Investigations on the specificity of thiophilic interaction for monoclonal antibodies of different subclasses

A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG_2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG_2a subclass indicated a restriction in the universal nature of thiophilic interaction.     

pcD-awte候选疟疾DNA疫苗制备及其质量相关性分析

本实验室构建的疟疾DNA疫苗经动物试验表明具有很好的免疫原性,为申请临床试验,进行了制备工艺的研究。本研究将含pcD-awte质粒的大肠杆菌DH5α在发酵罐中发酵培养,碱裂解法粗提质粒,再依次通过Sepharose 6FF分子筛层析、Plasmidselect 亲硫吸附层析和Source 30Q离子交换层析精制获得质粒纯品,并对纯品进行质量分析。结果每升培养液可获得质粒纯品43.9mg,质量符合Ferreira等推荐的药用标准。     

Thiophilic adsorption: a comparison of model protein behavior

A newly recognized type of protein-ligand interaction phenomenon has resulted in the preparation of simple, nonionic, and highly specific gel derivatives for selective adsorption chromatography. The essential structure of the immobilized ligand can be represented as agarose-CH2CH2SO2CH2CH2SCH2CH2OH, which was prepared by using mercaptoethanol to derivatize [0.9-1.0 mmol (g of dry gel)-1] divinyl sulfone activated agarose (thiophilic or T-gel). Proteins interacting with this ligand are provisionally termed "thiophilic" to recognize their affinity for the definitive sulfone-thioether constituents. To better understand the experimental variables affecting adsorption efficiency and selectivity, several well-characterized proteins with diverse physicochemical features have been evaluated for thiophilic properties. Thiophilic interaction chromatography was investigated as a function of pH as well as the type and concentration of water-structure-forming salts required to promote adsorption. The model proteins characterized varied distinctly in their individual thiophilic affinities. At acidic pH values, a salt-independent adsorption process was observed. Furthermore, a minimum in the salt-promoted thiophilic adsorption tendency at pH 5-6 was found, with varying magnitude, for each of the model proteins evaluated. Recovery of adsorbed proteins routinely varied from 90% to 100%. There does not appear as yet to be any easily recognized physicochemical property associated with either thiophilic or nonthiophilic behavior. These results suggest that thiophilic interaction chromatography is a process that utilizes a previously unrecognized protein-ligand interaction mechanism. We suggest that salt allows the protein into close proximity with the sulfone-thioether group where short-range forces are effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.     

Immunoaffinity Purification of Indole-3-acetamide Using Monoclonal Antibodies

A single-step immunoaffinity purification procedure using monoclonalantibodies was developed to isolate indole-3-acetamide fromplant extracts. Antibodies from a selected clone, raised againstIAA-Cl'-BSA, with pronounced ability to recognize indole-3-acetamide(IAM) were used to prepare an immunoaffinity absorbent. Antibodiespurified by thiophilic interaction chromatography were immobilizedon divinylsulfoneactivated agarose. This column shows a veryhigh selectivity towards IAM compared to IAA. This single stepof immunoaffinity purification gave plant extracts of sufficientpurity for direct quantification by on-line spectrofluorimetryafter an analytical ionsuppression-HPLC run. Successive approximationby a second analytical ion-pairing-HPLC run confirmed the validityof this analytical technique. (Received November 15, 1986; Accepted May 7, 1987)     

Identification and quantitative analysis of alkyl isocyanates as their urea derivatives

A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of short-chain alkyl isocyanates. The procedure is based upon measurement of urea derivatives formed from reaction of the isocyanates with amines. Gas chromatographic and thin-layer chromatographic separations of a number of urea derivatives are described. Application of the method is demonstrated in studies of the decomposition of and urea formation from isocyanates in aqueous solutions.     

Purification of aminophenyl mercuryacetate-activated human matrix metalloproteinase 1 and removal of the organomercurial in a single-step chromatography

Matrix metalloproteinases are secreted from different cells as inactive zymogens. For their activation in vitro organomercurials may be used, the presence of which, however, can falsify activity assays and modulate the effects of the proteases in subsequent investigations. Here, we demonstrate the binding of human matrix metalloproteinase 1 to a thiophilic resin (mercaptoethylquinazolinedione derivatized agarose) and take advantage of this thiophilic interaction for the purification of organomercurial activated matrix metalloproteinase 1 from the supernatant of a thyroid carcinoma cell line in connection with the simultaneous removal of the activator.This revised version was published online in October 2005 with corrections to the Cover Date.     

Purification of aminophenyl mercuryacetate-activated human matrix metalloproteinase 1 and removal of the organomercurial in a single-step chromatography

Matrix metalloproteinases are secreted from different cells as inactive zymogens. For their activation in vitro organomercurials may be used, the presence of which, however, can falsify activity assays and modulate the effects of the proteases in subsequent investigations. Here, we demonstrate the binding of human matrix metalloproteinase 1 to a thiophilic resin (mercaptoethylquinazolinedione derivatized agarose) and take advantage of this thiophilic interaction for the purification of organomercurial activated matrix metalloproteinase 1 from the supernatant of a thyroid carcinoma cell line in connection with the simultaneous removal of the activator.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Reexamination of spectroscopic properties of ceruloplasmin freshly isolated with a novel very rapid single-step procedure

A novel, single-step, chromatographic procedure for the purification of ceruloplasmin is described. The procedure consists of a single chromatographic step, leading in approximately 5 hours from 2 liters of plasma to an electrophoretically homogeneous ceruloplasmin preparation which exhibits different spectroscopic properties with respect to the protein obtained with more lengthy, currently available procedures. The method is suitable for either small or large scale purification from various vertebrate plasma.     

Purification of human B cell growth factor

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.     

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.     

Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.     

Measurement of plasma catecholamines by high-performance liquid chromatography with electrochemical detection in intensive care patients after dobutamine infusion

A procedure for the determination of plasma catecholamine concentrations in critical care patients after dobutamine infusion is presented. A modified chromatographic system is required with an additional washing procedure to achieve maximum sensitivity and stable chromatographic conditions. The influence of storage time on the catecholamine concentrations of plasma samples is reported in detail. A time-dependent decrease in catecholamine concentrations of up to 12 and 39% was found within two and ten months, respectively.     

An easy cAMP extraction method facilitating adenylyl cyclase assays.

We present a facile procedure for measuring adenylyl cyclase activity which circumvents the two-step chromatographic purification of 32P-labeled cAMP. cAMP produced by stimulated cell membrane preparations is easily purified by organic extraction and thus available for quantification using tritium-labeled tracer cAMP and commercially available cAMP-binding protein. The quantification of cAMP is unaffected by the extraction procedure and sample handling. Data obtained by this method were identical to those obtained by the chromatographic method. This procedure could be shown to be suitable for measuring receptor-mediated adenylyl cyclase modulation.     

Ways of collagen separation in pathologically altered tissue

A system of chromatographic methods using two successive DEAE-cellulose chromatographic steps and two successive separations on Bio-Gel A-1.5 m has been worked out for the separation of individual collagen types. The success of the procedure is based on the preliminary removal of proteoglycans during the first DEAE-cellulose run. Alternatively it is possible to replace chromatographic steps, following the removal of proteoglycans, with fraction precipitation.     

Determination of chlorhexidine in saliva using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of chlorhexidine in saliva is reported. The method developed includes a simple and short sample preparation with a one-step extraction procedure and a short total chromatographic run time of 5 min. In a preliminary pharmacokinetic study with a healthy volunteer the chlorhexidine concentration found in saliva after 12 h was 0.8 μg/ml.     

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.