A rapid method for monitoring in vitro glycoengineering

项目摘要 众所周知，治疗性蛋白质如抗体的糖基化会影响生物活性，然而， 当在生物反应器中生产糖蛋白时，实现均匀的糖基化是具有挑战性的。一种方法 获得明确的均一糖基化的方法是使用糖苷酶和糖基转移酶 （糖酶）修饰蛋白质的糖基化，后生产。这种方法经常被称为 体外糖工程。然而，与传统的小分子化学合成不同， 用于监测酶反应过程中酶反应进程的方便的成本有效的方法或试剂盒 糖工程。在这里，我们建议开发一种名为GlycoSense的创新技术， 体外糖酶反应将使用基于珠的聚糖的流式细胞术进行近实时监测， 特殊试剂选择流式细胞术作为优选平台，因为数据在统计学上是一致的。 坚固耐用，设备相对普通，分析消耗非常少量的样品， 将使G&D能够利用其在生产碳水化合物传感试剂方面的专有经验。 在第一阶段的应用中，我们将展示我们的GlycoSense技术监测 应用于商业治疗生物制剂的糖工程的糖酶，由主要的 生物制药公司。选择这些生物制剂进行当前的概念验证研究，因为 它们的医学重要性，以及由于糖工程在开发 生物仿制药和生物改善剂。

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.