CN107847549B - 用于增加细胞中的谷胱甘肽水平的方法 - Google Patents
用于增加细胞中的谷胱甘肽水平的方法 Download PDFInfo
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- CN107847549B CN107847549B CN201680044852.8A CN201680044852A CN107847549B CN 107847549 B CN107847549 B CN 107847549B CN 201680044852 A CN201680044852 A CN 201680044852A CN 107847549 B CN107847549 B CN 107847549B
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Abstract
本发明涉及磺基半胱氨酸和其衍生物增加细胞中的谷胱甘肽库的用途。
Description
本发明涉及磺基半胱氨酸和其衍生物增加细胞中的谷胱甘肽库(glutathionepool)的用途。
谷胱甘肽,一种由氨基酸L-甘氨酸、L-半胱氨酸和L-谷氨酸(亦称为L-谷氨酸盐)组成的三肽分子,是哺乳动物中主要的细胞内抗氧化剂之一。
谷胱甘肽以还原(GSH)和氧化(GSSG)状态存在,其中还原形式(GSH)占优势。增加的GSSG:GSH比率被视为表示氧化应激。
尽管谷胱甘肽在大多数细胞中产生,但许多疾病和其它病理与细胞内谷胱甘肽的水平降低有关。谷胱甘肽是一种受严格调节的细胞内成分,和其产生受其通过酶γ-谷氨酰基半胱氨酸合成酶的自身合成的负反馈抑制的限制,因此使任何过量可能性极大最小化。使用谷胱甘肽合成的前体的谷胱甘肽增加是一种经开发以解决谷胱甘肽缺乏、高氧化应激、免疫缺陷和异物过载的状态的策略,其中谷胱甘肽在所涉及的异物的解毒中起作用。在人中谷胱甘肽缺乏状态包括但不限于,HIV/AIDS、化学和感染性肝炎、肌痛性脑脊髓炎、慢性疲乏综合征、前列腺和其它癌症、白内障、阿尔茨海默病、帕金森病、慢性阻塞性肺病、哮喘、辐射中毒、营养不良状态、恶劣机体应激和衰老,和与亚最佳免疫反应有关。许多临床病理与氧化应激有关,和在许多医学参考资料中详细描述。
此外,公认的是谷胱甘肽系统的缺陷导致显著的细胞衰老,和最终导致细胞发病。细胞的谷胱甘肽的浓度对抗氧化剂功能具有显著影响;营养素限制、锻炼和氧化应激对细胞的谷胱甘肽浓度具有显著影响。
谷胱甘肽在一系列生物化学反应中,利用ATP、镁和三种氨基酸甘氨酸、谷氨酸和半胱氨酸合成。一般而言,γ-谷氨酰基半胱氨酸的合成速率决定了谷胱甘肽的合成速率,和半胱氨酸的巯基为谷胱甘肽提供了其生物学效力。因此,半胱氨酸利用率对于谷胱甘肽的利用率和其抗氧化功能是重要的。
谷胱甘肽的利用率可涉及治疗应用和美容应用,还有细胞培养应用。在培养的细胞中总GSH库的增加导致细胞内区室中的氧化反应性降低,因此导致延长的培养持续时间,通常还导致效价增加。
本发明的目的因此是发现增加细胞的GSH含量的方法。对此,主要在治疗领域中,数种方法是本领域已知的。
US 2014/0100283公开了GSH衍生物S-乙酰基-谷胱甘肽的用途。
US 2011/0077303公开了提供甘氨酸和N-乙酰基-半胱氨酸。
US 2013/0338165表明了某些半胱氨酸/半胱氨酸前药或N-乙酰基-半胱氨酸前药的用途。
对于细胞培养应用,这样的方法可行性有限,因为谷胱甘肽前体由于需要特定的运输系统而通常不被细胞吸收。对于细胞培养,更有效和通用的方法将是有利的。
已发现S-磺基半胱氨酸和其盐增加哺乳动物细胞的GSH含量。另外已发现,如果向细胞提供等量的半胱氨酸或S-磺基半胱氨酸,与接受半胱氨酸的细胞相比,已接受s-磺基半胱氨酸的细胞显示更高量的GSH。
本发明因此涉及增加细胞中的谷胱甘肽的水平的方法,包括向所述细胞加入有效增加细胞内谷胱甘肽水平的量的S-磺基半胱氨酸和/或S-磺基半胱氨酸盐。
在优选的实施方案中,所述方法通过以下来进行:在液体细胞培养基中培养所述细胞,和在培养的过程中在一个或多个时间点向细胞培养基加入有效增加培养物中的细胞的细胞内谷胱甘肽水平的量的S-磺基半胱氨酸和/或S-磺基半胱氨酸盐。
在优选的实施方案中,加入(S)-2-氨基-3-磺基硫烷基丙酸钠盐。
在另一优选的实施方案中,细胞培养基具有介于6.8和7.5之间的pH。
在另一优选的实施方案中,S-磺基半胱氨酸和/或S-磺基半胱氨酸盐以使得其在细胞培养物中的浓度介于0.4和50 mM之间的量加入。
在一个实施方案中,细胞在至少包含一种或多种糖组分、一种或多种氨基酸、一种或多种维生素或维生素前体、一种或多种盐、一种或多种缓冲剂组分、一种或多种辅因子和一种或多种核酸组分的细胞培养基中培养。
在一个实施方案中,本发明的方法通过以下进行:
a) 提供生物反应器;
b) 将待培养的细胞与包含S-磺基半胱氨酸和/或S-磺基半胱氨酸盐的细胞培养基混合;
c) 孵育步骤b)的混合物。
在另一优选的实施方案中,本发明的方法通过以下进行:
- 向生物反应器中加入细胞和液体细胞培养基;
- 在生物反应器中孵育细胞;
- 在生物反应器中的细胞的整个孵育时间内连续地,或在所述孵育时间内一次或数次,向生物反应器加入细胞培养基,在此情况下所述细胞培养基是进料培养基,
其中进料培养基包含S-磺基半胱氨酸和/或S-磺基半胱氨酸盐。
优选地,进料培养基包含浓度介于1和100 mmol/l之间、优选地介于5和20 mmol/l之间的S-磺基半胱氨酸和/或S-磺基半胱氨酸盐。
本发明还涉及S-磺基半胱氨酸和/或S-磺基半胱氨酸盐用于增加细胞内谷胱甘肽的量的用途。
图1显示用半胱氨酸或S-磺基半胱氨酸,用在生物反应器分批进料实验中培养的CHO细胞获得的IgG浓度的比较。进一步的细节可见于实施例。
图2显示用半胱氨酸或S-磺基半胱氨酸,在生物反应器分批进料实验中培养的CHO细胞的细胞内活性物质的比较。进一步的细节可见于实施例。
图3显示用半胱氨酸或S-磺基半胱氨酸,在生物反应器分批进料实验中培养的CHO细胞的谷胱甘肽水平的比较。进一步的细节可见于实施例。
S-磺基半胱氨酸,亦称为(S)-2-氨基-3-磺基硫烷基丙酸,是一种产物,例如通过缩合硫酸和半胱氨酸可获得的产物。合适的盐是碱金属或碱土金属盐,例如锂盐、钠盐、钾盐、钙盐或镁盐,或其混合物。优选的是钠盐、钾盐、钙盐和镁盐,最优选的是钠盐。
S-磺基半胱氨酸和其盐也可通过下式I和II显示:
其中R是
和X是H、Li、Na、K、½ Ca、½ Mg,优选地H、Na、K。术语丙酸(propanoic acid)还可代替术语丙酸(propionic acid)使用。
2-氨基-3-磺基硫烷基-丙酸,亦称为(S)-2-氨基-3-磺基硫烷基-丙酸、S-磺基-半胱氨酸或半胱氨酸-S-硫酸和其盐的合成公开于例如I.H.Segel和M.J.Johnson,Analytical Biochemistry 5 (1963), 330-337和J.S. Church, D.J. Evans,Spectrochimica Acta Part A 69 (2008) 256-262。钠盐另外可市售获自Bachem,Switzerland。
细胞培养基是维持和/或支持细胞的体外生长的任何组分混合物。其可能是复合培养基或化学成分确定的培养基。细胞培养基可包含维持和/或支持细胞的体外生长所必需的所有组分,或仅一些组分,使得单独添加另外的组分。细胞培养基的实例是完全培养基,其包含维持和/或支持细胞的体外生长所必需的所有组分,以及培养基补充物或进料。完全培养基,亦称为基础培养基,通常具有介于6.8和7.8之间的pH。进料培养基优选地具有低于8.5的pH。
通常,根据本发明的细胞培养基用于维持和/或支持在生物反应器中的细胞生长和支持所述细胞的IgG产生。
一些细胞培养基作为无菌水性液体提供。液体细胞培养基的缺点是它们减少的保存期限以及运输和贮存困难。因而,目前许多细胞培养基作为细磨干粉混合物提供。它们为在水和/或水性溶液中溶解的目的而制造,并且在溶解状态下经设计,通常与其它补充物一起,为细胞提供重要的生长营养基础和/或从所述细胞产生生物药物。
大多数生物药物产生平台基于分批进料细胞培养方案。目的通常是开发高效价细胞培养过程以满足日益增加的市场需求和减少制造成本。除了使用高效重组细胞系之外,需要细胞培养基和过程参数的改进以实现最大生产潜能。
在分批进料过程中,基础培养基支持初始生长和生产,和进料培养基防止营养物耗尽和维持生产阶段。选择培养基以适应在不同的生产阶段期间的不同的代谢需求。过程参数设定 — 包括进料策略和控制参数 — 定义了适合于细胞生长和蛋白产生的化学和物理环境。
进料或进料培养基是这样的细胞培养基,其不是在细胞培养中支持初始生长和生产的基础培养基,而是在后面的时期加入以防止营养物耗尽和维持生产阶段的培养基。与基础培养基相比,进料培养基可具有更高浓度的一些组分。例如,一些组分,例如营养物,包括氨基酸或碳水化合物,可以基础培养基中浓度的约5X、6X、7X、8X、9X、10X、12X、14X、16X、20X、30X、50X、100x、200X、400X、600X、800X或甚至约1000X存在于进料培养基中。
哺乳动物细胞培养基是维持和/或支持哺乳动物细胞的体外生长的组分的混合物。哺乳动物细胞的实例是人或动物细胞,优选地CHO细胞、COS细胞、I VERO细胞、BHK细胞、AK-1细胞、SP2/0细胞、L5.1细胞、杂交瘤细胞或人细胞。
化学成分确定的细胞培养基是不包含任何化学不确定的物质的细胞培养基。这意味着培养基中使用的所有化学品的化学组成是已知的。化学成分确定的培养基不包含任何酵母、动物或植物组织;它们不包含饲养细胞、血清、提取物或消化物或其它组分,这些可促使化学确定差的蛋白质进入培养基。化学不确定或确定差的化学组分是化学组成和结构是未知的,以变化的组成存在或仅以大量实验工作才能确定的那些 – 与蛋白质例如白蛋白或酪蛋白的化学组成和结构的评价相当。
粉状的细胞培养基或干粉培养基是通常自研磨过程或冻干过程产生的细胞培养基。这意味着粉状的细胞培养基是粒状的颗粒培养基 – 不是液体培养基。术语"干粉"可与术语"粉末"互换使用;然而,本文使用的"干粉"仅指粒状材料的总体外观,和不意味着该材料完全不含复合或聚集的溶剂,除非另外指明。粉状的细胞培养基也可是粒状的细胞培养基,例如通过碾压干燥成粒。
粉状的细胞培养基优选地通过混合所有组分和研磨它们来产生。组分的混合是通过研磨生产干粉细胞培养基的领域的技术人员已知的。优选地,所有组分充分混合,以致混合物的所有部分具有几乎相同的组成。组成的均匀性越高,在同质细胞生长方面,得到的培养基的质量越好。
研磨可用适合于生产粉状的细胞培养基的任何类型的研磨机进行。典型的实例是球磨机、针磨机、fitz磨机或喷射磨机。优选的是针磨机、fitz磨机或喷射磨机,非常优选针磨机。
本领域技术人员知道如何运行这样的研磨机。
对于研磨的粉状培养基的使用,溶剂,优选地水(最特别是蒸馏和/或去离子水或纯化水或注射用水)或水性缓冲剂加入培养基,和将组分混合,直至培养基全部溶于溶剂中。
溶剂还可包含盐水、提供合适的pH范围(通常在pH 1.0-pH 10.0的范围内)的可溶性酸或碱离子、稳定剂、表面活性剂、防腐剂和醇或其它极性有机溶剂。
还可能添加另外的物质,例如用于调节pH的缓冲物质、胎牛血清、糖等至细胞培养基和溶剂的混合物。得到的液体细胞培养基然后与待生长或维持的细胞接触。
用本发明的方法处理的细胞可以是正常细胞、永生细胞、患病细胞、转化细胞、突变细胞、体细胞、生殖细胞、干细胞、前体细胞或胚胎细胞,其任何可以是建立的或转化的细胞系,或获自天然来源。优选地,细胞是哺乳动物细胞,更优选BHK、VERO、HEK或CHO细胞,最优选是CHO-S、CHO dhfr- (DG44和Duxb11)、CHO-M和CHOK1细胞。
细胞培养基,特别是完全培养基,通常至少包含一种或多种糖组分、一种或多种氨基酸、一种或多种维生素或维生素前体、一种或多种盐、一种或多种缓冲剂组分、一种或多种辅因子和一种或多种核酸组分。
培养基还可包含丙酮酸钠、胰岛素、植物蛋白、脂肪酸和/或脂肪酸衍生物和/或普卢兰尼克酸和/或表面活性组分例如化学制备的非离子表面活性剂。合适的非离子表面活性剂的一个实例是以伯羟基封端的双官能嵌段共聚物表面活性剂,亦称为泊洛沙姆,例如可以商品名pluronic ®从BASF, Germany获得。
糖组分全部是单糖或二糖,例如葡萄糖、半乳糖、核糖或果糖(单糖的实例)或蔗糖、乳糖或麦芽糖(二糖的实例)。
根据本发明的氨基酸的实例是酪氨酸,蛋白质性氨基酸,特别是必需氨基酸,亮氨酸、异亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、苏氨酸、色氨酸和缬氨酸,以及非蛋白质性氨基酸,例如D-氨基酸。
酪氨酸意指L-或D-酪氨酸,优选地L-酪氨酸。
半胱氨酸意指L-或D-半胱氨酸,优选地L-半胱氨酸。
维生素的实例是维生素A (视黄醇、视黄醛、各种类视色素和四种类胡萝卜素)、维生素B1 (硫胺素)、维生素B2 (核黄素)、维生素B3 (烟酸、烟酰胺)、维生素B5 (泛酸)、维生素B6 (吡哆醇、吡多胺、吡哆醛)、维生素B7 (生物素)、维生素B9 (叶酸、亚叶酸)、维生素B12(氰钴胺、羟钴胺、甲钴胺)、维生素C (抗坏血酸)、维生素D (麦角钙化醇、胆钙化醇)、维生素E (生育酚、生育三烯酚)和维生素K (叶绿醌、甲萘醌)。还包括维生素前体。
盐的实例是包含无机离子例如碳酸氢根、钙、氯离子、镁、磷酸根、钾和钠或痕量元素例如Co、Cu、F、Fe、Mn、Mo、Ni、Se、Si、Ni、Bi、V和Zn的组分。实例是五水合硫酸铜(II)(CuSO4 .5H2O)、氯化钠(NaCl)、氯化钙(CaCl2 .2H2O)、氯化钾(KCl)、硫酸铁(II)、无水单碱式磷酸钠(NaH2PO4)、无水硫酸镁(MgSO4)、无水二碱式磷酸钠(Na2HPO4)、六水合氯化镁(MgCl2 .6H2O)、七水合硫酸锌。
缓冲剂的实例是CO2/HCO3 (碳酸盐)、磷酸盐、HEPES、PIPES、ACES、BES、TES、MOPS和TRIS。
辅因子的实例是硫胺素衍生物、生物素、维生素C、NAD/NADP、钴胺素、黄素单核苷酸和衍生物、谷胱甘肽、血红素、磷酸核苷酸和衍生物。
根据本发明的核酸组分是核碱基,例如胞嘧啶、鸟嘌呤、腺嘌呤、胸腺嘧啶或尿嘧啶;核苷,例如胞苷、尿苷、腺苷、鸟苷和胸苷;和核苷酸,例如腺苷单磷酸或腺苷二磷酸或腺苷三磷酸。
进料培养基可具有与完全培养基不同的组成。它们通常包含氨基酸、痕量元素和维生素。它们还可包含糖组分,但有时为生产原因,糖组分在单独的进料中添加。
合适的进料培养基可例如包含一种或多种以下化合物:
L-天冬酰胺一水合物 |
L-异亮氨酸 |
L-苯丙氨酸 |
L-谷氨酸钠一水合物 |
L-亮氨酸 |
L-苏氨酸 |
L-赖氨酸一盐酸盐 |
L-脯氨酸 |
L-丝氨酸 |
L-精氨酸一盐酸盐 |
L-组氨酸一盐酸盐一水合物 |
L-甲硫氨酸 |
L-缬氨酸 |
L-天冬氨酸一钠一水合物 |
L-色氨酸 |
氯化胆碱 |
肌醇 |
烟酰胺 |
D(+)泛酸钙 |
吡哆醇盐酸盐 |
氯化硫胺素盐酸盐 |
微粉化的维生素B12 (氰钴胺E) |
生物素 |
叶酸 |
核黄素 |
无水硫酸镁 |
五水合硫酸铜(II) |
七水合硫酸锌 |
1,4-二氨基丁烷二盐酸盐 |
四水合七钼酸铵 |
水合硫酸镉 |
四水合氯化锰(II) |
六水合氯化镍(II) |
偏硅酸钠 |
偏钒酸钠 |
二水合氯化锡(II) |
亚硒酸钠(约45% SE) |
磷酸二氢钠一水合物 |
柠檬酸铵铁(III)(约18% FE) |
本发明的目的是增加细胞中的总谷胱甘肽库。已发现,通过添加S-磺基半胱氨酸和/或其盐来增加细胞中谷胱甘肽的量,通常实现一种或多种以下积极效果:
- 更长的培养持续时间
- 更高的效价(产生的IgG的浓度)
- 更高的特异性生产率
- 更低的细胞内氧化潜能
这些效果非常有益于细胞培养,因为高效价、高生产率以及较长的培养持续时间全都增加细胞培养效率。
根据本发明用于S-磺基半胱氨酸和/或其盐处理的细胞通常是为生物药物生产目的在生物反应器中培养的细胞。合适的细胞培养过程的实例是分批进料过程或灌流细胞培养过程。
S-磺基半胱氨酸和/或其盐可在细胞培养的任何时期加入细胞。
其可在开始细胞培养时加入。在此情况下,S-磺基半胱氨酸和/或其盐优选地与用于开始细胞培养的基础培养基的其它成分混合和研磨。这种包含S-磺基半胱氨酸和/或其盐的干粉混合物然后通过混合粉末和溶剂溶于合适的溶剂,使得粉末溶解和产生具有需要的和均一的浓度的培养基组分的液体细胞培养基。
S-磺基半胱氨酸和/或其盐也可在细胞培养期间一次或多次加入。细胞培养通常进行1-3周。在此期间,持续或一次或多次加入进料培养基。S-磺基半胱氨酸和/或其盐可与其它进料培养基成分一起加入进料培养基,或其可在仅包含S-磺基半胱氨酸和/或其盐的单独进料中加入。另外,进料通常是液体,使得进料的所有组分在加入细胞培养物之前溶于合适的溶剂。
在优选的实施方案中,S-磺基半胱氨酸和/或其盐作为进料添加。优选地在细胞培养期间添加至少4次,优选地4-6次。
在一个实施方案中,S-磺基半胱氨酸和/或其盐每隔一天至每隔三天添加。
包含S-磺基半胱氨酸和/或其盐的进料的pH优选地介于6.8和7.5之间,最优选介于6.8和7.1之间。
已发现,如果S-磺基半胱氨酸和/或其盐以0.4-50 mM、优选地1-10 mM的浓度存在于细胞培养物中,谷胱甘肽的水平可最有效地增加。通常,在整个培养过程中添加至细胞培养物的进料的体积是已经存在于生物反应器中的细胞培养基的体积的约30%。在进料中S-磺基半胱氨酸和/或其盐的浓度优选地介于1和100 mmol/l之间,优选地介于5和20 mmol/l之间。
通常,细胞培养通过以下进行:
a) 提供生物反应器
b) 将待培养的细胞与液体细胞培养基在生物反应器中混合
c) 孵育步骤b)的混合物一定的时间
生物反应器是其中可培养细胞的任何容器、袋、管或池。进行细胞培养是本领域技术人员已知的。这通常通过在合适的条件(例如pH、渗透压、温度、搅拌、换气(氧气/CO2)等)下孵育生物反应器中的细胞,和任选在细胞培养期间一次或数次添加进料培养基进行。优选地,细胞培养作为分批进料细胞培养进行。
分批进料培养是其中在细胞培养期间一种或多种营养物(底物)被进料(提供)至生物反应器和其中产物保留在生物反应器中直至运行结束的细胞培养过程。该方法的可选描述是其中基础培养基支持初始细胞培养和添加进料培养基以阻止营养物耗尽的培养。分批进料培养的优点是可以随意需要的水平控制在培养液体中的进料底物的浓度。
一般而言,当控制一种或多种营养物的浓度影响所需代谢物的收率或生产率时,在此情况下例如S-磺基半胱氨酸和/或其盐,分批进料培养优于常规分批培养。
因此,优选地,本发明通过以下进行:
- 向生物反应器中加入细胞和液体细胞培养基
- 孵育生物反应器中的细胞
- 在生物反应器中的细胞的整个孵育时间内连续地,或在所述孵育时间内一次或数次,向生物反应器加入细胞培养基,在此情况下所述细胞培养基为进料培养基,
其中进料培养基优选地具有介于6.8和7.5之间的pH,和包含S-磺基半胱氨酸和/或其盐。
在另一个实施方案中,细胞培养作为灌流培养进行。
灌流培养是这样的培养,藉此细胞中通过例如过滤、包封、锚定至微载体等限制在培养物中,和培养基从生物反应器中连续或间断地引入和除去。在此情况下,S-磺基半胱氨酸和/或其盐优选地作为培养基的一部分引入。
在灌流培养中,在细胞培养过程中,细胞(生物质)自细胞培养物(细胞悬浮液)分离,一个方面用尽的培养基从过程中取出,另一个方面,通过新培养基使新的营养物对细胞而言是可用的。如果细胞在过程期间保持在培养系统中,这称为"灌流"。如果细胞在过程期间从含用尽的培养基的系统中除去,这称为"连续方法"。在灌流过程中,细胞也可以定义的时间间隔从培养系统中除去,使得能够维持最大细胞浓度。对于本领域技术人员,该操作被称为"溢泌(bleeding)"。细胞分离用各种技术进行,其中一些技术间接促进细胞分离。一些可能的细胞分离方法的实例是过滤、细胞包封、细胞粘附至微载体、细胞沉降或离心。
已发现,用本发明的方法,细胞内谷胱甘肽水平可有效地增加。通常,在至少一部分的细胞培养时间,该水平可增加两倍以上。优选地,与在同等条件下但未添加S-磺基半胱氨酸和/或其盐的细胞培养物相比,对于超过一半的细胞培养时间,谷胱甘肽的水平增加至少10%,优选地超过25%。
本发明通过以下附图和实施例进一步说明,然而不限于此。
上文和下文引用的所有申请、专利和出版物,以及2015年7月30日提交的相应EP15179065.6的整个公开内容,通过引用结合到本文中。
实施例
以下实施例提供了本发明的实际应用。
在1.2L玻璃生物反应器中,使用CHO悬浮细胞系,在Cellvento® CHO 220培养基和进料220中的生物反应器分批进料实验
将15 mM S-磺基-L-半胱氨酸(SS)整合至中性pH主进料-220 (n=5)。在第3天以3%(v/v)和在第5、7、9和14天以6% (v/v)添加进料。在对照条件下,以相同的比率添加不含SS的进料,而在碱性进料中单独添加150mM L-半胱氨酸,和以以下比率添加:第3天0.3% (v/v)和第5、7、9、14天6% (v/v) (n=2)。pH控制在6.95 +/- 0.15。通过开管喷头用纯氧和空气鼓泡,溶解氧浓度控制在50%空气饱和度。温度设定在37°C,和在培养的第5天从37°C变化至33°C。搅拌维持在140 rpm。用浊度计量方法,使用Cedex BioHT测量悬浮液中的IgG浓度。结果在图1中示出。可以见到,用SS的效价(IgG浓度)高于标准品。
在使用S-磺基半胱氨酸的分批进料中(对比对照过程),在CHO细胞中的细胞内活性物质
细胞内活性物质使用6-羧基-2',7'-二氯二氢二醋酸荧光素染色(羧基H2DCFDA,Life Technologies)量化。由于氧化反应导致的荧光增加使用Perkin Elmer荧光读出器测定。对于这些反应,与对照条件和15 mM SSC条件相比,在分批进料实验期间进行测量。每天获取样品和直接分析。简言之,将3x105个细胞离心(1200 rpm, 5 min),并重悬于PBS (阴性对照)或用50 µM羧基-H2DCFDA加载,和在37 °C和1000 rpm下孵育20 min。然后将细胞离心,重悬于PBS和使用读板器分析(对于两个条件,n=3)。当在进料中使用S-磺基半胱氨酸时荧光强度越低,表明细胞中的反应性越低,从而表明分子的抗氧化潜力。结果在图2中显示。
在使用S-磺基半胱氨酸的分批进料期间(对比对照过程),在CHO细胞中的细胞内总谷胱甘肽
为了定量细胞内总谷胱甘肽,细胞在冷PBS中洗涤三次和在-20°C下冷冻用于进一步分析。为了抑制潜在的Cys-转化酶的活性,将12x106个细胞在100 µl的phosphoSafe试剂(Merck Millipore)中裂解,所述试剂包含四种磷酸酶抑制剂:氟化钠、钒酸钠、β-甘油磷酸和焦磷酸钠,并补充有20 mM碘乙酰胺(在活性位点包含一个半胱氨酸的所有酶被烷基化)。谷胱甘肽浓度(GSH和GSSG)通过UPLC,使用柱前衍生化,依赖于AccQ Tag Ultra®试剂盒测定。衍生化、色谱和数据分析按供应商的建议进行(Waters, Milford, MA)。FB过程的每天,总谷胱甘肽通过GSH和GSSG相加获得,并标准化至在对照条件下获得的浓度。结果显示在图3中。
Claims (11)
1.增加在生物反应器中培养的CHO细胞中的谷胱甘肽的水平的方法,包括向所述细胞加入有效增加细胞内谷胱甘肽水平的量的S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐。
2.权利要求1的方法,特征在于所述方法通过以下进行:在液体细胞培养基中培养所述细胞,和在培养的过程中在一个或多个时间点向所述细胞培养基加入有效增加培养物中的细胞的细胞内谷胱甘肽水平的量的S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐。
3.权利要求1或2的方法,特征在于加入(S)-2-氨基-3-磺基硫烷基丙酸钠盐。
4.权利要求2的方法,特征在于所述细胞培养基具有介于6.8和7.5之间的pH。
5.权利要求1或2的方法,特征在于S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐以使得它们在细胞培养物中的浓度介于0.4和50 mM之间的量加入。
6.权利要求1或2的方法,特征在于所述细胞在至少包含一种或多种糖组分、一种或多种氨基酸、一种或多种维生素或维生素前体、一种或多种盐、一种或多种缓冲剂组分、一种或多种辅因子和一种或多种核酸组分的细胞培养基中培养。
7.权利要求1或2的方法,特征在于所述方法通过以下进行:
a) 提供生物反应器;
b) 将待培养的细胞与包含S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐的细胞培养基混合;
c) 孵育步骤b)的混合物。
8.权利要求1或2的方法,特征在于所述方法通过以下进行:
向生物反应器中加入细胞和液体细胞培养基;
孵育生物反应器中的细胞;
在生物反应器中的细胞的整个孵育时间内连续地,或在所述孵育时间内一次或数次,向生物反应器加入进料培养基,
其中所述进料培养基包含S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐。
9.权利要求8的方法,特征在于所述进料培养基包含浓度介于1和100 mmol/l之间的S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐。
10.权利要求1或2的方法,特征在于对于超过一半的细胞培养时间,谷胱甘肽的水平比未添加S-磺基-L-半胱氨酸和/或其盐的细胞培养物高超过25%。
11.S-磺基-L-半胱氨酸和/或S-磺基-L-半胱氨酸盐用于增加在生物反应器中培养的CHO细胞内谷胱甘肽的量的用途。
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