Phospholipid Microscale Glycan Sequencing: Linking Structure to Antibody Function

磷脂微量聚糖测序：将结构与抗体功能联系起来

• 批准号: 9316668
• 负责人: Lisa A Holland
• 金额: $ 28.5万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316668
• 关键词: Address; Antibodies; Binding; Biological Assay; Blood capillaries; Capillary Electrophoresis; Carrington dermal wound gel; Cell Death; Cells; Centers for Disease Control and Prevention (U.S.); Charge; Chromatography; Cleaved cell; Complex; Consumption; Devices; Drug usage; Electrophoresis; Electrophoretic Mobility Shift Assay; Enzymes; Evaluation; Exoglycosidases; Fc Receptor; Flow Cytometry; Generic Drugs; Glycoproteins; Goals; Heart; Immune response; Immune system; Incubated; Lectin; Legal patent; Light; Link; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Microfluidic Microchips; Microfluidics; Molecular Structure; NanoGel; Oligosaccharides; Pattern; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Phospholipids; Physiological; Play; Polysaccharides; Positioning Attribute; Property; Reaction; Recombinant Proteins; Research; Role; Safety; Sales; Sampling; Series; Specificity; Structure; System; Therapeutic; Therapeutic antibodies; Time; Variant; Viscosity; base; capillary; chemical binding; cost; cytotoxicity; glycosylation; immune function; immunogenicity; improved; instrument; migration; monomer; nanolitre; new technology; programs; public health relevance; receptor; screening; synergism; therapeutic effectiveness; therapeutic protein; tool

 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.

 描述：抗体疗法是一类快速生长的糖蛋白药物。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。必须建立几种抗体药物接近专利保护的终结，而生物仿制药的有效性和安全性必须建立。尽管聚糖仅占抗体药物质量的约3％，但糖基化显着影响抗体对刺激免疫系统破坏特定细胞的影响。迫切需要介绍抗体糖基化，但是对聚糖的分析受到挑战。这是因为聚糖的定义是由单体糖单元的类型变化，相邻糖单体之间的连锁位置和链分支的定义。拟议的研究产生了基于磷脂的酶迁移率分析，以迅速序列抗体糖基化和建立在抗体功能中暗示的聚糖单体的组成和连锁方向。两种分析策略支持一种系统的方法来快速序列聚糖。仅清除具有高特异性的末端单体的外糖苷酶被整合到显微镜分离通道中。聚糖在电泳上驱动到酶中，孵育几分钟，然后使用电泳在同一通道中分离。当聚糖的末端单体与酶的特异性匹配时，它会从聚糖中裂开。这降低了聚糖的电荷与大小比率，并导致迁移时间的变化，用于识别单体和链接。 AIM 1活性通过在可编程毛细管电泳仪器中整合纳米素体积来改善抗体的表征。糖可以通过一系列酶进行测序。这种自动化的方法可以测定femtolorallycans，并仅消耗几种酶的酶。 AIM 2活动通过简单地执行多种外糖苷酶反应来大大增加方法的吞吐量。这是在具有平行通道或无通道分离的微流体设备中完成的。该微观测序的心脏是独特的磷脂分离添加剂，是一种在25°C下粘度低的热响应材料，在30°C下凝胶状粘度。这些属性使使用自动仪器或并行的微流体设备在毛细管分离中进行微观测序变得更加容易，更实用。