Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93 mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89 % was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2 mg mL(-1), near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.1673632644Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Adsorption of glucagon and insulin on an immobilized metal ion affinity chromatography silica matrix

Glucagon is a hormone that increases blood glucose concentrations and is used as a pharmaceutical product mainly in the treatment of hypoglycaemia associated with diabetes. Given both its importance and high current cost, improved purification processes are in demand. By using immobilized metal ion affinity chromatography (IMAC), we conducted tests aimed at the purification of glucagon from complex mixtures. Adsorption studies of glucagon, insulin and a mixture of the two on adsorbents of silica-IDA-Me2+ (Cu2+, Ni2+ and Zn2+) were carried out. Fixed-bed chromatographic experiments were performed and the adsorption affinity verified for all three metals tested. The most promising condition for glucagon and insulin separation was achieved by using Ni2+ as the metal ligand and desorption with a pH step gradient. Two industrial insulin-processing fractions (one glucagon-rich and the other insulin-rich) were evaluated qualitatively under these conditions, resulting in an increase in glucagon purity for both fractions with a purification factor of five for the latter.O TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE FEVEREIRO DE 2015.211088389

In vitro evaluation of biospecific and pseudobiospecific ligands aimed at extracorporeal treatment for immunoglobulin E removal

This work investigated the potential use of an alternative adsorbent to anti-immunoglobulin E (IgE)-agarose for IgE selective adsorption therapy. A screening of several commercially available adsorbents (Concanavalin A, Lens culinaris [Lc], D-tryptophan, poly-L-lysine, and aminohexyl immobilized on agarose) was done through batch system assays, considering some criteria, such as adsorption capacity, selectivity, and biocompatibility. In the Lc-agarose adsorbent, total IgE, and specific IgE-for the airborne allergens Dermatophagoides pteronyssinus and Blomia tropicalis-were significantly better removed (63, 58, and 59%, respectively) than immunoglobulin G (19%), immunoglobulin A (33%), immunoglobulin M (9%), and albumin (18%). This adsorbent was packed into a column and the effect of superficial velocity, ratio of plasma volume to bed volume, number of perfusions, and temperature on IgE adsorption were evaluated. In vitro simulation of therapeutic adsorption (single perfusion) indicated that about 50% of total IgE could be eliminated.30860661

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-HCI 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. (C) 2002 Elsevier Science Ltd. All rights reserved.37657357

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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. (C) 2010 Elsevier B.V. All rights reserved.8782320872093Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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 (C) 2009 Elsevier B.V. All rights reserved.87841795557566Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Interaction of histidine-tagged human proinsulin with immobilized nickel ion: Effect of chelating ligand and thermodynamics analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The nature of the chelating ligand is one of various factors affecting the performance of IMAC. In this work we studied the effect of three chelating ligands on the adsorption of recombinant human proinsulin with His-tag (proinsulin-(His)(6)). The chelating ligands (complexed with Ni(II)) were two tetradentates (carboxymethylated aspartic acid, CM-Asp, and tris(2-aminoethyl)amine, TREN) and one pentadentate (tris (carboxymethyl) ethylenediamine, TED). Their performance were compared with the performance of one tridentate ligand (iminodiacetic acid, IDA) and the commercial adsorbent HisTrap both also complexed with Ni(11). Higher selectivities were achieved with Ni(II)-CM-Asp and Ni(II)-TED. The adsorption capacity decreased according to the order: TREN, HisTrap, IDA, CM-Asp, and TED (172.82, 167.98, 133.09, 110.18, and 69.22 mg of proinsulin/mL of gel, respectively, at 25 degrees C). Although TREN-agarose had the highest capacity, the ligand with the second highest capacity - HisTrap - showed better performance since proinsulin was eluted in a single, concentrated peak with imidazole, while for Ni(II)-TREN proinsulin was eluted with imidazole and EDTA, requiring an extra step to free the product from the EDTA. Therefore, the choice of an adsorbent for this separation depends on the priority: capacity (TREN or HisTrap for one step process) or selectivity (CM-Asp). Adsorption isotherms were determined for temperatures from 4 to 45 degrees C and the Langmuir model was fitted to the experimental data. The thermodynamics analysis showed positive Delta H(o) (from 18.83 to 86.05 kJ/mol), indicating that the adsorption of proinsuin-(His)(6) in all chelating ligands is an endothermic process. The negative change in Gibbs free energy (from -26.30 to -35.11 kJ/mol) indicated that the adsorption of the proinsulin-(His)(6) on all the adsorbents studied was a thermodynamically favorable process. (C) 2010 Elsevier B.V. All rights reserved.36941699176185Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Protein L-agarose for adsorption of autoantibodies: A potential tool for extracorporeal treatment

This work investigated the adsorption of autoantibodies such as anti-SS-A/Ro, anti-SS-B/La, anti-Sm, and anti-dsDNA on protein L-agarose gel. In order to determine better conditions for IgG adsorption on this matrix, some buffer systems were tested. Adsorption data were analyzed using the Langmuir and Langmuir-Freundlich isotherm models. The experimental isotherms were best described by the Langmuir-Freundlich model, which indicated negative and positive cooperativities for binding in the presence of PBS and HEPES buffers, respectively. The K-d values for phosphate buffered saline solution (PBS) and hydroxyethylpiperazine ethanesulfonic acid (HEPES) were 2.8 x 10(-7) M and 3.2 x 10(-7) M, respectively, which indicate a high affinity between IgG and the immobilized protein L. The amount of protein adsorbed per amount of protein loaded was high for anti-Sm (44%) and anti-dsDNA (46%), but low for anti-SS-B/La (9%). The amount of albumin adsorbed was lower than 0.06 mg/mL, which may remove the need for a plasma replacement solution in clinical apheresis.29431332

Depyrogenation of snake antivenom serum solutions by hollow fiber-based pseudobioaffinity filtration

The affinity filtration technique using histidine as the pseudobiospecific ligand immobilized on poly(ethylene vinyl alcohol) hollow fiber membranes (Histidine-PEVA) was used to remove endotoxin (ET) from snake antivenom serum solutions. Immobilized histidine bound to horse antibodies (F(ab')(2) fragments) and ET. The effects of solution conditions on the efficiency of ET removal and protein recovery were studied. The lowest antibody adsorption capacity was obtained with acetate pH 6.0 and phosphate pH 6.5 buffers. Under both conditions, ET was removed but the highest efficiency was found for the acetate buffer. This buffer was used to determine the optimal combination of ET concentration and Q(F)/Q(i) (filtrate flow rate/inlet flow rate) ratio for achieving a high rate of ET removal without a significant loss of antibodies. Removal of 93% of the ET and recuperation of 95% of the antibodies were obtained at a Q(F)/Q(i) ratio equal to 0.68 and an initial ET concentration of 675 EU/ml, A high clearance rate of 99% was obtained with initial concentrations of 65 and 351 EU/ml for lachesis-bothrops and bothrops antivenom sera, respectively, and an almost complete depyrogenation of bothrops antivenom serum was achieved, since the final ET content in solution was 0.8 EU/mL. (C) 2000 Elsevier Science B.V. All rights reserved.173223524