CN107099508A - 一种无血清杂交瘤细胞培养基 - Google Patents

一种无血清杂交瘤细胞培养基 Download PDF

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CN107099508A
CN107099508A CN201710484822.5A CN201710484822A CN107099508A CN 107099508 A CN107099508 A CN 107099508A CN 201710484822 A CN201710484822 A CN 201710484822A CN 107099508 A CN107099508 A CN 107099508A
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曲宝赤
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Abstract

本发明公开了一种无血清杂交瘤细胞培养基,主要成分包括氨基酸,无机盐成分,生长因子及白蛋白成分。本发明的无血清杂交瘤细胞培养基,适合工业抗体生产,可提高杂交瘤细胞在摇瓶生长的最大密度,扩增倍数为60~80倍,同时抗体表达量增高,是传统工艺的1倍以上;在产量提高的同时,使用较少的培养基,大大降低了生产成本。

Description

一种无血清杂交瘤细胞培养基
技术领域
本发明涉及一种无血清杂交瘤细胞培养基。
背景技术
自从1975年Kohler和Milstein建立杂交瘤技术以来,由杂交瘤细胞生产的单克隆抗体已应用到许多方面,特别是在疾病诊断和治疗及亲和层析分离方面,单克隆抗体具有十分诱人的应用前景,其中CD3单抗于1987年被美国FDA批准用于临床,1990年已达到1千万美元的年产值。美国制药工业协会调查报告显示,单抗位列所有医药生物技术产品之首。
单克隆抗体生产分体内法和体外法,体外细胞培养途径具有重要的优点,如:1.可以采用单元操作法培养,随着规模增大,降低生产成本;2.减少鼠类动物感染疾病给产物带来的污染,避免鼠的其它抗体的存在;3.可以生产人的单克隆抗体;4.过程可以工程化和自动控制,提高了重演性。
随着单克隆抗体的大量应用,杂交瘤细胞的大规模培养技术日趋成熟。英国Celltech公司的1万升气升式反应器已经开发成功,并应用于抗人ABO血型单抗的生产。目前,杂交瘤细胞的培养已不再以反应器培养规模作为重要的指标,而把更多的精力集中于杂交瘤细胞的代谢调控、反应器的操作模式和控制策略及无血清无蛋白培养技术等方面,以提高杂交瘤细胞密度和单抗的生产率,生产杂蛋白含量低、容易分离纯化的体内用单克隆抗体。
相对于贴壁依赖性细胞,杂交瘤细胞培养的放大可以更多地借助于微生物发酵的经验。八十年代初,已利用生物反应器大规模培养杂交瘤细胞。为了使培养放大工作更加有效,细胞高密度培养的研究引起重视,而首先遇到的则是培养条件的优化,并逐渐过渡到代谢的调控等方面。
发明内容
针对上述现有技术,本发明提供了一种无血清杂交瘤细胞培养基,适合工业抗体生产,配合反应器或摇瓶培养,可维持杂交瘤细胞的高密度生长。本发明的无血清培养基,在代谢调控,细胞抗体表达以及高密度维持做了很大改进。
本发明是通过以下技术方案实现的:
一种无血清杂交瘤细胞培养基,是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸100~300;
CuSO4·5H2O 0.0005~0.005;
L-天门冬酰胺25~75;
L-天门冬氨酸10~30;
L-谷氨酸10~30;
Ni(NO3)2·6H2O 0.00002~0.0002;
L-谷氨酰胺0~500;
ZnSO4·7H2O 0.06~0.6;
甘氨酸5~15;
CoCl2·6H2O 0.001~0.008;
L-组氨酸10~30;
NaSiO3·9H2O 0.001~0.01;
L-异亮氨酸25~75;
Na3VO4·12H2O 0.0005~0.005;
L-赖氨酸盐酸20~60;
SnC12·2H2O 0.00001~0.0001;
L-蛋氨酸10~30;
Na2SeO3 0.002~0.01;
L-苯丙氨酸10~30;
FeSO4·7H2O 0.2~1.6;
L-脯氨酸10~30;
葡萄糖1000~4000;
L-丝氨酸15~45;
维生素C O.176~0.704;
L-苏氨酸10~30;
对羟基苯甲酸0.5~1.5;
L-色氨酸5~15;
丙酮酸钠55~550;
L-缬氨酸1O~30;
亚油酸0.01~0.05;
L-亮氨酸25~75;
β-巯基乙醇0.8~4.0;
二水L-酪氨酸二钠144~432;
乙醇胺1~5;
L-胱氨酸二盐酸25~75;
地塞米松0.001~0.005;
人转铁蛋白5~50;
胰岛素:8~20
纯化人转铁蛋白:1~30;
胆固醇:20~60;
过氧化氢酶:20~50;
亚硒酸钠:17.3~35.0
1%二巯基乙醇溶液:0.35~1.0ml/L;
氯化锰0.0001~0.01;
偏钒酸铵0.0001~0.1;
氯化镍0.0001~0.1;
二水氯化锡0.0001~0.1;
四水钼酸铵0.0001~0.1;
余量为水。
本发明的无血清杂交瘤细胞培养基的制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH值至7.2,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
本发明的无血清杂交瘤细胞培养基,主要成分包括氨基酸,无机盐成分,生长因子及白蛋白成分,可提高杂交瘤细胞在摇瓶生长的最大密度,扩增倍数为60~80倍,同时抗体表达量增高,是传统工艺的1倍以上;在产量提高的同时,使用较少的培养基,大大降低了生产成本。本发明的制备工艺全部采用在线清洗及在线灭菌设备,保证批间差的稳定,同时生产中采用惰性气体氮气保护,避免不稳定性成分被氧化,该工艺生产的免疫细胞无血清培养基质量稳定,解决了大多数厂家生产质量不易控制的问题。
附图说明
图1:杂交瘤在无血清培养基中的生长图片(100×)。
图2:杂交瘤在无血清培养基中的生长图片(400×)。
图3:杂交瘤细胞的生长曲线示意图,其中,Yocon代表本发明的培养基(图中的1d是指12小时)。
图4:培养杂交瘤细胞所产生抗体量对比示意图,其中,位于上方的曲线代表采用本发明的培养基培养,位于下方的曲线代表采用GIBCO国外无血清培养基培养。
具体实施方式
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
实施例1制备无血清杂交瘤细胞培养基
是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸200;
CuSO4·5H2O 0.002;
L-天门冬酰胺50;
L-天门冬氨酸20;
L-谷氨酸20;
Ni(NO3)2·6H2O 0.0001;
L-谷氨酰胺250;
ZnSO4·7H2O 0.3;
甘氨酸10;
CoCl2·6H2O 0.005;
L-组氨酸20;
NaSiO3·9H2O 0.005;
L-异亮氨酸50;
Na3VO4·12H2O 0.003;
L-赖氨酸盐酸40;
SnC12·2H2O 0.00005;
L-蛋氨酸20;
Na2SeO3 0.005;
L-苯丙氨酸20;
FeSO4·7H2O 0.9;
L-脯氨酸20;
葡萄糖3000;
L-丝氨酸30;
维生素C O.500;
L-苏氨酸20;
对羟基苯甲酸1.0;
L-色氨酸10;
丙酮酸钠300;
L-缬氨酸20;
亚油酸0.03;
L-亮氨酸50;
β-巯基乙醇2.0;
二水L-酪氨酸二钠300;
乙醇胺3;
L-胱氨酸二盐酸50;
地塞米松0.003;
人转铁蛋白30;
胰岛素:15
纯化人转铁蛋白:15;
胆固醇:40;
过氧化氢酶:35;
亚硒酸钠:28.0
1%二巯基乙醇溶液:0.70ml/L;
氯化锰0.005;
偏钒酸铵0.005;
氯化镍0.005;
二水氯化锡0.005;
四水钼酸铵0.005;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH值至7.2,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实施例2制备无血清杂交瘤细胞培养基
是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸100;
CuSO4·5H2O 0.0005;
L-天门冬酰胺75;
L-天门冬氨酸30;
L-谷氨酸10;
Ni(NO3)2·6H2O 0.00002;
L-谷氨酰胺500;
ZnSO4·7H2O 0.6;
甘氨酸5;
CoCl2·6H2O 0.001;
L-组氨酸30;
NaSiO3·9H2O 0.01;
L-异亮氨酸25;
Na3VO4·12H2O 0.0005;
L-赖氨酸盐酸60;
SnC12·2H2O 0.0001;
L-蛋氨酸10;
Na2SeO3 0.002;
L-苯丙氨酸30;
FeSO4·7H2O 1.6;
L-脯氨酸10;
葡萄糖1000;
L-丝氨酸45;
维生素C 0.704;
L-苏氨酸10;
对羟基苯甲酸0.5;
L-色氨酸15;
丙酮酸钠550;
L-缬氨酸1O;
亚油酸0.01;
L-亮氨酸75;
β-巯基乙醇4.0;
二水L-酪氨酸二钠144;
乙醇胺1;
L-胱氨酸二盐酸75;
地塞米松0.005;
人转铁蛋白5;
胰岛素:8
纯化人转铁蛋白:30;
胆固醇:60;
过氧化氢酶:20;
亚硒酸钠:17.3
1%二巯基乙醇溶液:1.0ml/L;
氯化锰0.01;
偏钒酸铵0.0001;
氯化镍0.0001;
二水氯化锡0.1;
四水钼酸铵0.1;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH值至7.2,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实施例3制备无血清杂交瘤细胞培养基
是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸300;
CuSO4·5H2O 0.005;
L-天门冬酰胺25;
L-天门冬氨酸10;
L-谷氨酸30;
Ni(NO3)2·6H2O 0.0002;
L-谷氨酰胺100;
ZnSO4·7H2O 0.1;
甘氨酸15;
CoCl2·6H2O 0.008;
L-组氨酸10;
NaSiO3·9H2O 0.001;
L-异亮氨酸75;
Na3VO4·12H2O 0.005;
L-赖氨酸盐酸20;
SnC12·2H2O 0.00001;
L-蛋氨酸30;
Na2SeO3 0.01;
L-苯丙氨酸10;
FeSO4·7H2O 0.2;
L-脯氨酸30;
葡萄糖4000;
L-丝氨酸15;
维生素C O.176;
L-苏氨酸30;
对羟基苯甲酸1.5;
L-色氨酸5;
丙酮酸钠55;
L-缬氨酸30;
亚油酸0.05;
L-亮氨酸25;
β-巯基乙醇1.0;
二水L-酪氨酸二钠432;
乙醇胺5;
L-胱氨酸二盐酸25;
地塞米松0.001;
人转铁蛋白50;
胰岛素:20
纯化人转铁蛋白:1;
胆固醇:20;
过氧化氢酶:50;
亚硒酸钠:35.0
1%二巯基乙醇溶液:0.35ml/L;
氯化锰0.0001;
偏钒酸铵0.1;
氯化镍0.1;
二水氯化锡0.0001;
四水钼酸铵0.0001;
余量为水。
制备方法为:取上述除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如上所述,调节pH值至7.2,即得,工业滤芯过滤,同时氮气保护进行分装(避免不稳定性成分被氧化)。
实验
将实施例1制备的培养基置于T25细胞培养瓶中,将小鼠杂交瘤细胞(购自上海细胞保藏中心)以70cells/微升接种,在温度37℃下培养,培养图片如图1、图2所示(由图1、图2为细胞状态图,表明细胞生长状态良好。其生长曲线如图3所示,由图3可见,杂交瘤细胞密度最高为2.185x106cells/mL,倍增时间:14.53h。
同时,以GIBCO国外无血清培养基(市场购买得到)为对照培养杂交瘤细胞。
上述两种方式培养杂交瘤细胞,所得单抗的浓度对比如图4所示。由图4可见,与GIBCO国外无血清培养基对比,本发明的培养基培养的杂交瘤细胞,其抗体量有大幅度提高,抗体产量为国外培养基的1.37倍。另外,本发明的培养基培养的杂交瘤细胞时,无需驯化培养,避免了细胞培养时驯化的繁琐工作,而其他杂交瘤无血清培养均需要梯度驯化,驯化周期较长(传统驯化需要至少三周时间)。
上述虽然结合实施例对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。

Claims (6)

1.一种无血清杂交瘤细胞培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸100~300;
CuSO4·5H2O 0.0005~0.005;
L-天门冬酰胺25~75;
L-天门冬氨酸10~30;
L-谷氨酸10~30;
Ni(NO3)2·6H2O 0.00002~0.0002;
L-谷氨酰胺0~500;
ZnSO4·7H2O 0.06~0.6;
甘氨酸5~15;
CoCl2·6H2O 0.001~0.008;
L-组氨酸10~30;
NaSiO3·9H2O 0.001~0.01;
L-异亮氨酸25~75;
Na3VO4·12H2O 0.0005~0.005;
L-赖氨酸盐酸20~60;
SnC12·2H2O 0.00001~0.0001;
L-蛋氨酸10~30;
Na2SeO3 0.002~0.01;
L-苯丙氨酸10~30;
FeSO4·7H2O 0.2~1.6;
L-脯氨酸10~30;
葡萄糖1000~4000;
L-丝氨酸15~45;
维生素C O.176~0.704;
L-苏氨酸10~30;
对羟基苯甲酸0.5~1.5;
L-色氨酸5~15;
丙酮酸钠55~550;
L-缬氨酸1O~30;
亚油酸0.01~0.05;
L-亮氨酸25~75;
β-巯基乙醇0.8~4.0;
二水L-酪氨酸二钠144~432;
乙醇胺1~5;
L-胱氨酸二盐酸25~75;
地塞米松0.001~0.005;
人转铁蛋白5~50;
胰岛素:8~20
纯化人转铁蛋白:1~30;
胆固醇:20~60;
过氧化氢酶:20~50;
亚硒酸钠:17.3~35.0
1%二巯基乙醇溶液:0.35~1.0ml/L;
氯化锰0.0001~0.01;
偏钒酸铵0.0001~0.1;
氯化镍0.0001~0.1;
二水氯化锡0.0001~0.1;
四水钼酸铵0.0001~0.1;
余量为水。
2.根据权利要求1所述的无血清杂交瘤细胞培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸200;
CuSO4·5H2O 0.002;
L-天门冬酰胺50;
L-天门冬氨酸20;
L-谷氨酸20;
Ni(NO3)2·6H2O 0.0001;
L-谷氨酰胺250;
ZnSO4·7H2O 0.3;
甘氨酸10;
CoCl2·6H2O 0.005;
L-组氨酸20;
NaSiO3·9H2O 0.005;
L-异亮氨酸50;
Na3VO4·12H2O 0.003;
L-赖氨酸盐酸40;
SnC12·2H2O 0.00005;
L-蛋氨酸20;
Na2SeO3 0.005;
L-苯丙氨酸20;
FeSO4·7H2O 0.9;
L-脯氨酸20;
葡萄糖3000;
L-丝氨酸30;
维生素C O.500;
L-苏氨酸20;
对羟基苯甲酸1.0;
L-色氨酸10;
丙酮酸钠300;
L-缬氨酸20;
亚油酸0.03;
L-亮氨酸50;
β-巯基乙醇2.0;
二水L-酪氨酸二钠300;
乙醇胺3;
L-胱氨酸二盐酸50;
地塞米松0.003;
人转铁蛋白30;
胰岛素:15
纯化人转铁蛋白:15;
胆固醇:40;
过氧化氢酶:35;
亚硒酸钠:28.0
1%二巯基乙醇溶液:0.70ml;
氯化锰0.005;
偏钒酸铵0.005;
氯化镍0.005;
二水氯化锡0.005;
四水钼酸铵0.005;
余量为水。
3.根据权利要求1所述的无血清杂交瘤细胞培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸100;
CuSO4·5H2O 0.0005;
L-天门冬酰胺75;
L-天门冬氨酸30;
L-谷氨酸10;
Ni(NO3)2·6H2O 0.00002;
L-谷氨酰胺500;
ZnSO4·7H2O 0.6;
甘氨酸5;
CoCl2·6H2O 0.001;
L-组氨酸30;
NaSiO3·9H2O 0.01;
L-异亮氨酸25;
Na3VO4·12H2O 0.0005;
L-赖氨酸盐酸60;
SnC12·2H2O 0.0001;
L-蛋氨酸10;
Na2SeO3 0.002;
L-苯丙氨酸30;
FeSO4·7H2O 1.6;
L-脯氨酸10;
葡萄糖1000;
L-丝氨酸45;
维生素C 0.704;
L-苏氨酸10;
对羟基苯甲酸0.5;
L-色氨酸15;
丙酮酸钠550;
L-缬氨酸1O;
亚油酸0.01;
L-亮氨酸75;
β-巯基乙醇4.0;
二水L-酪氨酸二钠144;
乙醇胺1;
L-胱氨酸二盐酸75;
地塞米松0.005;
人转铁蛋白5;
胰岛素:8
纯化人转铁蛋白:30;
胆固醇:60;
过氧化氢酶:20;
亚硒酸钠:17.3
1%二巯基乙醇溶液:1.0ml;
氯化锰0.01;
偏钒酸铵0.0001;
氯化镍0.0001;
二水氯化锡0.1;
四水钼酸铵0.1;
余量为水。
4.根据权利要求1所述的无血清杂交瘤细胞培养基,其特征在于:是由以下浓度的组分组成的,各浓度单位若无特别说明,均为mg/L:
L-精氨酸300;
CuSO4·5H2O 0.005;
L-天门冬酰胺25;
L-天门冬氨酸10;
L-谷氨酸30;
Ni(NO3)2·6H2O 0.0002;
L-谷氨酰胺100;
ZnSO4·7H2O 0.1;
甘氨酸15;
CoCl2·6H2O 0.008;
L-组氨酸10;
NaSiO3·9H2O 0.001;
L-异亮氨酸75;
Na3VO4·12H2O 0.005;
L-赖氨酸盐酸20;
SnC12·2H2O 0.00001;
L-蛋氨酸30;
Na2SeO3 0.01;
L-苯丙氨酸10;
FeSO4·7H2O 0.2;
L-脯氨酸30;
葡萄糖4000;
L-丝氨酸15;
维生素C O.176;
L-苏氨酸30;
对羟基苯甲酸1.5;
L-色氨酸5;
丙酮酸钠55;
L-缬氨酸30;
亚油酸0.05;
L-亮氨酸25;
β-巯基乙醇1.0;
二水L-酪氨酸二钠432;
乙醇胺5;
L-胱氨酸二盐酸25;
地塞米松0.001;
人转铁蛋白50;
胰岛素:20
纯化人转铁蛋白:1;
胆固醇:20;
过氧化氢酶:50;
亚硒酸钠:35.0
1%二巯基乙醇溶液:0.35ml;氯化锰0.0001;
偏钒酸铵0.1;
氯化镍0.1;
二水氯化锡0.0001;
四水钼酸铵0.0001;
余量为水。
5.权利要求1~4中任一项所述的无血清杂交瘤细胞培养基的制备方法,其特征在于:取除水外的组分,根据其各自溶解特性分类溶解,然后混合,加入水使各组分终浓度如权利要求1~4中任一项所述,调节pH值至7.2,即得。
6.权利要求1~4中任一项所述的无血清杂交瘤细胞培养基在培养杂交瘤细胞中的应用。
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