Institute of Bioprocess Science and Engineering, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
Research and Development, Bilfinger Industrietechnik Salzburg GmbH, Salzburg, Austria.
Biotechnol Prog. 2021 May;37(3):e3124. doi: 10.1002/btpr.3124. Epub 2021 Jan 21.
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.
在存在还原糖的情况下（在生物工艺过程中），单克隆抗体（mAbs）的翻译后、非酶糖基化是一种广泛存在的现象，它会影响蛋白质的异质性，并可能对最终产品的质量、安全性和疗效产生影响。定量分析每种治疗性应用于人体的 mAb 的糖基化水平是强制性的。因此，我们提出了一种在生物工艺过程中监测 mAb 产品糖基化水平的分析方法。这是用于工艺设计考虑的有用工具，特别是涉及葡萄糖补料策略和温度作为蛋白质糖基化的主要驱动因素。在这项研究中，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。事实上，在发现上清液中存在复杂的基质后，它成为使用 BAC 的障碍，但通过一个简单的净化步骤，我们发现洗脱曲线可以得到显著改善，从而可以达到定性和定量的测定。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，我们建立了保留 mAb 峰（鉴定为糖基化抗体单体）的校准程序。对于这种方法，成功应用了一种现有的、完全表征的 mAb 标准品（Humira®），并最终对三个重复补料分批过程中的 mAbs 进行了连续监测。通过这种实用的新方法，我们获得了生物处理过程中糖基化水平的深入了解，同时还了解了葡萄糖水平和产品滴度随时间的变化情况，从而有助于高效的工艺开发和批次一致性监测。
