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: 8984617
• 负责人: Lisa A Holland
• 金额: $ 25.9万
• 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/8984617
• 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; Flow Cytometry; Generic Drugs; Glycoproteins; Goals; Heart; Immigration; 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; cost; cytotoxicity; glycosylation; immune function; immunogenicity; improved; instrument; migration; monomer; nanolitre; new technology; public health relevance; receptor; screening; therapeutic effectiveness; 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：抗体治疗药物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，引发或加速细胞死亡和消耗，一些抗体药物的专利保护和生物仿制药的功效和安全性已接近尾声。尽管聚糖仅占抗体药物质量的 3%，但糖基化会显着影响抗体刺激免疫系统破坏特定细胞的效果，因此迫切需要。来分析抗体糖基化，但聚糖的分析具有挑战性，这是因为聚糖是由单体糖单元的类型、相邻糖单体之间的连接位置以及链分支来定义的。基于酶迁移率变动分析，可快速对抗体糖基化进行测序，并确定与抗体功能有关的聚糖单体的组成和连接方向，两种分析策略支持系统方法。仅切割具有高特异性的末端单体的外切糖苷酶被整合到微型分离通道中，将聚糖通过电泳驱动到酶中，孵育几分钟，然后在同一通道中使用电泳分离末端单体。聚糖与其从聚糖上裂解的酶的特异性相匹配，这降低了聚糖的电荷与尺寸比，并导致用于迁移的时间发生变化。目标 1 通过将纳升体积的酶整合到可编程毛细管电泳仪中，改进抗体的表征。利用一系列酶对聚糖进行测序，这种自动化方法可以检测飞摩尔聚糖，并且只消耗飞摩尔聚糖。每次孵育需要几纳升的酶，Aim 2 活性通过同时执行多个外切糖苷酶反应显着提高了该方法的通量，这是在微流体装置中实现的。这种微量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 时具有低粘度，在 30°C 时具有类似凝胶的粘度。使用自动化仪器或并行微流体装置在毛细管分离中进行微量测序更容易、更实用。