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

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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