生物化学与分析生物化学

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2015 年生物技术大会：补料分批工艺中的化学修饰半胱氨酸及其对 CHO 特定生产率的影响 - Aline Zimmer Merck Millipore

艾琳·齐默

工业化补料分批培养哺乳动物细胞用于生产治疗性蛋白质，例如单克隆抗体。除了确保初始生长的培养基外，补料对于提高生长、活力和抗体产量也是必要的。已建立的商业系统包括微酸性浓缩主进料和单独的碱性氨基酸进料，其中含有 L-酪氨酸和 L-半胱氨酸。由于 L-半胱氨酸在空气和金属催化剂存在下会二聚化为半胱氨酸，因此不稳定，因此需要稳定的 L-半胱氨酸衍生物以将所有氨基酸包含在中性 pH 进料中。这些单一进料系统有利于简化进料方案并通过稳定 pH 和 DO 信号来提高整体工艺的稳健性。在这里，我们建议在工业化单一进料补料分批工艺中使用化学改性的半胱氨酸与磷酪氨酸二钠盐组合，该工艺适用于小规模以及生物反应器。细胞培养实验是在旋转管或生物反应器中进行的，其中 CHO 悬浮细胞系表达人类单克隆抗体。使用自动细胞计数仪测量活细胞密度和活力。在柱前衍生化和 UPLC 分析后对上清液进行废培养基分析，以检测氨基酸，并使用 LC-MS/MS 分析维生素。

使用 Cedex Bio HT 进行代谢物测量，依靠光度法和浊度法。使用 2-AB 标记进行聚糖分析、使用 cIEF 进行电荷变体分析和使用 LC-MS/MS 进行肽图分析实验来表征单克隆抗体。含有改性半胱氨酸衍生物的进料的稳定性研究表明，该分子是稳定的，并且在室温或 4°C 下储存三个月时不会释放 L-半胱氨酸或 L-胱氨酸。此外，没有观察到进料颜色随时间的变化。在小规模批量实验中，用相同数量的化学改性半胱氨酸替代 L-半胱氨酸，表明生长或活力概况没有变化。与已建立的双进料系统相比，在小规模补料分批工艺中使用改性半胱氨酸衍生物表明最大活细胞密度相当、活力延长和滴度增加。

Bioreactor experiments confirmed the increase in specific productivity described at small scale when the single feed strategy was compared to the two feed strategy. In depth characterization of the monoclonal antibody indicated no change in the glycosylation or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Mammalian took care of clump forms for monoclonal neutralizer (mAb) creation depend on vital taking care of a few supplements, for example, glucose, nutrients and amino acids to expand culture time and improve protein production[1].In real procedures, L-cysteine and L-tyrosine are taken care of independently at soluble pH due their low steadiness and low solvency at impartial pH, bringing about pH tops and precipitations[2]. To improve cutting edge forms, both amino acids have been artificially altered to upgrade their particular solidness and dissolvability profiles. Past work has exhibited that phosphotyrosine disodium salt (PTyr2Na) is a steady L-tyrosine subordinate and can be utilized in unbiased pH takes care of without detectably affecting the way of life execution or the mAb quality attributes[3]. Here, we present outcomes got utilizing a L-cysteine subsidiary in a nonpartisan pH, single-feed framework.

The dependability of the L-cysteine subsidiary was assessed in nonpartisan pH, CellventoTM Feed-220 during a quarter of a year at room temperature or 4°C.For took care of group societies, a CHO K1 clone communicating a human mAb was utilized. Development was acted in the Cellvento™ CHO-220 media framework as indicated by the item procedure direction. For controls, Cellvento™ Feed-220 and a different basic cysteine/tyrosine feed were utilized though in the single feed process, the L-cysteine subsidiary and PTyr2Na were legitimately solubilized in the unbiased pH, primary feed. Trials were acted in turn tubes and 1.2L bioreactors. Development and reasonability were observed utilizing a ViCell®,titer was resolved utilizing Cedex Bio HT and explicit profitability was determined dependent on titer, vital feasible cell thickness and weakening.

For spent media investigation, the L-cysteine subsidiary and amino corrosive measurement was performed by UPLCusing AccQ·TagTMUltra Reagent. To evaluate the responsive capability of the feed, H2DCFDA was added to the nonpartisan pH, Cellvento™ Feed-220 enhanced or not with the subordinate. For intracellular responsive species evaluation, cells were marked with carboxy-H2DCFDA and fluorescence was estimated. To assess the limit of the cells to use the subsidiary, cell lysates were spiked and shaped items were evaluated by UPLC. For mAb examination, N-glycosylation was measured utilizing HPLC after 2-AB marking, while charge variations were resolved utilizing cIEF.

对中性 pH 补料中 L-半胱氨酸副产物稳定性的分析表明，在室温或 4°C 下储存超过三个月时，副产物浓度和 L-半胱氨酸/L-胱氨酸释放均未发生变化。未观察到沉淀或颜色变化，表明副产物在测试条件下是稳定的。与对照条件相比，使用单补料方法在转管中控制的群体生长导致最终活力更高，从而导致最终滴度增加。在生物反应器中确认了转管效应，使用单补料方法导致最终活力更高、滴度增加和特异性产量更高。

将 H2DCFDA 扩增至中性 pH 后，检测到较低的色度氧化，与仅添加该物质相比，添加该物质的 Cellvento™ Feed-220 显示出较低的接受潜力。将羧基-H2DCFDA 扩增至单添加处理组群，检测到较低的细胞内色度氧化，在使用该物质时，显示出较低的细胞内接受物种年龄。当加入细胞裂解物中时，将半胱氨酸物质用于半胱氨酸。在递送的 mAb 中未观察到 N-糖基化或电荷变化的差异，表明该物质对递送的基本质量特性没有影响。该研究表明，L-半胱氨酸物质和 PTyr2Na 可以结合到中性 pH 处理中，并且可以用作处理组群形式中 L-半胱氨酸的来源，与同类方法相比，可产生更高的特异性效率，而不会影响 mAb 的基本质量特性。