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Quantification of glycated IgG in CHO supernatants: A practical approach

机译:CHO上清液中糖化IgG的定量:实用方法

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Post-translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process-design considerations, especially concerning glucose-feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean-up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira (R), was successfully applied, and continuous monitoring of mAbs across three repetitive fed-batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch-consistency monitoring.
机译:在还原糖(BioProcesses)存在下单克隆抗体(mAb)的翻译后非酶的甘露解度是一种广泛的已知现象,其影响蛋白质异质性,并且可能对最终产品的质量,安全性和功效产生影响。对于在人体中治疗施用的每种MAb的每个MAb的规定是强制性的。因此,我们提出了一种用于监测生物过程中MAB产品的糖化水平的分析方法。这是一个有用的过程设计考虑的工具,特别是关于葡萄糖饲料策略和温度作为蛋白质糖化的主要驱动因子。在该研究中,优化了硼酸硼亲和力色谱(BAC)以测定上清液中mAb的甘氨酸水平。实际上,在上清液中发现的复杂基质是使用BAC的潜在障碍,但是通过简单的清洁步骤,我们发现可以显着改善洗脱曲线,从而可以达到定性和定量的测定。互补分析方法证实了性能质量,包括结果的正确性和特异性。为了定量测定上清液中的MAb甘露化,我们建立了保留的MAB峰的校准程序,鉴定为糖化抗体单体。对于这种方法,已成功应用了可用的完全表征的MAB标准,Humira(R),最后进行了三种重复的补料批处理中的MAB的连续监测。通过这种实用的,新的方法,在生物处理期间获得了糖化水平的识别,与葡萄糖水平和产品滴度随时间,促进有效的过程开发和批量 - 一致性监测。

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