Covalent chromatography and salt-promoted thiophilic adsorption

Covalent chromatography on 3-(2-pyridyl disulfido)-2-hydroxypropyl agarose, abbreviated PyS2, turns out to involve more complex interactions than has been supposed heretofore. Unexpectedly, the sorption is highly salt dependent. The relative affinities for serum proteins have therefore been determined in the absence and presence of different types of salts at different salt concentrations and with different degrees of ligand substitution on the adsorbent. In the presence of water-structuring salts the PyS2-gel shows an adsorption pattern for serum proteins resembling that of the "thiophilic" T-gel (J. Porath; F. Maisano, and M. Belew (1985) FEBS Lett. 185, 306-310). Superimposed on thiophilic adsorption we have found, as expected, covalent attachment of thiol-containing proteins. Also the thiol-disulfide exchange increases from 4-5% in the absence of potassium sulfate or sodium chloride up to about 40% of the applied serum proteins when such a water-structuring salt is present. We have thus shown that the interaction of a protein with the ligand is greatly facilitated by a water-structuring salt--and in this case the product is a covalently as well as a thiophilically immobilized protein. A cautious interpretation of protein interaction phenomena is justified whenever ligands containing sulfide, disulfide, or pi-electron-rich structures such as aromatic moieties are involved.     

Novel heterocyclic ligands for the thiophilic purification of antibodies

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.     

Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Separation of the different plasmid isoforms is a major challenge in purifying plasmid DNA. We describe a new type of biochemical interaction that occurs in the presence of high concentrations of lyotropic salt and results in the selective adsorption of supercoiled plasmid DNA to aromatic thioether ligands. Under well-defined conditions, these ligands are capable of separating supercoiled plasmid DNA (ccc) from its isoform, i.e. open circular (oc) form. Integrated in a process, preceded by group separation and followed by anion-exchange chromatography, this new purification method may facilitate the production of highly purified supercoiled plasmid DNA for use in gene therapy and DNA vaccine applications.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.     

Salt-independent binding of antibodies from human serum to thiophilic heterocyclic ligands

Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and α₂-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.     

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area.     

Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody F(ab) fragment without the need for an affinity tag

Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant F(ab) fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli. Adsorption of the F(ab) fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a His(6) tag or Strep tag or of the human or murine nature of the C(H)1 and C(L) domains (subclass IgG1/kappa). Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F(ab) fragment at high purity. Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F(ab) fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer. Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F(ab) fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix. This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F(ab) fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system.     

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.     

Lymphoblastoid cell-produced immunoglobulins: preparative purification from culture medium by hydroxylapatite chromatography

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.     

Fermentative production of rhamnolipid and purification by adsorption chromatography

Glycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24?hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96?hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8?g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.     

Synthesis of oxytocin using iodine for oxidative cyclization and silica gel adsorption chromatography for purification.

Oxytocin (OT) was synthesized employing the solid phase method. Resins made of copolymers of polystyrene-1%-crosslinked with divinylbenzene gave better yields (73-95%) of Z-Cys(Bzl)-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) than 2%-crosslinked resins (10--56%). Reduction of I with Na-liq.NH3 and oxidation with I2-MeOH at -40 degrees minimized dimer and polymer formation, and resulted in good yields (49--54%) of OT. The large volumes of MeOH required when several grams of I are reduced and then oxidized were rapidly evaporated in vacuo, and the residue was desalted by dissolving the peptide in a small volume of glacial acetic acid and filtering to remove the salt. OT was purified by adsorption chromatography on a silica gel column with combinations of MeOH-CHCl3 of graded polarity. Oxytocin elutes with 33% MeOH-CHCl3. After two purification steps by adsorption chromatography, the resulting OT was found to be homogeneous. The hormone was characterized chemically and found to be active biologically.     

Behaviour of human immunoglobulin G subclasses on thiophilic gels: comparison with hydrophobic interaction chromatography

We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not adsorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for adsorption on thiophilic gels at low and high salt concentration.     

Novel synthesized colourless ligands in affinity chromatography for purification of alkaline phosphatase

Summary A 350-and a 460-fold one step purification of alkaline phosphatase from a crude calf intestine extract has been achived using two new synthesized colourless biomimetic compounds coupled to Agarose CL-4B.     

Simple method for large-scale isolation and purification of gangliosides by non-ionic adsorption chromatography

A type of non-ionic adsorption resin, X-5, was used in the isolation and purification of brain gangliosides. Hydrolysis with base treatment was carried out for the purpose of eliminating contaminants. The major advantages of the new procedure, compared to conventional methods, were the shorter separation time, higher loading capacity (80 micromol LBSA per gram resin), and recovery (98%) of separated ganglioside fractions with little solvent. It is a practical way for large-scale isolation and purification of gangliosides.     

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.     

Use of an immobilized human endogenous lectin for the purification of complementary ligands.

1. A galactoside-specific endogenous lectin isolated from human brain was covalently immobilized on divinylsulfone-activated agarose. This highly selective affinity adsorbent proved to be useful in purifying soluble protein ligands. 2. The maximum binding capacity of the adsorbent for complementary proteins was calculated to be 618 micrograms per g of gel (wet resin). 3. Sequential elutions using 0.1 M lactose, 0.3 M lactose and 0.5 M NaCl, and competition assays using incorporation in the presence 0.1 M lactose revealed differences in lectin-ligand interactions.