A new approach to monitoring glycosylation during glycoprotein production

项目总结 在治疗性生物制品的生产过程中，糖基化已被确定为一种关键的质量属性 (CQA)必须仔细监测，以确保所需的蛋白质稳定性、免疫原性、抗体 效应器功能，药理安全性和有效性，以及血清半衰期是一致的。许多 在生产过程中，影响糖链组成的因素，如介质、温度、 溶解气体、诱导时间等，以及随后的糖基化变化是领先的 药品批次拒收的原因。 目前，蛋白质糖基化的表征在很大程度上依赖于使用层析的方法。 和/或质谱学，这需要高水平的专业知识，既耗时又昂贵， 因为它们在生物制品生产过程中很难实现，所以通常只执行 后期制作。认识到对新的分析技术的持续需求，可在 治疗生产，美国食品和药物管理局(FDA)鼓励采用新工艺 分析方法(PAT)，提供设计、分析或促进过程测量的机制 影响CQA的参数。在这里，我们建议优化一种简化的方法并将其商业化 监测糖蛋白产生过程中的糖基化特征，我们称之为GlycoSenseTM，这是完全 与FDA的PAT倡议保持一致。 GlycoSense技术使用流式细胞术，将葡聚糖特异性试剂连接到多重珠子上 目的：提供一种快速、廉价、易用的蛋白质糖基化模式监测方法。流动 选择细胞仪作为首选平台是因为数据在统计上是稳健的，设备是 相对常见的是，分析消耗的样本量非常少，这将使Lectenz Bio能够 利用其在生产碳水化合物敏感试剂方面的专有专业知识。 在这里报告的初步研究中，GlycoSense性能指标是使用 糖链和糖蛋白的标准。在第二阶段，这项技术将被优化以监测糖基化。 在细胞培养过程中的变化。这将导致GlycoSense试剂盒可用于以下各种应用程序 基础研究和生物制药。

PROJECT SUMMARY During the production of therapeutic biologicals, glycosylation has been identified as a critical quality attribute (CQA) that must be carefully monitored to ensure that the desired protein stability, immunogenicity, antibody effector function, pharmacological safety and potency, and serum half-life are consistently obtained. Many factors can affect the composition of the glycan chains during production, such as media, temperature, dissolved gases, induction times, etc., and consequently variations in glycosylation are among the leading causes of pharmaceutical batch rejection. Currently, the characterization of protein glycosylation relies heavily on methods that employ chromatography and/or mass spectrometry, which require a high level of expertise, are time-consuming and costly, and, because they are challenging to implement during biologics production at-line, are generally performed only post-production. In recognition of the continuing need for novel analytical technologies that can be used during therapeutic production, the US Food and Drug Administration (FDA) encourages the adoption of new process analytical methods (PAT) that offer mechanisms to design, analyze, or facilitate the measurement of process parameters that affect CQAs. Here we propose to optimize and commercialize a simplified approach for monitoring glycosylation features during glycoprotein production, which we call GlycoSenseTM, that is fully aligned with the FDA PAT initiative. The GlycoSense technology uses flow cytometry with glycan-specific reagents conjugated to multiplex beads to provide a rapid, inexpensive, and easy-to-use method for monitoring protein glycosylation patterns. Flow cytometry was chosen as the preferred platform because the data are statistically robust, the equipment is relatively commonplace, the analyses consume very low quantities of sample, and it will enable Lectenz Bio to leverage its proprietary expertise in producing carbohydrate-sensing reagents. In preliminary studies reported here, GlycoSense performance metrics were established and published using glycans and glycoprotein standards. In phase II, the technology will be optimized for monitoring glycosylation changes during cell culture. This will result in GlycoSense kits that can be used for a variety of applications in basic research and in biopharma.