A rapid method for monitoring in vitro glycoengineering

项目总结 众所周知，治疗蛋白(如抗体)的糖基化会影响生物活性，然而， 当糖蛋白在生物反应器中产生时，要实现均匀的糖基化是具有挑战性的。一种方法 要获得明确的均一糖基化，需要使用糖苷酶和糖基转移酶 (糖酶)修饰蛋白质的糖基化，后期产生。这种方法通常指的是 就像在体外糖工程一样。然而，与传统的小分子化学合成不同，没有 方便、经济有效的方法或试剂盒，用于监测酶反应的进展 糖化工程。在这里，我们建议开发一种名为GlycoSense的创新技术，它将允许在 体外糖酶反应将被近实时使用基于珠基的糖链的流式细胞术监测- 特定的试剂。选择流式细胞仪作为首选平台是因为数据具有统计学意义 坚固耐用，设备相对普通，分析消耗的样本量非常少，而且 这将使G&D能够利用其在生产碳水化合物敏感试剂方面的专利经验。 在这个第一阶段的应用中，我们将展示我们的GlycoSense技术能够监控 糖酶应用于商业治疗性生物制品的糖工程，由一家主要的 生物制药公司。这些生物制品被选为本概念研究的证据是因为 它们在医学上的重要性，以及糖工程在发展糖工程方面的新潜力 生物仿制药和生物防腐剂。

PROJECT SUMMARY The glycosylation of therapeutic proteins, such as antibodies, is well known to affect bioactivity, however, uniform glycosylation is challenging to achieve when glycoproteins are produced in bioreactors. One approach to obtaining well-defined homogeneous glycosylation is to use glycosidases and glycosyl transferases (glycoenzymes) to modify the glycosylation of the protein, post-production. This approach is often referred to as in vitro glycoengineering. However, unlike traditional small-molecule chemical synthesis, there are no convenient cost-effective methods or kits for monitoring the progress of the enzyme reactions during glycoengineering. Here, we propose to develop an innovative technology called GlycoSense that will permit in vitro glycoenzyme reactions to be monitored in near real-time using flow cytometry with bead-based glycan- specific reagents. 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 G&D to leverage its proprietary experience in producing carbohydrate-sensing reagents. In this Phase I application, we will demonstrate the ability of our GlycoSense technology to monitor the effect of glycoenzymes applied to the glycoengineering of commercial therapeutic biologics, provided by a major biopharmaceutical company. These biologics were chosen for the present proof of concept studies because of their medical importance and because of the emerging potential of glycoengineering in the development of biosimilars and biobetters.