Affinity Chromatography Adsorbent Research Articles

An immobilized titanium (IV) ion affinity chromatography adsorbent for solid phase extraction of phosphopeptides for phosphoproteome analysis

In this study, we developed a centrifugation assisted solid phase extraction (SPE) method for the selective enrichment of phosphopeptides using a new Ti4+-IMAC material synthesized in-house. This new material has the feature of big size and large specific surface area which makes it more suitable to enrich phosphopeptides in a SPE way. The spin tips loaded with the Ti4+-IMAC material were applied to enrich phosphopeptides from the complex protein digests. It was found that phosphopeptides can be specifically enriched from tryptic digest of bovine serum albumin and β-casein at a molar ratio up to 1000:1. And about 4700 unique phosphorylated peptides can be identified with the specificity as high as 99% from the tryptic digest of HeLa cell proteins. This tip was demonstrated to have good column-to-column reproducibility. Furthermore, it is fitted to analyze minute amount of sample. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which making it a time-saving and convenient method for phosphopeptide enrichment. Compared with the commercial TiO2 material, this new materials yielded much more phosphopeptide identifications and much higher enrichment specificity.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8–15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

Equilibrium adsorption of poly(His)-tagged proteins on immobilized metal affinity chromatographic adsorbents

A systematic analysis on the adsorption of homo-oligomeric, poly(His)-tagged recombinant proteins on a hydroxyapatite-based immobilized metal affinity chromatography adsorbent was performed. Under non-denaturing conditions, the Langmuir–Freundlich isotherm model was found ideal for predicting the adsorption behavior of the model proteins. The dissociation constants, as low as 10 −9 M, decreased with the number of poly(His) tags, suggesting the presence of multi-point attachment. The maximum adsorption capacities, ranging from 79.1 nmol/g for the 88 kDa epimerase to 42.8 nmol/g for the 320 kDa racemase, were inversely proportional to the contact surface areas of the proteins. Under denaturing conditions, the Langmuir isotherm model fitted well with the experimental data. The maximum adsorption capacities for and the dissociation constants of the three model proteins were essentially identical, as the subunits of the model proteins were of similar dimensions and behaved similarly in the absence of complex tertiary or quaternary structure. Phosphate buffer at a concentration of 500 mM, pH 8.0 was found to be effective for the elution of the model proteins with a recovery yield more than twofold higher than that obtained with 500 mM imidazole. The results suggest the hydroxyapatite-based adsorbent is a promising alternative for large scale applications.

Immobilized-metal affinity chromatography adsorbent with paramagnetism and its application in purification of histidine-tagged proteins

A new method for synthesis of an Immobilized-Metal Affinity chromatography (IMAC) adsorbent with superparamagnetism (Fe 3O 4/SiO 2-GPTMS-Asp-Co) was reported in this paper. Fe 3O 4 nanoparticles were first modified by SiO 2 to form the core–shell Fe 3O 4/SiO 2 with superparamagnetism, the core–shell microspheres were then successively treated by 3-glycidoxypropyltrimethoxysilane (GPTMS), l-aspartic acid ( l-Asp) and 2-bromoacetic acid to form Fe 3O 4/SiO 2-GPTMS-Asp nanoparticles with tetradentate ligands. Finally, the IMAC adsorbent with superparamagnetism, Fe 3O 4/SiO 2-GPTMS-Asp-Co, was finally obtained by the coordination of Co 2+ with the resulting nanoparticles. The intermediates and product obtained from the process mentioned above were characterized by TEM, SEM, XRD, XPS, FT-IR, TGA, AAS, EDS, element analysis and magnetic hysteresis loop. Fe 3O 4/SiO 2-GPTMS-Asp-Co nanoparticles used as IMAC adsorbent to separate the recombinant 6-histidine-tagged gp41 protein were investigated. The result indicates that the IMAC adsorbent we prepared has outstanding advantages in the separation of the 6-histidine-tagged proteins from the crude bacterial lysate, such as simple operation, high selectivity and capacity.

Specific Phosphopeptide Enrichment with Immobilized Titanium Ion Affinity Chromatography Adsorbent for Phosphoproteome Analysis

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti (4+)-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti (4+) through the chelating interaction between phosphate groups on the polymer and Ti (4+). The resulting Ti (4+)-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti (4+)-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti (4+)-IMAC to other enrichment methods including Fe (3+)-IMAC, Zr (4+)-IMAC, TiO 2 and ZrO 2, and demonstrate superior selectivity and efficiency of Ti (4+)-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti (4+)-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.

A poly(2-hydroxyethyl methacrylate)-based immobilized metal affinity chromatography adsorbent for protein purification

Poly(HEMA) microbeads were prepared by suspension polymerization of 2-hydorxyethylmethacrylate and ethyleneglycoldimethacrylate (EGDMA). The water content, ligand density, and selectivity for poly(His)-tagged d-hydantoinase of the poly(HEMA)-based adsorbents were affected by the concentration of EDGMA used during polymerization. The Ni(II)-loaded poly(HEMA) adsorbent exhibited an adsorption capacity of 1.0 mg/g for poly(His)-tagged d-hydantoinase under optimal conditions with buffer containing 100–300 mM NaCl at pH 6.0. One-step purification protocol with the adsorbent gave a purity of at least 92%. The adsorption capacity of adsorbent declined by 54% after 7 cycles, due to the leaching of Ni(II) from the adsorbent. However, upon regeneration the adsorption capacity can be restored. Given the ease of preparation and the chemical and microbial resistance, the poly(HEMA)-based IMAC adsorbent could be a promising substitute for the polysaccharide-based IMAC adsorbents.

Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents

The equilibrium adsorption of four homo-oligomeric recombinant proteins containing up to eight poly(histidine) affinity tags on a silica-based Cu(II)-IDA immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The equilibrium adsorption data are well fitted with the three-parameter Langmuir–Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins and the degree of cooperativity seems to increase with the number of poly(His) tags. This phenomenon is also supported by the concave-downward curves in the Scatchard analysis. The maximum capacities the IMAC adsorbent for the model proteins, declining from 31.79 nmol/g gel for 88 kDa epimerase to 21.50 nmol/g gel for 320 kDa racemase, are in principle affected by the size of the molecule, and the binding strengths, expressed in term of association constants K a increasing from 1.47 μM −1 for the dimeric epimerase to 50.01 μM −1 for the octameric racemase, are dictated by the number of affinity tags. Nevertheless, some structure-specific interactions observed among native N-carbamoylase molecules may promote multilayer adsorption and thus lead to a much higher adsorption capacity, 715 nmol/g gel and a lower compounded binding affinity, 0.42 μM −1. The observed positive cooperativity for the model proteins and the structure-specific interactions among N-carbamoylase molecules are eliminated under denaturing conditions, resulting in a homogeneous adsorption well fitted by the Langmuir isotherm model.

Porous poly(hydroxyethyl methacrylate) based monolith as a new adsorbent for affinity chromatography

A new approach was developed to obtain an efficient and cost effective purification of IgG from human plasma. Porous monoliths were obtained by the bulk polymerization of 2-hydroxyethyl methacrylate (HEMA) and N-methacryloyl-( l)-histidinemethylester (MAH) conducted in a glass tube. Poly(HEMA-MAH) monolith had a specific surface area of 145.8 m 2/g. Poly(HEMA-MAH) monolith was characterized by swelling studies, FTIR, scanning electron microscopy and elemental analysis. Poly(HEMA-MAH) monolith with a swelling ratio of 68%, and containing 47.8 μmol MAH/g were used in the adsorption/desorption of IgG from aqueous solutions and human plasma. The maximum amount of IgG adsorption from aqueous solution in phosphate buffer was 9.8 mg/g at pH 7.4. A higher adsorption value was obtained from human plasma (up to 96.5 mg/g) with a purity of 95.3%. It was observed that IgG could be repeatedly adsorbed and desorbed with Poly(HEMA-MAH) monolith without significant loss in the adsorption capacity.

1H high-resolution magic-angle spinning (HR-MAS) NMR analysis of ligand density on resins using a resin internal standard.

We recently attempted to generate an affinity chromatography adsorbent to purify cytochrome P450 4A1 by coupling 11-(1'-imidazolyl)-3,6,9-trioxaundecanoic acid to Toyopearl AF-Amino 650 M resin. Variations in ligand density for several resin batches were quantified by high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy using a novel resin internal standard. The uniquely designed ImQ internal resin standard yields its signature resonance in a transparent region of the analyte spectrum making suppression of the polymer background unnecessary. This method enabled us to target a reasonable ligand density for enzyme purification and provides an advantageous alternative to quantitation against soluble standards or protonated solvent.

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu 2+/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l- p-hydroxyphenylhydantoin ( d,l-HPH) to N-carbamoyl- d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.

Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose

A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5α cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent – Co 2+-carboxymethylaspartate agarose Superflow – was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co 2+-carboxymethylaspartate agarose for each protein was determined.

Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.

Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 micromol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 micromol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 microM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 microM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 micromol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.

Design and study of peptide‐ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Design and study of peptide‐ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Preparation of new synthetic hydrogels containing galactose, applicable in affinity chromatography

AbstractNew biospecific affinity chromatography adsorbents were prepared from epoxy reactive matrices with subsequent coupling of galactose as the ligand and were afterward characterized. Their usefulness in the sorption of peanut lectin, a protein specifically retained by the galactose residue, is reported. © 1994 John Wiley & Sons, Inc.

An affinity adsorbent derived from aminopropyl silica for serine protease chromatography

AbstractA silica‐based adsorbent for affinity chromatography of serine protease was prepared by bonding p‐aminobenzamidine (pABZ) to aminopropyl silica. Silanization of silica with both γ‐aminopropyltrimethoxysilane and γ‐amino‐propyltriethoxysilane under anhydrous conditions led to a monolayer density of primary amino groups. The aminopropyl silica was converted to primary hydroxylcontaining silica via diazotization, and consequently activated with p‐nitrophenyl chloroformate and immobilized with ligand pABZ. The resultant silica–pABZ has a ligand density of 13.8 μmol g−1. Pyridine was indicated by the data to increase the reactivity of the chloroformate toward the hydroxyl group more efficiently than 4‐dimethylaminopyridine. Two stages of adsorption were found in batch adsorption of trypsin with an equilibrium adsorption isotherm of the Langmuir type. When the chromatographic column packed with this silica–p ABZ was operated under a higher flow rate (2·33 cm3 min−1) and with 3 g dm−3 of crude urokinase as the influence, the yield was 55%. Both the flow rate and the concentration of the crude protein were shown, by measuring the dynamic binding capacity from chromatographic experiments, to be the factors which influenced the chromatographic efficiency.

Preparation of anhydrochymotrypsin diol silica as selective adsorbent for affinity chromatography of peptides with aromatic amino acids at C-termini

Anhydrochymotrypsin (AHC), a catalytically inert derivative of chymotrypsin in which the serine-residue active site has been converted chemically to a dehydroalanine residue, was immobilized on diol silica by activation with trifluoroethanesulfonyl chloride. A AHC-diol-silica column was used for high-performance affinity chromatographic separation of peptides with aromatic amino acids at their C-termini from other peptides. Faster separations were achieved.

New developments in affinity chromatography

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.

Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M.

The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.

Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M

The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.