Affinity Sorbent Research Articles

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.

Newly synthesized bentonite–histidine (Bent–His) micro-composite affinity sorbents for IgG adsorption

Bentonite has several advantages for use as a support, including its lack of toxicity, chemical reactivity and hydrophilicity allowing easy fixation of biomolecules. A new approach was developed to obtain an efficient and cost effective purification of IgG from human plasma. This study focused on developing bentonite–histidine micro-composite affinity sorbents (Bent–His) (38–105 μm) for immunoglobulin-G (IgG) adsorption from human plasma. The pseudo-biospecific affinity ligand l-histidine was reacted with covalent binding onto the bentonite structures. X-ray diffraction and FT-IR analysis of Bent–His composite affinity sorbents were performed. The surface area of the bentonite and Bent–His micro-composite structures were found as 33.00 ± 0.30 and 24.70 ± 0.30, respectively. An elemental analysis of immobilized l-histidine for nitrogen was estimated to be 541.3 μmol/g bentonite. The non-specific IgG adsorption onto bentonite structures was negligible (ca. 0.3 mg/g). Higher adsorption values up to 15.6 mg/g were obtained in which Bent–His micro-composite affinity sorbents were used from aqueous solutions. Isotherm studies were carried out to evaluate the effect of initial concentration of IgG, pH and the desorption characteristics of Bent–His micro-composite affinity sorbents. The maximum amount of IgG adsorption from aqueous solution in phosphate buffer was 15.6 mg/g at pH 7.0. A higher adsorption value was obtained from human plasma (up to 89.6 mg/g) with a purity of 94.5%. IgG molecules could be repeatedly adsorbed and desorbed with these micro-composite affinity sorbent systems without noticeable loss in their IgG adsorption capacity.

Novel method for human serum albumin adsorption/separation from aqueous solutions and human plasma with Cibacron Blue F3GA-Zn(II) attached microporous affinity membranous capillaries

A novel affinity sorbent system for human serum albumin (HSA) adsorption from aqueous solutions and human plasma was developed. The affinity capillary sorbents were prepared by Cibacron Blue F3GA and Zn(II) derived microporous membranous poly(tetrafluoroethylene) (PTFE) capillaries which are modified by poly(vinyl alcohol) (PVA) coating. The maximum PVA adsorption capacity was 23.5 mg/g polymer, the average density of CB F3GA attached to the capillary is 89.8 μmol/g polymer and the amount of Zn(II) ions adsorbed on the CB F3GA immobilized capillaries was 292 μmol/g polymer. Optimal adsorptions could be achieved at a suitable flow rate, pH, and ionic strength. The maximum adsorption was observed at pH 5.0 which is the isoelectric pH of HSA. Desorption ratio was up to 92% in 30 min using 1.0 M NaSCN (pH 8.0) as the desorption agent. Metal-chelate affinity capillaries could be reused without significant decrease in the HSA adsorption capacity.

“Click Chemistry” in the Preparation of Porous Polymer-Based Particulate Stationary Phases for μ-HPLC Separation of Peptides and Proteins

With the use of the copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an element of "click chemistry," stationary phases carrying long alkyl chains or soybean trypsin inhibitor have been prepared for use in HPLC separations in the reversed-phase and affinity modes, respectively. The ligands were attached via a triazole ring to size monodisperse porous beads containing either alkyne or azide pendant functionalities. Alkyne-containing beads prepared by direct copolymerization of propargyl acrylate with ethylene dimethacrylate were allowed to react with azidooctadecane to give a reversed-phase sorbent. Azide-functionalized beads were prepared by chemical modification of glycidyl methacrylate particles. Subsequent reaction with a terminal aliphatic alkyne produced a reversed-phase sorbent similar to that obtained from the alkyne beads. Soybean trypsin inhibitor was functionalized with N-(4-pentynoyloxy)succinimide to carry alkyne groups and then allowed to react with the azide-containing beads to produce an affinity sorbent for trypsin. The performance of these stationary phases was demonstrated with the HPLC separations of a variety of peptides and proteins.

Activity-Based Matrix Metallo-Protease Enrichment Using Automated, Inhibitor Affinity Extractions

An automated inhibitor affinity extraction method for the activity-based enrichment of matrix metallo-proteases (MMPs) is presented. Samples containing purified MMP-12 were first extracted at different flow rates in a syringe pump setup, using cartridges packed with an MMP inhibitor affinity sorbent based on an immobilized hydroxamic acid containing peptide (PLG-NHOH) with mumol/L MMP affinity. Faster extractions, a reduced number of manual manipulations, and higher extraction yields (98.9%-99.3%) were obtained over the whole flow rate range compared to batch extractions. Application of the method to synovial fluid from a rheumatoid arthritis patient followed by gelatin-zymography revealed a strong enrichment of distinct MMPs from this biological sample that were not clearly visible in the original sample. The use of an auto-sampler and a solid-phase extraction (SPE) workstation allowed full automation of the extraction procedure with the potential for on-line coupling to further sample preparation and analytical steps. MMP-12 extractions were optimized showing that ligand density is an important factor with a clear extraction yield optimum around 5 to 7.5 mmol/L. Conditioning of the stationary phase for 1 week prior to use resulted in a further slight increase in extraction yield. Under optimal conditions, an extraction yield of 99.5% was reached with a cartridge contact time of only 13 s for MMP-12. The efficacy of the extraction method for activity-based MMP profiling was further improved by the use of a broad-spectrum MMP inhibitor with nmol/L affinity (TAPI-2). This resulted in an increased extraction yield for all tested MMPs. For MMP-1, -7, -8, -10, -12, and -13 extraction yields of at least 98.8% were obtained, while for MMP-9 (full length and catalytic domain) an extraction yield of at least 96.1% was reached.

Separation Media in Affinity Chromatography of Proteins - A Critical Review

The rapid development of proteomics has been made possible primarily by the progress in analytical and preparative separation methods. Biological systems are so complex that the separation procedures employed must be highly efficient as well as highly selective. The latter requirement is best met by affinity separations based on molecular recognition. The present review critically discusses the properties of the affinity sorbents employed in proteomics, namely, agarose gels, dextrans, modified methacrylate and acrylamide polymers, porous and nonporous silica, porous glass beads, monoliths, affinity membranes and magnetic beads. The physico-chemical properties of these materials, their preparation, application, approaches to their modification and their relative advantages and drawbacks are discussed. Keywords: Affinity chromatography, proteins, supports, proteomic applications

Comparison of protein A affinity sorbents III. Life time study

Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000 l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1–3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30–40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000–4000 ppm was achieved.

Comparison of protein A affinity sorbents: II. Mass transfer properties

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed. The agarose media MabSelect Xtra and MabSelect SuRe are recent developments of the existing protein A affinity medium MabSelect. MabSelect Xtra is designed to exhibit a higher binding capacity for IgG, and MabSelect SuRe is functionalized with an alkaline stabilized protein A. ProSep-vA Ultra is a porous glass medium with a pore size of 70 nm, also developed to improve the binding capacity. Adsorption was measured in a finite and infinite bath. Mass transfer in these systems could be well described by a model including film and pore diffusion. Mass transfer parameters were used to accurately predict IgG breakthrough in packed bed mode. The dynamic binding capacity of all three media did not change when residence time was at least 4 min. All three media are suited for capture of feed stocks with high antibody content.

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 μmol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6–7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.

Characterization of Charcoal Adsorption Sites for Aromatic Compounds: Insights Drawn from Single-Solute and Bi-Solute Competitive Experiments

Charcoal, the residue of incomplete biomass burning that is found in many soils and sediments, is considered a high affinity sorbent for organic pollutants. However, little is known about the microscopic processes controlling sorption. The purpose of this study was to gain molecular-scale insight into the sorption on a charcoal of three weakly soluble aromatic compounds [benzene (BEN), toluene (TOL), and nitrobenzene (NBZ)] by conducting both single-solute and bi-solute experiments. The charcoal (420 m2 g(-1)) was produced from maple wood shavings by oxygen-limited pyrolysis at 673 K. Solute affinity for charcoal followed the order NBZ > TOL > BEN. Commonly employed sorption models did not adequately describe the single-solute isotherms. Competition in both TOL-BEN and the TOL-NBZ bi-solute systems was strong. Normalization of the isotherms for the hydrophobic driving force by using an existing free energy correlation between sorption and partitioning to an inert solvent (benzene or n-hexadecane) with a nonpolar aromatic compound calibration set resulted in a finding of enhanced sorption of NBZ relative to the coalesced BEN and TOL isotherms, indicating some specificity in the interaction of NBZ. The competitive data indicated 1:1 molar competition between BEN and TOL and between NBZ and TOL, showing conclusively that this specificity was not due to a subpopulation of sorption sites unique to NBZ. H-bonding was ruled out, as the relative affinity for the sorbent among the solutes did not change at all when increasing the solution pH from 6.5 to 11. 1H NMR experiments showed molecular complexation in chloroform between NBZ and model graphene polycyclic aromatic units (naphthalene, phenanthrene, and pyrene) which was absentfor BEN and TOL. This result, in combination with the results of a companion study (Zhu and Pignatello, Environ. Sci. Technol. (in press)), is used to support the existence of pi-pi electron donor-acceptor interactions between NBZ (electron acceptor) and the polycyclic aromatic charcoal surface (electron donor) as the cause of enhanced NBZ sorption.

Solid phase peptide synthesis on epoxy‐bearing methacrylate monoliths

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.

Determinação eletroanalítica de corante reativo presente como contaminante em proteínas purificadas por cromatografia de afinidade

Procion Green HE-4BD is a reactive dye currently used in affinity purification, and commonly present as a contaminant in the final biological preparation. An assay method is described to determine trace amounts of the dye in the presence of human serum albumin(HSA) and leakage from agarose as affinity sorbent by cathodic stripping voltammetry. The proposed method is based on the reductive peak at -0.55V in B-R buffer pH 3 (E=0V and t= 240s), obtained when samples of HSA 2% (m/v) containing dye concentrations in sodium hydroxide pH 12 are submitted to a heating time of 330 min at 80 oC. Linear calibration curves can be obtained for RG19 dye concentrations from 5x10-9 mol L-1 to 8 x10-8 mol L-1. The detection limit (3s) is 1x10-9 mol L-1.

Antibodies specific to modified glyceraldehyde-3-phosphate dehydrogenase induce inactivation of the native enzyme and change its conformation

The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained. They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent. Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms. Differential scanning calorimetry of the purified [GAPDH]·[antibody] complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature. Again, the effect appeared to be different with the apoenzyme. Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody. The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM ® Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM ® affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.

Activity-based enrichment of matrix metalloproteinases using reversible inhibitors as affinity ligands

Matrix metalloproteinases (MMPs) are zinc dependent metalloproteases characterized by the ability to cleave extracellular matrix and many other extracellular proteins. MMP activity is tightly regulated but disturbances in this regulation can contribute to various disease processes characterized by a progressive destruction of the extracellular matrix. The ability to profile classes of enzymes based on functionally related activities would greatly facilitate research about the involvement of MMPs in physiological and/or pathological states. Here we describe the characterization of an affinity sorbent using an immobilized reversible inhibitor as a stationary phase for the activity-based enrichment of MMPs from biological samples. With a ligand density of 9.8 m M and binding constant of 58 μmol/l towards MMP-12, the capturing power of the affinity sorbent was strong enough to extract MMP-12 spiked into serum with high selectivity from relatively large sample volumes. Experiments with endogenous inhibitors revealed that MMP-12 extraction is strictly activity-dependent, offering powerful means to monitor MMP activities in relation to physiological and/or pathological events by using affinity extraction as a first step in an MMP profiling method.

Immobilized-metal affinity sorbents based on hydrophilic methacrylate polymers and their interaction with immunoglobulins

Hydrophilic homogeneous poly[2-(2-hydroxyethoxy)ethyl methacrylate- co-ethylene dimethacrylate] poly(DEGMA- co-EDMA) beads have been prepared as precursors for introducing the chelating groupings of ethylenediaminetriacetic acid, quinolin-8-ol, or N-(2-pyridylmethyl)glycine. The effect of the chelating ligand structure on coordination with Ni 2+ ions and that of the metal complexes on binding the model protein (horseradish-peroxidase-specific immunoglobulin IgG 1) was analyzed. It was found that the protein binding affinity and selectivity was the highest for IMA sorbents with quinolin-8-ol groups.

Comparison of protein A affinity sorbents

Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes.

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 °C.

Comparison of protein A affinity sorbents

Comparison of protein A affinity sorbents

N-methacryloly-(L)-histidinemethylester carrying a pseudospecific affinity sorbent for immunoglobulin-G isolation from human plasma in a column system

N-methacryloly-(L)-histidinemethylester (MAH) as a pseudospecific ligand was synthesized by using methacryloyl chloride and histidine. Spherical beads with an average size of 63-75 μm were obtained by suspension polymerization of ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA) and MAH conducted in an aqueous dispersion medium. The specific surface area of the beads was found to be 18.3 m2/g. Poly(EGDMA-HEMA-MAH) beads were used in the separation of immunoglobulin-G (HIgG) from aqueous solutions and/or human plasma in a packed-bed column system. HIgG adsorption capacity of the beads decreased with an increase in the flow-rate of plasma. The maximum HIgG adsorption on the poly(EGDMA-HEMA-MAH) sorbents was observed at pH 7.4. HIgG adsorption onto the poly(EGDMA-HEMA) sorbents was negligible. Higher adsorption values (up to 135 mg/g) were obtained when the poly(EGDMA-HEMA-MAH) sorbents were used from aqueous solutions. HIgG adsorption increased with decreasing temperature and the maximum adsorption achieved at 4°C. MAH incorporation significantly affected HIgG adsorption capacity (135 mg/g). Higher amounts of HIgG were adsorbed from human plasma (up to 165 mg/g). Adsorption capacities of other blood proteins were obtained as 8.7 mg/g for fibrinogen and 14.6 mg/g for albumin. The total protein adsorption was determined as 191 mg/g. The pseudospecific affinity beads allowed one-step separation of HIgG from human plasma. HIgG molecules could be repeatedly adsorbed and desorbed with these sorbents without noticeable loss in their HIgG adsorption capacity.