Everson Alves Miranda

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.

Aqueous Extraction of Recombinant Human Proinsulin from Transgenic Maize Endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.

Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography

Protein expression in transgenic plants is considered one of the most promising approaches for producing pharmaceutical proteins. As has happened with other recombinant protein production schemes, the downstream processing (dsp) of these proteins produced in plants is key to the technical and economic success of large-scale applications. Since dsp of proteins produced transgenically in plants has not been extensively studied, it is necessary to broaden the investigation in this field in order to more precisely evaluate the commercial feasibility of this route of expression. In this work, we studied the substitution of an IMAC chromatographic step, described in previous work (Azzoni et al., 2002), with ion-exchange chromatography on SP Sepharose Fast Flow resin as the second step in the purification of recombinant aprotinin from transgenic maize seed. The main goal of this second purification step is to separate the recombinant aprotinin from the native corn trypsin inhibitor. Analysis of the adsorption isotherms determined at 25°C under different conditions allowed selection of 0.020 M Tris pH 8.5 as the adsorption buffer. The cation-exchange chromatographic process produced a high-purity aprotinin that was more than ten times more concentrated than that generated using an IMAC step.

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 immunoglobulin G onto ethacrylate and histidine-linked methacrylate

The adsorption of human IgG onto GMA (a semirigid methacrylate-based chromatography matrix) and His-GMA adsorbents was studied by chromatography and batch equilibrium binding analysis. IgG molecules adsorbed onto GMA gel by nonspecific hydrophobic interactions and the specificities were similar for both adsorbents. Adsorption data were analyzed using three isotherm models, namely the Langmuir, Freundlich and Langmuir-Freundlich models, and the adsorption parameters were computed. The experimental isotherms were best described by a combined Langmuir-Freundlich model, which indicated the presence of unequal binding sites on both adsorbents and/or positive cooperativity in the binding of the IgG molecules.

Evaluation of operational parameters on the precipitation of endoglucanase and xylanase produced by solid state fermentation of Aspergillus niger

In order to develop cost effective processes for converting biomass into biofuels, it is essential to improve enzyme production yields, stability and specific activity. In this context, the aim of this work was to evaluate the concentration of two enzymes involved in the hydrolysis of biomass, endoglucanase and xylanase, through precipitation. Statistical experimental design was used to evaluate the influence of precipitant agent concentration (ammonium sulfate and ethanol), aging time, and temperature on enzyme activity recovery. Precipitant agent concentration and aging time showed a statistically significant effect at the 95% confidence level, on both enzyme activity recoveries. The recovery of endoglucanase with ammonium sulfate and ethanol reached values up to 65 and 61%, respectively. For xylanase, the recovery rates were lower, 27 and 25% with ammonium sulfate and ethanol, respectively. The results obtained allowed the selection of the variables relevant to improving enzyme activity recovery within operational conditions suitable for industrial applications.

Kinetics of Gas-Phase Hydrolysis of Ethyl Acetate Catalyzed by Immobilized Lipase

Reactions catalyzed by supported enzymes present important advantages when compared with those in aqueous media or organic solvents: separation of enzymes from substrate is easily accomplished, enzyme stability may be improved, and control of the reaction products is more accurate. We present the experimental results of the kinetic study of ethyl acetate hydrolysis in gaseous phase catalyzed by a commercial immobilized lipase (Lipozyme IM; Novo Nordisk). The hydrolysis reaction was studied as a function of ethyl ester and water partial pressure at a constant temperature of 318 K. The amount of biocatalyst used was varied between 100 and 300 mg, and the reaction was studied in a flow-through glass microreactor. Under the conditions used, water was an important parameter in the gas-phase reaction. Activation energy was 24.8 kJ/mol and the overall order of reaction was one. Finally a Bi-Bi reaction mechanism is proposed.

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Abstract Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.

Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant β-Glucuronidase

Soybean is one of the plant species with potential to be used as seed-based bioreactor. As part of the downstream processing (DSP) of this technology, extraction is a key step, since it defines the composition of the solution from which the recombinant product will be purified. In the present work, the characteristics of soybean seeds used as a bioreactor were evaluated from a process engineering standpoint through analysis of the influence of pH and ionic strength on the extraction of recombinant β-glucuronidase (rGUS). Concentrations of recombinant protein and native soybean compounds were analyzed and compared with similar data from extraction studies using transgenic corn seeds as bioreactor. Efficient rGUS extraction was obtained at pH of around 5.5 with no addition of salt. Soybean seed extracts had lower levels of co-extracted native compounds, than corn seed extracts, and should be considered as a potential plant bioreactor in terms of DSP.

Precipitation of porcine insulin with carbon dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO2) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO2 as an acid‐precipitating agent. The solubility of porcine insulin in NaHCO3 solutions in pressurized CO2 was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self‐association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO2, although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909–919.