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

Negative chromatography on agarose-TREN as a technique for purification of protein spiked in soybean seeds extract

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Alkyl amines and polyamines have been used as ligands for protein purification by mixed-mode chromatography. The adsorption of proteins onto these ligands seems to be governed by multiple effects such as electrostatic, hydrophobic, and affinity interactions. In this work we investigated the adsorption of proteins extracted from soybean onto the adsorbent agarose-Tris(2-aminoethyl)amine (TREN). The effects of flow rate, buffer system, and extract concentration on the capture of proteins extracted from soybean were evaluated. Experiments using Mes at pH 6.5 as adsorption buffer allowed the adsorption of almost the totality of native soybean protein with a dynamic adsorption capacity of 13.50 mg mL(-1) adsorbent. Experiments with human IgG (pl in the range of 5.8-9.0) and human serum albumin (HSA, pl of 4.9) spiked into these extracts lead to the conclusion that electrostatic forces play a major role in the interaction between protein and agarose-TREN. Based on this work, negative chromatography with agarose-TREN should be considered as a method for purification of basic recombinant protein produced in transgenic soybean seeds. (C) 2012 Elsevier Ltd. All rights reserved.471222552261Coordenação 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

IgG purification by negative chromatography in amine-based ligands: A comparison of L-lysine and poly-L-lysine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Agarose gels with immobilized amine-based ligands L-lysine (Lys) and poly-L-lysine (PLL) exhibited the ability to adsorb human serum proteins and to separate immunoglobulin G (IgG) in nonretained chromatographic fractions (negative chromatography). The effect of the buffer system and pH on IgG purification on Lys-agarose and PLL-agarose showed that PLL was the most selective ligand. PLL-agarose was able to purify IgG from human serum solution diluted in Bis-Tris buffer at pH 6.0 with 79% yield and 88.7% purity (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). Human serum albumin (HSA) and IgG adsorption data showed that the first followed the Langmuir model whereas the second followed the Langmuir-Freundlich model, due to the presence of a positive cooperativity effect (n = 1.60), with the maximum adsorption capacities of 76.4 mg mL(-1) for HSA and 23.6 mg mL(-1) for IgG. The Delta G(max) values obtained by non-linear regression of HSA and IgG adsorption data of Temkin model were similar (-7.0 kcal mol(-1)), indicating that the process is spontaneous in nature. The joint analysis of these data shows that PLL-agarose can be considered an alternative adsorbent for the isolation of IgG from human serum with the potential to be integrated to a purification process on a large scale. (C) 2011 Elsevier Ltd. All rights reserved.461222772285Fundação 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

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

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow-fiber membranes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Iminodiacetic acid (IDA) and tris(2-aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow-fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA-Cu(II) or TREN-Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini-cartridge in a cross-flow filtration mode (52.5 and 298.4mgg(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3mgg(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively). When mini-cartridges were used, the dynamic adsorption capacity of IDA-Cu(II) was the same for both mini-cartridge and agarose gel. Copyright (c) 2013 John Wiley & Sons, Ltd.2610514520Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação 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

Evaluation of Immobilized Metal-Ion Affinity Chromatography (IMAC) as a Technique for IgG(1) Monoclonal Antibodies Purification: The Effect of Chelating Ligand and Support

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand-CM-Asp-was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.160721482165Fundação 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

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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. (C) 2008 Elsevier B.V. All rights reserved.877416711723Conselho 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)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP