CN113403281B - 一种提高抗体半乳糖基化水平的细胞培养方法 - Google Patents
一种提高抗体半乳糖基化水平的细胞培养方法 Download PDFInfo
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Abstract
本发明公开了一种提高抗体半乳糖基化水平的细胞培养方法,该方法以CHO细胞为表达系统,通过在细胞培养过程中添加半乳糖、尿嘧啶核苷和四水氯化锰后能有效提高抗体半乳糖基化水平,并且可以提高抗体的活性。
Description
技术领域
本发明涉及一种细胞培养方法,具体涉及一种提高抗体半乳糖基化水平和抗体活性的细胞培养方法。
背景技术
CHO细胞(中国仓鼠卵巢细胞,Chinese Hamster Overy)是生产抗体药物最为广泛的宿主细胞之一,相对于其他细胞表达系统,CHO细胞表达系统能够与外源基因稳定地整合,同时细胞内部含有真核细胞复杂的翻译后修饰系统,使得表达的抗体药物在抗体结构、免疫原性、糖基化类型和方式等与人源单克隆抗体较为相似,因此能够保证抗体的药性和药效,同时CHO细胞自身分泌的内源性蛋白较少,有利于抗体后续的分离纯化,常见的CHO细胞包括CHO-S、CHO-K1、CHO-GS以及CHO-DG44细胞等。
抗体(包括Fc融合蛋白)药物发挥靶向性消灭靶细胞(如肿瘤细胞)的重要作用方式(效应功能)主要有两种:ADCC(antibody-dependent,cell-mediated cytotoxicity,抗体依赖的细胞介导的细胞毒性作用)和CDC(complement dependent cytotoxicity,补体依赖的细胞毒性作用)。其中,CDC是通过特异性抗体与细胞膜表面相应抗原结合,形成复合物而激活补体经典途径,所形成的攻膜复合物对靶细胞发挥裂解效应。
因此,CDC的作用机制是抗体药物研发的体外研究、评价、筛选的重要指标。此外该效应与糖基化修饰水平和类型还具有直接关系。目前,针对CDC抗体效应功能的抗体工程主要集中在对Fc的改造上,以增强抗体与FcγR的相互作用,最终增加效应功能。
然而,通过基因手段对Fc的改造来改变抗体的糖型或糖基化水平的方法周期较长,并且对于表达的抗体的安全性、稳定性和有效性都需要长时间的验证。除了通过对抗体Fc的基因改造改变CDC效应外,还可以依赖上游或下游生产工艺的改进来提高抗体的CDC活性。相对比基因手段来说,改进上游或下游生产工艺的开发时间更短,并且可以降低总体开发成本。
因此,目前仍需开发一种改进的抗体生产工艺,以改变糖基化谱,提高抗体的CDC活性,满足实际生产的需求。
发明内容
本发明提供了一种改进的工艺方法,该方法采用CHO表达系统的细胞培养工艺,通过在细胞培养的补料过程中添加半乳糖、四水氯化锰和尿嘧啶核苷,能够提高抗体的半乳糖基化水平,提高抗体CDC活性,且简便易操作。
为了实现上述目的,在一些实施方案中,本发明以CHO细胞为表达系统,在补料过程中添加半乳糖、四水氯化锰和尿嘧啶核苷;
进一步地,在一些实施方案中,本发明以CHO-S、CHO-K1、CHO-GS或CHO-DG44细胞为表达系统,在补料过程中添加半乳糖、四水氯化锰和尿嘧啶核苷;
更进一步地,在一些实施方案中,本发明以CHO-K1细胞为表达系统,在补料过程中添加半乳糖、四水氯化锰和尿嘧啶核苷。
在一些实施方案中,半乳糖在补料过程中添加总量为3.30-9.40g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为0.92-2.60g/kg初始培养液、四水氯化锰在补料过程中添加总量为1.48-4.20mg/kg初始培养液;
进一步地,所述半乳糖在补料过程中添加总量为3.45-9.20g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为0.94-2.51g/kg初始培养液、四水氯化锰在补料过程中添加总量为1.51-4.03mg/kg初始培养液;
更进一步地,所述半乳糖在补料过程中添加总量为5.18-9.20g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为1.41-2.51g/kg初始培养液、四水氯化锰在补料过程中添加总量为2.27-4.03mg/kg初始培养液;
优选地,在一些实施方案中,半乳糖在补料过程中添加总量为3.45g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为0.94g/kg初始培养液、四水氯化锰在补料过程中添加总量为1.51mg/kg初始培养液。
优选地,在一些实施方案中,半乳糖在补料过程中添加总量为5.18g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为1.41g/kg初始培养液、四水氯化锰在补料过程中添加总量为2.27mg/kg初始培养液。
优选地,在一些实施方案中,半乳糖在补料过程中添加总量为6.90g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为1.88g/kg初始培养液、四水氯化锰在补料过程中添加总量为3.02mg/kg初始培养液。
优选地,在一些实施方案中,半乳糖在补料过程中添加总量为9.20g/kg初始培养液,尿嘧啶核苷在补料过程中添加总量为2.51g/kg初始培养液、四水氯化锰在补料过程中添加总量为4.03mg/kg初始培养液。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入摇瓶或细胞反应器中,接种任一上述细胞进行培养;
b)将上述半乳糖、尿嘧啶核苷和四水氯化锰均分成五等份,分别在培养的第3、5、7、9、11天加入,并添加补料培养基;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第13-15天结束;
所述方法能使收获的抗体糖基化水平提高至少199%;
进一步地,所述方法能使收获的抗体糖基化水平提高218%;
再进一步地,所述方法能使收获的抗体糖基化水平提高227%;
更进一步地,所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,本发明所述补料培养基为一种或多种的组合。
在一些实施方案中,本发明所述补料培养基为补料培养基A和补料培养基B。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加补料培养基A、补料培养基B和3.30-9.40g/kg初始培养液的半乳糖,0.92-2.60g/kg初始培养液的尿嘧啶核苷、1.48-4.20mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高至少199%;
进一步地,所述方法能使收获的抗体糖基化水平提高218%;
再进一步地,所述方法能使收获的抗体糖基化水平提高227%;
更进一步地,所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和3.30-9.40g/kg初始培养液的半乳糖,0.92-2.60g/kg初始培养液的尿嘧啶核苷、1.48-4.20mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高至少199%;
进一步地,所述方法能使收获的抗体糖基化水平提高218%;
再进一步地,所述方法能使收获的抗体糖基化水平提高227%;
更进一步地,所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和3.45-9.20g/kg初始培养液的半乳糖,0.94-2.51g/kg初始培养液的尿嘧啶核苷、1.51-4.03mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高至少199%;
进一步地,所述方法能使收获的抗体糖基化水平提高218%;
再进一步地,所述方法能使收获的抗体糖基化水平提高227%;
更进一步地,所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和5.18-9.20g/kg初始培养液的半乳糖,1.41-2.51g/kg初始培养液的尿嘧啶核苷、2.27-4.03mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高218%;
再进一步地,所述方法能使收获的抗体糖基化水平提高227%;
更进一步地,所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和3.45g/kg初始培养液的半乳糖,0.94g/kg初始培养液的尿嘧啶核苷、1.51mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高199%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和5.18g/kg初始培养液的半乳糖,1.41g/kg初始培养液的尿嘧啶核苷、2.27mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高218%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和6.90g/kg初始培养液的半乳糖,1.88g/kg初始培养液的尿嘧啶核苷、3.02mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高227%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入到摇瓶中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和9.20g/kg初始培养液的半乳糖,2.51g/kg初始培养液的尿嘧啶核苷、4.03mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束;
所述方法能使收获的抗体糖基化水平提高232%。
在一些实施方案中,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入细胞反应器中,接种细胞进行培养;
b)添加25.0%(w/w)初始培养液重量的补料培养基A,1.5%(w/w)初始培养液重量的补料培养基B和6.90g/kg初始培养液的半乳糖,1.88g/kg初始培养液的尿嘧啶核苷、3.02mg/kg初始培养液的四水氯化锰;第3、5、7、9、11天,每天将上述物质均分成5等份进行补加;
c)培养至第5天降温到33.0℃,维持这一温度继续培养至第14天结束。
在一些实施方案中,上述基础培养基选自Dynamis AGT Medium或ActiPro。
在一些实施方案中,上述补料培养基A选自Cell Boost 7a,补料培养基B选自CellBoost 7b。
在一个实施方案中,所述抗体为单特异性抗体或双特异性抗体或多特异性抗体。
在一个实施方案中,所述抗体为单克隆抗体,进一步地,所述单克隆抗体为抗CD20单克隆抗体。
在一个实施方案中,本发明抗CD20单克隆抗体包含重链可变区VH和轻链可变区VL,其中所述VH包含互补决定区域HCDR1、HCDR2和HCDR3,其中所述的HCDR1包含SEQ ID NO:5所示的氨基酸序列、HCDR2包含SEQ ID NO:6所示的氨基酸序列和HCDR3包含SEQ ID NO:7所示的氨基酸序列;其中所述VL包含互补决定区域LCDR1、LCDR2和LCDR3,其中所述的LCDR1包含SEQ ID NO:8所示的氨基酸序列、LCDR2包含SEQ ID NO:9所示的氨基酸序列和LCDR3包含SEQ ID NO:10所示的氨基酸序列。
在一个实施方案中,本发明的抗CD20单克隆抗体所述HCDR1包含SYNMH(SEQ IDNO:5)所示的氨基酸序列、HCDR2包含AIYPGNGDTSYNQKFKG(SEQ ID NO:6)所示的氨基酸序列和HCDR3包含STYYGGDWYFNV(SEQ ID NO:7)所示的氨基酸序列;其中所述LCDR1包含RASSSVSYIH(SEQ ID NO:8)所示的氨基酸序列、LCDR2包含ATSNLAS(SEQ ID NO:9)所示的氨基酸序列和LCDR3包含QQWTSNPPT(SEQ ID NO:10)所示的氨基酸序列。
在一个实施方案中,本发明的抗CD20单克隆抗体为利妥昔单抗。
本发明例示的抗CD20单克隆抗体的重链:
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)
本发明例示的抗CD20单克隆抗体的轻链:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2)
本发明例示的抗CD20单克隆抗体的重链可变区:
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YYCARSTYYGGDWYFNVWGAGTTVTVSA(SEQ ID NO:3)。
本发明例示的抗CD20单克隆抗体的轻链可变区:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIY ATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGG GTKLEIK(SEQ ID NO:4)
发明详述
在详细描述本发明之前,应了解,本发明不受限于本说明书中的特定方法及实验条件,因为所述方法以及条件是可以改变的。另外,本文所用术语仅是供说明特定实施方案之用,而不意欲为限制性的。
定义
除非另有定义,否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的,下文定义了以下术语。
术语“初始培养液”是指首次加入培养基并完成细胞接种时的培养液,其中细胞接种密度通常可以为0.8-1.2×106细胞/ml。
术语“单克隆抗体”指具有单一氨基酸组成的抗体分子的制备物,而不指其产生的方法。单克隆抗体或其抗原结合片段可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或此类或其它本领域已知的技术的组合来产生。
术语“单特异性”抗体表示这样的抗体,其具有一个或多个结合位点,每一结合位点结合至相同抗原的相同表位。
术语“双特异性”意指抗体能够特异性结合至少两种不同的抗原决定簇,例如各自由一对抗体重链可变结构域(VH)和抗体轻链可变结构域(VL)形成的两个结合位点与不同抗原或同一抗原上的不同表位结合。此类双特异性抗体为1+1形式。其他双特异性抗体形式是2+1形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的一个结合位点)或2+2形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的两个结合位点)。通常,双特异性抗体包含两个抗原结合位点,每个抗原结合位点特异于不同的抗原决定簇。
术语“多特异性”抗体指具有至少两个抗原结合位点的抗体,所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链的结构域或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构,其中每个可变结构域包含四个保守的构架区(FR)和三个互补决定区。(参见,例如,Kindt等Kuby Immunology,6th ed.,W.H.FreemanandCo.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外,可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体,以分别筛选互补VL或VH结构域的文库。参见,例如,Portolano等,J.Immunol.150:880-887(1993);Clarkson等,Nature 352:624-628(1991)。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用),是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3,从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3,而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中,各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定,所述指派系统包括例如:基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883,Al-Lazikani等人,“Standard conformations for the canonical structures ofimmunoglobulins”,Journal of Molecular Biology,273,927-948(1997)),基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London),国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/),以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
除非另有说明,否则在本发明中,当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时,是指根据Kabat编号系统(Kabat等人,Sequences ofProteins of Immunological Interest,5th Ed.Public Health Service,NationalInstitutes of Health,Bethesda,Md.(1991))的编号位置。
附图说明
图1是实施例1中的细胞补料批次实验的细胞活率。
图2是实施例1中的细胞补料批次实验的糖基化含量图。
图3是实施例中抗体的糖基结构图。
图4实施例2中的细胞补料批次实验的细胞活率。
图5是实施例2中的细胞补料批次实验的活细胞密度。
图6是实施例2中的细胞补料批次实验的抗体表达量图。
图7是实施例2中的细胞补料批次实验的糖基化含量图。
具体实施方式
通过以下实施例向本领域普通技术人员提供如何制备和使用本发明的方法和组合物的完整公开和描述,并且不旨在限制本发明所涵盖的范围。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本发明实施例中的中国仓鼠卵巢细胞系CHO-K1购自ATCC,货号:CCL-61TM。
本发明中所用的基础培养基Dynamis AGT Medium购自Gibco,货号A26175-03;ActiPro购自HyClone,货号SH31037.04;
本发明中所用的补料培养基:Cell Boost 7a购自HyClone,货号:SH31026.05;Cell Boost 7b购自HyClone,货号:SH31027.04CN;
本发明中所用的半乳糖购自美国Spectrum,货号:G5924C;氯化锰购自美国Spectrum,货号:M1106;尿嘧啶核苷购自美国Ameresco,货号:0975;
本发明中实施例中使用的抗CD20抗体为利妥昔单抗。
实施例1:一种表达利妥昔单抗的CHO-K1细胞摇瓶培养
在细胞培养试验中,第0天将80mL基础培养基(Dynamis AGT Medium)分别加入到250mL摇瓶中,将CHO-K1细胞以1.0×106cells/mL进行接种,在36.5℃±0.5℃,6%±1%CO2,130±10r/min条件下培养,培养第5天开始降低温度并控制在33℃,维持该温度至细胞培养的第14天,然后结束培养,进行收获。在第3、5、7、9、11天分别进行补料,5天共添加25.0%(w/w)初始培养液重量的补料培养基A(Cell Boost 7a),1.5%(w/w)初始培养液重量的补料培养基B(Cell Boost 7b)和3.45g/kg初始培养液的半乳糖,0.94g/kg初始培养液的尿嘧啶核苷,1.51mg/kg初始培养液的氯化锰(A组);或5.18g/kg初始培养液的半乳糖,1.41g/kg初始培养液的尿嘧啶核苷,2.27mg/kg初始培养液的氯化锰(B组);或6.90mg/kg初始培养液的半乳糖,1.88g/kg初始培养液的尿嘧啶核苷,3.02mg/kg初始培养液的氯化锰(C组);或9.20g/kg初始培养液的半乳糖,2.51g/kg初始培养液的尿嘧啶核苷,4.03mg/kg初始培养液的氯化锰(D组)。第3、5、7、9、11天,每天将上述物质均分成5等份进行补加。在第3、5、7、9、11天根据生化分析仪检测葡萄糖含量,葡萄糖低于3.0g/L时,补加葡萄糖浓缩液使细胞液中葡萄糖浓度提高到4.0g/L。在第3、5、7、9、11天测定细胞活率,第14天检测糖基化(G0F、G1F、G2F)水平及CDC活性。
细胞活率用细胞活力分析仪(厂家:美国BECKMAN,货号:Vi-CELL XR)进行测定;抗体糖基用PNGase酶进行酶解,按照试剂包的说明书(厂家NEB,货号:P0704L)进行酶解,然后采用HPLC-MS(厂家:美国Agilent,货号:Agilent 1260)检测糖基化(G0F、G1F、G2F)水平;抗体CDC活性用酶标仪(厂家:美国Thermo、货号:MULTISKAN FC)进行检测,G0F、G1F和G2F的结构图见图3。本发明中糖基化水平主要考察G0F、G1F和G2F的含量,其中G0F表示抗体Fc没有半乳糖基化,G1F表示Fc段糖基化末端有1个半乳糖基化修饰,G2F表示Fc糖基化末端有2个半乳糖基化修饰。已有研究表明,增加抗体半乳糖基化(即提高G1F和G2F的含量,降低G0F的含量)可以提高CDC活性,原因可能是由于糖基化修饰后的糖链结构能够维持抗体的空间构想,稳定抗体的结构,保护抗体不受某些酶的水解。(Jason Hodoniczky,Yuan Zhi Zhengand David C.James,Control of Recombinant Monoclonal Antibody EffectorFunctions by Fc N-Glycan Remodeling in Vitro,Biotechnol.Prog.2005及王冲,现代免疫学,单克隆抗体糖基化修饰研究进展,2017年第37卷第3期)。
本实验得出,与对照(补料过程不添加半乳糖、氯化锰、尿嘧啶核苷)相比,补料过程中随着半乳糖、氯化锰、尿嘧啶核苷的添加量的增大,G2F和G1F含量逐渐升高,G0F含量逐渐降低,抗体CDC的相对活性逐渐增强(见图2和表1)。此外,随着半乳糖、氯化锰、尿嘧啶核苷的添加量的增大,与对照相比,细胞活率无显著的差异,细胞活率在培养结束的第14天仍能维持90%以上(见图1)。综上,在补料过程中加入半乳糖、氯化锰、尿嘧啶核苷可以显著提高G1F和G2F的含量,降低G0F的含量,半乳糖基化水平A组提高了199%,B组提高了218%,C组提高了227%,D组提高了232%,并且抗体CDC活性均有所提高(见表1);此外,补料过程中加入半乳糖、氯化锰、尿嘧啶核苷不影响细胞的活性。
表1:抗体糖基化水平及CDC相对活性
注:表中各物质含量误差允许范围为±2%;摇瓶培养初始培养液重量为0.08kg。
实施例2:一种表达利妥昔单抗的CHO-K1细胞反应器培养
本实验选自实施例1的C组进行放大试验。
在细胞培养试验中,第0天在细胞培养实验中,按密度1.0×106个/ml接种CHO-K1细胞至2L细胞反应器(厂家:德国Sartrious,货号:BIOSTATR A)中,DO设置为40%,pH控制为6.90±0.25,培养温度36.5℃,培养至第5天降温到33.0℃,维持该温度至细胞培养的第14天,然后结束培养,进行收获。培养至第3、5、7、9、11天分别进行补料,5天共添加25.0%(w/w)初始培养液重量的补料培养基A(Cell Boost 7a),1.5%(w/w)初始培养液重量的补料培养基B(Cell Boost 7b)和6.90g/kg初始培养液的半乳糖,1.88g/kg初始培养液的尿嘧啶核苷,3.02mg/kg初始培养液的氯化锰。第3、5、7、9、11天,每天将上述物质均分成5等份进行补加。每天根据生化分析仪检测葡萄糖含量,在葡萄糖低于3.0g/L时,补加葡萄糖浓缩液使细胞液中葡萄糖浓度提高到4.0g/L。根据通气产生的泡沫情况,泡沫高于液面5cm时手动加入10% ADCF Antifoam Solution(厂家:美国Hyclone,货号:SH30897.01)总量不能超过50ppm。每天测定细胞活率和细胞密度,从第6天开始检测抗体表达量,第14天检测糖基化(G0F、G1F、G2F)水平。
细胞活率和细胞密度用细胞活力分析仪(厂家:美国BECKMAN,货号:Vi-CELL XR)进行测定,抗体浓度通过生化分析仪(厂家:瑞士Roche,货号:CEDEX BIO HT)进行测定,抗体糖基用PNGase酶进行酶解,按照试剂包的说明书(厂家NEB,货号:P0704L)进行酶解,然后采用HPLC-MS(厂家:美国Agilent,货号:Agilent 1260)检测糖基化(G0F、G1F、G2F)水平。实验重复三次,对应图4-7中的实验一、实验二和实验三,实验一、二和三的初始培养液重量为1.2kg。
表2三批实验G0F、G1F和G2F的含量
实验得出,在反应器中三批实验细胞生长情况基本一致,最高活细胞密度均在29.0×106个/ml左右,无显著差异(见图4),细胞活率的变化趋势也基本一致,直至培养结束时细胞活率均维持在90%以上,三批实验无显著差异(见图5)。抗体最终表达量最终均能达到4.0mg/mL以上,且批次间抗体表达量并没有出现明显的差异(见图6)。此外,添加一定比例的半乳糖、氯化锰、尿嘧啶核苷后,G1F+G2F与G0F含量的比值与放大前实验相同,三批放大实验间无差异(见图7和表2),且能够维持细胞密度、活率、抗体产量等不受到影响。进而验证了该方法具有很好的稳定性,能够满足实际放大生产的要求。
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Claims (2)
1.一种提高抗体半乳糖基化水平的细胞培养方法,其特征在于,所述细胞培养方法以CHO-K1细胞为表达系统,在补料过程中添加半乳糖、尿嘧啶核苷和四水氯化锰;所述抗体为利妥昔单抗;所述半乳糖添加总量为9.20g/kg初始培养液,尿嘧啶核苷添加总量为2.51g/kg初始培养液、四水氯化锰添加总量为4.03mg/kg初始培养液;
并将所述半乳糖、尿嘧啶核苷和四水氯化锰均分成五等份,分别在培养的第3、5、7、9、11天加入,并添加补料培养基CellBoost 7a和补料培养基CellBoost 7b。
2.根据权利要求1所述的方法,其特征在于,所述的细胞培养方法包括以下步骤:
a)将基础培养基加入摇瓶或细胞反应器中,接种细胞进行培养;
b)培养至第5天降温到33.0℃,维持这一温度继续培养至第13-15天结束;
所述方法能使收获的抗体糖基化水平提高至少199%。
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