Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

<p>Abstract</p> <p>Background</p> <p>Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered <it>Escherichia coli </it>(Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in <it>E. coli</it>. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in <it>E. coli </it>cells.</p> <p>Results</p> <p>Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in <it>E. coli </it>BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, <it>in vivo</it>, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.</p> <p>Conclusion</p> <p>The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.</p

Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Recombinant human interferon beta 1b (rhIFNβ−1b) is clinically used to treat multiple sclerosis. A reversed−phase liquid chromatography (RP−LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE−LC) method was carried out on a BioSep−SEC−S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1−200 µg/mL (r2 = 0.9998) and 0.50−200 µg/mL (r2 = 0.9999), respectively, for RP−LC and SE−LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNβ-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Antiinflammatory activity and biochemical parameters of the ethanol extract of Nopalea cochenillifera (L.) Salm-Dyck (Cactaceae)

We evaluated the antiinflammatory activity of ethanol 70 % extract of Nopalea cochenillifera in a model of induction of granulomatous tissue and the kidney and liver toxicity through serum dosage in rats. During 7 days were administered orally 1.5 ml, 3 times a day, of the ethanol extract of cladodes of N. cochenillifera. We used nimesulide 5 mg/kg/day as positive control and 20 % propylene glycol as a negative control. After the treatment period, we assessed the formation of granulomas and the serum levels of AST, ALT, albumin, creatinine and urea in all groups, noting that the animals treated with the extract showed 53.5 % inhibition formation of granulomatous tissue while the positive control group showed 58.5 %, confirming a significant antiinflammatory activity. There was not a significant elevation of biochemical markers in relation to negative control.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Morpho-anatomy of Glechon spathulata Benth. (Lamiaceae) leaves

Glechon spathulata Benth. (Lamiaceae) is a native plant in Brazil popularly known as "mangerona-do-campo". This plant is a perennial sub-shrub with aromatic leaves used in South America as a condiment and in traditional medicine for different purposes, such as inflammations, dyspepsia, diaphoretic, expectorant and antiseptic in catarrhal affections of the respiratory tract, colds, bronchitis and laryngitis. In this study, morpho-anatomical parameters of leaves of this plant were determimed, by macro and microscopic analysis, aiming to help its diagnosis as a pharmaceutical ingredient. The leaves are sub-sessile with spatulate shape and subrevolute margin, finely crenulate in the middle-upper. The uniseriate epidermis shows straight to slightly sinuous cells, diacytic stomata and non-glandular and glandular trichomes. The mesophyll is dorsiventral, displaying an uniseriate palisade parenchyma and a little compact spongy parenchyma, with an average of 4 cell layers. The vascular bundles are collateral closed. There was no presence of crystals. These morphological and anatomical features, when taken together, contribute to quality control of plant leaves of G. spathulata as a pharmaceutical ingredient.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 ºC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v) as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 &#956;g/mL (r²=0.9999) and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenobarbital 100 mg after single oral dose administration to healthy human volunteers

Avaliação de pirogênios em produtos de uso veterinário pelos testes da hipertermia em coelhos e do lisado de amebócitos do Limulus Pyrogens in veterinary products by the rabbit pyrogen test and the Limulus amoebocyte lysate test

Realizou-se a avaliação de pirogênios em produtos veterinários de uso parenteral, pelo método da hipertermia em coelhos, calculando-se, para o teste das amostras, doses com concentrações de três a sete vezes superiores à terapêutica. Preconizou-se como resposta positiva o aumento de temperatura de 0,6&deg;C. Utilizou-se também o ensaio do lisado de amebócitos do Limulus (LAL) por geleificação, semiquantitativo, executando o teste de interferentes, validando o procedimento e estabelecendo a máxima diluição válida para a análise de cada produto. Paralelamente, efetuou-se avaliação comparativa de amostras com o método do LAL cromogênico, quantitativo, demonstrando correlação e reprodutibilidade dos resultados. Avaliaram-se vinte e oito produtos de diferentes classes farmacológicas, observando-se que dois não cumpriram as especificações, sendo reprovados. Sugere-se que as especificações estudadas sejam adotadas, contribuindo para aprimorar o controle de contaminantes, garantindo a qualidade e a segurança dos produtos veterinários.The rabbit pyrogen test was used to evaluate veterinary products, suggesting the temperature rise of 0.6&deg;C as the ending point for the positive results. The test doses were calculated based on the therapeutic dose increased from three to seven times. The semi-quantitative Limulus amoebocyte lysate (LAL) gel clot test was performed and compared to the LAL spectrophotometric chromogenic, quantitative assay. The comparative evaluation of the samples showed correlation and reproducibility of the results. The interference test was carried out, the procedure validated and the maximum valid dilution established for the analysis of the products without Pharmacopoeial specifications. Two of the twenty-eight products of different pharmacological groups evaluated didn't meet the requirements and were reproved. The specifications investigated are suggested to be used for the purity evaluation of the veterinary parenteral products, as a contribution to assure the quality and safety of the products

Desarrollo y validación de método por HPLC para la cuantificación y estudios de disolución de Valdecoxib

En el presente trabajo se desarrolló y validó un método por cromatografía líquida de alta eficacia (CLAE) para los estudios de disolución y la estimación cuantitativa de valdecoxib en tabletas y en el ingrediente farmacéutico activo. En la técnica de CLAE se utilizó una columna cromatográfica Synergi® fusión de fase reversa RP-18 (150 x 4,6 mm) de 4 µm a 30 ºC. La fase móvil estuvo compuesta por agua de pH 7,0-acetonitrile (52:48, v/v), velocidad de flujo fue de 1,0 mL/min y se utilizó un detector ultravioleta con longitud de onda fijada a 210 nm. En la validación del método analítico se determinaron los siguientes parámetros de validación: linealidad (r2 =0.9999), curva de calibración, precisión, exactitud, robustez y especificidad; el método rindió buenos resultados con un límite de cuantificación de 50 ng/mL y un límite de detección de 10 ng/mL. El perfil de disolución se realizó en agua conteniendo 0,5% de lauril sulfato de sodio como medio de disolución a 75 rpm. El método desarrollado se empleó con éxito para el análisis de las tabletas, del ingrediente farmacéutico activo y para los estudios de disolución.An isocratic high-performance liquid chromatography (HPLC) procedure was developed for the dissolution rate studies and quantitative determination of valdecoxib in solid dosage forms and in active pharmaceutical ingredient. HPLC separation was carried out by reversed phase chromatography on a Synergi® fusion C18 column (150 mm x 4.6 mm i.d.; 4 µm particle size), held at 30 ºC. The mobile phase consisted of water, pH 7.0/acetonitrile (52:48, v/v), run at a flow rate of 1.0 mL/min and with UV detection at 210 nm. Method validation investigated parameters such as the linearity (r2 =0.9999), range, precision, accuracy, robustness and specificity. The method yielded good results with a quantitation limit of 50 ng/mL and a detection limit of 10 ng/mL. The dissolution test conditions and the dissolution medium was chosen as 0.5% of sodium lauryl sulfate in water at a stirring rate of 75 rpm. The described method can be successfully applied for the analysis of tablets, active pharmaceutical ingredient and drug dissolution studies.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Desarrollo y validación de método por HPLC para la cuantificación y estudios de disolución de Valdecoxib

En el presente trabajo se desarrolló y validó un método por cromatografía líquida de alta eficacia (CLAE) para los estudios de disolución y la estimación cuantitativa de valdecoxib en tabletas y en el ingrediente farmacéutico activo. En la técnica de CLAE se utilizó una columna cromatográfica Synergi® fusión de fase reversa RP-18 (150 x 4,6 mm) de 4 µm a 30 ºC. La fase móvil estuvo compuesta por agua de pH 7,0-acetonitrile (52:48, v/v), velocidad de flujo fue de 1,0 mL/min y se utilizó un detector ultravioleta con longitud de onda fijada a 210 nm. En la validación del método analítico se determinaron los siguientes parámetros de validación: linealidad (r2 =0.9999), curva de calibración, precisión, exactitud, robustez y especificidad; el método rindió buenos resultados con un límite de cuantificación de 50 ng/mL y un límite de detección de 10 ng/mL. El perfil de disolución se realizó en agua conteniendo 0,5% de lauril sulfato de sodio como medio de disolución a 75 rpm. El método desarrollado se empleó con éxito para el análisis de las tabletas, del ingrediente farmacéutico activo y para los estudios de disolución.An isocratic high-performance liquid chromatography (HPLC) procedure was developed for the dissolution rate studies and quantitative determination of valdecoxib in solid dosage forms and in active pharmaceutical ingredient. HPLC separation was carried out by reversed phase chromatography on a Synergi® fusion C18 column (150 mm x 4.6 mm i.d.; 4 µm particle size), held at 30 ºC. The mobile phase consisted of water, pH 7.0/acetonitrile (52:48, v/v), run at a flow rate of 1.0 mL/min and with UV detection at 210 nm. Method validation investigated parameters such as the linearity (r2 =0.9999), range, precision, accuracy, robustness and specificity. The method yielded good results with a quantitation limit of 50 ng/mL and a detection limit of 10 ng/mL. The dissolution test conditions and the dissolution medium was chosen as 0.5% of sodium lauryl sulfate in water at a stirring rate of 75 rpm. The described method can be successfully applied for the analysis of tablets, active pharmaceutical ingredient and drug dissolution studies.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Determination of phenytoin in human plasma by a validated liquid chromatography method and its application to a bioequivalence study

A sensitive and specific method based on liquid chromatography was developed and validated for the determination of phenytoin in human plasma using phenobarbital as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a Phenomenex Synergi MAX-RP C12 column (150x 4.6 mm i.d.), with water: acetonitrile: methanol(58.8:15.2:26, v/v/v) as mobile phase. Detection was carried out using photodiode array detector set at 205 nm. The chromatographic separation was obtained within 12 min and was linear in the concentration range of 50-2500 ng/mL (r2 = 0.9999). The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenytoin 100 mg after single oral dose administration to 28 healthy human volunteers. The 90% confidence intervals were calculated for the Cmax, AUC(0-t) and AUC(0-∞), giving values between 99.97–118.40% demonstrating the bioequivalence of the two formulations.Colegio de Farmacéuticos de la Provincia de Buenos Aire