Preparation And Applications Of Composite Cryogels

Polyacrylamide-based supermacroporous monolithic cryogels have large-sized pores of several to several hundreds of microns,which permit direct capture of biomolecules from microbial fermentation feedstocks at high flow velocities with very low back pressure.Their main properties such as tissue-like elasticity,low flow resistance,high selectivity and biocompatibility,render these cryogels interesting for a broad range of applications in bioseparation areas.Besides these advantages,the mechanical strength of composite cryogel is improved significantly.The properties of pure lysozyme adsorption/elution onto the cation-exchange cryogel with sulfo groups have been reported in our previous work,but the practical application of cation-exchange cryogel on lysozyme purification from chicken egg white has not been studied so far.In this paper,we presented a new method for chromatographic separation of lysozyme from chicken egg white solution using cation-exchange cryogel.With regard to the problem that the powder of silica nanoparticles is not well dispersed in the monomer solution,affecting the porous structure morphologies and properties of the composite cryogel,chemical reaction was conducted to prepare uniform size distribution of silica particles suspension,then the cryogel embedded with silica particles was prepared by the radical cryogenic copolymerization under freezing-temperature variation conditions.The anion-exchange functional groups were introduced to the composite cryogel pore wall surface by in-situ graft copolymerization of monomers ion-exchange groups using suitable initiator.Then we explored the possibility of separating plasmid DNA(pDNA)from alkaline lysate of Escherichia coli(DH5a)cells using this anion-exchange composite cryogel at high flow velocities.The results showed that one-step isolation of lysozyme from chicken egg white was performed using cation-exchange chromatographic cryogel by sequential elution with different salt concentrations in sodium phosphate buffer.In the elution procedure,lower concentration of NaCl was used for eluting the weakly bound protein impurities and lysozyme was subsequently eluted by that with higher concentration.The maximum purity of the lysozyme eluted with 0.5 M NaCl in sodium phosphate buffer(pH 7.8)was about 96%.Chromatography of lysozyme,using lysozyme as a model protein,in the composite cryogel after grafted with 2-acrylamido-2-methyl-l-propanesulfonic acid(AMPSA)was carried out to reveal the protein breakthrough and elution characteristics.The superficial velocity in the process can reach 12 cm/min,indicating the improvement of the mechanical strength due to the embedded silica particles.The one-step isolation of pDNA from non-clarified bacterial lysate using anion-exchange composite cryogel could be achieved at the high velocities of 5 and 10 cm/min.The maximal purity of the obtained pDNA by the one-step separation method was 89.2%using 20 mM sodium phosphate as running buffer in the[2-(methacryloyloxy)ethyl]-trimethylammonium chloride(META)anion-exchange chromatography.Due to the large pore size in macroporous composite cryogel the particulate material presented in non-clarified feeds did not block the column.The purity of pDNA obtained at 10 cm/min was higher than that at lower velocities such as 2 and 5 cm/min.This chromatographic method is an efficient time-saving one.