RESUMO

Antivenom therapy is a critical intervention for treating the more than 5.000.000 envenomation accidents that occur each year around the world. These immunotherapeutic drugs are mostly produced following techniques developed more than fifty years ago with minor changes. Aggregate content has been described as one of the main causes of early adverse effects after intravenous administration of antivenoms. In this work we propose the introduction of a final polishing step to traditional antivenom manufacturing processes aimed at lowering the aggregate content in the final product. The refinement step proposed in this work is based on the selective capture of immunoglobulin aggregates by a cation exchange monolithic stationary phase. We show that this media can effectively remove aggregates in the final product under isotonic ion-strength and mildly acidic conditions following a negative chromatography strategy, thus making it a useful technique for producing higher quality products.

Assuntos

RESUMO

N6-(2-(2-Furanyl-2-oxoethyl))-l-lysine (furosine) is a deteriorative reaction product that is produced during heat treatment and storage of milk. This compound affects the quality of commercial dairy products. Accurate determination of furosine is necessary as it may serve as a measure of the degree of protein degradation in dairy products. In this article, two HPLC based methods (1. a novel ion-pairing reagent 2. a strong cation exchange column) are proposed to quantify furosine. These methods were optimized and validated for their application to analyze fluid milk and dried milk powder. •Two methods that can be used for routine milk quality control, including heat damage and adulteration, were developed.•Compared to previous methods, the modified procedures herein using aromatic sulfonic acids (a pairing agent or covalently bound to a matrix on a strong cation exchange column) provide less expensive and more sensitive determinations.•The identification and quantification of the furosine chromatographic signal was successfully achieved during analysis of commercial and spiked samples.

RESUMO

Composting is a sustainable approach to manage animal and vegetal waste generated in the Fundação Parque Zoológico de São Paulo. The resulting compost is often used in ZOO's premises as an organic fertilizer for the production of vegetables, which is further used to feed the animals. The composting product provides many forms of mineral and also amino acids (AA) that are absorbed by plants as nutrients. Since most amino acids absorb only slightly or not at all in the UV wavelengths, we developed a method for the determination of AA of agricultural interest in the composting samples. Due to the complexity of samples, we used ion exchange chromatography for the purification of AA prior to analysis. The proposed CZE-C4 D method allowed a separation of the AA in a short analysis time (less than 3.0 min), with great linearity (with R2 ranging from 0.993 to 0.998). Using a BGE of 10 mmol/L TEA, reduction of high-frequency noise and lower baseline fluctuations were obtained. The LOQ for the five AA were around 35 µmol/L, and were adequate for our purpose. In addition, the method showed good precision (RSD of peak area and migration time less than 1.55 and 1.16%, respectively).

Assuntos

RESUMO

Two methods of purification of the plantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31 are used in this study - the method of ammonium sulfate precipitation, Sep-pack C18 cartridge and reverse-phase HPLC chromatography on C18 Nucleosil column, and the method of direct purification by cation exchange SP Sepharose Fast Flow column Amersham (Pharmacia Biotech). The purity of the products from the two experimental protocols are examined for their molecular weight, aminoacid composition and sequence. Comparison of results show that the plantaricins purified with the two methods are identical. Both methods may be used to purify plantaricin ST31. Comparison of the yield in the purification protocols is 0.8% in the HPLC experimental protocol and 5.9% in the cation-exchange chromatography method.

Dois métodos de purificação de plantaricin ST31, uma bacteriocina produzida por Lactobacillus plantarum ST31 foram usados neste estudo - o método de precipitação pelo sulfato de amônia usando cartucho Sep-pack C18 para a filtração e HPLC de fase reversa em coluna de C18 Nucleosil, e o método de purificação direta por troca catiônica SP Sepharose "Fast Flow column Amersham" (Pharmacia Biotech). A pureza dos produtos obtidos pelos dois protocolos foi examinada através da determinação dos pesos moleculares, composição e seqüência dos aminoácidos. A comparação destes resultados revelou que, em termos da pureza dos produtos, não havia diferenças entre os dois métodos de purificação podendo-se, portanto, utilizar qualquer um dos protocolos de purificação testados. No entanto, o rendimento da purificação pelo método da troca catiônica foi de 5.9% enquanto o do método HPLC foi de 0.8%.

RESUMO

Two methods of purification of the plantaricin ST31, a bacteriocin produced by Lactobacillus plantarum ST31 are used in this study - the method of ammonium sulfate precipitation, Sep-pack C18 cartridge and reverse-phase HPLC chromatography on C18 Nucleosil column, and the method of direct purification by cation exchange SP Sepharose Fast Flow column Amersham (Pharmacia Biotech). The purity of the products from the two experimental protocols are examined for their molecular weight, aminoacid composition and sequence. Comparison of results show that the plantaricins purified with the two methods are identical. Both methods may be used to purify plantaricin ST31. Comparison of the yield in the purification protocols is 0.8% in the HPLC experimental protocol and 5.9% in the cation-exchange chromatography method.

Dois métodos de purificação de plantaricin ST31, uma bacteriocina produzida por Lactobacillus plantarum ST31 foram usados neste estudo - o método de precipitação pelo sulfato de amônia usando cartucho Sep-pack C18 para a filtração e HPLC de fase reversa em coluna de C18 Nucleosil, e o método de purificação direta por troca catiônica SP Sepharose "Fast Flow column Amersham" (Pharmacia Biotech). A pureza dos produtos obtidos pelos dois protocolos foi examinada através da determinação dos pesos moleculares, composição e seqüência dos aminoácidos. A comparação destes resultados revelou que, em termos da pureza dos produtos, não havia diferenças entre os dois métodos de purificação podendo-se, portanto, utilizar qualquer um dos protocolos de purificação testados. No entanto, o rendimento da purificação pelo método da troca catiônica foi de 5.9% enquanto o do método HPLC foi de 0.8%.