Impact of double cryogelation process on a macroporous dye-affinity hydrogel

Cryogels with interconnected channels allow high flow-through properties and mass transfer when dealing with complex mixtures such as non-clarified crude extracts. However, their mechanical strength can be challenged due to a large void volume inside the polymeric network. We have addressed this problem by forming a double-layer cryogel applied as a dye-affinity chromatography gel. In this study, poly(acrylamide-co-allyl glycidyl ether) cryogel was prepared at sub-zero temperature. The second layer was then prepared inside the primary cryogel under the same conditions to form a double-layer network. Cibacron Blue F3GA, a dye molecule, was immobilized on the surface of the cryogels. Bovine serum albumin was used as a model molecule to study the adsorption/elution procedure in batch and continuous modes. The maximum batch binding capacity and the dynamic binding capacity for the single-layer cryogel were 18 and 0.11, and for the double-layer cryogel were 7.5 and 0.9 mg/g of gel, respectively. However, the mechanical stability of the double-layer cryogel increased 7-fold (144 kPa). It was found that the kinetic and adsorption isotherms follow pseudo-second-order and Freundlich models, respectively. The regeneration of the columns after adsorption/elution cycles was evaluated, and no significant loss of capacity was observed after 10 cycles.     

染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究

采用反相悬浮交联法制备壳聚糖微球,对微球进行羟丙基氯化及氨基化,并偶联色素配体Cibacron Blue F3GA,得到一种新型染料亲和吸附剂.以牛血清白蛋白(BSA)为目标蛋白,考察了该染料亲和吸附剂的吸附性能,发现其对BSA有较高的吸附量(95.2mg/g),吸附行为满足Langmuir吸附等温式.负载牛血清白蛋白的微球容易洗脱,洗脱率高达99％.     

Adsorption of Human Serum Albumin onto PVA-coated Affinity Microporous PTFE Capillary

Affinity dye-ligand Cibacron Blue F3GA（CB F3GA） was covalently coupled with poly（vinyl alcohol）（PVA） coated on the inner surface of microporous poly（tetra-fluoroethylene）（MPTFE） membranous capillary. The PVA-coated PTFE capillary surface was characterized by XPS and FESEM. The grafting degree of PVA and the amount of CB F3GA immobilized onto the membranous capillary were 23.5 mg/g and 89.6 pmol/g, respectively. These dyed membranous capillaries were chemically and mechanically stable, and could be reproducibly prepared. Human serum albumin（HSA） was selected as model protein. The saturation adsorbance of the dye attached membranous capillary was 85.3 mg HSA/g, while the capacity of non-specific adsorption for HSA was less than 0.3 mg/g.     

染料壳聚糖微球的制备及其对人血清白蛋白吸附性能研究

人血清白蛋白(Human Serum Albumin,HSA)是血浆中含量最丰富的蛋白质,约占血浆总蛋白的60%.在人体内,HSA有许多重要的生理功能[1],临床上广泛应用于手术输血和危重病人补液,治疗创伤休克、烧伤、水肿和低白蛋白血症等,而且能增强人体抵抗能力,是迄今为止产量最大、临床用量最大     

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H₂O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.     

Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes

Cibacron Blue F3GA, Procion Red HE-3B and Procion Blue MX-R were immobilized on macroporous chitosan and chitin membranes with concentrations as high as 10–200 μmol/ml membrane. These dyed membranes were chemically and mechanically stable, could be reproducibly prepared, and operated at high flow rates. Human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins, and their adsorption on and desorption from the dyed chitosan membranes investigated. The Cibacron Blue F3GA membranes had a higher protein adsorption capacity, much greater for HSA than BSA, than the other dyed membranes. About 8.4 mg HSA/ml membrane were adsorbed at saturation by Cibacron Blue F3GA–chitosan membranes from a 0.05 M Tris–HCl/0.05 M NaCl, pH 8 solution. The chitin membranes had a lower dye content and hence a lower protein adsorption capacity than the chitosan membranes. The effects of important operation parameters (flow rate, protein concentration and loading) were also investigated. Cibacron Blue F3GA–chitosan membranes were employed for the separation of HSA from human plasma and high purity HSA thus obtained. This suggests that these membranes could be used for large-scale plasma fractionation.     

Oriented immobilized anti‐hIgG via Fc fragment‐imprinted PHEMA cryogel for IgG purification

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F_c fragment‐imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti‐hIgG for IgG purification from human plasma. Non‐imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti‐hIgG to compare the adsorption capacities of oriented (MIP/anti‐hIgG) and random (NIP/anti‐hIgG) cryogel columns. The amount of immobilized anti‐hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti‐hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti‐hIgG column (29.7 mg/g) for the MIP/anti‐hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti‐hIgG cryogel column. Adsorbed IgG was eluted using 1.0 m NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti‐hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.     

Protein C recognition by ion‐coordinated imprinted monolithic cryogels

The protein C imprinted monolithic cryogel was synthesized using 2‐hydroxyethyl methacrylate by redox cryo‐polymerization method. The prepared monolithic cryogel was characterized by Fourier transform infrared spectroscopy, swelling test, surface area measurements, and scanning electron microscopy. The nonimprinted cryogel was prepared as well for control. Adsorption of protein C from aqueous solutions was investigated in a continuous mode and several parameters affecting adsorption performance were optimized. The maximum protein C adsorption amount was 30.4 mg/g. The selectivity studies were performed by monolithic column studies and fast protein liquid chromatography, using hemoglobin and human serum albumin as competing proteins. The relative selectivity coefficients were 2.37 and 8.89 for hemoglobin and human serum albumin, respectively. Reusability was tested for ten consecutive adsorption–desorption cycles, and no significant change in adsorption capacity was recorded. A pseudo‐second‐order model was suitable to interpret kinetic data, and the Langmuir model suited the adsorption isotherms well.     

白蛋白在以染料为配基的醋酸纤维滤棒上的等温吸附行为(英文)

 以醋酸纤维滤棒为基质 ,染料CibacronBlueF3GA为配基 ,合成了一种新的染料亲和介质 ;分别以牛血清白蛋白 (BSA)和人血清白蛋白 (HSA)为对象 ,用静态法进行了吸附实验 ,得到了相应的亲和等温吸附曲线 ;对曲线按Langmuir模型和Freundlich模型分别进行拟合 ;结果表明 ,醋酸纤维滤棒染料亲和介质对BSA和HSA的等温吸附遵循Freundlich模型。采用该亲和介质装柱并分离实际样品人血浆 ,可得到纯化的人血清白蛋白。     

Application of Supermacroporous Monolithic Hydrophobic Cryogel in Capturing of Albumin

Supermacroporous poly{2-hydroxyethyl methacrylate-co-[N,N-bis(2,6-diisopropylphenyl)-perylene-3,4,9,10-tetracarboxylic diimide]} [poly(HEMA-co-DIPPER)] monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with DIPPER as functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50 μm size. Poly(HEMA-co-DIPPER) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA-co-DIPPER) cryogel was 14.7 g H₂O/g dry cryogel. Poly(HEMA-co-DIPPER) cryogel was used in the adsorption/desorption of albumin from aqueous solutions. The nonspecific adsorption of albumin onto plain poly(HEMA) cryogel was very low (3.36 g/g polymer). The maximum amount of albumin adsorption from aqueous solution in acetate buffer was 40.9 mg/g polymer at pH 5.0. It was observed that albumin could be repeatedly adsorbed and desorbed with the poly(HEMA-co-DIPPER) cryogel without significant loss of adsorption capacity.     

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.     

Preparation of Cibacron Blue F3GA Bonded Poly (styrenedivinylbenzene) (PSDVB) Microbeads Used for High Performance Affinity Chromatography

Recentlyhighperformanceliquidaffinitychr0mat0graphy(HPLAC)hasdevel0pedveryquickly.HPLACcombinesthespeedandres0lvingp0werofHPLCwithbiol0gicalspecificityofaffinitychromatographyandhasbeenwidelyusedasananalyticalt00linbiochemicalresearch.CibacronBIueF3GAisthem0stwideIyusedreactivetriazine-baseddyewhichhasspecificinteracti0nwithpyridinenucleotide-dependentdehydr0genase,kinase,blo0dproteinsandotherpr0teinsandenzymes'.ltisasuitabIeHPLACligandbecauseofitsreactivityandchemicaIstability.Inthi…     

Comparison of adsorption performances of metal–chelated polyamide hollow fibre membranes in lysozyme separation

Commercially available microporous polyamide hollow fibres are modified by acid hydrolysis to activate the reactive groups and subsequently binding of the ligand, i.e. Cibacron Blue F3GA. Then the Cibacron Blue F3GA-derived hollow fibres were loaded with different metal ions (i.e. Zn(II), Cu(II), Ni(II)) to form the metal chelate. The internal polymer matrix was characterised by scanning electron microscopy. The effects of pH, initial concentration of lysozyme, metal type and temperature on the adsorption of lysozyme to the metal–chelated hollow fibres were examined in a batch reactor. The non-specific adsorption of lysozyme onto the polyamide hollow fibres was 1.8 mg/g. Cibacron Blue F3GA immobilisation increased the lysozyme adsorption up to 62.3 mg/g. Metal–chelated hollow fibres showed a significant increase of the adsorption efficiency. Lysozyme adsorption capacities of Zn(II), Cu(II) and Ni(II)-chelated hollow fibres were different. The maximum capacities of Zn(II), Cu(II) or Ni(II)-chelated hollow fibres were 144.2, 75.2 and 68.6 mg/g, respectively. Significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. Repeated adsorption–desorption process showed that this novel metal–chelated polyamide hollow fibres are suitable for lysozyme adsorption.     

A novel adsorbent for protein chromatography: supermacroporous monolithic cryogel embedded with Cu2+-attached sporopollenin particles

The aim of this study is to prepare supermacroporous cryogels embedded with Cu(2+)-attached sporopollenin particles (Cu(2+)-ASP) having large surface area for high protein adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Cu(2+)-ASP was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for affinity purification of human serum albumin (HSA). Firstly, Cu(2+) ions were attached to sporopollenin particles (SP), then the supermacroporous PHEMA cryogel with embedded Cu(2+)-ASP was produced by free radical polymerization using N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Embedded particles (10 mg) in PHEMA-based cryogel column were used in the adsorption/desorption of HSA from aqueous solutions. Optimum conditions of adsorption experiments were performed at pH 8.0 phosphate buffer, with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of HSA adsorption from aqueous solution was very high (677.4 mg/g SP) with initial concentration 6 mg/mL. It was observed that HSA could be repeatedly adsorbed and desorbed to the embedded Cu(2+)-ASP in PHEMA cryogel without significant loss of adsorption capacity.     

Hydrophobic cryogels for DNA adsorption: Effect of embedding of monosize microbeads into cryogel network on their adsorptive performances

As alternative hydrophobic adsorbent for DNA adsorption, supermacroporous cryogel disks were synthesized via free radical polymerization. In this study, we have prepared two kinds of cryogel disks: (i) poly(2‐hydroxyethyl methacrylate‐N‐methacryloyl‐l ‐tryptophan) [p(HEMA‐MATrp)] cryogel containing specific hydrophobic ligand MATrp; and (ii) monosize p(HEMA‐MATrp) particles synthesized via suspension polymerization embedded into p(HEMA) cryogel structure to obtain p(HEMA‐MATrp)/p(HEMA) composite cryogel disks. These cryogel disks containing hydrophobic functional group were characterized via swelling studies, Fourier transform infrared spectroscopy, elemental analysis, surface area measurements and scanning electron microscopy. DNA adsorption onto both p(HEMA‐MATrp) cryogel and p(HEMA‐MATrp)/p(HEMA) composite cryogels was investigated. Maximum adsorption of DNA on p(HEMA‐MATrp) cryogel was found to be 15 mg/g polymer. Otherwise, p(HEMA‐MATrp)/p(HEMA) composite cryogels significantly increased the DNA adsorption capacity to 38 mg/g polymer. Composite cryogels could be used repeatedly without significant loss on adsorption capacity after 10 repetitive adsorption–desorption cycles. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecularly imprinted supermacroporous cryogels for cytochrome c recognition

Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity.     

活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用

以3.0 μm无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂为基质,与三嗪染料活性蓝F3GA(Cibacron Blue F3GA)反应,制备出 一种固定化染料聚合物高效亲和色谱填料。考察了应用该填料时流动相中的盐离子浓度、有机溶剂及流速等对牛血清白蛋白(BSA)和 溶菌酶(Lys)保留行为的影响。实验结果表明,该染料亲和填料具有良好的色谱性能。利用前沿色谱法测定了染料与溶菌酶之间的表观 解离常数为5.26×10-5 mol/L。使用该填料成功地从鸡蛋清和小牛血清中分别分离纯化了Lys和BSA,十二烷基硫酸钠-聚丙烯酰胺凝胶 电泳(SDS-PAGE)分析显示Lys和BSA的纯度分别为95%和92%。     

Gelatin-Immobilised Poly(hydroxyethyl methacrylate) Cryogel for Affinity Purification of Fibronectin

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10–200 μm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30 mg/ml, while much higher adsorption values, up to 38 mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92 % of the adsorbed fibronectin was eluted using 2 M urea containing 1 M NaCl as elution agent. The adsorption–elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.     

辛巴蓝F-3GA修饰的啤酒废酵母菌亲和吸附溶菌酶

利用j嗪染料辛巴蓝F-3GA修饰经戊二醛交联的啤酒废酵母菌,得到一种新型染料亲和吸附剂.辛巴蓝F-3GA的固载量为161.1 mg/g.以溶菌酶为研究对象,考察吸附时间、酶初始浓度、pH值、离子强度等因素对吸附率的影响.结果表明:当pH=7.0时,其对溶菌酶有较高的吸附量(229.1 mg/g),吸附性能明显优于未接枝...     

Poly(HEMA-co-NBMI) Monolithic Cryogel Columns for IgG Adsorption

Supermacroporous poly(2-hydroxyethyl methacrylate-co-1,5-naphthalene bismaleimide) [poly(HEMA-co-NBMI)] monolithic cryogel column was prepared by free radical cryo-copolymerization of HEMA with NBMI as a hydrophobic functional comonomer and N,N′-methylene-bisacrylamide as cross-linker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix which has interconnected pores of 10–100 μm size. Poly(HEMA-co-NBMI) cryogel was characterized by swelling studies, FTIR and scanning electron microscopy. The equilibrium swelling degree of the poly(HEMA-co-NBMI) cryogel was 10.5 g of H₂O/g dry cryogel. Poly(HEMA-co-NBMI) cryogel was used in the adsorption/desorption of IgG from aqueous solutions. The maximum amount of IgG adsorption from aqueous solution in phosphate buffer was 98.20 mg/g polymer at pH 7.0. The nonspecific adsorption of IgG onto plain poly(HEMA) cryogel was very low (2.79 g/g polymer). It was observed that IgG could be repeatedly adsorbed and desorbed with the poly(HEMA-co-NBMI) cryogel without significant loss of adsorption capacity.