Superporous agarose anion exchangers for plasmid isolation

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.     

Preparation and evaluation of polymer-coated adsorbents for the expanded bed recovery of protein products from particulate feedstocks

A novel prototype polymer-coated adsorbent (PCA) has been developed for the effective expanded bed recovery of protein products from particulate feedstocks. The adsorbents were manufactured using the three-phase emulsification process by which the selected core phases (anion- and cation-exchangers and a custom-assembled pseudo-affinity adsorbent) were coated by an agarose gel. This new non-stick exterior coating acts as a sieve reducing the non-specific binding of cell and cell debris without diminution of selective capture of target protein from complex feedstocks such as whole microbial broths and cell disruptates. The new coated adsorbents were subjected to physical and hydrodynamical comparison with the performance of their uncoated adsorbents. Hydrodynamic characteristics (e.g. axial dispersion coefficient (D(axl)) and Bodenstein number (B(o))) of PCA demonstrated a marked robustness in the face of biomass loading disrupted yeast cells. In addition, each adsorbent was compared with its uncoated native form during the expanded bed adsorption of one of two intracellular proteins (i.e. glyceraldehyde 3-phosphate dehydrogenase and cytochrome c) from a 20% (ww/v) yeast disruptate. The performance parameters of efficiency of washing, purification factor, turbidity of the eluted product and protein recovery in all analysed cases were favourable to the coated materials. In particular, exploiting PCA reduced significantly undesirable adsorption of cells without significant loss of binding capacity for the target product. The generic application of such adsorbents and their potential for the recovery of target products from complex feedstock is discussed, whilst other application such as the subtractive purification of nanoparticles were detailed in our previous publication.     

Preparation and some properties of a polyethyleneimine-agarose metal adsorbent

An adsorbent for metal ions has been prepared by reacting high molecular weight polyethyleneimine (PEI) with a crosslinked and activated agarose gel, Novarose. The synthesis variables, i.e. time, temperature, pH, PEI concentration and PEI/Novarose ratio, were optimized in order to obtain a high metal binding capacity of the adsorbent. The binding capacity for Cu(2+) is 500 micromol/ml packed adsorbent. A number of properties of the adsorbent relevant for metal ion accumulation has been investigated for Cu(2+), Ni(2+), Cd(2+) and Zn(2+). Adsorption capacities, adsorption isotherms, distribution coefficients, recoveries and relative rates of accumulation were determined. The adsorbent can be used for preconcentration and for separation of interfering alkali and alkaline earth metals in analytical applications.     

Phenylboronate-chitosan resins for adsorption of β-amylase from soybean extracts

Isolation and purification of bioproducts from crude extracts can be obtained by affinity methods based on reversible binding of a specific molecule to ligand immobilized in a porous matrix. In the present work, nicrospheres based on chitosan matrix, which incorporated aminophenylboronic acid as a derivative, were prepared and characterized, aimed at developing a β-amylase adsorption process. Kinetic curves and adsorption isotheriru of the crude extracts as well as the breakthrough curves for a frontal chromatographic separation method of a commercial sample of β-amylase from soybean are presented. These results were compared to similar data obtained with a comercial microspheres gel based-on agarose.     

Evaluation and applications of a new dye affinity adsorbent

The basic properties of a new dye affinity adsorbent Toyopearl AF-Blue HC-650M and its applications to the purification of proteins were studied. The binding capacity for human serum albumin (HSA) was greater than 18 mg per ml gel. The dye leakage from Toyopearl AF-Blue HC-650M in 0.5 M NaOH and 0.5 M HCI was less compared with an agarose adsorbent. Caustic stability study also demonstrated this material withstood exposure to 0.1 M NaOH for 1 month with no significant loss of binding capacity for HSA. We purified human albumin from human serum and lactate dehydrogenase (LDH) from rabbit muscle extract in a single step. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that human albumin and LDH were highly purified.     

Direct capture of product from fermentation broth using a cell-repelling ion exchanger

A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Fabrication and Characterization of a Dye-Immobilized Yttria-Stabilized Zirconia Pellicular Adsorbent for Expanded Bed Adsorption Chromatography

This study deals with the fabrication and characterization of a pellicular adsorbent appropriate for the expanded bed adsorption (EBA) process. The synthesized adsorbent has an yttria-stabilized zirconia nucleus coated with agarose. Morphological analysis of the coated particles was performed by light-scattering microscopy and showed an average diameter of 197.54 and 202.25 µm, for the nucleus and coated particle, respectively. A screening for the reactive dyes reactive blue 19 (RB19), reactive blue 21 (RB21) and reactive orange 107 (RO107) was performed after immobilization onto the pellicular adsorbent by changing the pH, aiming at finding the binding capacity of these to adsorb bovine serum albumin (BSA). The reactive orange 107 was selected and it was more stable at pH 4.5. Study of the kinetics between BSA and the dye-immobilized particle showed that equilibrium is reached before 1 h. The adsorption isotherm of BSA onto RO107-immobilized adsorbent fitted the Langmuir model showing a q_m = 102.328 mg BSA/mL of adsorbent. The pellicular adsorbent also showed good expansion even at a high operating flow rate. Therefore, at a linear velocity as high as 2725 cm/h, a dynamic capacity of 15.7 mg of BSA/mL of adsorbent was obtained.     

Galactosyl-biomimetic dye-ligands for the purification of Dactylium dendroides galactose oxidase

Two anthraquinone galactosyl-biomimetic dye-ligands comprising, as terminal biomimetic moiety, galactose analogues (1-amino-1-deoxy-beta-D-galactose and D(+)-galactosamine) were designed for the enzyme galactose oxidase (GAO), using molecular modelling, synthesized and characterized. The biomimetic ligands were immobilized on agarose beads and the affinity adsorbents, together with a non-biomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their ability to purify GAO from Dactylium dendroides. Both biomimetic adsorbents showed higher purifying ability for GAO compared to the non-biomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising, as terminal biomimetic moiety, 1-amino-1-deoxy-beta-D-galactose (BM1) exhibited the highest purifying ability for GAO. This affinity adsorbent did not bind galactose dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, or glucose oxidase. The dissociation constant (K(D)) of the immobilized BM1 ligand with GAO was found to be equal to 45.8 microM, whereas the binding capacity was equal to 709 U per ml adsorbent. Therefore, the BMI adsorbent was integrated in a facile two-step purification procedure for GAO. The purified enzyme showed a specific activity equal to 2038 U/mg, the highest reported so far, approximately 74% overall recovery and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis.     

Modification of aluminum chips for LDI mass spectrometry of proteins

Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOFMS) combined with affinity chromatography on immobilized phenylboronic acid agarose gels was used for selective enrichment and detection of specifically modified proteins such as glycated proteins in complex biological samples. Physicochemical grafting of hydrophilic polymers on aluminum surface was developed to reduce nonspecific protein sorption and to create a proper support layer for a three-dimensional affinity hydrogel. Grafted agarose allowed the fixation of three-dimensional agarose hydrogel on the chip surface. Both pinched polymers and hydrogels were effectively derivatized. 3-Aminophenylboronic acid (mPBA) was covalently immobilized as an affinity ligand to achieve specific binding of glycated plasma proteins. Alternatively, the affinity sorbent was immersed into the hydrogel to increase binding capacity. MALDI TOFMS was used to evaluate binding efficiency and molecular mass changes of human serum albumin due to glycation. Glycated proteins were captured directly on the chip with high selectivity and efficacy, and low nonspecific binding. Thus they could easily be characterized by MALDI TOFMS.     

Application of the FCP-activation procedure to the synthesis of a biospecific adsorbent for trypsin

The synthesis of a biospecific adsorbent for trypsin was chosen as a model to investigate the applicability of FCP activation in affinity chromatography.p-Aminobenzamidine was chosen as a ligand, directly suitable for immobilization. The nonspecific binding properties of the first series of synthesized agarose derivatives were obviated either by FCP activation of the ligand instead of the matrix, or by modifying the initial FCP-activation procedure. The adsorbents prepared in this way, however, demonstrated no selectivity between trypsin and chymotrypsin. The introduction ofe-aminocaproic acid as a spacer was ineffectual. These problems were solved by the application of glycylglycine as a spacer. The final affinity matrices had a degree of substitution of approximately 4 μ.mol of ligand per gram gel (100 μmol ligand per gram dry adsorbent). The specific activity of a current trypsin preparation was increased by 58% in a single cycle. The biospecificity of these adsorbents was demonstrated.     

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.     

Tris(hydroxymethyl)aminomethane-functionalized agarose particles: parameters affecting the binding of bovine serum albumin

A new protein adsorbent is introduced based on the coupling of the common buffer ion, tris(hydroxymethyl)aminomethane, to the agarose gel Sepharose HP from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. The article describes the synthesis of the new adsorbent and the use of BSA as a model in a binding study. By optimization of the coupling procedure, a maximum ligand density of 63.5 μmol/mL gel could be obtained. Adsorption equilibria were investigated in the pH range 5.0-8.0 and at salt concentrations of 0-0.4 mol/L. Binding of BSA under different conditions indicated that both electrostatic interaction and hydrogen bonding were involved in the adsorption process where the former played a major role.     

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: role of water activity

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)-iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)-nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A20, G20, C20 and T20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.     

IgA肾病免疫吸附剂的研究(Ⅲ)

在碱性条件下,用环氧氯丙烷对琼脂糖载体进行活化,并通过戊二醛法引入手臂,偶联羊抗人IgA的抗体,清除IgA肾病病人体内高含量的IgA分子,从而达到治疗病症的目的.通过体外实验发现,新研制的免疫吸附剂最高吸附率可以达到70%左右.     

Chemisorption tobacco mosaic virus removal from aqueous solutions and study of virus interaction with active chemisorbent surface by infrared spectroscopy

The interaction of the tobacco mosaic virus with the active surface of an iron-containing adsorbent obtained from lignin by the plasma-catalytic method was studied by IR spectroscopy. The results revealed decomposition of the virus into protein molecules and RNA reacting with the lignin surface via oxygen atoms during the binding of the virus to the sorbent surface. It was assumed that oxygen carboxylate groups interrelate with nanosized iron clusters incorporated into the adsorbent surface structure.     

Solid-phase thiolsulfinates for the reversible immobilization of thiols

A new method for the reversible immobilization of thiol-containing substances on agarose beads is presented. It is based on the use of thiolsulfinate (disulfide monoxide) as a solid-phase reactive group. The thiolsulfinate groups are introduced by controlled oxidation of thiol agarose. The method comprises two steps: First, mild oxidation of the agarose thiol groups to disulfide structures with potassium ferricyanide. Second, the oxidation of the so-formed agarose disulfide groups to thiolsulfinate groups by use of a stoichiometric amount of the oxidizing agent magnesium monoperoxyphtalate. The solid-phase thiolsulfinate groups react very easily with thiols, which, as a result of the reaction, will be bound to the agarose beads by disulfide bonds. The adsorbent derivative is very suitable for the reversible immobilization of low as well as high-mol-wt thiols as demonstrated with reduced glutathione, penicillamine, mercaptoethanesulfonic acid, thiolated bovine serum albumin,β-galactosidase, and ±₁-antitrypsine. Since treatment of the agarose derivatives with an excess of low-mol-wt thiols (e.g., dithiothreitol) leads to release of the bound molecules and regeneration of the original thiol groups, the reactive thiolsulfinate groups can easily be regenerated by the mentioned two-step procedure. The cycle of oxidation, binding, reduction, and reoxidation can be performed several times while retaining thiol binding capacity.     

Equilibrium and kinetic properties of a fast iminodiacetate based chelating ion exchanger and its incorporation in a FIA-ICP-AES system

The equilibrium and kinetic properties of an iminodiacetate (IDA) based chelating ion exchanger with a crosslinked agarose, Novarose, as support has been investigated. The second and third acidity constants and some complexation constants of the ligand were determined for adsorbents with metal binding capacities of 140, 55 and 18 micromol ml(-1), respectively. The adsorbent of medium capacity showed fast adsorption and desorption of Cu(II), Cd(II), Ni(II) and Ca(II) both in the batch and column mode. It was found to be about 50 times faster than Chelex-100 (50-100 mesh) in accumulation of these metal ions in the batch mode. Studies of the adsorbent in a flow system, using a 5 mm x 6 mm i.d. column, indicated quantitative accumulation of Cu(II), Cd(II), and Ni(II) at volumetric flow rates up to 110 ml min(-1). Linear calibration curves with r > 0.999 and signal enhancement factors up to 1300 were obtained. Preconcentration by a FIA system connected to an ICP-AES instrument will make simultaneous measurement of ultratrace concentrations of a number of metal ions possible within reasonable cycle times due to the high flow rates which can be used with the adsorbent. Trace amounts of cadmium and copper in tap water were determined successfully at 60 ml min(-1). However, copper and nickel in tap water are strongly complexed and do not accumulate quantitatively even at low flow rates. Hence a sample pretreatment is needed. Copper was completely adsorbed after UV-treatment of the sample.     

Manufacturing of agarose-based chromatographic adsorbents--effect of ionic strength and cooling conditions on particle structure and mechanical strength

The effect of ionic strength of agarose solution and quenching temperature of the emulsion on the structure and mechanical strength of agarose-based chromatographic adsorbents was investigated. Solutions of agarose containing different amounts of NaCl were emulsified at elevated temperature in mineral oil using a high-shear mixer. The hot emulsion was quenched at different temperatures leading to the gelation of agarose and formation of soft particles. Analysis of Atomic Force Microscopy (AFM) images of particle surfaces shows that pore size of particles increases with ionic strength and/or high quenching temperature. Additionally it has been found that the compressive strength of particles measured by micromanipulation also increases with ionic strength of the emulsion and/or high quenching temperature but these two parameters have no significant effect on the resulting particle size and particle size distribution. Results from both characterization methods were compared with Sepharose 4B, a commercial agarose-based adsorbent. This is the first report examining the effect of ionic strength and cooling conditions on the microstructure of micron-sized agarose beads for bioseparation.     

An agar-microchamber cell-cultivation system: flexible change of microchamber shapes during cultivation by photo-thermal etching

A new type of cell-cultivation system based on photo-thermal etching has been developed for the on-chip cultivation of living cells using an agarose microchamber array. The method can be used to flexibly change the chamber structure by photo-thermal etching, even during the cultivation of cells, depending upon the progress in cell growth. We used an infrared (1064 nm) focused laser beam as a heat source to melt and remove agar gel at the heated spot on a thin chromium layer. The melting of the agar occurred just near the chromium thin layer, and the size of the photo-thermally etched area depended almost linearly on the power of the irradiated laser beam from 2 microm to 50 microm. Thus by using photo-thermal etching with adequate laser power we could easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar-layer even during cell cultivation. After 48 h of cultivation of nerve cells, the nerve cells in two adjacent chambers made fiber connections through the fabricated narrow tunnel-shaped channels. These results suggest that photo-thermal etching occurred only in the area where an absorbing material was used, which means that it is possible to photo-thermally etch lines without damaging the cells in the microchambers. The results also suggest that the agar-microchamber cell cultivation system in combination with photo-thermal etching can potentially be used for the next stage of single cell cultivation including the real-time control of the interaction of cells during cell cultivation.