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®, 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.

中文翻译：

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CHO 上清液中糖化 IgG 的定量：一种实用的方法

单克隆抗体 (mAb) 在还原糖存在下（在生物过程中）的翻译后非酶糖基化是一种广为人知的现象，它会影响蛋白质的异质性，并可能对最终产品的质量、安全性和功效产生影响。对于在人类中治疗应用的每种 mAb，个体糖基化水平的量化是强制性的。因此，我们提出了一种在生物过程中监测 mAb 产物糖基化水平的分析方法。这是工艺设计考虑的有用工具，特别是关于葡萄糖进料策略和温度作为蛋白质糖化的主要驱动因素。在本研究中，硼酸盐亲和层析 (BAC) 被优化用于测定上清液中 mAb 的糖基化水平。实际上，在上清液中发现的复杂基质是使用 BAC 的潜在障碍，但通过简单的清理步骤，我们发现可以显着改善洗脱曲线，从而实现定性和定量测定。补充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 糖基化，我们为保留的 mAb 峰建立了校准程序，被确定为糖化抗体单体。对于这种方法，一个可用的完全表征的 mAb 标准 Humira® 被成功应用，并最终在三个重复的分批补料过程中连续监测 mAb。通过这种实用、新颖的方法，可以深入了解生物加工过程中的糖基化水平，