Kejian Yao

Microchannel liquid-flow focusing and cryo-polymerization preparation of supermacroporous cryogel beads for bioseparation

Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0 cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0 cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500 μm with supermacropores in sizes of about 3-50 μm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248 μm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29 × 10⁻¹⁰ m²) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78-92% at the flow velocities of 0.5-15 cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation.

Chromatographic separation of cytidine triphosphate from fermentation broth of yeast using anion-exchange cryogel.

A novel separation method was developed to isolate directly cytidine triphosphate (CTP) from fermentation broth of yeast using anion-exchange supermacroporous cryogel. The anion-exchange cryogel with tertiary amine groups was prepared by graft polymerization. The breakthrough characteristics and elution performance of pure CTP in the cryogel bed were investigated experimentally and the CTP binding capacity was determined. Then the separation experiments of CTP from crude fermentation broth of yeast using the cryogel column were carried out using deionized water and 0.01 M HCl as washing buffer, respectively. The chromatographic behavior was monitored and analyzed. The purity and concentration of the obtained CTP in these processes were determined quantitatively by HPLC. The maximal purity of CTP obtained at the condition of 0.01 M HCl as washing buffer and 0.5 M NaCl in 0.01 M HCl as elution buffer reached 93%.

Isolation of Lysozyme from Chicken Egg White Using Polyacrylamide-based Cation-exchange Cryogel

Abstract An effective cation-exchange chromatographic method for lysozyme isolation from chicken egg white is presented, using supermacroporous cryogel grafted with sulfo functional groups. The chromatographic processes were carried out by one-step and sequential elution, respectively. Sodium phosphate buffer (pH 7.8) containing different concentrations of NaCl is used as elution agent. The corresponding breakthrough characteristics and elution behaviors in the cryogel bed were investigated and analyzed. Purity of lysozyme in the elution effluent was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The maximum purity of the obtained lysozyme was about 96%, and the cryogel is demonstrated as a potential separation medium for purification of high-purity lysozyme from chicken egg white.

Preparation of Supermacroporous Composite Cryogel Embedded with SiO2 Nanoparticles

Abstract Supermacroporous composite cryogels embedded with SiO 2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide (AAm) and N, N -methylene-bis-acrylamide (MBAAm) containing SiO 2 nanoparticles (mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08) under the freezing-temperature variation condition in glass columns. The properties of these composite cryogels were measured. The height equivalent to theoretical plate (HETP) of the cryogel beds at different liquid flow rates was determined by residence time distribution (RTD) using tracer pulse-response method. The composite cryogel matrix embedded with the mass fraction of SiO 2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization. Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was carried out to evaluate the protein breakthrough and elution characteristics. The chromatography can be carried out at relatively high superficial velocity, i.e ., 15 cm·min −1 , indicating the satisfactory mechanical strength due to the embedded nanoparticles.

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin-G and albumin from human serum. Immunoglobulin-G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.

Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities.

This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities.

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)‐based anion‐exchange cryogel

Bovine milk whey contains several bioactive proteins such as α-lactalbumin, β-lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2-hydroxyethyl methacrylate)-based anion-exchange cryogel. The monolithic cryogel was prepared by grafting 2-(dimethylamino) ethyl methacrylate onto the poly(2-hydroxyethyl methacrylate)-based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high-purity IgG from bovine milk whey.

Numerical Simulation and Analysis of Gas Flow Field in Serrated Valve Column

Abstract A three-dimensional computational fluid dynamics (CFD) model for gas flow through a serrated valve tray was presented. The flow field, as well as the dry-pressure drop of the valve under the full-opening condition was simulated based on the proposed model by using FLUENT 6.0 software. Compared with the values of dry-pressure drop in different turbulent models, the simulated results using RNG k -ɛ model are in reasonable agreement with experimental data, indicating that RNG k -ɛ model is suitable in simulating gas flow through the serrated valve tray. Then the CFD model combining RNG k -ɛ model was used to study the three-dimensional gas flow through the considered serrated valve tray. The simulated results showed that various eddies existed on the serrated valve tray, and both the eddy and the non-eddy areas were nearly equal. The existence of addendum can decrease the eddy area caused by gas passing through the lateral outlet slots. The size of eddies can be reduced by optimizing the distance between valves.

Cryo-copolymerization preparation of dextran-hyaluronate based supermacroporous cryogel scaffolds for tissue engineering applications

Dextran-hyaluronate (Dex-HA) based supermacroporous cryogel scaffolds for soft tissue engineering were prepared by free radical cryo-copolymerization of aqueous solutions containing the dextran methacrylate (Dex-MA) and hyaluronate methacrylate (HA-MA) at various macromonomer concentrations under the freezing condition. It was observed that the suitable total concentration of macromonomers for the preparation of Dex-HA cryogel scaffold with satisfied properties was 5% (w/w) at the HA-MA concentration of 1% (w/v), which was then used to produce the test scaffold. The obtained cryogel scaffold with 5% (w/w) macromonomer solution had high water permeability (5.1 × 10−12 m2) and high porosity (92.4%). The pore diameter examined by scanning electron microscopy (SEM) was in a broad range of 50–135 μm with the mean pore diameter of 91 μm. Furthermore, the cryogel scaffold also had good elastic nature with the elastic modulus of 17.47±1.44 kPa. The culture of 3T3-L1 preadipocyte within the scaffold was investigated and observed by SEM. Cells clustered on the pore walls and grew inside the scaffold indicating the Dex-HA cryogel scaffold could be a promising porous biomaterial for applications in tissue engineering.

Chromatographic adsorption of serum albumin and antibody proteins in cryogels with benzyl-quaternary amine ligands.

The preparation and characterization of mixed-mode adsorbents for a typical separation purpose are of great importance in bioseparation areas. In this work, we prepared a new monolithic cryogel with a combination of ion-exchange and hydrophobic functions by employing benzyl-quaternary amine groups. The fundamental cryogel properties, protein equilibrium adsorption isotherm and chromatographic adsorption in the cryogel were measured experimentally. The results showed that, by using bovine serum album as the model protein, the dual functional cryogel has protein binding capability even in salt solution and the buffer with pH close or below the protein isoelectric point due to both the electrostatic and hydrophobic interactions. A capillary-based adsorption model was developed, which provided satisfied insights of the microstructure, axial dispersion, mass transfer as well as protein adsorption characteristics within the cryogel bed. The chromatographic isolation of bioactive proteins from rabbit blood serum was carried out by the cryogel. Immunoglobulin G antibody with a purity of 98.2% and albumin with a purity of 96.8% were obtained, indicating that the cryogel could be an interesting and promising adsorbent in bioseparation areas.