CN115386536A - 用于培养Vero细胞的化学限定培养基、扩增病毒的方法和制备疫苗的方法 - Google Patents
用于培养Vero细胞的化学限定培养基、扩增病毒的方法和制备疫苗的方法 Download PDFInfo
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Abstract
本申请提出了一种用于培养Vero细胞的化学限定培养基,其包括:MEM基础培养基,所述MEM基础培养基中含有添加剂。根据本申请实施例,通过采用本申请不含血清和蛋白、化学成分明确的培养基进行Vero细胞培养和病毒的扩增尤其是疫苗病毒的扩增生产,能够实现低密度接种,在提高产能效率的同时降低血清等动物源性添加物的安全风险,并减少生产过程中杂蛋白对病毒疫苗纯化制剂的压力,本发明对于无血清培养基的工业化应用具有极强的现实意义。
Description
技术领域
本发明涉及生物技术领域,更具体的,本申请涉及针对特定细胞的培养基,尤其是用于培养Vero细胞的化学限定培养基、扩增病毒的方法和制备疫苗的方法。
背景技术
为了培养细胞、特别是真核细胞、更具体地说哺乳动物细胞,通常必须使用配有所需营养物质的特定培养基,这些营养物质用于实现细胞的有效生长并用于生产生物制品例如生物药物,诸如重组体蛋白、抗体、病毒、病毒抗原以及病毒状颗粒。
细胞培养基配方通常包括类似胎牛血清(FCS)、 数种动物衍生蛋白和/或牛源蛋白质水解物、以及来源于植物或者酵母的蛋白质水解物等添加剂。
但是,当细胞培养是为了人类或者动物的医学目的时,胎牛血清等所含有的成分会不可避免地引发不必要的免疫反应。另外,由于胎牛血清等成分复杂,批件差异比较明显,不利于进行大规模的产业化生产。所以,人们曾经进行了许多尝试,以实现不需要血清或者其他的动物性蛋白质化合物。
WO 96/15231公开了一种无血清培养基,由合成的最低必需培养基和酵母提取物组成, 用于脊椎动物细胞的增殖和病毒的生产工艺。
然而,酵母水解物是寡肽和其他未知组分及杂质的不明确的混合物。另外, 市场上可买到的许多水解物的品质变化极大。其结果是,在重组体蛋白或者病毒产品的生产中有很大的变化这与使用的许多水解物(“批间偏差”)有关。这种不利因素影响到细胞的增殖以及各细胞的蛋白表达。
为此,目前针对应用于人类或者动物医学的细胞培养技术仍有待改进。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选择。为此,本发明的一个目的在于提出一种能够有效地用于对Vero细胞进行培养的化学限定培养基。
由此,在本申请的第一方面,本申请提出了一种用于培养Vero细胞的化学限定培养基,其包括:MEM基础培养基,所述MEM基础培养基中含有下列添加剂:
组分 | 终浓度(mg/L) |
L-丙氨酸 | 41 |
L-盐酸精氨酸 | 98 |
L-天门冬氨酸 | 32.5 |
L-门冬酰胺 | 31 |
L-半胱氨酸盐酸盐一水 | 8 |
L-胱氨酸二盐酸盐 | 27.5 |
L-谷氨酸 | 47.5 |
L-谷氨酰胺 | 292.5 |
甘氨酸 | 23.08 |
L-盐酸组氨酸一水 | 24.5 |
L-羟脯氨酸 | 14 |
L-异亮氨酸 | 36 |
L-亮氨酸 | 39 |
L-盐酸赖氨酸 | 50 |
L-甲硫氨酸 | 15.5 |
L-苯丙氨酸 | 21 |
L-脯氨酸 | 25.5 |
L-丝氨酸 | 42 |
L-苏氨酸 | 30 |
L-色氨酸 | 7.5 |
L-缬氨酸 | 32 |
二水氯化钙 | 260 |
四水硝酸钙 | 25 |
无水硫酸镁 | 120 |
五水硫酸铜 | 0.000975 |
六水氯化镁 | 31.425 |
D-生物素 | 0.0675 |
次黄嘌呤 | 1.3425 |
亚油酸 | 0.0465 |
硫辛酸 | 0.1575 |
1,4-丁二胺二盐酸盐 | 0.045 |
维生素B2 | 0.1725 |
胸苷 | 0.2925 |
对氨基苯甲酸 | 0.195 |
氯化铝 | 0.000150056 |
乙酸钡 | 0.000431411 |
六水氯化钴 | 0.00018757 |
四水氯化锰 | 1.50056E-05 |
氟化钠 | 0.00056271 |
氯化亚锡 | 0.000131299 |
氯化镉 | 0.000131299 |
二氧化锗 | 5.6271E-05 |
亚硒酸钠 | 0.00375 |
九水偏硅酸钠 | 0.00075028 |
氯化镍 | 1.8757E-05 |
偏钒酸铵 | 3.7514E-05 |
溴化钾 | 4.69E-06 |
碘化钾 | 1.50056E-05 |
氯化铷 | 7.50E-07 |
钼酸铵 | 9.3785E-06 |
硝酸银 | 5.63E-07 |
腺嘌呤 | 0.056 |
盐酸鸟嘌呤 | 0.019 |
维生素D2 | 0.009 |
维生素A 醋酸酯 | 0.015 |
吐温80 | 3.803 |
5-氨基-4-甲酰胺咪唑 | 0.002 |
烟酸 | 0.004 |
2D-脱氧核糖 | 0.056 |
氢化可的松 | 0.002 |
肉豆蔻酸 | 0.015 |
油酸 | 0.015 |
棕榈酸 | 0.015 |
硬脂酸 | 0.015 |
D-无水葡萄糖 | 3000 |
重组人胰岛素 | 0.75 |
柠檬酸铁 | 3.75 |
在本文中所使用的术语“化学限定培养基”是指化学成分完全明确,组成和浓度固定的培养基。化学限定培养基具有批间差异小、可重复性强、结果稳定、无动物源风险等优点。然而,化学限定培养基的通用性差,针对不同的细胞系,通常需要开发不同的培养基配方,才能实现有效地细胞扩增或者培养。在本申请的完成过程中,本申请的发明人进行了大量的优化和筛选实验,发现在调整添加剂的配方时,并无规律可以遵循,结果也是无法预测的。根据本申请实施例,通过采用本申请不含血清和蛋白、化学成分明确的培养基进行Vero细胞培养和病毒的扩增尤其是疫苗病毒的扩增生产,能够实现低密度接种,能够在提高产能效率的同时降低血清等动物源性添加物的安全风险,并减少生产过程中杂蛋白对病毒疫苗纯化制剂的压力,本发明对于无血清培养基的工业化应用具有极强的现实意义。
在本申请的第二方面,本申请提出了一种扩增病毒的方法,其包括:
将Vero细胞接种在前面所描述的化学限定培养基中;
对所述Vero细胞进行培养,以便实现对所述Vero细胞的扩增;
在所述Vero细胞中接种病毒,以便实现对病毒进行扩增。
根据本申请的实施例,所述病毒包括选自狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒、SARS病毒、 甲型流感病毒、乙型流感病毒、慢病毒、罗斯河病毒、西尼罗河病毒、黄热病毒、FSME病毒、以及甲型肝炎病毒的至少之一。
在本申请的第三方面,本申请提出了一种制备疫苗的方法,其包括:
按照前面所描述的方法,对病毒进行扩增;
对所述病毒进行减毒或灭活处理,以便获得所述疫苗。
在本申请的第四方面,本申请提出了一种疫苗,所述疫苗是根据前面所述的方法制备的。
另外,本申请还提出了第四方面所描述的疫苗在制备药物中的用途,所述药物用于治疗或者预防病毒相关疾病。
需要说明的是,本申请是基于发明人的下列发现而完成的:
如前所述,Vero细胞作为哺乳动物细胞可以用于病毒的扩增,这些病毒可以用来制备疫苗来预防或者治疗人类或者动物的相关疾病。在采用常见的基础培养基例如MEM对Vero细胞进行培养时,需要添加血清,本发明的发明人发现采用血清培养会由于血清中蛋白成分的存在会造成不必要的免疫反应,同时其培养效率也并不高,倍增时间较长。为此本申请的发明人致力于开发出一款新型的化学限定培养基,即不存在任何蛋白质成分,以避免在应用于疫苗领域时不会引发不必要的免疫反应。为此目的,发明人结合之前在细胞培养领域积累的丰富经验,进行了大量的筛选和优化工作,意外地获得了一组配方,可以作为Vero细胞的化学限定培养基,该培养基能够有效地对Vero细胞进行培养,同时能够在多项指标上获得比血清培养更优异的表现。
如前所述,通过采用本申请不含血清和蛋白、化学成分明确的培养基进行Vero细胞培养和病毒的扩增尤其是疫苗病毒的扩增生产,能够在提高产能效率的同时降低血清等动物源性添加物的安全风险,并减少生产过程中杂蛋白对病毒疫苗纯化制剂的压力,本发明对于无血清培养基的工业化应用具有极强的现实意义。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
具体实施方式
下面详细描述本发明的实施例,这些实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
一、配制培养基
通过市售可得的方式,获取MEM培养基,并按照下表所列的成分和用量在MEM培养基中添加额外成分,配制化学限定培养基(分别命名为化学限定培养基1和化学限定培养基2,其中化学限定培养基1为本发明的化学限定培养基,化学限定培养基2为对照化学限定培养基),同时配制MEM+10%血清的培养基作为血清培养基对照,配制商品培养基1(健顺Vero细胞无血清培养基)、商品培养基2(源培Vero细胞无血清培养基)作为对照。其中,MEM培养基的配方如下所示:
原料名称 | mg/L |
L-丙氨酸 | 8.9 |
L-盐酸精氨酸 | 126 |
L-门冬酰胺 | 13.2 |
L-天门冬氨酸 | 13.2 |
L-胱氨酸二盐酸盐 | 31 |
L-谷氨酸 | 14.7 |
L-谷氨酰胺 | 292 |
甘氨酸 | 7.5 |
L-盐酸组氨酸一水 | 42 |
L-异亮氨酸 | 52 |
L-亮氨酸 | 52 |
L-盐酸赖氨酸 | 73 |
L-甲硫氨酸 | 15 |
L-苯丙氨酸 | 32 |
L-脯氨酸 | 11.5 |
L-丝氨酸 | 10.5 |
L-苏氨酸 | 48 |
L-色氨酸 | 10 |
L-酪氨酸二钠盐无水 | 52 |
L-缬氨酸 | 46 |
苯酚红 | 10 |
二水氯化钙 | 265 |
氯化钾 | 400 |
无水硫酸镁 | 98 |
氯化钠 | 6800 |
一水磷酸二氢钠 | 140 |
D-无水葡萄糖 | 1000 |
D-泛酸钙 | 1 |
叶酸 | 1 |
氯化胆碱 | 1 |
肌醇 | 2 |
烟酰胺 | 1 |
盐酸吡哆醛 | 1 |
维生素B2 | 0.1 |
维生素B1 | 1 |
化学限定培养基1和化学限定培养基2的具体配方如下表所示:
二、细胞培养和检测
在前面得到化学限定培养基1、化学限定培养基2、血清培养基、商品培养基1、商品培养基2后,按照下列方式对Vero细胞进行培养:
将Vero细胞(P171)按照1.5×106cells接种于T75方瓶(透气盖),置于37℃、5%CO2的培养箱内进行培养。培养5d后用TrypLE™ Express Enzyme (1X)胰酶进行消化计数,随即再按上述细胞数接种至T75方瓶中,进行连续传代,计算细胞倍增时间。细胞倍增时间(doubling time,又称DT),细胞倍增所需要的时间,是判断细胞生长是否旺盛的重要指标,可以用公式(DT=t*[lg2/(lgNt-lgN0)])计算。t为培养时间,N0为接种细胞数,Nt为t时间后的细胞数。
三、结果和分析
采用化学限定培养基1、化学限定培养基2、血清培养基、商品培养基1、商品培养基2进行五代培养的相关参数检测结果总结如下:
通过上表可以看出,当进行低密度细胞接种时,采用化学限定培养基1进行细胞培养的DT时间为32.7h,而采用血清培养基的DT时间为37.21h,相较于血清培养基,化学限定培养基1的DT时间缩短了13.79%;商品培养基1比血清培养基缩短了11.04%,商品培养基2比血清培养基缩短了9.28%;化学限定培养基2的DT时间比商品培养基1、2都长。化学限定培养基1和化学限定培养基2采用了相同的成分,但化学限定培养基1和化学限定培养基2中各成分的配比不同,与化学限定培养基2(DT时间为35.19)相比,化学限定培养基1的DT时间缩短了7.61%。
在不添加血清的情况下,化学限定性培养基1实现了比添加血清还要好的培养效果,尤其在低密度接种的条件下,细胞能持续进行扩增,达到疫苗生产过程中细胞扩增要求,对于疫苗生产行业具有重要的意义。
综上,可进一步证明,本申请的化学限定培养基1是经过大量的试验筛选得到的,其组成对于培养效果(例如DT时间)而言十分重要,同时也是没有规律可以遵循的。因此,采用本申请的化学限定培养基1,可以显著提高Vero细胞的培养效率,缩短倍增时间。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、 “示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (6)
1.一种用于培养Vero细胞的化学限定培养基,其特征在于,包括:
MEM基础培养基,所述MEM基础培养基中含有下列添加剂:
终浓度为41mg/L的L-丙氨酸,
终浓度为98mg/L的L-盐酸精氨酸,
终浓度为32.5mg/L的L-天门冬氨酸,
终浓度为31mg/L的L-门冬酰胺,
终浓度为8mg/L的L-半胱氨酸盐酸盐一水,
终浓度为27.5mg/L的L-胱氨酸二盐酸盐,
终浓度为47.5mg/L的L-谷氨酸,
终浓度为292.5mg/L的L-谷氨酰胺,
终浓度为23.08mg/L的甘氨酸,
终浓度为24.5mg/L的L-盐酸组氨酸一水,
终浓度为14mg/L的L-羟脯氨酸,
终浓度为36mg/L的L-异亮氨酸,
终浓度为39mg/L的L-亮氨酸,
终浓度为50mg/L的L-盐酸赖氨酸,
终浓度为15.5mg/L的L-甲硫氨酸,
终浓度为21mg/L的L-苯丙氨酸,
终浓度为25.5mg/L的L-脯氨酸,
终浓度为42mg/L的L-丝氨酸,
终浓度为30mg/L的L-苏氨酸,
终浓度为7.5mg/L的L-色氨酸,
终浓度为32mg/L的L-缬氨酸,
终浓度为260mg/L的二水氯化钙,
终浓度为25mg/L的四水硝酸钙,
终浓度为120mg/L的无水硫酸镁,
终浓度为0.000975mg/L的五水硫酸铜,
终浓度为31.425mg/L的六水氯化镁,
终浓度为0.0675mg/L的D-生物素,
终浓度为1.3425mg/L的次黄嘌呤,
终浓度为0.0465mg/L的亚油酸,
终浓度为0.1575mg/L的硫辛酸,
终浓度为0.045mg/L的1,4-丁二胺二盐酸盐,
终浓度为0.1725mg/L的维生素B2,
终浓度为0.2925mg/L的胸苷,
终浓度为0.195mg/L的对氨基苯甲酸,
终浓度为0.000150056mg/L的氯化铝,
终浓度为0.000431411mg/L的乙酸钡,
终浓度为0.00018757mg/L的六水氯化钴,
终浓度为1.50E-05mg/L的四水氯化锰,
终浓度为0.00056271mg/L的氟化钠,
终浓度为0.000131299mg/L的氯化亚锡,
终浓度为0.000131299mg/L的氯化镉,
终浓度为5.63E-05mg/L的二氧化锗,
终浓度为0.00375mg/L的亚硒酸钠,
终浓度为0.00075028mg/L的九水偏硅酸钠,
终浓度为1.88E-05mg/L的氯化镍,
终浓度为3.75E-05mg/L的偏钒酸铵,
终浓度为4.69E-06mg/L的溴化钾,
终浓度为1.50E-05mg/L的碘化钾,
终浓度为7.50E-07mg/L的氯化铷,
终浓度为9.38E-06mg/L的钼酸铵,
终浓度为5.63E-07mg/L的硝酸银,
终浓度为0.056mg/L的腺嘌呤,
终浓度为0.019mg/L的盐酸鸟嘌呤,
终浓度为0.009mg/L的维生素D2,
终浓度为0.015mg/L的维生素A 醋酸酯,
终浓度为3.803mg/L的吐温80,
终浓度为0.002mg/L的5-氨基-4-甲酰胺咪唑,
终浓度为0.004mg/L的烟酸,
终浓度为0.056mg/L的2D-脱氧核糖,
终浓度为0.002mg/L的氢化可的松,
终浓度为0.015mg/L的肉豆蔻酸,
终浓度为0.015mg/L的油酸,
终浓度为0.015mg/L的棕榈酸,
终浓度为0.015mg/L的硬脂酸,
终浓度为3000mg/L的D-无水葡萄糖,
终浓度为0.75mg/L的重组人胰岛素,
终浓度为3.75mg/L的柠檬酸铁。
2.一种扩增病毒的方法,其特征在于,包括:
将Vero细胞接种在权利要求1所述的化学限定培养基中;
对所述Vero细胞进行培养,以便实现对所述Vero细胞的扩增;
在所述Vero细胞中接种病毒,以便实现对病毒进行扩增。
3.根据权利要求2所述的方法,其特征在于,所述病毒包括选自狂犬病毒、流感病毒、冠状病毒、法氏囊病毒、登革热病毒、脑炎病毒、痘病毒、正粘病毒、副粘病毒、反向病毒、披膜病毒、黄病毒、肠道病毒、细小核糖核酸病毒、嵌沙样病毒、疱疹病毒、腺病毒、牛痘病毒、SARS病毒、甲型流感病毒、乙型流感病毒、慢病毒、罗斯河病毒、西尼罗河病毒、黄热病毒、FSME病毒、以及甲型肝炎病毒的至少之一。
4.一种制备疫苗的方法,其特征在于,包括:
按照权利要求2或3的方法,对病毒进行扩增;
对所述病毒进行减毒或灭活处理,以便获得所述疫苗。
5.一种疫苗,其特征在于,所述疫苗是根据权利要求4所述的方法制备的。
6.根据权利要求5所述的疫苗在制备药物中的用途,所述药物用于治疗或者预防病毒相关疾病。
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