High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain

Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.     

Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.     

Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma

To replace conventional affinity ligand like protein A or protein G, a pseudobioaffinity ligand seems to be an alternative for the purification of immunoglobulin G (IgG). In this study, sulfamethazine (SMZ) was chosen as novel affinity ligand for investigating its affinity to human IgG. Monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) (PGMA) beads were employed as the support for high-performance affinity chromatography. SMZ was immobilized on PGMA beads using bisoxirane (ethanediol diglycigyl ether) as spacer. The resultant affinity media presented minimal non-specific interaction with other proteins. Results of high-performance frontal analysis indicated that the media showed specific affinity to human IgG with a dissociation constant on the order of 10(-6) M. The SMZ affinity column proved useful for a very convenient one-step purification of IgG from human plasma. Antibody purity after a one-step purification was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction under reducing condition. The results obtained indicate that SMZ is a valuable affinity ligand for purification of human IgG.     

Protein A immobilized affinity cartridge for immunoglobulin purification

Recombinant Protein A was immobilized on a cellulose and acrylic composite matrix through Schiff base formation. Various factors that could affect the binding of immunoglobulin by the Protein A molecules immobilized on the solid matrix were studied to achieve optimum affinity purification. The spacer arm length and ligand concentration of Protein A were verified as factors crucial to optimized IgG purification. Liquid-phase environmental conditions such as pH and salt concentration also play important roles in adsorption capacity by affecting the molecular interaction between IgG and the immobilized Protein A. The rate of interaction between Protein A and IgG is rather fast, with minimal differences observed at 10-fold increases in the cartridge loading rate. This paper describes a cellulose/acrylic composite matrix for immobilizing Protein A, at an optimized ligand concentration, installed on a spacer arm of adequate length, to purify immunoglobulins from animal plasma. The fast-flow property of the cartridge made from such a matrix and its simplicity in operation provide effective means for purifying immunoglobulins on a relatively large scale.     

Production of polygalacturonase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.     

The chemistry of human transcortin: Improved affinity matrices for the purification of transcortin

While spacer arms have been shown to play an important role in affinity chromatography, no systematic investigations of spacer arms in the purification of transcortin have been reported. Among the five cortisol-agaroses, cortisol-21-succinyl-1,6-hexanediamidoagarose achieved the highest extraction efficiency of transcortin from plasma. The optimal length of the spacer arm for extraction is ca. 12–13 Å. Cortisol-succinyl-agaroses having hydrophobic spacer arms extract transcortin better then those having hydrophilic arms of approximately equal length. Affinity supports are usually synthesized sequentially; cortisol-agaroses thus prepared were found to complicate the purification of transcortin. The problems of nonspecificity and instability associated with these agaroses were eliminated by using reverse addition. A complete ligand-spacer arm, synthesized in a single step by displacing the tosyl group from cortisol-21-tosylate with 1,6-hexanediamine, was coupled with cyanogen bromide-activated agarose. Although the 21-deoxy-21-(ω-amidohexyl) aminocortisol-agarose ranked second in extraction efficiency, its superior stability and low nonspecific adsorption of other proteins make it the prime choice for affinity chromatography of transcortin.     

Screening of suitable immobilized metal chelates for adsorption of monoclonal antibodies against mutant amidase from Pseudomonas aeruginosa

The chromatographic behaviour of monoclonal antibodies (MAbs) of IgM class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated. The effect of ligand concentration, the length of spacer arm and the nature of metal ion were investigated on immobilized metal ion affinity chromatography (IMAC). MAbs against mutant amidase adsorbed to Cu (II), Ni (II), Zn (II), Co (II) and Ca (II)-IDA agarose columns. The adsorption of MAbs onto immobilized metal chelates was pH dependent because an increase in the binding of MAbs was observed as the pH was raised from 6.0 to 8.0. The adsorption of MAbs to metal chelates was due to coordination of histidine residues which are available in the 3rd constant domain of heavy chain (CH3) of immunoglobulins since the presence of imidazole in the equilibration buffer abolished the adsorption of MAbs to the column packed with commercial IDA-Zn(II) agarose at pH 8.0. The combination of tailor-made stationary phases for IMAC and a correct choice of the adsorption conditions permitted to design a one-step purification procedure for MAbs of IgM class. Culture supernatants containing MAbs of IgM class against mutant amidase (T103I) were chromatographed by IMAC Co (II) column at pH 8.0. The results strongly suggest that one-step purification of MAbs of IgM class by IMAC is a cost-effective and process-compatible alternative to the other purification procedures.     

A蛋白亲和层析法纯化动物血清抗体的效果比较

用ＨｉｔｒａｐＰｒｏｔｅｉｎ－ＡＳｅｐｈａｒｏｓｅ亲和层析系统,对不同动物血清或抗血清抗体的纯化效率进行了比较,为选择抗体纯化方法提供依据。该方法简便快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。层析柱反复使用４０多次,纯化抗体效率不变。但Ｐｒｏｔｅｉｎ－Ａ对不同动物血清的ＩｇＧ吸附能力不同：对兔和豚鼠的血清抗体吸附能力强     

Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study

Polyvinyldiene fluoride (PVDF)-based affinity membranes with immobilized copper ions were developed in this study. The resulting membranes were tested for their adsorption properties using a model protein, lysozyme, in batch mode. First, different lengths of diamine were utilized as spacer arms to immobilize the metal ions onto the membranes. It was found that the application of 1,8-diaminooctane as the spacer arm led to the highest adsorption capacity. Moreover, the effects of pH and salt concentration were investigated to distinguish the proportion of specific and nonspecific interactions. A big fraction of lysozyme adsorption capacity for the immobilized metal affinity membranes was considered to come from nonspecific electrostatic interactions, which could be reduced by increasing salt concentration. Lastly, the purification of hepatocyte growth factor (HGF) from insect cell supernatant was performed using the immobilized metal affinity membranes in batch mode. HGF was found in the elution condition using EDTA, indicating the successful purification of HGF.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Examination of protein adsorption in fluidized bed and packed bed columns at different temperatures using frontal chromatographic methods

The influences of various experimental parameters on the dynamic adsorption capacity (DAC) and the dynamic adsorption rate (DAR) of a biomimetic affinity silica-based adsorbent in fluidized and packed bed columns operated under plug flow conditions and at different temperatures have been investigated with different inlet concentrations of hen egg white lysozyme (HEWL) and human serum albumin (HSA). The DACs as well as the DARs of both the fluidized and packed beds were examined at 10% saturation (i.e., at the QB value) and the experimental data compared with the corresponding data obtained from batch equilibrium adsorption procedures. Parameters examined included the fluid superficial velocity and protein concentration and their effect on the binding capacity and column efficiency. Consistent with various results reported from this and other laboratories on the behavior of biospecific affinity adsorbents derived from porous silica and zirconia particles, adsorbents prepared from Fractosil 1000 were found to exhibit appropriate rheological characteristics in fluidized bed systems under the experimental conditions. Moreover, changes in temperature resulted in a more significant effect on the breakthrough profiles of HSA compared to HEWL with the immobilized Cibacron Blue F3G-A with Fractosil 1000 adsorbent. This result suggests that temperature effects can possibly be employed profitably in some processes as part of a strategy to enhance column performance with fluidized bed systems for selective recovery of target proteins. At relatively low superficial velocities of the feed, the DARs with HEWL and HSA were similar for both the fluidized and packed bed column systems, whereas, at high superficial velocities, the DARs for these proteins were larger with the packed bed columns.     

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC–MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called “sponge effect” was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC–MALDI-TOF/TOF-MS analysis was less than 17.5%.     

Optimal spacer arm microenvironment for the immobilization of recombinant Protein A on heterofunctional amino-epoxy agarose supports

In this study, recombinant Staphylococcus Protein A (rSPA) was immobilized on three different amino-epoxy agaroses: traditional amino-epoxy, butanediol diglycidyl-amino and glycidyl-amino agarose (coded as AE, BDA and GA agarose, respectively), for obtaining affinity adsorbents to bind human immunoglobulin G (hIgG). The effects of the spacer arm microenvironment of the support on the rSPA immobilization were investigated. Compared with the AE agarose, the GA agarose presents ionized amino groups far from the support. Therefore, the rSPA immobilization efficiency of 92 % is slightly higher than that of 88 % on AE agarose due to the weak steric hindrance. Moreover, the BDA agarose exhibited the lowest immobilization efficiency of 58 %, attributing to the existence of hydrophobic butylidene groups on the BDA agarose. Ethanolamine was used as the blocking agent to obtain three affinity adsorbents. The hIgG-binding capacity from the human plasma was determined to be 18.7, 34.7 and 38.7 mg/mL for rSPA-BDA, rSPA-AE and rSPA-GA, respectively. Furthermore, the maximum hIgG-binding capacity was calculated by the Langmuir model of adsorption isotherm to be 25.1, 44.8 and 52.2 mg/mL for rSPA-BDA, rSPA-AE and rSPA-GA, respectively. Therefore, the GA agarose bears the optimal spacer arm microenvironment for preparing the rSPA adsorbent with high hIgG-binding capacity.     

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma

Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.     

Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel

Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 μm size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles.     

Thermodynamic Stability and Functional Activity of Tumor-Associated Antibodies

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2–CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.     

Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci.

A novel approach for production of small polypeptides, using a staphylococcal protein A vector, is described. This system is used to express, secrete and purify human insulin-like growth factor I (IGF-I). A fusion protein consisting of protein A and IGF-I is recovered in high yield by passing the culture medium through an IgG affinity column. Using site-specific mutagenesis an acid labile asp-pro cleavage site was introduced at the fusion point between the two proteins. The protein A "tail" can thereby be removed from the affinity purified fusion protein by chemical cleavage releasing biologically active IGF-I molecules.     

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.