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

下游亲和力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，只提供有限的扩散传质能力，因为大的蛋白质亲和配体在多孔介质中占据了大量的孔内空间，从而阻碍了吸附生物分子扩散到它们在多孔基质中的结合位点。低分子质量配体更适合满足下游加工的操作要求。此外，目前基于蛋白质的亲和配体还有其他缺点：它们通常在极端pH下通过变性缓冲液洗脱，并且容易从柱基质中滤出，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配体发育的理想候选结构，因为它们体积小，无毒，免疫原性低，并且仍然达到一定的复杂性，可能具有与其他碳水化合物结构高度特异性和多价结合的潜力。因此，本项目的目的是验证低聚糖结构作为生物制药糖蛋白亲和配体的潜力。本项目提出的低分子量碳水化合物配体有望合成具有更高配体密度和每柱床体积动态结合能力的色谱载体。本项目的具体目标如下：1。证明硼酸共轭和金属配位的低聚糖结构可以以多价方式结合到蛋白质连接的聚糖上。确定最佳粘结剂和粘结条件。研究是否可以观察到配体亲和力的加性和协同性增加。评价配体结合的选择性生理条件下配体洗脱优化验证柱浸出液的低免疫原性潜力

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