Phospholipid Microscale Glycan Sequencing: Linking Structure to Antibody Function

 描述：抗体治疗是一类快速增长的糖蛋白药物。许多抗体药物与细胞上的受体结合，并启动或加速细胞死亡和耗竭。几种抗体药物的专利保护即将结束，必须确定生物仿制药的有效性和安全性。虽然聚糖仅占抗体药物质量的约3%，但糖基化显著影响抗体对刺激免疫系统破坏特定细胞的作用。迫切需要对抗体糖基化进行分析，但聚糖的分析具有挑战性。这是因为聚糖由单体糖单元的类型、相邻糖单体之间的键合位置和链分支的变化来定义。拟议的研究产生了一种基于磷脂的酶迁移率变化测定，以快速测序抗体糖基化，并建立与抗体功能有关的聚糖单体的组成和连接方向。两种分析策略支持快速测序聚糖的系统方法。将仅以高特异性切割末端单体的外切糖苷酶整合到微尺度分离通道中。聚糖被电泳驱动进入酶中，孵育几分钟，然后在同一通道中使用电泳分离。当聚糖的末端单体与酶的特异性匹配时，它从聚糖上被切割下来。这降低了聚糖的电荷-尺寸比，并导致用于鉴别单体和连接的迁移时间发生变化。目的1通过在可编程毛细管电泳仪中整合纳升体积的酶来提高抗体的活性表征。用一系列酶对聚糖进行测序。这种自动化方法可以测定飞摩尔聚糖，每次孵育仅消耗几纳升酶。目的2活性通过同时进行多个外切糖苷酶反应来显著增加该方法的通量。这在具有平行通道或无通道分离的微流体装置中实现。这种微型测序的核心是一种独特的磷脂分离添加剂，它是一种热响应材料，在25°C下具有低粘度，在30°C下具有凝胶状粘度。这些特性使得使用自动化仪器或平行微流体装置在毛细管分离中进行微量测序变得更容易和更实用。

 DESCRIPTION: Antibody therapeutics are a rapidly growing class of glycoprotein pharmaceuticals. Many antibody drugs bind to receptors on cells and either initiate or accelerate cell death and depletion. Several antibody drugs are nearing the end of patent protection and the efficacy and safety of biosimilars, which are the generic replacements, must be established. Although glycans comprise only ~3% of the mass of an antibody drug, glycosylation significantly impacts the effect the antibody has on stimulating the immune system to destroy specific cells. There is a critical need to profile antibody glycosylation, but the analysis of glycans is challenging. This is because glycans are defined by the variation in the type of monomeric saccharide unit, the position of the linkage between adjacent saccharide monomers, and chain branching. The proposed research generates a phospholipid-based enzyme mobility shift assay to rapidly sequence antibody glycosylation and establish both the composition and linkage orientation of glycan monomers that are implicated in antibody function. Two analytical strategies support a systematic approach to rapidly sequence glycans. Exoglycosidase enzymes that cleave only the terminal monomers with high specificity are integrated into a microscale separation channel. The glycan is electrophoretically driven into the enzyme, incubated for several minutes and then separated in the same channel using electrophoresis. When the terminal monomer of a glycan matches the specificity of the enzyme it is cleaved from the glycan. This decreases the charge-to-size ratio of the glycan and results in a shift in migration time that is used to identify both the monomer and the linkage. Aim 1 activities improve the characterization of antibodies by integrating nanoliter volumes of enzymes in a programmable capillary electrophoresis instrument. Glycans are subject to sequencing with a series of enzymes. This automated method can assay femtomolar glycans and consumes only a few nanoliters of enzyme for each incubation. Aim 2 activities dramatically increase the throughput of the approach by performing multiple exoglycosidase reactions simultaneously. This is accomplished in microfluidic devices with parallel channels or channel-free separations. The heart of this microscale sequencing is a unique phospholipid separation additive that is a thermally-responsive material with low viscosity at 25°C, and gel- like viscosity at 30°C. These properties make it easier and more practical to perform microscale sequencing in capillary separations with an automated instrument or parallel microfluidic device.