸、L-酪氨酸、L-半胱氨酸组成;所述添加剂包括总浓度0.103-1.251g/L的以下组分:丙酮酸钠、乙醇胺、谷胱甘肽、精胺、柠檬酸铁铵、亚硒酸钠。
优选的,所述添加剂由以下用量的组分组成:丙酮酸钠0.1-1.0g/L、乙醇胺0.001-0.050g/L、谷胱甘肽0.0005-0.05g/L、精胺0.001-0.05g/L、柠檬酸铁铵0.001-0.1g/L、亚硒酸钠0.000001-0.0005g/L。
作为优选的技术方案,所述补料培养基共混的基础培养基包括如下体积份数的组分:
进一步的,上述由补料培养基共混的基础培养基,还包括缓冲剂、渗透压调节剂和消泡剂。
所述缓冲剂、渗透压调节剂和消泡剂均为本领域的常用材料,例如缓冲剂可以选用HEPES和NaHCO3、渗透压调节剂可以选用NaCl,消泡剂可以选用P188(泊洛沙姆188)。上述助剂的用量可以根据实际应用需要进行调节。
进一步优选的,所述补料培养基B中各组分具体含量如下:
所述补料培养基B中各组分更优选的含量如下:
所述补料培养基B中各组分含量最好如下:
作为优选的技术方案,所述补料培养基A的具体成分及其含量如下:
氨基酸部分
无机盐和微量元素部分
维生素部分
其它成份
进一步优选的,所述补料培养基A的具体成分及其含量如下:
氨基酸部分
无机盐和微量元素部分
维生素部分
其它成分
本发明还进一步公开了上述由补料培养基共混的基础培养基的制备方法,包括如下步骤:按比例将补料培养基A、补料培养基B以及添加剂加入适量水中,搅拌混匀,然后依次加入缓冲剂、消泡剂和渗透压调节剂,混匀;用HCl调节pH值至7.0-7.4后加水定容,用滤膜过滤,即得所述基础培养基。
所述滤膜优选0.22μm滤膜。
其中所述补料培养基A的制备方法包括如下步骤:
1)按配制体积定量称取补料培养基A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,水温控制在20-30℃,并加入氢氧化钠调pH促进溶解;
3)每升培养基中加入75g葡萄糖,搅拌均匀;
4)调整pH在6.7-6.9之间,并用超纯水定容;
5)采用0.22μm滤膜过滤,2-8℃保存。
所述补料培养基B的制备方法包括如下步骤:
1)按配制体积定量称取补料培养基B中各成分,混匀;
2)加入配制体积70%的水,水温控制在20-30℃;
3)加入配制体积20%的6N氢氧化钠溶液;
4)调节pH在10.80-11.80之间,并用超纯水定容;
5)采用0.22μm滤膜过滤,2-8℃保存。
本发明还进一步提供所述由补料培养基共混的基础培养基在细胞培养中的应用,可广泛应用于诸如CHO细胞株、293F细胞株、Expi293细胞株等细胞株的培养。
与现有技术相比,本发明提供的由补料培养基共混的基础培养基具有如下有益效果:
1、本发明基础培养基能够支撑工业界和实验室主流细胞株的生长代谢,具有广谱的适用性。
2、基础培养基中的组分明确且不含有动植物来源成分,能确保批次间的一致性。
3、本发明的设计思路新颖,由补料培养基开发基础培养基的成功,能给其他科研工作者提供借鉴。
4、本发明基础培养基的原料价格低廉,工艺操作简单,适合大批量工业生产。
附图说明
图1为各实施例和对比例基础培养基培养细胞的活细胞密度随时间变化图;
图2为各实施例和对比例基础培养基培养细胞的细胞活率随时间变化图;
图3为各实施例和对比例基础培养基培养细胞的细胞直径随时间变化图。
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。本发明配方中各组分如无特别说明,均为常规市售产品。
实施例中,补料培养基A的成分及配方:
配制补料培养基A的方法如下:
(1)按配制体积定量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,水温控制在20-30℃,并加入氢氧化钠调pH促进溶解;
(3)每升培养基中加入75g葡萄糖,搅拌均匀;
(4)调整pH在6.7-6.9之间,并用超纯水定容;
(5)采用0.22μm滤膜过滤,2-8℃保存。
补料培养基B的成分及配方:
氨基酸 | 含量mg/L | 氨基酸 | 含量mg/L |
L-色氨酸 | 28000 | L-半胱氨酸 | 24400 |
L-酪氨酸 | 48300 |
配制补料培养基B的方法如下:
(1)按配制体积定量称取补料培养基中各成分,混匀;
(2)加入配制体积70%的水,水温控制在20-30℃;
(3)加入配制体积20%的6N氢氧化钠溶液;
(4)调节pH在10.80-11.80之间,并用超纯水定容;
(5)采用0.22μm滤膜过滤,2-8℃保存。
添加剂(B-9添加剂)由以下用量的组分经简单混合定容而成:丙酮酸钠(0.25g/L)、乙醇胺(0.016g/L)、谷胱甘肽(0.002g/L)、精胺(0.015g/L)、柠檬酸铁铵(0.05g/L)、亚硒酸钠(0.0002g/L)。
实施例1
1、B-9基础培养基配制步骤:
(1)取800mL超纯水,加入100mL补料培养基A,10mL补料培养基B,以及10mL B-9添加剂将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-9基础培养基。
2、细胞培养
从细胞库中复苏CHO-K1工程细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-9基础培养基重悬细胞,调整细胞密度为3×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如表1和图1、2、3所示。三次传代结果显示,使用B-9基础培养基后,CHO-K1工程细胞株传代2天后活细胞密度维持在1.59×106cell/mL左右,细胞活率在97.4%左右,细胞直径在14.7μm左右。
实施例2
1、B-9基础培养基配制步骤:
(1)取800mL超纯水,加入100mL补料培养基A,10mL补料培养基B,以及10mL B-9添加剂将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-9基础培养基。
2、细胞培养
从细胞库中复苏CHO-ZN工程细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-9基础培养基重悬细胞,调整细胞密度为5×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如表1和图1、2、3所示。三次传代结果显示,使用B-9基础培养基后,CHO-ZN工程细胞株传代2天后活细胞密度维持在1.16×106cell/mL左右,细胞活率在97.0%左右,细胞直径在14.5μm左右。
实施例3
1、B-9基础培养基配制步骤:
(1)取800mL超纯水,加入100mL补料培养基A,10mL补料培养基B,以及10mL B-9添加剂将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-9基础培养基。
2、细胞培养
从细胞库中复苏293F细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-9基础培养基重悬细胞,调整细胞密度为5×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如表1和图1、2、3所示。三次传代结果显示,使用B-9基础培养基后,293F细胞株传代2天后活细胞密度维持在1.31×106cell/mL左右,细胞活率在95.8%左右,细胞直径在18.6μm左右。
实施例4
1、B-9基础培养基配制步骤:
(1)取800mL超纯水,加入100mL补料培养基A,10mL补料培养基B,以及10mL B-9添加剂将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-9基础培养基。
2、细胞培养
从细胞库中复苏Expi293细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-9基础培养基重悬细胞,调整细胞密度为5×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如表1和图1、2、3所示。三次传代结果显示,使用B-9基础培养基后,Expi293细胞株传代2天后活细胞密度维持在1.94×106cell/mL左右,细胞活率在98.3%左右,细胞直径在19.2μm左右。
对比例1
1、B-9-0基础培养基(未加入B-9添加剂)配制步骤:
(1)取800mL超纯水,加入100mL补料培养基A,10mL补料培养基B,以及10mL B-9添加剂将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-9基础培养基。
2、细胞培养
从细胞库中复苏Expi293细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-9-0基础培养基重悬细胞,调整细胞密度为5×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如表1和图1、2、3所示。三次传代结果显示,使用B-9-0基础培养基后,Expi293细胞株随着传代次数增加,活细胞密度逐渐下降,细胞活率也不断降低。
对比例2
1、B-10基础培养基(采用Hyclone公司的CB7a、CB7b补料培养基替代本发明补料培养基A和补料培养基B,未加入B-9添加剂)配制步骤:
(1)取800mL超纯水,加入100mL CB7a(Hyclone),10mL CB7b(Hyclone),将搅拌器的转速调节至800r/min,磁力搅拌10min;
(2)向步骤(1)中加入1.8g HEPES,搅拌10min,使其完全溶解;
(3)向步骤(2)中加入1.8g P188,搅拌10min;
(4)向步骤(3)中加入NaHCO3的2.1g,搅拌10min;
(5)向步骤(4)中加入NaCl的2g,搅拌10min;
(6)利用6N的HCl调节步骤(5)中溶液的pH值,使其在7.0-7.4范围内;
(7)加水定容至1L;
(8)测量步骤(7)得到的溶液的渗透压,其值应该在280-320mOsm/kg;
(9)采用0.22μm滤膜过滤,即得B-10基础培养基。
2、细胞培养
从细胞库中复苏Expi293细胞株,传代3次后,取样计数,离心去除初始培养基,随后用30mL的B-10基础培养基重悬细胞,调整细胞密度为5×105cell/mL,接种到125mL摇瓶中进行传代培养。摇床的温度设置为37℃,转速110r/min,CO2浓度控制在8%。每两天取样,用countstar自动细胞计数仪(产品型号:IC 1000;生产厂家:上海睿钰生物科技有限公司)检测活细胞密度、细胞活率和细胞直径,同样的密度再次传代。传代3次后结束培养。
活细胞密度、细胞活率和细胞直径分别如图1、2、3所示。三次传代结果显示,使用B-10基础培养基后,Expi293细胞株传代2天后活细胞密度维持在1.65×106cell/mL左右,细胞活率在98.6%左右,细胞直径在19.1μm左右。
各实施例和对比例的细胞培养结果如下表:
表1培养细胞的活细胞密度、细胞活率、细胞直径随时间变化数据
本发明以补料培养基为主体,通过添加缓冲剂HEPES和NaHCO3,消泡剂P188对配方进行优化,制得基础培养基。实验结果显示,本发明基础培养基能很好的支持当前工业界和实验室常用细胞株的生长代谢,具有广谱的应用价值。未加入添加剂的基础培养基,其活细胞密度随着传代次数增加而逐渐下降,细胞活率也不断降低,表明本发明添加剂具有显著的协同增效作用。
需要说明的是,在本文中,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、组分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、组分或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (10)
1.一种由补料培养基共混的基础培养基,其特征在于,包括补料培养基A、补料培养基B和添加剂,所述补料培养基B的体积用量是补料培养基A的5-15%;所述补料培养基A包含有总浓度为12900-124000mg/L的氨基酸,总浓度为1210-12560mg/mL的无机盐和微量元素,总浓度为780-10260mg/L的维生素,以及总浓度为1300-8020mg/mL的其它成分;所述补料培养基B包含有总浓度为18000-180000mg/L的氨基酸,所述补料培养基B中的氨基酸由L-色氨酸、L-酪氨酸、L-半胱氨酸组成;所述添加剂包括总浓度0.103-1.251g/L的以下组分:丙酮酸钠、乙醇胺、谷胱甘肽、精胺、柠檬酸铁铵、亚硒酸钠。
2.根据权利要求1所述的由补料培养基共混的基础培养基,其特征在于,所述添加剂由以下用量的组分组成:丙酮酸钠0.1-1.0g/L、乙醇胺0.001-0.050g/L、谷胱甘肽0.0005-0.05g/L、精胺0.001-0.05g/L、柠檬酸铁铵0.001-0.1g/L、亚硒酸钠0.000001-0.0005g/L。
3.根据权利要求2所述的由补料培养基共混的基础培养基,其特征在于,所述补料培养基共混的基础培养基包括如下体积份数的组分:补料培养基A 100份、补料培养基B 5-15份、添加剂5-15份、余量为水,总量共计1000份。
4.根据权利要求1-3任一项所述的由补料培养基共混的基础培养基,其特征在于,所述补料培养基共混的基础培养基还包括缓冲剂、渗透压调节剂和消泡剂。
9.一种如权利要求1-8任一项所述由补料培养基共混的基础培养基的制备方法,其特征在于,包括如下步骤:按比例将补料培养基A、补料培养基B以及添加剂加入适量水中,搅拌混匀,然后依次加入缓冲剂、消泡剂和渗透压调节剂,混匀;用HCl调节pH值至7.0-7.4后加水定容,用滤膜过滤,即得所述基础培养基。
10.一种如权利要求1-8任一项所述由补料培养基共混的基础培养基在细胞培养中的应用。
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