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

Abstract

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 (C -C ), 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.

Preparation of supermacroporous cryogels with improved mechanical strength for efficient purification of lysozyme from chicken egg white

“…Due to their interconnected, macroporous 3D structure, cryogels have been widely used in bioseparation‐related applications [1–10], tissue engineering [10–14], and other relevant bioengineering and sensor applications [15–17]. In particular, monolithic cryogels have been used as a new generation of chromatographic matrices for the separation of cells (mammalian, bacterial, and yeast), proteins, viruses, and plasmids [18–22].…”

Preparation and characterization of large-format macroporous cryogel disks for use in affinity chromatography and biotechnological applications

“…To introduce reactive groups, allyl-glycidylether (AGE) is added as additional monomer in the polymerization solution. As evaluated earlier [12] optimal structural conditions and optimal AGE concentrations are observed at a molar acrylamide/AGE ratio of 10. These conditions are used for preparation of all further cryogel gels.…”

“…In the present work we have focused on the preparation and characterization of polyacrylamide-epoxy based cryogel discs with an ≥ 2.5 cm Ø. Discs with different acrylamide concentrations were made and compared in function of ligand binding and target binding capacity. As in earlier work [12] we determined the beneficial effect on the yield using spacers to immobilize selected phage, we here will combine upscaling the column format by using larger discs columns in combination with the introduction of spacers to obtain a higher ligand binding allowing to further optimize the yield of the target of interest.…”

Organized cryogel composites with 3D hierarchical porosity as an extraction adsorbent for nucleosides

“…After cryogelation, the templates of ice crystals are simply removed by thawing at room temperature without any pollution to obtain the cryogel macropores [9]. However, the inherent drawbacks of cryogels are scarce functional monomers or crosslinkers with high activity and good water solubility for direct application or functionalization, difficulty in adjusting macropore structure, and the rather low specific surface area, which have been considered as the major challenges to broaden their applications [10]. Presently, most cryogels are used for purification of large biomolecules [11], such as DNA or dehydrogenase, chromatographic separation or capturing of proteins [12], tissue engineering [13], cell culturing [14], and so on.…”