Multivalent, carbohydrate-based chromatography ligands for specificand selective capture of glycoproteins

用于特异性和选择性捕获糖蛋白的多价碳水化合物色谱配体

• 批准号: 320841447
• 负责人: Professor Dr. Hans Henning von Horsten
• 金额: --
• 依托单位: Studiengang Life Science Engineering
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/320841447?language=en
• 关键词: Multivalent; carbohydrate; based; chromatography; ligands

The downstream affinity capture step has been recognized as a major bottleneck to productivity increases in biopharmaceutical manufacturing. Current commercial affinity resins, such as protein A resins, offer only a limited capacity for diffusive mass transfer because large proteinaceous affinity ligands occupy a large amount of intrapore space in porous media and thereby hinder diffusion of adsorbate biomolecules to their binding site within the porous matrix. Low molecular mass ligands are much better suited to meet operational requirements in downstream processing. Also, current protein-based affinity ligands have other disadvantages: They are typically eluted by denaturing buffers at extreme pH and tend to leach off from the column matrix thereby causing a risk to patient safety. Thus there is a need for alternative affinity ligands. Carbohydrate structures of glycoproteins are an ideal candidate structure for ligand development since they are small, non toxic, low immunogenic and still reach a level of complexity that may potentially enable highly specific and multivalent binding to other carbohydrate structures. Therefore, the aim of this project is to verify and validate the potential of oligosaccharide structures as affinity ligands for biopharmaceutical glycoproteins. Low molecular weight carbohydrate ligands proposed in this project are expected to enable the synthesis of chromatography supports with higher ligand density and increased dynamic binding capacity per column bed volume. Specific objectives of this project are the following:1. Proof that boronic acid conjugated and metal coordinated oligosaccharide structures can bind to protein-linked glycans in a multivalent fashion. Identify optimal binders and binding conditions. Investigate whether an additive and synergistic increase in ligand affinity can be observed.2. Evaluate the selectivity of ligand binding 3. Optimize ligand elution under physiological conditions4. Verify the low immunogenic potential of column leachables

下游亲和捕获步骤已被认为是生物制药生产中生产率提高的主要瓶颈。目前的商业亲和树脂，如蛋白A树脂，仅提供有限的扩散传质能力，因为大的蛋白质亲和配体占据多孔介质中的大量孔内空间，从而阻碍吸附物生物分子扩散到其在多孔基质内的结合位点。低分子量配体更适合于满足下游加工中的操作要求。此外，目前的基于蛋白质的亲和配体具有其他缺点：它们通常在极端pH下通过变性缓冲液洗脱，并且倾向于从柱基质中浸出，从而对患者安全造成风险。因此，需要替代的亲和配体。糖蛋白的碳水化合物结构是用于配体开发的理想候选结构，因为它们是小的、无毒的、低免疫原性的，并且仍然达到可以潜在地实现与其他碳水化合物结构的高度特异性和多价结合的复杂性水平。因此，本项目的目的是验证和确认寡糖结构作为生物制药糖蛋白的亲和配体的潜力。本项目中提出的低分子量碳水化合物配体预计能够合成具有更高配体密度和增加的动态结合容量/柱床体积的色谱支持物。本项目的具体目标如下：1. 证明硼酸共轭和金属配位寡糖结构可以以多价方式与蛋白质连接聚糖结合。确定最佳粘合剂和粘合条件。研究是否可以抑制配体亲和力的相加和协同增加。 评估配体结合的选择性3. 在生理条件下优化配体洗脱4. 验证色谱柱稀释物的低免疫原性潜力