Igor Tadeu Lazzarotto Bresolin

Isolation and purification of bromelain from waste peel of pineapple for therapeutic application

The aim of this work was to isolate and purify bromelain extracted from the pineapple peel by ammonium sulfate precipitation (40-80%), followed by desalting and freeze-drying with a 75% activity recovery and 2.2 fold increased specific activity. Ion exchange chromatography on DEAE-Sepharose was able to separate the polysaccharides from the enzyme, which was recovered in the elution step, maintaining its enzymatic activity. The batch adsorption of bromelain was evaluated in terms of total protein and enzymatic activity using Langmuir and Langmuir-Freundlich models. Results showed that the process could be suitable for the recovery and purification of the enzyme, maintaining its specific activity.

Dye Ligand Epoxide Chitosan/Alginate: A Potential New Stationary Phase for Human IgG Purification

Cibacron Blue F3GA immobilized as ligand onto epoxide chitosan/alginate composite was prepared and evaluated for the purification of human immunoglobulin G (IgG) isolated from human serum by dye-ligand affinity chromatography. The effect of buffer pH on IgG adsorption, kinetic and isothern adsorption of high-purity IgG were studied by batch adsorption. The adsorption isotherm data were well fitted (using 25 mmol/L sodium phosphate buffer at pH 6.0) by the Langmuir–Freundlich model with a maximum adsorption capacity of 99 mg/g, apparent dissociation constant of 1.2 × 10−6 mol/L and n equal to 4.86. Fixed-bed experiments were performed using high-purity IgG, achieving a recovery rate of 53%. Human serum diluted ten times was also injected into the column. An enrichment of IgG in the elution fraction with traces of human serum albumin was observed. According to the study results, this material can be applied in the purification of human IgG under optimized conditions.

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

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 298.4 mg g−1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g−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

Phosphorylated-tyrosine based pseudobioaffinity adsorbent for the purification of immunoglobulin G

The present study evaluated the phosphorylated-tyrosine (P-Tyr) based pseudobioaffinity adsorbent for the purification of human immunoglobulin G (IgG). P-Tyr was selected as a ligand to mimic the natural interactions that occur between the immunoreceptor tyrosine-based activation motif and the IgG. The ligand was coupled to bisoxirane-activated agarose gel and the effect of buffer system, pH, and conductivity was performed to elucidate the nature of IgG-P-Tyr interactions. P-Tyr-agarose was able to purify IgG from human plasma solution in HEPES buffer at pH 7.0 exhibiting a purification factor of 9.1 with IgG purity of 91% (based on ELISA analysis of albumin, transferrin, and immunoglobulins A, G, and M). The evaluation of different functional groups of P-Tyr on the adsorption of human IgG indicated the predominance of electrostatic interactions with phosphate groups, although the contributions of aromatic and carboxylic groups also play a role. The thermodynamic parameters (ΔH°, ΔS°, ΔG°) for IgG adsorption onto P-Tyr-agarose were determined from the temperature dependence. The maximum IgG binding capacity at 20°C was 273.51±12.63mgg-1 and the dissociation constant value of the complex IgG-P-Tyr was in the order of 10-5molL-1 indicating low-affinity.

Preface: 10th Brazilian meeting on adsorption

In 2014, Brazil held the FIFA World Cup, which was the most evidenced event in the worldwide community. However, we were also proud to host some other meetings that is boosting Brazil into a world class scientific production. Two months earlier, the Brazilian adsorption community promoted the 10th Brazilian Meeting on Adsorption (EBA10), the most expressive national conference in the field. This edition was chaired by Federal University of Sao Paulo (UNIFESP) Diadema, held from April 27th to 30th, in Guaruja, Sao Paulo. Reaching its tenth edition, EBA has experienced a growing interest by Brazilian and international scientific and business community. To understand this growing, we need to know a little bit of history: the first EBA happened in Fortaleza, Ceara on June 1996, organized by an initiative of a group of professors at the Federal University of Ceara, led by Celio Loureiro Cavalcante Jr, PhD. This first meeting showed that the initiative was timely, not only because it relied on the presence of Prof. Douglas Morris Ruthven, PhD, but also by the number of participants (about 100) and quality of the 65 papers presented. After six editions more (Table 1), EBA’s growth came along breaking the country’s frontiers. For this reason, the city of Campina Grande/PB received the EBA7 on June 2008, in which the 1st South American Symposium on Adsorption, Science and Technology and the 1st South American School on Adsorption were held in parallel. The 9th edition, in Recife/PE, held the 1st Ibero-American Symposium on Adsorption (IBA1), which was a longstanding demand of many researchers on Adsorption area who met sporadically at conferences like the Fundamentals of Adsorption (FOA). The simultaneous occurrence of events led to a massive participation of researchers from the Ibero-Latin American region. In this meeting, it was decided that the IBAs will be held every three years. Prof. Juan Carlos Moreno-Pirajan, PhD, from Universidad de los Andes (Colombia) is the chairman of IBA2, which will be held from 26th to 30th April, 2015, in Cartagena de Indias—a World Cultural Heritage by UNESCO. This growth trend that EBA experienced was the driving force that motivated a group of young professors from the Chemical Engineering undergraduate program at UNIFESP to accept the invitation to organize the EBA10. The Enseada Beach in Guaruja, a beautiful coastal city 90 km far from Sao Paulo was chosen as the place to host this milestone tenth edition. The development of human resources and the interest of undergraduate and graduate students were highlighted by carrying out the School of Adsorption during EBA10. As its name implies, in the school lessons on various areas of adsorption are taught by professors from educational and research institutions and companies. International invited speakers were responsible for five plenary lectures. Nine keynotes also took place held by international and Brazilian researchers, from academia and business community in order to foster research partnerships. Distributed in eight oral sessions and two poster sessions, 228 papers were presented. The conference was attended by 334 participants from Brazil, South America and Europe. We thank our invited speakers (Gino Baron, Alois Jungbauer, Alirio Rodrigues, Abdelhamid Sayari, Juan Carlos I. T. L. Bresolin (&) Department of Exact and Earth Sciences, Federal University of Sao Paulo (UNIFESP), Diadema, SP 09972-270, Brazil e-mail: igor.bresolin@gmail.com

Phospho-l-tyrosine-agarose chromatography: Adsorption of human IgG and its proteolytic fragments

The behavior of human immunoglobulin G (IgG) and antigen-binding fragment (Fab fragment) adsorption onto phospho-l-tyrosine immobilized on agarose (P-Tyr-agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis-Tris, Tris-HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L-1 NaP buffer at pH 6.0. The capture of IgG by the P-Tyr-agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L-1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo-second-order model. The adsorption isotherm data were well described by the Langmuir-Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P-Tyr-agarose from digested human IgG in HEPES 25 mmol L-1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.

Purification of Anti-Interleukin-6 Monoclonal Antibody Using Precipitation and Immobilized Metal-Ion Affinity Chromatography

Monoclonal antibodies (MAbs) have been widely used in immunodiagnostic and clinical tests, whose applications require a high level of purity. As a result, several stages of purification are required in the downstream processing of MAbs. The objective of this study was to recover and purify an anti-interleukin-6 MAb from cell culture supernatant by a combined method involving precipitation and immobilized metal-ion affinity chromatography (IMAC), aiming its subsequent application in diagnostic kits. Experimental results suggest that it is possible to purify MAbs by precipitation with ammonium sulphate at 20–40% saturation, followed by adsorption on IMAC with immobilized Zn(II). The MAbs were obtained with a purity of 100% and a purification factor of 24.2, proving the feasibility of the method.