Sonia Maria Alves Bueno

Separation of immunoglobulin G from human serum by pseudobioaffinity chromatography using immobilized l-histidine in hollow fibre membranes

L-Histidine, intended as a pseudobiospecific ligand, was immobilized on poly(ethylenevinyl alcohol) hollow fibre membranes after their activation with epichlorohydrin or butanediol diglycidyl ether. The affinity membranes obtained allowed the one-step separation of immunoglobulin G (IgG) from untreated human serum. Elution was possible under mild conditions with discontinuous pH or salt gradients. IgM was also adsorbed to a certain extent and partially separated from IgG by pH gradient elution. The bound IgG fractions showed pI values between 8 and 9.5 and contained IgG1 and IgG3. The dissociation constants for IgG on the bisoxirane- and epichlorohydrin-activated membranes coupled with histidine were determined by equilibrium binding analysis to be 2.5 x 10(-5) and 2.0 x 10(-5) M, respectively. The maximum binding capacity of the affinity hollow fibre membranes was 80 and 70 mg of IgG per gram of support, respectively. With a cartridge of surface area 1 m2 (about 19 g of fibres), during a 60-min run, theoretically up to 1.5 g of IgG can be removed from human serum. The histidine affinity membranes are very stable owing to the simple nature of the ligand and the coupling via an ether linkage. Reproducible results were obtained over more than 1 year even with untreated human serum being used regularly.

IMAC of human IgG: studies with IDA-immobilized copper, nickel, zinc, and cobalt ions and different buffer systems

Human IgG is an important plasma protein produced worldwide on a large scale. Affinity chromatographic processes are not commercially used for the production of IgG since the ligands tried so far hinder their application to pharmaceutical products. Immobilized ion affinity chromatography (IMAC) has the potential to circumvent these problems. The adsorption of human IgG onto IDA-Sepharose immobilized Cu2+, Ni2+, Zn2+, and Co2+ with MOPS, phosphate, MMA, and Tris–HCl adsorption buffering systems is reported. Adsorption of high purity IgG was high for all metals irrespective of the buffer system used. Elution of IgG was similar for all buffer systems except for the case of pH elution when copper was the ligand. Isoeletrocfocusing showed the presence of molecules of low pI in the flowthrough of the chromatographic runs with Ni2+-phosphate-acetate, Ni2+-MOPS-imidazole and Zn2+-MOPS-imidazole systems. Chromatography runs with human plasma indicated the potential of this technique for IgG purification.

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes

L-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their Fab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in KD with increasing temperature and a positive entropy value between 20 and 35 degrees C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.

Experimental kinetic aspects of hollow fiber membrane-based pseudobioaffinity filtration: process for IgG separation from human plasma

The pseudobiospecific affinity ligand l-histidine was immobilized through an ether linkage onto poly(ethylene vinyl alcohol) hollow fiber cartridge to obtain an affinity support for IgG purification or IgG removal from human plasma with a view to clinical apheresis for the treatment of immune disorders, alloimmunization and cancer. The kinetics and mass transfer aspects were studied in order to determine the optimum loading flow rate for IgG adsorption. Experiments were run at various filtrate flow rates (various residence times) in cross flow mode with recirculation of both the retentate and the permeate in the reservoir. The results showed that this affinity membrane had the potential of fractionating plasma proteins at fast flow rates (residence times below 10 s). However, protein trapping into the membrane was enhanced by residence times below 5 s. Comparison of equilibrium and dynamic maximum capacities determined using Langmuir isotherms showed that dynamic capacity (190 μg/cm2) was somewhat higher than the equilibrium capacity (148 μg/cm2). The dissociation constants for IgG were determined using the Langmuir isotherm equation to be 3.7 × 10−6 M (dynamic mode) and 9.8 × 10−6 M (equilibrium binding analysis), indicating medium affinity, which was typical for pseudobiospecific affinity ligands. The cartridge was loaded with human serum or human plasma to test its ability to fractionate these protein solutions. The affinity membrane showed high selectivity for IgG from two fold diluted human plasma or serum. IgM was also adsorbed to a certain extent. When the serum or plasma was not diluted with equilibration buffer before loading the cartridge, the amount of adsorbed proteins was significantly (50%) smaller than with the 2 or 10 fold dilutions, but the specificity remained.

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidines ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG1 (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS of Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m2 histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 l of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.

A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto ω-aminodecyl-agarose

The adsorbent omega-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n=2.30+/-0.38) and negative cooperativity (n=0.63+/-0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25 mmol L(-1) pH 6.8 feeding human serum. These results indicate that the use of omega-aminodecyl-agarose is a potential technique for purification of IgG from human serum.

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

The omega-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69-76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir-Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL(-1) of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing omega-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

Immobilized Metal Ion Affinity Cromatography - IMAC - is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.

Adsorption of human serum proteins onto TREN-agarose: Purification of human IgG by negative chromatography

Tris(2-aminoethyl)amine (TREN) - a chelating agent used in IMAC - immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90-95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.

In vitro removal of human IgG autoantibodies by affinity filtration using immobilized L-histidine onto PEVA hollow fiber membranes.

Histidine was immobilized onto PEVA membrane to obtain an affinity support for human IgG removal from serum with a view to clinical apheresis for the treatment of autoimmune diseases. These membranes were able to remove in vitro several autoantibodies from the serum of SLE patients.