Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents

Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel? (n-hexyl amine) and PPA HyperCel? (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel? and PPA HyperCel? sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50–60 mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50–60 mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions.     

Adsorption of phytochrome on several substituted sepharose gels

Phytochrome from Avena sativa shows strong adsorption with hydrophobic ligands such as octyl and phenyl Sepharose. The same behaviour was observed for undegraded (MW 400 000) and degraded (MW 60 000) phytochromes in the P_r, or P_fr, form as well. The pigment is photoreversible after adsorption on those gels. Chromatography with amino acid ligands of degraded phytochrome was also tested. The chromoprotein showed the same strong adsorption on tryptophyl Sepharose. A more specific adsorption could be achieved on histidyl Sepharose but with loss of 70% of photoreversibility. This can be interpreted by the accessibility and perturbation of the chromophoric site by the histidyl ligands     

Comparison of affinity membranes and conventional affinity matrices with regard to protein purification

Summary Binding capacities for purified malate dehydrogenase from pig heart and malate dehydrogenase from Escherichia coli were determined for different gel matrices and a Cibacron Blue modified membrane. During batch adsorption the membrane has nearly the same capacity as Blue Sepharose. During filtration the effective capacity of the membrane was increased in contrast to Blue Sepharose. With cell homogenates, when used under cross-flow conditions, the membrane displayed better performance than Blue Sepharose.     

Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures

Direct product sequestration of extracellular proteins from microbial batch cultures can be achieved by continuous or intermittent broth recycle through an external extractive loop. Here, we describe the development of a fluidisable, mixed mode adsorbent, designed to tolerate increasing ionic strength (synonymous with extended productive batch cultures). This facilitated operations for the integrated recovery of an extracellular acid protease from cultures of Yarrowia lipolytica. Mixed mode adsorbents were prepared using chemistries containing hydrophobic and ionic groups. Matrix hydrophobicity and titration ranges were matched to the requirements of integrated protease adsorption. A single expanded bed was able to service the productive phase of growth without recourse to the pH adjustment of the broth previously required for ion exchange adsorption. This resulted in increased yields of product, accompanied by further increases in enzyme specific activity. A step change from pH 4.5 to 2.6, across the isoelectric point of the protease, enabled high resolution fixed bed elution induced by electrostatic repulsion. The generic application of mixed mode chemistries, which combine the physical robustness of ion-exchange ligands in sanitisation and sterilisation procedures with a selectivity, which approaches that of affinity interactions, is discussed.     

Assessment of the use of cyanogen bromide-activated Sepharose in analytical and preparative immunoadsorption

Procedures are described for the analytical and preparative purification of antigens based on their specific interaction with their complementary antibody immunoadsorbents prepared from cyanogen bromide-derivatized macroporous agarose matrices. In principle, the antigen to be purified in the affinity chromatography/immunoadsorption process should bind specifically and reversibly to the attached antibody, while other proteins pass through unretarded. In the case of tight binding, elution of the antigen is achieved by the use of eluting solutions of very high or very low pH, or with the use of chaotropic solutions such as 3 m KSCN. The performance of immunoadsorbents prepared from Sepharose 4B have been studied with the aim of improving the efficient utilization of immunoadsorption techniques. As a model, human serum was applied serially to several columns of Sepharose 4B sheep anti-human IgG which were then subjected to a number of successive adsorption/desorption cycles. Loading the columns with increasing amounts of serum showed that the performance was best when the antigen load was approximately threefold the ideal binding capacity. By limiting the amount of immobilized protein and carefully controlling the antigen load, significant improvements in yield and purity have been achieved. Antigen loads of threefold the potential binding capacity of the immunoadsorbent column results in the optimal yield of antigen with high purity and significant concomitant reduction in non-specific interference from other serum proteins. The non-specific adsorption which is an inherent problem and which leads to considerable inactivation of the covalently coupled antibody is highlighted. Although the popularity of such matrices is probably unsurpassed, it is clear that use has been made of them very frequently without an examination of quantitative aspects or side reactions.     

Plasma protein fractionation with advanced membrane adsorbents

High capacity membrane adsorbents have been used as a stationary phase for the preparative chromatographic purification of human serum albumin. A two-step ion exchange fractionation scheme yields albumin with 98% purity from clarified, microfiltrated, and desalted human plasma. Experiments with laboratory and pilot scale membrane modules are compared to literature data obtained with conventional Fast Flow Sepharose in a similar purification protocol. Increased productivity in combination with excellent reproducibility and stability was found using the membrane adsorbents. Scale-up of the process based on standard microfiltration equipment was successful but resulted in reduced capacity and productivity due to deteriorated flow characteristics of the module. This was attributed to the effects of substantial axial dispersion in the pilot scale module. Methods to reduce this limitation were identified. The concept of membrane adsorption chromatography for the fast purification of proteins is illustrated and engineering aspects important for the process design are discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 181-189, 1997.     

Adsorption-desorption of BSA to highly substituted dye-ligand adsorbent: quantitative study of the effect of ionic strength

Cibacron Blue 3GA was immobilized on Sepharose CL-6B to obtain a highly substituted dye-ligand adsorbent which dye concentration was 17.4?μmol dye per gram wet gel. This adsorbent had a highly binding capacity for bovine serum albumin (BSA). The effects of ionic strength on the adsorption and desorption of BSA to the adsorbent were studied. Adsorption isotherms were expressed by the Langmuir model. The quantitative relationships between the model parameters and the ionic strength were obtained. The desorptions were performed by adding salt to the BSA solutions in which adsorption equilibria had been reached. Adding salt to the solution resulted in the desorption of the bound protein. It was found that the isotherm obtained from the desorption experiments agreed well to the isotherm obtained from the adsorption experiments at the same ionic strength. The result demonstrated that the adsorption of BSA to the highly substituted adsorbent was reversible.     

Use of immobilized cytochrome c as a ligand for affinity chromatography of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans

Three matrices were used for immobilizing the cytochrome c: Sepharose CL-4B, Silasorb SPH amine and a laboratory-prepared new matrix based on crosslinked triazine (2,4,6-tris(aminoethylamine)-1,3,5-triazine) (TAT). Cytochrome c was immobilized on the matrices by several procedures and the amount of incorporated cytochrome c was determined. Cytochrome c immobilized on Sepharose CL-4B with periodate activation, cytochrome c immobilized on Silasorb-amine with carbodiimide activation and cytochrome c immobilized on crosslinked triazine were suitable for purification of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans. The yield with all matrices was about 90%. The purification factor of the above matrices was about 15. A new matrix based on TAT with cytochrome c represented a suitable way for thiosulfate dehydrogenase purification.     

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow‐fiber membranes

Iminodiacetic acid (IDA) and tris(2‐aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow‐fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA‐Cu(II) or TREN‐Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini‐cartridge in a cross‐flow filtration mode (52.5 and 298.4 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively). When mini‐cartridges were used, the dynamic adsorption capacity of IDA‐Cu(II) was the same for both mini‐cartridge and agarose gel. Copyright © 2013 John Wiley & Sons, Ltd.     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

Lumped parameter model for prediction of initial breakthrough profiles for the chromatographic capture of antibodies from a complex feedstock

A simple mathematical model to predict initial breakthrough profiles from preparative chromatographic separations of biological macromolecules has been developed. A lumped parameter approach was applied, employing Langmuirian adsorption kinetics to describe the rate of mass transfer (MT) from the bulk liquid in the column to the bound state. Equilibrium and kinetic adsorption data were determined for six different packed bed chromatographic adsorbents: two derivatised with rProtein A; and four functionalised with synthetic low molecular weight ligands. All adsorption isotherms were well described by the Langmuir model, whereas the data fitting to kinetic batch experiments showed that the model was inadequate after the first approximately 25 min of adsorption for four of the six adsorbents. The model underestimated the dynamic Ig breakthough on packed beds of rProtein A Sepharose FF, MabSelect, MBI HyperCel, and MabSorbent A1P, applying a feedstock of 20-100% (v/v) clarified rabbit antiserum. However, when employing a maximum adsorption capacity 25% greater than that determined in batch binding studies, excellent agreement was obtained at all antiserum strengths for most adsorbents. Useful insights into scale-up and process design can be obtained by applying the model, without determining tentative parameters specific for each adsorbent and target protein concentration. However, the model parameters are solvent dependent so a prerequisite for its true applicability is that binding is both Langmuirian and essentially independent of the ionic strength of the feedstock applied.     

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine

Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.     

General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes.

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.     

A novel process of cyclodextrin production by the use of specific adsorbents: Part I. Screening of specific adsorbents

Novel adsorbents that are composed of ligand, spacer and support were chemically synthesized and the two consecutive screenings of them made it possible to determine the adsorbents that were most recommendable for α- and β-cyclodextrin (CD) production. First, ligands of high adsorption selectivity for each CD were screened from among the candidates (carboxylic acids) that are tied in ionic bonds to two types of strongly basic anion exchange resins as support. Secondly, ligand derivatives (as ligand and spacer) were bound in covalent bonds onto chilosan beads as support and then, the most suitable spacer length for the CDs' adsorption selectivity and capacity were investigated. An optimal mol ratio of ligand to amino group of the beads was also examined. Stearic acid was the most effective ligand for α-CD, whereas cyclohexanepropanamide-n-caproic acid was the best for β-CD. Adsorption selectivity of adsorbents derived from carboxylic acids (stearic and/or palmitic) and chitosan beads was almost 100%, while their adsorption capacities were large enough to meet the demand for economic production and purification of CDs on an industrial scale.     

Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow.

The binding capacity of protein A Sepharose 4 Fast Flow for mouse IgG1 monoclonal antibodies (mabs) appears to be highly dependent on the buffer composition with respect to both concentration and ion type. Depending on the particular mab dynamic binding capacities up to 20 mg mab per ml gel could be obtained, when these mabs were isolated from supernatants of protein free hollow fibre cell culture systems. Variation of linear flow rate from 10 up to 300 cm/h and temperature (4 degrees C versus 25 degrees C) had a slight effect on the dynamic binding capacity, when a high ionic strength buffer was used during adsorption. Applying optimum binding conditions, final IgG fractions with a purity of more than 95% monomeric IgG were obtained. However, as side effect of the use of binding buffers with high ionic strength, the binding of acid proteases was also promoted.     

Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immunoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rProtein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent A1P, MabSorbent A2P, FastMabsA and Kaptiv-GY. The general experimental approach taken was to sequentially challenge packed beds of each matrix with a series of different strengths of a clarified antiserum; beginning with the weakest and ending with the strongest. Marked differences in performance (principally evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.