CN108624549B - 一种cho dg44培养基及其应用 - Google Patents
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Abstract
本发明属于生物技术领域,具体涉及一种CHO DG44培养基及其应用。所述培养基的成分中含有:OPM‑CHO CD07、L‑Glutamine、F‑68、次黄嘌呤、胸腺嘧啶核苷、LONG®R3IGF‑I、亚油酸、吡哆胺二盐酸盐。本发明的CHO DG44培养基,用于培养CHO DG44细胞时,从细胞倍增时间,细胞最高密度和细胞培养时间上,都优于国外某知名品牌的CD DG44培养基,完全可以替代国外某知名品牌CD DG44培养基,更有利于CHO DG44细胞的培养。
Description
技术领域
本发明属于生物技术领域,具体涉及一种CHO DG44培养基及其应用。
背景技术
动物细胞表达的单克隆抗体药物销售额迅速增长,作为商业化生产单抗的核心技术之一的大规模动物细胞悬浮培养成为抗体药物发展的新瓶颈。其中之一主要技术限制就是国内没有自己的高效能商业化动物悬浮培养基,而SIGMA、Invitrogen、Hyclone和Lonza等国际知名培养基,每升高达上千元,且配方保密,不利于后续的培养工艺优化。故研制出自主品牌且低成本的无血清无动物来源培养基,对工艺优化与反应器放大,以满足大量表达单克隆抗体的需求,对于抗体药物的产业化的意义是非常重大的。
市场上抗体药物70%以上都是由CHO DG44宿主细胞表达,是二氢叶酸还原酶缺陷型(DHFR-)中国仓鼠卵巢细胞。目前CHO DG44转染前培养基主要使用某知名品牌CD DG44培养基,是化学成分确定的、无蛋白培养基,专门设计用于DG44细胞的生长和悬浮培养条件下蛋白质的表达。
现希望发明一种国产新型CHO DG44培养基。
发明内容
为了克服现有技术中所存在的问题,本发明的目的在于提供一种CHO DG44培养基及其应用。
为了实现上述目的以及其他相关目的,本发明采用如下技术方案:
所述OPM-CHO CD07可通过商购途径从上海奥浦迈生物科技有限公司获得,货号为P081307-001。
所述L-Glutamine为L-谷氨酰胺,分子式为C5H10N2O3,分子量为146.14,CAS号为56-85-9。所述L-Glutamine可通过商购途径获得,例如,可从味之素或GIBCO购买获得。
所述F-68可通过商购途径获得,例如,可从GIBCO购买获得,货号为24040-032。
所述次黄嘌呤(hypoxanthine)可通过商购途径获得,例如可从SIGMA购买获得,货号为H9636-1G。
所述胸腺嘧啶核苷(thymidine)可通过商购途径获得,例如可从SIGMA购买获得,货号为T1895-1G。
所述亚油酸的分子量为280.44、CAS号为60-33-3。所述亚油酸可通过商购途径获得,例如可从SIGMA购买获得,货号为L1012-100MG。
所述吡哆胺二盐酸盐的分子量为241.11、CAS号为524-36-7。所述吡哆胺二盐酸盐可通过商购途径获得,例如可从SIGMA购买获得,货号为P9158-1G。
优选地,每1LCHO DG44培养基中含有4~8mmol的L-Glutamine。
优选地,每1L CHO DG44培养基含有100g~250g的F-68。
优选地,每1L CHO DG44培养基含有50~200umol的次黄嘌呤。
优选地,每1L CHO DG44培养基含有8~32umol的胸腺嘧啶核苷。
优选地,每1L CHO DG44培养基含有10~100umol的亚油酸。
优选地,每1L CHO DG44培养基含有0.05~3mg的吡哆胺二盐酸盐。
本发明的第二方面,提供了前述CHO DG44培养基的制备方法,包括步骤:将各组分按配比混合,即可。
本发明的第三方面,提供了前述CHO DG44培养基用于CHO DG44细胞悬浮培养的用途。
本发明的第四方面,提供了一种悬浮培养CHO DG44细胞的方法,包括步骤:将CHODG44细胞置于前述CHO DG44培养基中进行培养。
与现有技术相比,本发明具有如下有益效果:
本发明的CHO DG44培养基,用于培养CHO DG44细胞时,从细胞倍增时间,细胞最高密度和细胞培养时间上,都优于某知名品牌的CD DG44培养基,完全可以替代CD DG44培养基,更有利于CHO DG44细胞的培养。
附图说明
图1:OPM-DG44 CDM1驯化CHO DG44细胞生长图。
图2:比较CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44CDM3培养基中培养生长活细胞图。
图3:CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44CDM3培养基中培养生长活率图。
图4:CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44CDM3培养基中倍增时间。
图5:有限流加培养比较CHO DG44细胞在培养基中的生长-VCD。
图6:有限流加培养比较CHO DG44细胞在培养基中的生长-VIA。
具体实施方式
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS INENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
实施例1
一、材料与方法
1.1实验试剂与细胞株
1.2主要仪器设备
1.3培养基的配方与配制
1.4实验方法
1.4.1细胞驯化
(1)将CHO DG44细胞复苏于DG44-1培养基中,观察细胞生长至稳定;
(2)CHO DG44细胞在DG44-1培养基生长至2±0.5x106cells/ml,将细胞用DG44-1/OPM-CDM1按1:1稀释细胞,在此混合培养基中进行过度适应。
(3)CHO DG44细胞在DG44-1/OPM-DG44 CDM1混合培养基中适应良好(PDT≈24h,VIA>90%),细胞按0.7x106cells/ml密度传到OPM-CDM1进行驯化,至细胞活率恢复>90%。
(4)CHO DG44细胞在OPM-DG44 CDM1培养基中适应完成后,接种至OPM-DG44 CDM2和OPM-DG44 CDM3培养基中传代检测是否增殖。
(5)绘图比较CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44 CDM3培养基中生长的细胞密度和活率。
1.4.2比较CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44 CDM3培养基中有限流加培养
(1)用于流加培养实验的种子密度约为2x106cells/ml,按5x105cells/ml接种密度接种摇瓶。
(2)接种D3,D5,D6,D7,D8和D9天取样检测细胞密度,活率和生化分析。当糖溶度低于2g/L的时候补加1%培养体积的450g/L葡萄糖浓缩液;
(3)细胞活率在60%左右结束培养。
(4)绘制细胞生长曲线图和活率图,比较CHO DG44细胞CD DG44-1、OPM-DG44CDM1、OPM-DG44 CDM2和OPM-DG44 CDM3培养基中表现。
二、实验结果
2.1OPM DG44 CDM1驯化传代图
如图1所示,CHO DG44细胞复苏于DG44-1培养基中传代5代后生长稳定,将细胞按7x105cells/ml密度接种于DG44-1:OPM-CDM1=1:1(体积比)的混合培养基中适应两代后生长较好,便按7x105cells/ml密度接种于OPM-CDM1培养基中驯化适应。经过96天OPM-CDM1的驯化培养,细胞活率可达到95%。
2.2CHO DG44细胞在CD DG44-1、OPM-DG44 CDM1、OPM-DG44 CDM2和OPM-DG44 CDM3培养基中传代培养
分别复苏DG44-1和OPM-DG44 CDM1培养的CHO DG44细胞于各自对应的培养基中,按5x105cells/ml分别接种DG44-1,OPM-DG44 CDM1,OPM-DG44 CDM2和OPM-DG44 CDM3传代培养。由于刚复苏的前两代细胞增殖不稳定,计算P4-P9代的倍增时间(PDT),根据PDT公式(PDT=Δt×Lg2/(LgNt-LgN0))。PDT(DG44-1)=34±4h,PDT(OPM-DG44 CDM1)=28±4h,PDT(OPM-DG44 CDM2)=31±3h,PDT(OPM-DG44 CDM3)=31±4h;细胞增殖越快及倍增时间越短,OPM-DG44 CDM1<OPM-DG44 CDM2<OPM-DG44 CDM3<DG44-1。即OPM-DG44 CDM1、OPM-DG44CDM2和OPM-DG44 CDM3比DG44-1更适合CHO DG44细胞增殖。
细胞在DG44-1仅流加葡萄糖的培养中维持到第6天活率便降为60%,细胞最高密度达1.94x106cells/ml;在OPM-DG44 CDM1中培养第9天活率降为60%,细胞最高密度达3.28x106cells/ml,OPM-DG44 CDM2和OPM-DG44 CDM3培养基中最高细胞密度分别为1.97x106cells/ml和2.99x106cells/ml;根据细胞密度最高峰可见OPM-DG44 CDM1>OPM-DG44 CDM3>OPM-DG44 CDM2>DG44-1更适应于CHO DG44细胞生长。
综上,经过OPM CDM1驯化的细胞,从细胞倍增时间,细胞最高密度和细胞培养时间上,OPM DG44 CDM1、OPM DG44 CDM2、OPM DG44 CDM3培养基都优于某知名品牌的CD DG44培养基,完全可以替代CD DG44培养基,更有利于CHO DG44细胞的培养。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
Claims (9)
3.根据权利要求1所述的CHO DG44培养基,其特征在于,每1L CHO DG44培养基中含有4~8mmol的L-Glutamine。
4.根据权利要求1所述的CHO DG44培养基,其特征在于,每1L CHO DG44培养基含有100~250g的F-68。
6.根据权利要求1所述的CHO DG44培养基,其特征在于,每1L CHO DG44培养基含有10~100umol的亚油酸。
7.根据权利要求1所述的CHO DG44培养基,其特征在于,还包括以下特征中的任一项或多项:(1)每1L CHO DG44培养基含有0.05~3mg的吡哆胺二盐酸盐;(2)每1L CHO DG44培养基含有50~200umol的次黄嘌呤;(3)每1L CHO DG44培养基含有8~32umol的胸腺嘧啶核苷。
8.如权利要求1~7任一所述CHO DG44培养基用于CHO DG44细胞悬浮培养的用途。
9.一种悬浮培养CHO DG44细胞的方法,包括步骤:将CHO DG44细胞置于如权利要求1~7任一所述CHO DG44培养基中进行培养。
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