Development and screening of epoxy-spacer-phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide-expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost-effective one-step chromatographic set-up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di-amino-alkanes (C₂ -C₁₀ ), and branched polyethyleneimine spacers with different molecular weights (800 Da-10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15-mer and a cyclic 6-mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.