CN101052708B - 哺乳动物细胞的无血清细胞培养基 - Google Patents
哺乳动物细胞的无血清细胞培养基 Download PDFInfo
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Abstract
本发明涉及在无血清培养基中培养哺乳动物细胞制造蛋白质的方法。
Description
发明领域
本发明涉及在无血清培养条件下培养哺乳动物细胞的领域,具体指生产重组蛋白质,例如人生长激素(hGH)的细胞的培养。
发明背景
本发明涉及用无血清培养基培养哺乳动物细胞使之生长和维持。
目前已广泛采用细胞培养来生产多种生物学活性产品,如病毒疫苗、单克隆抗体,非抗体免疫调节剂、多肽生长因子、激素、酶、肿瘤特异性抗原等。这些产品可通过正常的或转化的和经基因工程改造的细胞产生。
以往,用于细胞培养的培养基需添加血清,作为生产生物活性产品的所有哺乳动物细胞系生长和维持所需的通用营养物质。血清含有激素、生长因子、载体蛋白质、粘附和扩散因子、营养成分、微量元素等。培养基通常含有高达约10%的动物血清,例如胎牛血清(FBS),也称为胎犊血清(FCS)。
虽然广泛采了用血清,但其有许多缺点。细胞产生的感兴趣所需蛋白质的有限的量受到血清所含的许多高水平蛋白质的干扰。在下游加工中,例如感兴趣蛋白质纯化时,必须将源于血清的这些蛋白质与所述蛋白质产物分离,因而这使得工艺复杂化并提高了成本。
一种具有长时间潜伏期或孵化期的牛传染性神经退行性疾病BSE(牛海绵状脑病)的出现,引起了对利用动物来源血清生产生物活性产物的重新关注。
因此,极其需要开发无动物血清的替代培养基来支持生产生物活性产品的细胞生长和维持该细胞。
通常,细胞培养基包含多种不同种类的成分,例如氨基酸、维生素、盐、脂肪酸和其它成分:
-氨基酸:例如美国专利US6,048,728(Inlow等)公开了可用于细胞培养基中的以下氨基酸:丙氨酸、精氨酸、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙胺酸、脯氨酸、丝氨酸、色氨酸、酪氨酸、苏氨酸和缬氨酸。
-维生素:例如美国专利US2003/0096414(Ciccarone等)或US5,811,299(Renner等)描述了可用于细胞培养基的以下维生素:生物素、泛酸盐、氯化胆碱、叶酸、肌醇、烟酰胺、维生素B6、核黄素、维生素B12、硫胺、腐胺。
-盐:例如美国专利US6,399,381(Blum等)公开的一种培养基包含:CaCl2、KCl、MgCl2、NaCl、NaH2PO4、Na2HPO4、Na2SeO3、CuSO4、ZnCl2。另一美国专利文献US2003/0153042(Arnold等)例子中公开了可用于培养基的无机盐,该培养基包含:CaCl2、KCl、MgCl2、NaCl、NaH2PO4、Na2HPO4、CuCl2·2H2O、ZnCl2。
-脂肪酸:已知可用于培养基的脂肪酸有:花生四烯酸、亚油酸、油酸、月桂酸、肉豆蔻酸和甲基-β-环糊精。例如参见美国专利US5,045,468(Darfler)。应注意环糊精本身不是脂肪,但是它能够与脂质形成复合物,因此在细胞培养基中用它来溶解脂肪。
-其它成分,具体指用于构成无血清细胞培养基的化合物,例如包括葡萄糖、谷氨酰胺、丙酮酸钠、胰岛素或乙醇胺(例如欧洲专利EP274445),或保护剂如Pluronic F68。PluronicF68(也称为泊洛沙姆(Poloxamer)188)是一种环氧乙烷(EO)与环氧丙烷(PO)的嵌段共聚物。
标准的“基础培养基”是本领域技术人员熟知的。这些培养基包含上述培养基诸成分。广泛应用的这种培养基的例子有达尔伯克改良伊格尔培养液(Dulbecco’s Modified Eagle’s Medium,DMEM),DMEMF12(1:1),Ham’s Nutrientmixture F-10,Roswell Park Memorial Institute Medium(RPMI),MCDB 131,或William’s Medium E。这些商品化培养基可以从例如Gibco、Invitrogen获得。
金属元素,例如锌(Zn)和铜(Cu)参与代谢反应(Vallee和Falchuk,1993,或Lindner,1991)。
锌是许多大分子的结构与功能和许多酶反应所必须的元素,其在蛋白质正确折叠中发挥着催化、助催化或结构性作用。在生物胺合成和单胺氧化酶代谢所必须的两种辅酶:5’-磷酸吡哆醛和黄素腺嘌呤二核苷酸(FAD)的合成中,Zn-ATP是必需的。
锌在保护生物结构免受自由基损伤中具有活性,可能是由于以下几种因素:它能保持足够水平的金属蛋白质也即自由基清除剂、它构成超氧化物歧化酶的一种主要成分、构成巯基保护剂、和能防止化学基团与铁反应形成自由基。
此外,Zn的存在防止了脂质的过度氧化。锌也是微管蛋白质聚合的效应物并在肌动蛋白质丝的体外形成和稳定中起作用。锌还是DNA结合蛋白质锌指结构基序的一种成分,该基序是转录蛋白质的共同基序。
锌离子主要以与蛋白质、核酸复合形式存在,它参与中间代谢、遗传信息的传递和表达调节、肽激素的贮藏、合成和作用、及染色质和生物膜结构的维持。
铜也是对许多细胞酶功能有重要作用的微量元素。铜可采取不同的氧化还原状态,氧化态Cu(II),或还原态Cu(I),在酶的氧化还原化学反应中作为催化辅助因子而在细胞生理学中发挥关键作用。它在一组铜氧化酶中发挥作用,包括细胞色素C氧化酶、酪氨酸酶、多巴胺-β-单加氧酶、胺氧化酶、赖氨酰氧化酶。铜还参与线粒体呼吸、作为血浆铜蓝蛋白的组分参与铁稳态、自由基清除和弹性蛋白交联(elsatin crosslining)。
无血清培养基包含金属离子例如锌或铜是本技术领域熟知的,例如美国专利US6,048,728(Inlow等),US4,767,704(Cleveland等)或国际专利WO01/16294(Life Technologies Inc.)。但是这些文献中并没有描述在标准的生产培养基中添加特定浓度的这些离子会有提高产量的作用。
开发和提供生物学活性产品,例如治疗蛋白质或疫苗,需要大量生产。广泛用于生产多肽的合适细胞为中国仓鼠卵巢细胞(CHO)。CHO细胞首先由Puck(J.Exp.Med.108:945,1958)从一雌性中国仓鼠卵巢活体组织中分离得到并培养。从这些原始细胞制备了多种具有不同特征的细胞亚系。这些CHO细胞系中的一个系CHO-K1,需要脯氨酸并且是二氢叶酸还原酶(DHFR)基因的二倍体。从该细胞系衍生的另一细胞系为DHFR缺陷型CHO细胞系(CHO DUKB11)(PNAS 77:4216-4220,1980),该细胞系的特征是由于DHFR基因中有一个突变导致DHFR功能丧失,以及随后其它基因的缺失。
常用于生产人用蛋白质的其它细胞系有人细胞系,例如人纤维肉瘤细胞系HT1080或人胚肾细胞系293。
鼠C127细胞系也很适合用于生产重组蛋白质(Carter等.,1989;Oka andRupp,1990)。
一种感兴趣的治疗蛋白质为生长激素。人生长激素(hGH)也称为somatropin(INN)或促生长素(somatotropin),是由垂体前叶生长激素(somatotropic)生成细胞产生和分泌的一种蛋白质激素。人生长激素通过对蛋白质、碳水化合物和脂类代谢的作用在儿童身体生长和成人新陈代谢中起着关键作用。
人生长激素是由191个氨基酸(Bewly等,1972)构成的单链多肽,具有两个二硫键,位于Cys-53和Cys-165之间的二硫键在分子内形成一个大环,位于Cys-182和Cys-189之间的二硫键形成靠近C-末端的一个小环。Martial等(1979)报道了证实该氨基酸序列的DNA序列。纯化hGH的冻干形式为白色无定形粉末。人生长激素易溶于(浓度>10mg/L)pH范围6.5-8.5的水性缓冲液中。
hGH在溶液中主要以单体存在,有少量二聚体和高分子量寡聚体。在特定条件下,hGH可经诱导形成大量的二聚物、三聚体和更高的寡聚物。
已知有几种hGH衍生物,包括自然产生的衍生物、变体和代谢产物,主要是生物合成hGH的降解产物和通过遗传方法产生的hGH的工程衍生物。hGH天然产生的衍生物的一个例子是胎盘中发现的生长激素变体GH-V。该基因座的其它成员在Chen等(1989)的文章有描述。hGH的衍生物,包括经设计能在机体内长期保持的衍生物都可用于本发明,只要这些衍生物保留了hGH的生物活性。
甲二磺酰(基)hGH是通过重组DNA技术产生的第一种形式的hGH。该化合物实际上是N-末端具有一额外甲硫氨酸残基的hGH衍生物(Goeddel等,1979)。
有报道(Lewis等,1978;Lewis等,1980)称天然产生的hGH变体20-K-hCG见于垂体和血液中。该化合物由于mRNA的交替剪接缺失了从Glu-32到Gln-46的15个氨基酸残基(DeNoto等,1981)。此化合物具有hGH的众多但不是全部的生物学性能。
20-K-hGH在垂体中产生分泌到血液中。它占成人生长激素输出量的约5%和儿童生长激素输出量的约20%。它具有与22kD生长激素相同的促生长活性,据报道它具有与22kD生长激素相等或更高的溶脂活性。它与生长激素受体结合的亲和力与22kD生长激素相同,催乳(促乳素-样)生物学活性为22kD生长激素的十分之一。与22kD生长激素不同的是20-K-hGH的抗胰岛素活性弱。
许多hGH衍生物是通过该分子的蛋白水解修饰而产生。hGH代谢的主要途径包括蛋白质水解。hGH的130-150残基区域对蛋白质水解极其敏感,已有报道(Thorlacius-Ussing,1987)几种hGH衍生物在该区域有缺口或缺失。此区位于hGH的大环内,切割此处肽键导致产生通过Cys-53和Cys-165之间二硫键连接的两条链。据报道许多该双链形式的生物学活性增强(Singh等,1974)。通过采用酶人工产生了许多人生长激素衍生物。已利用胰蛋白酶和枯草杆菌蛋白酶等在不同位点上修饰整个hGH分子(Lewis等,1977;Graff等,1982)。其中一类衍生物,称为双链合成代谢蛋白质(2-CAP),是通过使用胰蛋白酶控制水解hGH而形成的。发现此2-CAP具有与完整hGH分子非常不同的生物学性能,它主要保留了hGH的促生长活性但丧失了其对碳水化合物代谢的大部分活性。
在适当条件下,蛋白质中天冬酰胺和谷氨酰胺残基易发生脱酰胺反应。已证明垂体hGH发生该反应后,导致Asn-152转变为天冬氨酸,和较低程度Gln-137转变为谷氨酸(Lewis等,1981)。已证明脱酰胺后的hGH对枯草杆菌蛋质酶的水解易感性改变,提示脱酰胺对指导hGH蛋白质水解切割具有生理学意义。已知生物合成的hGH在某些贮藏条件下能降解而导致另一天冬氨酸(Asn-149)脱酰胺。该位点为脱酰胺主要位点,但也发现Asn-152发生脱酰胺(Becker等,1988)。生物合成hGH的Gln-137脱酰胺未见报道。
蛋白质的甲硫氨酸残基易于氧化,主要被氧化成亚砜。垂体产生的和生物合成的hGH都在Met-14和Met-125发生磺化氧化(sulfoxidation)(Becker等,1988),垂体中Met-170的氧化有报道但未见于生物合成的hGH。已发现脱酰胺hGH和Met-14磺化氧化hGH都显示具有完全的生物学活性(Becker等,1988)。
可通过酶作用或遗传学方法产生截短形式的hGH。通过控制的胰蛋白酶作用而产生的2-CAP去除了hGH的N-末端前8个残基。hGH的其它截短形式可通过基因修饰然后在合适宿主中表达而产生。将前13个残基除去,以产生具有与未切割多肽链不同的生物学性能的衍生物。
虽然人生长激素最初获自尸体脑垂体,但这些制品电泳上不具有均一性,用级别50%纯制品治疗的病人血液中出现了抗体,其免疫源性归因于非活性成分。重组DNA技术可在一些不同系统中产生无限制提供的hGH。非常少量的污染蛋白质的存在有利于从培养基中纯化hGH。事实上,已证明能在实验室规模上用反相HPLC柱一步纯化hGH(Hsiung等(1989)。
Serono International S.A.生产的重组人生长激素(rhGH)(SEROSTIM)已获FDA加快批准用于治疗爱滋病人体重减轻或消瘦。SAIZEN是重组人生长激素可用于儿童生长激素缺乏、女孩Turner综合症、和儿童慢性肾功能衰竭。Genentech,Inc.(South SanFrancisco,CA)生产的PROTROPIN与天然序列hGH在结构上略有不同,其N末端具有一额外甲硫氨酸残基。重组hGH通常含有冻干形式的hGH和添加的赋形剂(例如糖胶、甘露醇)的小瓶销售。提供了稀释液小瓶,允许病人在按剂量给药前重建该产品至需要的浓度。重组hGH也可以其它熟知的方式,例如预填装注射器等销售。
通常,没有观察到重组的天然序列hGH、重组的N-端甲硫氨酰hGH、或人垂体来源物的药物动力学或生物学活性有明显的差异(Moore等,1988;Jorgensson等,1988)。
鉴于生长激素可用于多种医学适应症,需要一种有效和安全的细胞培养方法,尤其是无血清细胞培养方法来生产足够数量的生长激素。
无血清培养基在本领域内已有描述。
例如美国专利US6,162,643描述了一种无血清基础培养基,命名为HECK-109,该培养基含有微量硫酸铜和氯化锌。这种特定设计的培养基用于正常人细胞,例如角化细胞的原代和次代培养,目的是人移植组织的传代。该美国专利没有提到在体外细胞系中表达重组人蛋白质。
美国专利US5,324,656公开了称为MCDB120和MCDB131M的无血清基础培养基,该培养基含有微量硫酸铜和氯化锌。该培养基特定设计用于体外培养人肌肉卫星细胞,目的是防止这些细胞分化。该文献也未注意到重组人蛋白质的生产。
英国专利GB2196348描述了一种用于体外培养杂交瘤细胞和骨髓瘤细胞的合成培养基。这种培养基含铜、锌和铁离子。在该培养基中培养杂交瘤细胞和骨髓瘤细胞,专用于制备单克隆抗体。
美国专利US6,103,529提供了一种用于体外培养动物细胞的无血清细胞培养基配方。可利用动物细胞来生产病毒、单克隆抗体、激素或生长因子。但是该文献未提到生长激素的生产。
美国专利US6,048,728公开了一种含有铜、锌和铁离子的无蛋白质细胞培养基,用于培养动物细胞,以生产如天然或重组产品,例如抗体。所培养的细胞产生的产品不包括专门提到的作为无血清培养基成分以增强培养细胞生长的生长激素或生长因子。
美国专利US5,316,938公开了一种用于培养中国仓鼠卵巢细胞(CHO)的生化成分明确的培养基,其含有柠檬酸铁、硫酸锌和硫酸铜,命名为WCM5。该培养基专门设计用于在CHO细胞中生产抗体和tPA。
美国专利US5,122,459描述了一种在含有锌和铁离子的无血清培养基中生产重组蛋白质的方法,该方法尤其适合培养CHO细胞。但该文献中未提及生长激素的生产。
因此,本发明需要解决的问题是提供一种无血清细胞培养基来有效生产生长激素,特别是人生长激素(hGH)。
发明概述
本发明依据于开发的一种细胞培养基,该培养基不含源自动物血清的成分但同时能使培养的哺乳动物细胞高效生长和维持,特别是使其产生重组蛋白质。
因此,本发明第一方面涉及一种不含源自动物血清的成分的细胞培养基,该培养基含有微量锌和/或铜。优选本发明培养基还含有微量铁离子。
本发明第二方面涉及一种蛋白质生产方法,包括在本发明培养基中培养表达感兴趣蛋白质的细胞的步骤。
本发明第三方面涉及使用本发明的培养基来生产感兴趣的蛋白质。
本发明第四方面涉及在感兴趣多肽的生产期间使用本发明的培养基来维持所培养的细胞。
附图简要说明
图1显示在连续添加10μM的各种元素到DMEM培养基生产阶段中获得的rhGH生成状况(RB=转瓶,H1-H14=生产阶段的天数1-14);
图2显示连续添加10μM金属元素(镍、钡、钴、铬)到DMEM培养基生产阶段中获得的rhGH平均产生值,表示为每转瓶产生的rhGH的毫克(mg)数;
图3显示在检测锌(Zn 0.5μM)、铜(Cu 0.02μM)、硒(Se 0.050μM)、锰(Mn0.001μM)和柠檬酸铁(4.8μM)组合的因子设计试验中,与对照相比获得的rhGH产量增加的平均值;
图4显示检测金属微量元素(Zn 0.5μM、Cu 0.02μM和柠檬酸铁4.8μM)与氨基酸谷氨酰胺(Gln 4.8mM)、丝氨酸(Ser 0.49mM)、半胱氨酸(Cys 0.29mM)混合物效果的因子设计试验结果;
图5显示DMEM中不同浓度的铜与锌和柠檬酸铁(Zn 0.5μM、柠檬酸铁4.8μM)联用对rhGH产量的影响;
图6显示DMEM中不同浓度的锌与铜和柠檬酸铁(Cu 0.02μM、柠檬酸铁4.8μM)联用对rhGH产量的影响;和
图7显示利用产生rhGH的C127细胞进行不同实验时,添加铜、锌和柠檬酸铁(Zn:1.5和0.5μM、Cu:0.02μM和柠檬酸铁:4.8μM)作为DMEM补充成分,对rhGH产量增加影响的总结。
发明详述
本发明的基础是开发一种无动物血清的细胞培养基。
本发明的无动物血清细胞培养基含有:
浓度范围0.2-1.75μM的锌(Zn),和/或
浓度范围10-75nM的铜(Cu),和/或
浓度范围3-10μM的铁(Fe)。
如以下实施例中所证明,在标准培养基中添加微量锌和/或铜可导致表达感兴趣分泌性蛋白质的细胞的生产能力提高。
添加上述鉴定浓度的Zn和Cu两者导致C127细胞的重组人生长激素(GH)产量比对照(同样细胞用标准DMEM培养)高60%。当生长激素表达细胞在含有上述鉴定浓度的Zn、Cu和Fe离子的培养基中培养时,产量比对照增加约70%。
本发明培养基金属离子的优选浓度范围如下:
-含约0.2、0.25、0.30、0.35、0.40、0.45、0.5、0.55、0.60、0.65、0.70、0.75、0.80、0.85、0.90、0.95、1、1.05、1.1、1.15、1.2、1.25、1.3、1.35、1.4、1.45、1.5、1.55、1.6、1.65、1.7、1.75μM的锌。
优选含约0.5μM或约1.5μM的锌。还优选所述锌成分是硫酸锌。
-含约10、15、20、25、30、35、40、45、50、55、60、65、70、或75nM的铜。
优选含约25nM的铜。还优选所述铜成分是硫酸铜。
-含约1、1.5、2、2.5、3、3.5、4、4.5、4.8、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5或10uM的铁离子
优选铁离子成分约5或6μM。优选铁离子成分是柠檬酸铁和/或硝酸铁,更优选柠檬酸铁占该细胞培养基中的铁离子的大部分。该培养基可包含例如约5μM的柠檬酸铁和约1μM的硝酸铁。
在一优选实施方案中,该培养基还包含基础培养基的组分。该培养基优选包含Dulbecco’s改良Eagle’s培养基(DMEM)作为基础培养基。标准培养基DMEM的组成报道见以下实施例。
本发明第二方面涉及一种生产蛋白质的方法,包括在本发明培养基中培养表达感兴趣蛋白质的细胞的步骤。
本发明方法的培养步骤可在任何合适的环境中实施,例如在皮氏培养皿(Petri dishes)、T-瓶或转瓶中实施,也可在大体积容器,如生物反应器中实施。
本发明蛋白质的表达可采用适合所用特定细胞类型的任何启动子。在一非常优选的实施方案中,蛋白质表达采用金属硫蛋白质(MT)启动子。若用鼠细胞生产蛋白质时,启动子优选鼠MT-1启动子。
本发明方法优选包括收集含有感兴趣蛋白质的培养基的步骤。
在一优选实施方案中,本发明方法还包括分离感兴趣的蛋白质。
在一更优选的实施方案中,本发明方法还包括将分离的蛋白质与药学上可接受的运载体配制成药物组合物。
本发明第三方面涉及本发明培养基生产感兴趣多肽的用途。
本发明第四方面涉及在感兴趣多肽的生产期间用本发明的培养基维持培养的细胞。
在本发明各个方面框架内所用的细胞优选哺乳动物细胞。该细胞可以是人来源或动物来源。可根据本发明方法进行培养的哺乳动物细胞的例子包括:例如鼠C127细胞、3T3细胞、COS细胞、人骨肉瘤细胞、MRC-5细胞、BHK细胞、VERO细胞、CHO(中国仓鼠卵巢)细胞、HEK293细胞、rHEK293细胞、正常人成纤维细胞、基质细胞、肝细胞或PER.C6细胞。可根据本发明方法培养的杂交瘤的例子包括例如DA4.4细胞、123A细胞、GAMMA细胞和67-9-B细胞。
优选根据本发明培养鼠C127细胞。
本发明培养的细胞可以是悬浮生长,或对于锚定依赖性细胞,则附着于固体支持物。微载体和Fibra-Cel盘可在哺乳动物细胞培养中用于锚定依赖性细胞的生长,并可在已建立的技术平台中用于蛋白质产业化生产(例如参见Bohak等.1987;Petti等.1994)。
本发明方法优选用于生产感兴趣多肽。因此,可用本发明培养基小规模或大规模生产感兴趣的多肽或蛋白质,该多肽或蛋白质可以是需要生产的任何多肽。
所述感兴趣多肽可以是例如天然分泌的蛋白质、正常胞质蛋白、正常跨膜蛋白、或人或人源化抗体。当感兴趣蛋白质为天然胞质蛋白或天然跨膜蛋白时,优选工程改造该蛋白质成为可溶性分泌蛋白质,例如在该蛋白质或其片段(可溶性或胞外片段)前安置一信号肽。
感兴趣多肽可以是任何来源,优选感兴趣多肽为人来源,更优选感兴趣蛋白质为治疗性蛋白质。
优选所述感兴趣蛋白质选自:激素、细胞因子结合蛋白质、干扰素、可溶性受体或抗体。
可根据本发明方法生产的治疗性蛋白质包括:例如绒毛膜促性腺激素、促卵胞激素、促黄体-绒毛膜促性腺激素、促甲状腺激素、生长激素(尤其是人生长激素)、干扰素(如干扰素β-1a、干扰素β-1b)、干扰素受体(如干扰素γ受体)、肿瘤坏死因子受体p55和p75和它们的溶解形式、TAC受体及其Fc融合蛋白、白介素(如白介素-2、白介素-11)、白介素结合蛋白(如白介素-18结合蛋白)、抗-CD11a抗体、(促)红细胞生成素、粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子、垂体肽激素、绝经期促性腺激素、胰岛素样生长因子(如生长调节素-C)、角质细胞生长因子、胶质细胞系衍生的神经营养因子、凝血调节蛋白质、碱性成纤维细胞生长因子、胰岛素、因子VIII、促生长素、骨形态发生蛋白质-2、血小板衍生生长因子、水蛭素、红血球生成素(epoietin)、重组LFA-3/IgG1融合蛋白、葡糖脑苷脂酶、和它们的变体蛋白、片段、溶解形式、功能衍生物、融合蛋白质。
在优选的实施方案中,所述多肽选自:绒毛膜促性腺激素(CG)、促卵胞激素(FSH)、促黄体-绒毛膜促性腺激素(LH)、促甲状腺激素(TSH)、人生长激素(hGH)、干扰素(如干扰素β-1a、干扰素β-1b)、干扰素受体(如干扰素γ受体)、肿瘤坏死因子受体p55和p75、白介素(如白介素-2、白介素-11)、白介素结合蛋白(如白介素-18结合蛋白)、抗-CD11a抗体,和它们的变体蛋白、片段、溶解形式、功能衍生物、融合蛋白质。
更优选感兴趣多肽包括:例如(促)红细胞生成素、粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子、垂体肽激素、绝经期促性腺激素、胰岛素样生长因子(如生长调节素-C)、角质细胞生长因子、胶质细胞衍生神经营养因子、凝血调节蛋白、碱性成纤维细胞生长因子、胰岛素、因子VIII、促生长素、骨形态发生蛋白质-2、血小板衍生生长因子、水蛭素、红血球生成素、重组LFA-3/IgG1融合蛋白、葡糖脑苷脂酶,和它们的变体蛋白、片段、溶解形式、功能衍生物、融合蛋白质。
按照本发明方法生产的感兴趣蛋白质可与药学上可接受的运载体配制成为药物组合物。
定义“药学上可接受的”意为包括不会干扰所述活性成分的生物活性效力,和对给予的宿主无毒性的任何运载体。例如肠胃道外给药时,可在媒介物如盐水、葡萄糖溶液、血清白蛋白和林格氏溶液中将所述活性蛋白质配制成注射用的单位剂型。
按照本发明配制的药物组合物可通过多种途径给予个体。给药途径包括:皮内、透皮(如在缓释制剂中)、肌肉内、腹膜内、静脉内、皮下、口服、颅内、硬脑膜外、局部、经直肠和鼻内途径。也可采用任何其它的治疗有效给药途径,例如通过上皮和内皮组织吸收或通过基因治疗将编码活性成分的DNA分子(例如通过载体)给予病人,基因治疗能使活性成分在体内表达和分泌。另外,本发明的蛋白质可与其它生物活性药物的组分,例如药学上可接受的表面活性剂、赋形剂、运载体、稀释剂和媒介物一起给药。
对于肠胃道外给药(如静脉内、皮下、肌肉内),可将活性蛋白质与药学上可接受的肠胃道外媒介物(如水、盐水、葡萄糖溶液)和能维持等渗(例甘露醇)或化学稳定性(如防腐剂和缓冲液)一起配制成溶液、悬浮液、乳液或冻干粉。可采用常规的技术对该制剂消毒。
本发明更优选采用本发明的培养基来生产生长激素。
GH可以是天然GH,例如天然产生的GH。优选生产的GH为人源GH。由于GH是可溶性分泌蛋白质,所以可通过其天然的信号肽或通过异源信号肽(即衍生自另一分泌蛋白质的可能在所用的特定表达系统中更为有效的信号肽)而释放到细胞培养液中。
本文所用术语“生长激素”与“GH”同义。该术语包括自然或天然的GH或重组产生的GH,及在“发明背景”中详述的GH变体。本文所用术语“GH”还包括:GH的突变蛋白、功能衍生物、活性片段、融合蛋白、循环性完全变化的蛋白(circularly permutated protein)和盐。GH优选人源,但也可是其它物种来源,特别是哺乳动物来源。
本文所用术语“变体蛋白质”指天然GH序列中的一个或多个氨基酸残基为不同氨基酸残基取代、或缺失、或加入了一个或多个氨基酸残基而产生的与野生型GH相比活性无明显减少的GH类似物。这些变体蛋白质可用已知的合成方法和/或定点诱变技术,或其它已知的合适技术制备。
本发明的突变蛋白质包括能在严谨条件下与DNA或RNA杂交的编码GH的核酸(如DNA或RNA)编码的蛋白质。编码GH的DNA序列本领域先前熟知的。术语“严谨条件”指本领域普通技术人员通常所称的“严谨”杂交条件和随后的洗涤条件。参见Ausubel等,Current Protocols in Molecular Biology,同上,Interscience,N.Y.,§§6.3和6.4(1987,1992)。严谨条件的非限制性例子包括在所研究杂交物的计算Tm值以下12-20℃,例如用2xSSC和0.5%SDS处理5分钟,2xSSC和0.1%SDS处理15分钟;0.1xSSC和0.5%SDS 37℃处理30-60分钟和随后68℃用0.1xSSC和0.5%SDS处理30-60分钟。本领域普通技术人员明白严谨条件还取决于DNA序列、寡核苷酸探针(例如10-40个碱基)或混合寡核苷酸探针的长度。如采用混合探针,优选采用四甲基氯化铵(TMAC)替代SSC。参见Ausubel,同上。
通过序列比较检测到的相同性反映了两条或多条多肽序列或两条或多条多聚核苷酸序列之间的关系。相同性通常指相对于所比较的序列的长度而言,两条多聚核苷酸或两条多肽序列的核苷酸与核苷酸,或氨基酸与氨基酸的精确对应关系。
对于不存在精确对应关系的序列而言,可测定其“相同性百分比”。通常,将要比较的两序列进行排列使二序列之间最大相联。排列包括在一条或二条序列中插入“空格”以提高排列对比程度。可基于所比较的各序列的全长来测定相同性百分比(所谓全长排列对比),这特别适用于相同或非常相似的序列,或可基于较短的、特定长度(所谓局部排列对比)的序列来测定该相同性百分比,这更适合于不等长的序列排列对比。
比较两条或多条序列相同性和同源性的方法是本领域熟知的。例如可使用在Wisconsin Sequence Analysis Oackage 9.1版中存在的程序(DevereuxJ等,1984),例如BESTFIT和GAP来检测两条多聚核苷酸之间的相同性百分比,和两条多肽序列之间的相同性和同源性百分比。BESTFIT采用Smith和Waterman的“局部同源”算法(1981)可发现两序列之间的一个最佳相似区域。其它检测两序列之间相同性和/或相似性的程序也是本领域熟知的,例如BLAST程序家族(Altschul S F等,1990,Altschul S F等,1997,从NCBI主页www.ncbi.nlm.nih.gov可以获得)和FASTA程序(Pearson W R,1990).
优选所有这种突变蛋白质的一段氨基酸序列与GH的氨基酸序列充分相同,例如具有与GH基本上相似的活性。GH的活性之一是能结合GH受体。只要该突变蛋白实质上能结合GH受体(GHR),即可认为其具有与GH基本上相似的活性。因此可通过常规实验来测定某给定的突变蛋白是否具有与GH基本上相同的活性,该实验包括对该突变蛋白质进行简单的夹心竞争试验,检测其是否能结合适当标记的GHR或表达GHR的细胞,例如可采用放射性免疫试验或ELISA试验。
在一优选实施方案中,这类突变蛋白与GH的氨基酸或DNA序列至少具有40%相同性或同源性。这些序列是本领域熟知的,例如见DeNoto等,1981或Martial等,1979所述。
更优选该突变蛋白具有至少50%、至少60%、至少70%、至少80%、或最优选至少90%或95%相同性或同源性。
本发明突变蛋白中优选的变化是所谓的保守性置换。GH多肽的保守氨基酸置换包括理化特性充分相似的同组成员中的同义氨基酸置换因而保留了该分子的生物学功能(Grantham,1974)。清楚的是,在上述序列中也可有氨基酸插入和缺失而不会改变它们的功能,尤其是插入或缺失只涉及很少的氨基酸,例如30个以下,优选10个以下,但不去除或置换功能构型的关键氨基酸(如半胱氨酸残基)。通过这种缺失和/或插入而产生的蛋白质和突变蛋白属于本发明的范围。
术语“融合蛋白质”指含有与另一蛋白质(如能延长体液中存留时间的蛋白)融合的GH、或其突变蛋白或片段的多肽。因此可将GH与另一蛋白质、多肽等,例如免疫球蛋白质或其片段融合。IgG的Fc部分适合于制备免疫球蛋白融合蛋白质。Ig融合蛋白的例子在欧洲专利EP314,317A1(Genentech)或EP0,325,224A2(Zymogenetics Inc.)中有描述。
作为GH或其突变蛋白和融合蛋白的“活性片段”,本发明包括单独的或与其相连的结合的分子或残基(例如糖或磷酸残基)一起的蛋白分子的多肽链的任何片段或前体,或该蛋白质分子或其糖基的凝聚物,只要所述片段具有与GH基本上相似的活性。
本发明的GH应以药物组合物使用,可用该组合物治疗和/或预防许多疾病或病症。这些疾病和病症优选涉及内源GH生产不足。纯化的GH可用于如治疗和/或预防GH不足、AIDS消瘦、脂肪代谢障碍(也称为HARS-HIV-相关畸变/代谢障碍综合症),或短肠综合征,尤其是儿科疾病。其它需要给予生长激素的适应症包括:肝硬化、成人生长激素缺乏症(adult growth deficiency)、动脉硬化症、克罗恩(节段性回肠炎)病和溃疡性结肠炎、骨关节炎、心源性恶病质、充血性心力衰竭、慢性肾功能不全、血细胞重建和动员、男性不育、造血干细胞转移、多发性硬化症、中风、多系统萎缩症、或癌症。
现已充分描述了本发明,本领域技术人员知道在不偏离本发明的精神和范围的情况下,不需要过多的试验即可以大范围的同等参数、浓度和条件实施本发明。
虽然本发明已结合具体实施方案进行了描述,但应知道仍能对其作进一步修改。本申请包括遵循本发明的原理作出的任何变化、应用或修改,包括例如本发明所属领域内的已知或常规实践范围内的偏差变动(departure)以及可能会被应用于上文所述必要特征的那些变化、应用或修改这些都属于本发明附带权利要求书的范围内。
本文所引用的所有参考文献,包括期刊杂志论文或摘要、发表或未发表美国或外国专利申请、出版的美国或外国专利或其它文献均纳入本文参考文献,包括引用文献中的所有数据、表格、图形和文本。另外,本文引用文献中所引用文献的全部内容也纳入作为参考。
参见已知的方法步骤、常规方法步骤、已知方法或常规方法,并非承认本发明的任何方面、描述或实施方案已在相关领域作了公开、说明或提出。
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实施例:开发用于C127细胞表达hGH的新型无血清培养基产品
1.简介
本研究描述的实验工作涉及开发加有微量金属元素的培养基产品(DMEM,“Dulbecco’s Modified Eagle’s Medium)。开发该产品的结果是,在C127细胞培养物中r-hGH产量的显著提高。
下表1小结了本实施例框架内所提供的DMEM的组成和及开发。
表1:生长培养基,DMEM和生产培养基配方
组分 | 生长培养基mg/L | DMEMmg/L | 生产培养基mg/L |
氨基酸 | |||
L-丙氨酸 | 49.45 | ||
L-精氨酸HCl | 227.5 | 84.0 | 84.0 |
L-天冬酰胺H2O | 27.50 | ||
L-天冬氨酸 | 21.65 | ||
L-半胱氨酸HCl H2O | 17.56 | ||
L-半胱氨酸2HCl | 31.29 | 62.0 | 62.0 |
L-谷氨酸 | 40.35 | ||
L-谷氨酰胺 | 693.5 | 584.0 | 584.0 |
甘氨酸 | 43.75 | 30.0 | 30.0 |
L-组氨酸HCl,H2O | 41.48 | 42.0 | 42.0 |
L-羟脯氨酸 | 6.50 | ||
L-异亮氨酸 | 114.5 | 105.0 | 105.0 |
L-亮氨酸 | 119.1 | 105.0 | 105.0 |
L-赖氨酸-HCL | 151.3 | 146.0 | 146.0 |
L-甲硫氨酸 | 52.24 | 30.0 | 30.0 |
L-苯丙氨酸 | 70.48 | 66.0 | 66.0 |
L-脯氨酸 | 37.25 | ||
L-丝氨酸 | 39.25 | 42.0 | 42.0 |
L-苏氨酸 | 93.45 | 95.0 | 95.0 |
L-色氨酸 | 15.02 | 16.0 | 16.0 |
L-酪氨酸2Na 2H2O | 75.79 | 104.0 | 104.0 |
L-缬氨酸 | 92.85 | 93.0 | 93.0 |
盐 | |||
CaCl2 | 121.0 | 200 | 200 |
Fe(NO3)3·9H2O | 0.050 | 0.1 | 0.1 |
FeSO4.7H2O | 1.251 | ||
KCl | 334.2 | 400 | 400 |
MgCl2 | 40.94 | ||
无水MgSO4 | 48.84 | 97 | 97 |
Na2HPO4 | 71.02 | ||
Na2SeO3.5H2O | 0.030 | ||
NaCl | 7500 | 6400 | 6400 |
NaH2PO4H2O | 62.5 | 125 | 125 |
NaHCO3 | 1200 | 3700 | 3700 |
LiCl | 1.00 | ||
ZnSO4·7H2O | 0.432 | 0.144 | |
碳水化合物 | |||
D-葡萄糖 | 3650 | 4500 | 4500 |
维生素 | |||
抗坏血酸 | 0.500 | ||
生物素 | 0.1035 | ||
氯化胆碱 | 11.48 | 4.00 | 4.00 |
维生素B12 | 1.68 | ||
泛酸(D Ca PANTHOTENATE) | 2.34 | 4.00 | 4.00 |
叶酸 | 3.65 | 4.00 | 4.00 |
I-肌醇 | 17.1 | 7.00 | 7.00 |
烟酰胺 | 2.27 | 4.00 | 4.00 |
对氨基苯甲酸 | 1.00 | ||
盐酸吡哆醛 | 0.25 | 4.00 | 4.00 |
盐酸吡哆醇 | 4.531 | ||
核黄素 | 0.419 | 0.40 | 0.40 |
盐酸硫胺 | 2.92 | 4.00 | 4.00 |
脂质 | |||
亚油酸 | 0.042 | ||
硫辛酸 | 0.205 | ||
亚油酸甲酯 | 0.1 | ||
杂项 | |||
柠檬酸铁 | 122.5 | 1.22 | |
HEPES | 3600 | ||
次黄嘌呤 | 2.900 |
二水合d-葡萄糖酸亚铁 | 4.820 | ||
L-谷胱苷肽 | 0.500 | ||
巯基乙醇 | 0.234 | ||
丙酮酸钠 | 57.00 | 110.0 | 110.0 |
腐胺,2HCl | 0.241 | ||
胸苷 | 0.505 | ||
去污剂 | |||
乙醇胺(作为碱) | 6.25 | ||
微量元素 | |||
AgCl | 0.0000044 | ||
BaCl2.2H2O | 0.002 | ||
CoCl2.6H2O | 0.002 | ||
KCr(SO4)2 | 0.001 | ||
KBr | 0.0001 | ||
KI | 0.0001 | ||
钼酸(钼酸铵·4H2O) | 0.0001 | ||
NaF | 0.004 | ||
偏钒酸铵 | 0.0006 | ||
硝酸镍·6H2O | 0.0002 | ||
RbCl | 0.00001 | ||
SnCl2·2H2O | 0.0001 | ||
CuSO4·5H2O | 0.0064 | 0.0064 | |
MnCl2·4H2O | 0.0001 | ||
氧化钛(TiO2.) | 0.001 | ||
补充成分 | |||
人重组胰岛素 | 10mg/L | ||
物理调节 | |||
pH | 7.1±0.1 | 7.0±0.2 | 7.0±0.2 |
克分子渗透压浓度(mosm/kg) | 325±25 | 330±30 | 330±30 |
2.材料和方法
试剂和溶液
所有化学试剂购自Merck:硫酸锌(ZnSO4·7H2O)、硫酸铜(CuSO4·5H2O)、氯化钡(BaCl2·2H2O)、氯化钴(CoCl2·6H2O)、硫酸铬钾(K[Cr(SO6H4)2(H2O)2]·6H2O)、硝酸镍(Ni(NO3)2·6H2O)、亚硒酸钠(Na2SeO3·5H2O)。柠檬酸铁(FeC6H5O7 Sigma目录号F3388)作为铁离子来源。
所有待检测的微量元素用蒸馏水配成浓缩液并用0.2μm滤膜过滤除菌。
不同实验中将不同的测试添加物(金属溶液等)直接加入到新鲜培养基中。
细胞培养
用遗传改造的C127鼠细胞(ATCC CRL 1616)表达重组人生长激素(rhGH)。载体基于BPV69T,含有pBR322多克隆位点和在小鼠金属硫蛋白-1(MT-1)启动子控制下的1.6kb rhGH微小基因。实验中使用产生自用不同rhGH生产批次获得的工作细胞库的培养物。
将细胞培养物保持在36℃±0.5℃以0.4rpm培育的含375mL±15mL培养基的2125cm2转瓶或含300mL培养基的1700cm2转瓶中。
培养基
rhGH生产阶段所用培养基为DMEM,含4.5g/L葡萄糖,用碳酸氢钠溶液(3.7g/L)缓冲。
RHGH滴定
每天用反相HPLC滴定检测培养基中的rhGH含量:
材料
Resource RPC 1mL,30mm×6.4mm内径,15μm,art 17-1181-01,AmershamBiosciences。
Symmetry300,50mm×4.6内径.,C4 5μm,P/N186000287(Waters)。
试剂
三氟乙酸(TFA)(Pierce,Cat#28904或等同物)。
乙腈(ACN)(Merck 1.00030或等同物)。
纯水,PW(如MilliQ水,或等同物)。
氦气
溶液
流动相A:TFA 0.08%,用水配制(v/v)
用量筒量取PW水并按下表加入TFA。搅拌并标记。
相A体积 | MILLIQ水体积 | TFA体积 |
1L | 1L | 0,8M1 |
0,5L | 0,5L | 0,4mL |
0,25L | 0,25L | 0,2mL |
流动相B:TFA 0.08%的乙腈溶液(v/v)
用容量瓶量取PW水并按下表加入TFA。搅拌并标记。
相B体积 | ACN体积 | TFA体积 |
1L | 1L | 0,8mL |
0,5L | 0,5L | 0,4mL |
0,25L | 0,25L | 0,2mL |
层析条件:
-梯度洗脱:以60/40比例的相A/相B混合液开始,以20/80比例的相A/相B混合液结束。5分钟内完成梯度洗脱(可根据所用仪器做轻微改变)
-上样体积:50μl
-检测:215nm处的UV吸光度
-标准曲线:10,50,100,120和150μg/ml的r-hGH标准液
通过与标准r-hGH浓度比较确定样品的r-hGH浓度。
GH产量
产量表示为每转瓶rhGH的毫克数。初始数据为收获时的rhGH浓度(HPLC检测)和转瓶总数。通常转瓶在收获期含约109个细胞。
3.结果
在第一系列实验中,测定了在培养基中间歇性加入高浓度钴(20μMCoCl2.6H2O)的作用。在一批培养的最初步骤(清洗,PM=生产培养基,即收获期的0点)和生产阶段的中间步骤中(收获6和7)两种培养基互换时加入。结果(没有显示)提示此启动子具有活性并可调节。
提高产量用其它金属元素得到进一步证实。将10μM浓度的钡(BaCl2·2H2O)、钴(CoCl2·6H2O)、铬(K[Cr(SO6H4)2(H2O)2]·6H2O)和镍(Ni(NO3)2·6H2O)各自连续加入到培养基中进行检测。本实验结果见图1和图2描述。
图1显示在实验期间(14天),当在含有测试元素的培养基中培养时表达hGH的C127细胞分泌的hGH量。图2显示各个测试元素所达到的产量平均值。与无微量元素的对照值相比较,获得的平均产量增加百分比范围是钡1.1%和钴32.4%。还注意到当试验中连续或间歇性加入钴时,rhGH产量与对照值相比增加了9%-32.5%。测试浓度的钡和铬在DMEM中都不显示产量增加。
各微量元素的检测
在DMEM按下表2中所列浓度加入微量元素Zn、Cu、Se和Co。这些浓度与生长培养基中的金属元素浓度相对应。表2显示在DMEM中添加微量元素导致rhGH产量获得提高。
表2
当在DMEM中测试所示浓度(μM)的金属离子时,rhGH产量增加百分数
离子 | 浓度(μM) | 产量增加百分数 |
Zn | 1.50300 | 12.10% |
Cu | 0.02560 | 37.50% |
Se | 0.11400 | -3.10% |
Co | 0.00840 | -3.20% |
应注意1.5μM测试浓度的锌可导致细胞“剥脱”现象,即细胞单层从转瓶塑料底层脱离。在一些情况下,这种剥脱可导致在最后收获步骤(通常介于收获12-14)时培养物的损失。采用适当的细胞培养容器,例如购自Corning公司商标名Cellbind的转瓶可以避免这种细胞剥脱现象。
检测金属(离子)之间的相互作用
根据铜和锌对hGH生产产量的影响,进行了因子设计实验以检测金属组合的可能影响。
结果的统计学分析见表3所示。发现联用Cu和Zn对产量有很强的协同作用(p≤0.0001)。
表3:三因子因子设计实验的统计学分析
产量变化分析
R-平方=98.1139%
R-平方(基于d.f.调整)=96.8565%
无论是否有铜存在,所有经1.5μM浓度锌处理的培养物均观察到细胞剥脱现象。
进行了随后的因子设计实验以研究当降低锌浓度(Zn0.5μM)和加入其它微量元素(例如可提高产量和培养物粘附基底层的锰、硒、柠檬酸铁)到生长培养基中时同时加入锌与铜对生长培养基的影响。
持续培养获得从起始时间点的每转瓶约20mg rhGH普通产量,达到终点值每转瓶接近70mg rhGH,而与之比较标准DMEM(未显示)对照培养物为30mgrhGH。与对照相比GH产量(即第14天时检测的每转瓶mg数)增加平均值见图3。
在本系列实验中,细胞剥脱离现象减少。
添加氨基酸
进行了氨基酸分析以检测hGH产量增加是否受到任何氨基酸的可用性的限制。表4显示用加和不加锌和铜培养两天后收获的培养基的氨基酸百分比。
表4:培养两天后收获的粗培养基中氨基酸浓度百分比,得自用标准DMEM培养基进行的培养或添加了0.5μM Zn、0.02μM Cu和4.8μM柠檬酸铁的DMEM培养基进行的培养。
氨基酸 | 标准DMEM | 添加的DMEM |
L-谷氨酰胺 | 31% | 13% |
L-丝氨酸 | 28% | 11% |
L-胱氨酸 | 20% | 13% |
其余L-氨基酸 | 50%-100% | 39%-86% |
检测了铜、锌和柠檬酸铁混合物(Zn 0.5μM,Cu 0.02μM和柠檬酸铁4.8μM)和以最高比例消耗的那些氨基酸的组合:L-谷氨酰胺(Gln 4.8mM),L-丝氨酸(Ser0.49mM)和L-胱氨酸(Cys 0.29mM)。结果见图4所示。
任何测试的氨基酸单独或联合都未见到对产量有正面作用。
当金属元素与氨基酸一起加入时,观察到对产量只有微小影响:谷氨酰胺对产量有轻微正面影响(78.2%升至81.1%)和胱氨酸对产量有负面影响(78.2%降至69.9%)。这种差异认为不显著。
使用含有金属混合物的DMEM所测得的结果(每转瓶每次收获的平均值约为50mg rhGH)与使用不含金属元素DMEM的培养(每转瓶每次收获的平均值约30mg rhGH)的产量差异显著。鉴于这些结果,认为不需要对生产培养基中的原先氨基酸成分进行修改。
滴定铜和锌的作用
为了检测铜的最佳浓度,保持柠檬酸铁浓度(4.8μM)和锌浓度(0.5μM)不变而改变铜的浓度进行了试验(图5)。
如图5所示,剂量反应实验证实,当锌和柠檬酸铁为所示浓度时,DMEM培养基中铜的最佳添加浓度为25.6nM
在另一实验中,保持柠檬酸铁浓度(4.8μM)和铜浓度(0.025μM)不变而改变锌的浓度(图6):锌浓度大于200nM时观察到最高产量水平(平台),产量稳定地高出对照平均值约60%。
结果总结
图7总结了微量锌和铜离子,联用或不联用铁离子获得的结果
以下数值基于为提高产量通过在DMEM培养基中添加金属微量元素而获得的数据:
锌 1.5μM:10.3%±5.0%,锌 0.5μM:16.8%±1.6%
铜 0.02μM:32.1%±9.4%,
锌 1.5μM+铜0.02μM:66.3%±16%
锌 0.5μM+铜0.02μM:61.2%(±10%)
锌 0.5μM+铜0.02μM+柠檬酸铁4.8μM:69.4%±19%.
鉴于以上数据,以下混合物为DMEM培养基的最佳添加物:
·以硫酸铜(CuSO4·5H2O)形式提供的铜,25nM铜,硫酸铜
·以硫酸锌(ZnSO4·7H2O)形式提供的锌,50nM-1500nM,优选浓度范围为200-500nM的锌
·柠檬酸铁,4.8μM,鉴于EMEM中已有硝酸铁,该离子的最终浓度为6μM。
结论
与标准DMEM培养基相比,在DMEM培养基中使用微量的铜、锌和柠檬酸铁作为补充,rhGH产量提高了50%以上。
参考文献
1.Altschul S F等,J Mol Biol,215,403-410,1990.
2.Arnold等.US2003/0153042
3.Ausubel等.,Current Protocols in Molecular Biology,同上,Interscience,N.Y.,§§6.3和6.4(1987,1992).
4.AltschulSF等,Nucleic Acids Res.,25:389-3402,1997.
5.Becker等,Biotechnol.Appl.Biochem.10:326(1988).
6.Bewly等,Int.J.Peptide and Protein Res.4:281-287(1972).
7.Blum等.US6,399,381
8.Bohak等.1987 Bohak Z,Kadouri A等.(1987)“用于悬浮培养哺乳动物细胞的新型锚定基质”,Biopolymers.26 Suppl:S205-213.
9.Carter M J,Facklam T J,Long P C,Scotland R A,Dev.Biol.Stand.1989;70:101-7.
10.Chen等,Genomics 4:479-497(1989).
11.Ciccarone等.US2003/0096414
12.Cleveland等.,US4,767,704
13.Darfler US5,045,568
14.DeNoto等,Nucleic Acids Res.9:3719(1981).
15.DevereuxJ等,Nucleic Acids Res,12,387-395,1984.
16.Gertler等,Endocrinology118:720(1986).
17.Goeddel等Nature,281:544(1979).
18.Graff等,J.Biol.Chem.257:2365(1982),
19.Grantham等.,Science,Vol.185,pp.862-864(1974).
20.Hsiung等,Biotechnology 7:267(1989).
21.Inlow等.US6,048,728
22.Jorgensson等,Pharmacol.Toxicol.63:129(1988).
23.Lewis等,Endocrinology 101:1587(1977).
24.Lewis等,J.Biol.Chem.253:2679(1978).
25.Lewis等,Endocrinology 104:1256(1979).
26.Lewis等,Biochem.Biophys.Res.Comm.92:511(1980).
27.Lewis等,J.Biol.Chem.256:11645(1981).
28.Lindner MC 1991.Biochemistry of copper.New York,Plenum press(教材).
29.Martial等,Science 205:602-607(1979).
30.Moore等,Endocrinology 122:2920(1988).
31.Novick,D,Kim,S-H,Fantuzzi,G,Reznikov,L,Dinarello,C,和Rubinstein,M(1999).Immunity 10,127-136.
32.OkaM S,Rupp RG.,Bioprocess Technol.1990;10:71-92
33.Pearson,Methods Enzymol.1990;183:63-98.
34.Petti SA,Lages AC等.(1994)“哺乳动物细胞在无纺聚酯纤维盘上的三维生长”,Biotechnol Prog.10(5):548-550.
35.Puck等.,J.Exp.Med.108,945,1958.
36.Renner等.,US5,811,299
37.Singh等,Endocrinology 94:883(1974).
38.Thorlacius-Ussing,Neuroendocrinology 43:233(1987).
39.ValleeBLandFalchukKH1993.锌生理学的生化基础,Physiologicalreviews73,79-118.
40.EP274445
41.EP314317A1
42.EP0325224A2
43.WO01/16294
Claims (12)
1.一种生产生长激素的方法,其特征在于,该方法包括在无得自动物血清的组分的细胞培养基中培养表达生长激素的细胞系细胞的步骤,所述培养基含有浓度为0.2μM-0.5μM的锌,浓度为25nM的铜和浓度为5或6μM的铁离子。
2.如权利要求1所述的方法,其中所述培养基所含的锌是硫酸锌。
3.如权利要求1所述的方法,其中所述培养基所含的铜是硫酸铜。
4.如权利要求1所述的方法,其中所述培养基含有的铁离子为柠檬酸铁和/或硝酸铁的铁离子。
5.如权利要求1所述的方法,其中所述培养基还含有基础培养基的成分。
6.如权利要求5所述的方法,其中所述基础培养基是Dulbecco’s改良Eagle’s培养基(DMEM)。
7.如权利要求1所述的方法,其中所述生长激素在金属硫蛋白启动子的控制下表达。
8.如权利要求7所述的方法,其中所述金属硫蛋白启动子是小鼠MT-1启动子。
9.如权利要求1所述的方法,所述方法还包括从细胞培养物收集生长激素的步骤。
10.如权利要求9所述的方法,所述方法还包括纯化生长激素。
11.如权利要求10所述的方法,所述方法还包括将纯化的生长激素与药学上可接受的运载体制备成药物组合物。
12.如权利要求1所述的方法,其中所述生长激素为人生长激素。
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Families Citing this family (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20090127326A (ko) | 2007-03-02 | 2009-12-10 | 와이어쓰 | 폴리펩티드의 생산을 위한 세포 배양물 중에서 구리 및 글루타메이트의 용도 |
WO2008136398A1 (ja) * | 2007-04-26 | 2008-11-13 | Chugai Seiyaku Kabushiki Kaisha | 高濃度アミノ酸含有培地を用いた細胞の培養方法 |
CN101386837B (zh) * | 2007-09-12 | 2011-06-29 | 北京清大天一科技有限公司 | 一种动物细胞培养方法 |
CA2720980C (en) * | 2008-04-17 | 2014-02-25 | Wyeth Llc | Methods for enhanced production of bone morphogenetic proteins |
MX2012012525A (es) * | 2010-04-26 | 2012-11-23 | Novartis Ag | Medio de cultivo mejorado. |
CN102234627B (zh) * | 2010-04-30 | 2015-06-03 | 中国科学院广州生物医药与健康研究院 | 一种培养基添加剂及其应用 |
ES2761692T5 (es) * | 2010-07-08 | 2023-07-05 | Takeda Pharmaceuticals Co | Método de producción de vWF recombinante de alto peso molecular en cultivo celular |
DK2702164T3 (en) * | 2011-04-29 | 2016-02-01 | Biocon Res Ltd | METHOD FOR REDUCING heterogeneity OF ANTIBODIES AND METHOD OF PRODUCING THESE ANTIBODIES |
CA2854780A1 (en) | 2011-11-11 | 2013-05-16 | Essential Pharmaceuticals, Llc | Kit comprising serum replacement and labile factors |
EP2970980B2 (en) | 2013-03-15 | 2022-07-27 | Janssen Biotech, Inc. | Manufacturing methods to control c-terminal lysine, galactose and sialic acid content in recombinant proteins |
USD752617S1 (en) * | 2013-10-23 | 2016-03-29 | Ares Trading S.A. | Display screen with graphical user interface |
KR20160125352A (ko) | 2013-12-20 | 2016-10-31 | 이센셜 파마수티컬스 엘엘씨 | 세포 배양용 배지 |
LT3110961T (lt) * | 2014-02-27 | 2020-02-10 | F. Hoffmann-La Roche Ag | Ląstelių augimo ir glikozilinimo moduliavimas, gaminant rekombinantinį glikoproteiną |
USD779504S1 (en) * | 2014-11-14 | 2017-02-21 | Dexcom, Inc. | Display screen or portion thereof with graphical user interface for presentation of analyte data |
USD779505S1 (en) | 2014-11-14 | 2017-02-21 | Dexcom, Inc. | Display screen or portion thereof with graphical user interface for analyte data presentation |
KR101867134B1 (ko) * | 2015-03-23 | 2018-06-12 | 한양대학교 산학협력단 | 포유류 세포를 이용하여 목적 물질을 고효율로 생산하기 위한 세포 배양 배지, 이를 이용한 세포 배양 방법 및 목적 물질의 생산 방법 |
CA3019574A1 (en) * | 2016-03-31 | 2017-10-05 | Sanbio, Inc. | Medium, methods, cells and secreted factors for stem cell culture and therapy |
CN110042137B (zh) * | 2019-05-14 | 2023-02-28 | 上海赛迈生物科技有限公司 | 高密度灌注培养重组cho细胞生产人促卵泡激素的方法、培养基及其应用 |
CN114075540B (zh) * | 2020-08-21 | 2023-10-13 | 苏州新微溪生物医药有限公司 | 全化学成分hek293细胞培养基及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0369458A2 (en) * | 1988-11-18 | 1990-05-23 | Phillips Petroleum Company | Bovine metallothionein regulatory region |
Family Cites Families (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4579821A (en) * | 1981-11-23 | 1986-04-01 | University Patents, Inc. | Control of DNA sequence transcription |
JPS60500358A (ja) * | 1982-12-23 | 1985-03-22 | アメリカ合衆国 | マウスの細胞内で組み換えdnaによって生産されたヒト成長ホルモン |
US4767704A (en) | 1983-10-07 | 1988-08-30 | Columbia University In The City Of New York | Protein-free culture medium |
WO1986004920A1 (en) * | 1985-02-13 | 1986-08-28 | Biotechnology Research Partners, Limited | Human metallothionein-ii promoter in mammalian expression system |
US4935350A (en) * | 1985-11-18 | 1990-06-19 | Amgen | Materials and methods for controlling plasmid copy number and stability |
CS262822B1 (en) | 1986-10-03 | 1989-04-14 | Kovar Jan | Synthetic medium for the cultivation of myelomic cells |
US5045468A (en) | 1986-12-12 | 1991-09-03 | Cell Enterprises, Inc. | Protein-free culture medium which promotes hybridoma growth |
NO162160C (no) | 1987-01-09 | 1989-11-15 | Medi Cult As | Serumfritt vekstmedium, samt anvendelse derav. |
NZ226414A (en) | 1987-10-02 | 1992-07-28 | Genentech Inc | Cd4 peptide adhesion variants and their preparation and use |
DE68926888T2 (de) | 1988-01-22 | 1997-01-09 | Zymogenetics Inc | Verfahren zur Herstellung von sekretierten Rezeptoranalogen |
US6048728A (en) * | 1988-09-23 | 2000-04-11 | Chiron Corporation | Cell culture medium for enhanced cell growth, culture longevity, and product expression |
US5143842A (en) * | 1988-11-01 | 1992-09-01 | The University Of Colorado Foundation, Inc. | Media for normal human muscle satellite cells |
US5834312A (en) * | 1990-01-29 | 1998-11-10 | Hy-Gene, Inc. | Process and media for the growth of human epithelia |
JP3008208B2 (ja) * | 1990-06-01 | 2000-02-14 | 武田薬品工業株式会社 | 新規ハイブリドーマ,その製造法および生理活性物質の製造法 |
US5122469A (en) * | 1990-10-03 | 1992-06-16 | Genentech, Inc. | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins |
GB9022545D0 (en) * | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
EP0666312A1 (en) | 1994-02-08 | 1995-08-09 | Wolfgang A. Renner | Process for the improvement of mammalian cell growth |
DE69623109T2 (de) | 1996-04-19 | 2002-12-12 | Nestle Sa | Menschliche immortalisierte Colon-Epithelialzellen |
JP2000517188A (ja) * | 1996-08-30 | 2000-12-26 | ライフ テクノロジーズ,インコーポレイテッド | 無血清哺乳動物細胞培養培地およびその使用 |
JP4543402B2 (ja) * | 1996-10-10 | 2010-09-15 | ライフ テクノロジーズ コーポレーション | 植物由来栄養素を含む動物細胞培養培地 |
US20030153042A1 (en) | 1998-07-28 | 2003-08-14 | California Institute Of Technology | Expression of functional eukaryotic proteins |
EP1210410B1 (en) | 1999-08-27 | 2008-01-16 | Invitrogen Corporation | Metal binding compounds and their use in cell culture medium compositions |
CA2438148A1 (en) * | 2001-02-15 | 2002-08-29 | Centocor, Inc. | Chemically defined medium for cultured mammalian cells |
US20030096414A1 (en) | 2001-03-27 | 2003-05-22 | Invitrogen Corporation | Culture medium for cell growth and transfection |
-
2005
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0369458A2 (en) * | 1988-11-18 | 1990-05-23 | Phillips Petroleum Company | Bovine metallothionein regulatory region |
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