GlycoFlow Glycoengineering of therapeutic antibodies by flow biocatalysis.

生物制药的出现正在彻底改变医疗保健，为传统药物难以治疗的疾病开辟了全新的途径。市场上销售的治疗性抗体是识别并结合病变细胞上的特定分子的蛋白质，从而引发有益的免疫反应。一个鲜为人知的事实是，抗体通常不是单一的同质蛋白质。它们还需要糖基化，这意味着抗体与各种糖（即聚糖）的复杂结构相连。这种糖基化对于产生有效的治疗性抗体是必不可少的。因此，这些生物制药是使用复杂而昂贵的蛋白质表达系统制造的，该系统携带安装这些糖所需的生物机械。由于使用活细胞，所得到的产物包括具有多种聚糖结构的异质群体，这些结构可以显示出显著的差异，当聚糖被聚焦（用聚焦糖装饰）时，功效降低高达50倍。这种异质性给治疗性抗体的开发和使用带来了沉重的负担，因为它影响了有效性和安全性、制造成本、批次可变性以及监管要求。因此，需要更好的方法来控制与抗体相连的聚糖的结构，以及聚糖种群的一致性。目前的策略利用转基因表达细胞系（例如，降低聚焦化水平），仍然产生高度可变的糖基化谱。另外，体外批量化学酶修饰已被证明可以成功地产生均质糖基化抗体，但它们存在复杂的纯化过程和可扩展性问题。我们提出了一个糖流平台，在该平台中，治疗性抗体可以使用流动生物催化进行糖工程。通过将抗体溶液通过模块化的糖基化酶试剂盒，我们的目标是在连续流动中直接操纵糖的结构，产生优化的、均匀的糖基化抗体。连续流技术是具有高效率和一致性的模块化合成药物成分的游戏规则改变者，但尚未被用于生物制药的糖工程。为了实现这一目标，我们在糖科学、聚糖加工酶、抗体生物化学和蛋白质工程方面组建了一支年轻而经验丰富的专家团队。我们的团队将利用UdeM在流动化学方面的历史专业知识，再加上来自Concordia和McMaster的世界领先的研究人员，为下一代生物制药提供创新的解决方案。具体来说，我们将开发生产均质糖基化抗体的流动技术，以及针对集中和唾液化聚糖的更简单的解决方案，这两种方法都显示出更高的功效和改善的抗炎特性。

The advent of biopharmaceuticals is revolutionizing health care, opening up entirely new avenues against diseases that are difficult to treat using traditional drugs. Marketed therapeutic antibodies are proteins that recognize and bind specific molecules on diseased cells, thereby eliciting a beneficial immunological response. A lesser-known fact is that antibodies are usually not single homogenous proteins. They also require glycosylation, which means the antibody is linked to a complex structure of various sugars (i.e. glycans). This glycosylation is essential for generating efficacious therapeutic antibodies. Therefore, these biopharmaceuticals are manufactured using elaborate and costly protein expression systems that carry the biological machinery needed to install these sugars. Because living cells are used, the resulting product comprises a heterogenous population with multiple glycan structures that can show significant differences, with up to a 50-fold decrease in efficacy when the glycans are fucosylated (decorated with a fucose sugar). Such a heterogeneity imposes a significant burden on the development and use of therapeutic antibodies, insofar as it impacts efficacy and safety profiles, manufacturing costs, batch variability as well as regulatory requirements.Consequently, better approaches are needed to control the structures of glycans linked to antibodies, as well as the consistency of the glycan population. Current strategies exploit genetically modified expression cell lines (e.g. to reduce the level of fucosylation) that still produce highly variable glycosylation profiles. Alternatively, in vitro batch chemo-enzymatic modifications have proven successful at generating homogenously glycosylated antibodies, but they suffer from complex purification processes and scalability issues.We propose a GlycoFlow platform in which therapeutic antibodies could be glycoengineered using flow biocatalysis. By passing a solution of antibodies through modular cartridges of glycan-processing enzymes, we aim to manipulate the structures of sugars directly in continuous flow, generating optimized and homogenous glycosylated antibodies. Continuous flow technologies are game-changers for the modular synthesis of pharmaceutical ingredients with high efficiency and consistency, but have yet to be leveraged for glycoengineering of biopharmaceuticals.To achieve this goal, we have assembled a diverse team of young and experienced experts in glycosciences, glycan-processing enzymes, antibody biochemistry, and protein engineering. Our team will take advantage of the historical expertise in flow chemistry at UdeM, coupled with world-leading researchers from Concordia and McMaster to deliver innovative solutions for the next-generation of biopharmaceuticals. Specifically, we will develop flow technologies that yield homogenously glycosylated antibodies as well as simpler solutions towards afucosylated and sialylated glycans, both of which have showed increased efficacy and improved anti-inflammatory properties.