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

Biological potency evaluation and characterization of rhG-CSF in pharmaceutical products

The identification of rhG-CSF was carried out in pharmaceutical preparations by non-reducing polyacrylamide gel electrophoresis and western blotting with specific antibodies, showing a single band in the 19 kDa region. The potency was assessed by the neutropenia mouse bioassay giving values between 88.4 and 122.4% of the stated potency. The precision index expressed by weight was between 141 and 432 for the independent assays. Batch-to-batch, the samples met the requirements for the safety test and bacterial endotoxins test. The biological and immunological results showed the quality of the products in clinical use and the specifications established contribute to assuring the safety and efficacy of biological medicines.Realizou-se a identificação do fator estimulador da colônia de granulócitos humanos recombinante em produtos farmacêuticos por eletroforese em gel de poliacrilamida não redutora e imunodetecção com anticorpos específicos, que apresentaram banda única na região de, aproximadamente, 19 kDa. A avaliação de potência baseada na contagem do número de neutrófilos em camundongos com neutropenia forneceu valores entre 88,4 - 122,4 % em relação à potência declarada. A precisão expressa pela ponderação, calculada nos ensaios independentes, forneceu valores entre 141 e 432. As amostras lote-a-lote cumpriram os requisitos dos testes de toxicidade e endotoxinas bacterianas. Os resultados dos ensaios biológicos e imunológicos demonstram a qualidade dos produtos farmacêuticos em uso clínico, e as especificações sugeridas contribuem para assegurar a inocuidade e eficácia terapêutica dos produtos biológicos

Avaliação dos efeitos do valdecoxibe sobre os parâmetros hemostáticos de ratos Wistar

The effects of the cyclooxygenase (COX)-2 selective inhibitor, valdecoxib, on blood coagulation parameters were evaluated, along with aspirin in male Wistar rats. Groups of animals were administered a daily oral dose of 10 mg/kg rat of valdecoxib, 100 mg/kg rat of aspirin and the vehicle alone during 4 weeks. Blood samples were collected at the end of 1, 2, 3 and 4 weeks of administration period and the plasma concentrations of valdecoxib were determined by RP-HPLC giving mean values of 101.1, 113.5, 164.0 and 184.6 ng/mL, respectively. The same plasma samples were used for the analysis of hematological parameters and the results compared to the controls. Valdecoxib induced significant activated partial thromboplastin time reduction (18%) after 2 weeks and prothrombin time reduction (12.2%) after 3 weeks (P<0.05). There were no significant changes in the platelet count and fibrinogen levels. The anti-factor Xa and anti-factor IIa activities showed slight reductions, but only significant for the anti-factor Xa on the 3rd week (6.7%). The results showed that valdecoxib at the dose tested with the plasmatic concentrations induced some changes in the hemostatic function of rats, which can be helpful to understand the side effects and the safe use of the drug.Avaliaram-se os efeitos do valdecoxibe, um inibidor seletivo da cicloxigenase-2 (COX-2), sobre os parâ&shy;metros sangüíneos da coagulação em ratos Wistar utilizando-se paralelamente a aspirina. Os animais foram divididos em grupos e submetidos à administração oral diária de 10 mg/kg animal para o valdecoxib, 100 mg/kg animal para a aspirina e veículo para o grupo controle, durante quatro semanas. A coleta do sangue foi efetuada após uma, duas, três e quatro semanas, e as concentrações plasmáticas do valdecoxibe determinadas por cromatografia líquida, obtendo-se valores médios de 101,1 - 113,5 - 164 e 184,6 ng/ml, respectivamente. As amostras de plasma foram também usadas para as análises hematológicas e os resultados comparados aos dos controles. O valde&shy;coxibe apresentou redução significativa no TTPA (18%) após duas semanas, e redução do TP (12,2%) após três semanas (P<0.05). Os efeitos observados na contagem de pla&shy;quetas e nos níveis plasmáticos de fibrinogênio não foram significativos. As atividades anti-fator Xa e anti-fator IIa apresentaram redução, porém os resultados foram significativos somente para o anti-fator Xa na terceira semana (6,7%). Os resultados experimentais com as concentrações plasmáticas alcan&shy;çadas demonstram que o valdecoxibe pode induzir alterações nos parâ&shy;metros hematológicos dos ratos. Esses dados poderão contribuir para a melhor compreensão dos efeitos colaterais e uso do fármaco com segurança

Biological potency evaluation and physicochemical characterization of unfractionated heparins Avaliação biológica de potência e caracterização físico-química de heparinas não fracionadas

Unfractionated heparins are used clinically as anticoagulants. The biological potency of thirteen samples of raw material and pharmaceutical formulations were assessed utilizing the 5th International Standard of heparin using the sheep plasma coagulation inhibition assay, activated partial thromboplastin time, anti-factor Xa assay, and anti-factor IIa assay, resulting in mean potencies of 101.15%, 96.15%, 98.15% and 99.37%, respectively. The samples were also evaluated by the protamine neutralization test giving results within the range of 92 - 138 IU/mg. The anti-factor IIa assay was performed showing reproducibility and significant correlation with the pharmacopoeial assays, thus demonstrating it to be a feasible alternative to the sheep plasma coagulation inhibition assay. Moreover, an analysis by nuclear magnetic resonance and capillary electrophoresis showed some peaks attributable to oversulfated chondroitin sulfate. The results show that batch-to-batch variations and the quality of samples contributed to improvements in the quality control of pharmaceutical products and to assure the safe use and clinical efficacy of this biological medicine.As heparinas não fracionadas são utilizadas clinicamente como anticoagulantes. A potência biológica de 13 amostras de matérias-primas e produtos farmacêuticos foram avaliadas em relação ao 5ª Padrão Internacional de heparina pelos ensaios da inibição da coagulação do plasma ovino, tempo de tromboplastina parcial ativada, anti-fator Xa e anti-fator IIa, que forneceram potências médias de 101,15%, 96,15%, 98,15% e 99,37%, respectivamente. As amostras foram também submetidas ao teste de neutralização pela protamina que apresentou resultados entre 92-138 UI/mg. Demonstrou-se reprodutibilidade e correlação significativa do ensaio do anti-fator IIa com os farmacopeicos, constituindo-se em alternativa ao ensaio da inibição da coagulação do plasma ovino. Além disso, as análises realizadas por ressonância magnética nuclear e eletroforese capilar mostraram picos correspondentesà condroitina supersulfatada. Os resultados mostraram variações lote-a-lote e a qualidade das amostras contribuindo para aprimorar o controle de qualidade dos produtos farmacêuticos e garantir a segurança e eficácia terapêutica desses produtos biológicos