Study On The Preparation Of Supermacroporous Cryogel For Continuous Bed Chromatography And Its Characteristics

The continuous bed chromatography using supermacroporous cryogel is suggested as a novel bioseparation technique in downstream processes in recent years. The cryogel has interconnected supermacropores with diameter of several to hundreds micrometers,which allow large bioparticles such as cell debris and even the whole cells passing through without being blocked.Thus,the back pressure and the mass transfer resistance are low and convective mass transfer of biomolecules always dominates within the cryogel beds.In addition,fast adsorption equilibrium can also be achieved in cryogels,which makes it possible to directly separate and purify target biomolecules or bioparticles from unclarified particulate-contained feedstock or crude fermentation broth without being pretreated at high flow rate.It is of great importance to reveal the mechanism of the formation of supermacropores in the preparation of cryogel matrix,the immobilization of functional binding groups on the matrix,the protein adsorption characteristics in cryogel columns and the related applications of cryogels.In this paper,the cryogel matrix was produced by radical co-ploymerization of acrylamide(AAm)-based reactive system under frozen conditions.The liquid-solid phase transition characteristics of the aqueous solution system containing AAm,AGE and MBAAm for the production of polyacrylamide-based cryogels were studied and the pore formation mechanisms were discussed.The basic properties and the chromatographic performances of the cryogels prepared under various conditions were measured and the effects of preparation condition on the microstructures and properties of the cryogels were also investigated.Then,metal ions were immobilized on polyacrylamide-based cryogel matrix to produce the metal-chelate affinity cryogels and the functional binding groups of sulfoacid and amine groups,were grafted onto the cryogel matrixes in an in-situ manner to prepare ion-exchange cryogels.The anion-exchange chromatography using cryogel was also applied to directly isolate cytidine triphosphate(CTP)and adenosine triphosphate(ATP)from the yeast fermentation broths and the corresponding behaviors were obtained and discussed.From the results of the liquid-solid phase transition characteristics of the reactive solution,the actual initial solvent crystallization temperature T_c and the freezing point temperature T_mcchanged with the monomer concentration,the freezing rate and the freezing terminated temperature.Freezing rate had a limited effect on T_c and T_mc,while the values of T_c and T_mcdecreased with the decrease of freezing terminated temperature at a constant freezing rate and a given monomer concentration.T_m decreased with the increase of monomer concentration at a constant freezing rate.From the present work,the process of supermacropores formation was a complex process combining both solvent freezing crystallization and momomer copolymerization.The cryogel structure,the pore morphology and the cryogel bed properties were influenced by several factors,i.e.,the column diameter,the terminated freezing temperature,the freezing rate,the monomer concentration as well as the catalyst and its amount used.A cryogel bed with well interconnected supermacropores of diameters of 10 to 100 micrometers,porosity of 82-85%and height equivalent to theoretic plate(HETP)of 0.5-1.1 mm was obtained under suitable preparations.The terminated freezing temperature of less than -15℃was found to be effective for a suitable freezing rate in a column with given inner diameter.The measuremental results of the properties of the Cu²⁺-IDA(iminodiacetic acid), Ni²⁺-IDA and Zn²⁺-IDA metal-chelate affinity cryogels showed that there are no obvious variations for the pore morphology,HETP and permeabilities between the metal-chelate affinity cryogels and the corresponding basic cryogel matrixes without immobilized metal ions.The adsorption and elution behaviors of bovine serum albumin(BSA)in these metal-chelate affinity cryogels were influenced by the buffer pH,the ionic strength,the flow rate and the eluent composition.The binding of BSA on the metal-chelate affinity cryogels was depended upon the cooperative effects of coordination and electrostatic interaction.The liquid flow velocity had a weak influence,while the buffer pH had an obvious effect on the binding capacity of protein in these cryogels.The protein binding capacity reached the maximum when the buffer pH was near the isoelectric point of BSA and decreased with the adding of salt to the loading liquid.The bound protein molecules were eluted effectively using imidazole solution and a low elution liquid flow velocity was found to be benefited to the elution process.The graft polymerization experiments showed that 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt(AHPSA)with sulfoacid group and 2-(dimethylamino)ethyl methacrylate(DMAEMA)with amine group can be successfully grafted onto the pore wall surface of the polyacrylamide-based cryogels to get the cation- and anion-exchange cryogels,respectively.The graft polymerization can be initiated by potassium diperiodatocuprate(K₅[Cu(HIO₆)₂])in an in-situ manner.For the cation-exchange cryogels grafted with AHPSA,the HETP was not obviously influenced by the graft reaction time and monomer concentration,while the permeability slightly decreased with the increase of graft reaction time.The protein binding capacity of lysozyme increased linearly with the increase of the concentration of AHPSA and had no obvious change under different graft reaction times.For the anion-exchange cryogels grafted with DMAEMA,the binding capacity of BSA decreased with the increase of ionic strength.This parameter was observed to decrease linearly in the existing of NaCl or CH₃COONa and exponentially in the existing of C₆H₅Na₃O₇ or Na₂SO₄,with the increase of ionic strength respectively.The isolations of CTP and ATP from unclarified yeast fermentation broths were achieved successfully by one-step chromatography using the anion-exchange cryogel with amine group as chromatographic matrix at high flow rate(2 to 10 cm/min).The results showed that high purify of CTP and ATP can be obtained by this method.The purity of CTP reached 93.4%and the recovery rate of CTP was 35%,while the purity of ATP reached 98.3%and the recovery rate of ATP was 58%.At high chromatographic flow rate of 10 cm/min,the purity of ATP still reached 97.4%and the recovery rate of ATP reached 49%after one-step separating operation in the present work.