Development of therapeutic anti-hTNFα.

O objetivo do projeto foi desenvolver linhagens celulares para um anticorpo terapêutico anti-hTNFα e comprovar sua funcionalidade. Os genes anti-hTNFα foram clonados em células CHO para seleção da população estável mista, demonstrando expressão de anticorpo com reconhecimento de hTNFα em estrutura tridimensional. A população de transfectantes de maior produtividade específica foi escolhida para geração de linhagem monoclonal utilizando a tecnologia robótica ClonePix FL. Não houve diferença estatística entre o anti-hTNFα purificado e o produto de referência na cinética de ligação ao TNFα e reconhecimento diferencial por FcγRs em ensaios por SPR O ensaio de atividade funcional mostrou que o anti-TNFα desenvolvido pôde neutralizar a citotoxicidade induzida em células L929 e inibir a expressão de ELAM-1 em HUVEC. Os resultados finais permitiram identificar os três melhores clones, estáveis por 60 gerações. A comparabilidade entre o anti-TNFα desenvolvido e a referência permite admiti-lo como não inferior, um dos requisitos para o desenvolvimento de biossimilar.The aim of the project was to develop a therapeutic anti-hTNFα antibody and evaluate its functionality. The anti-hTNFα synthezised genes were cloned into CHO cells and stable pools were selected, producing antibodies able to hTNFα three-dimensional structure recognition. The stable pools displaying higher antibody yields were the source for the generation of monoclonal lineage by ClonePix FL robotic technology. The clones selection proceeded using different criteria as cell density, specific productivity, fed-batch performance, kinetics measured by surface plasmonic resonance, hTNFα binding through ELISA, western-blotting and SPR, FcγRs binding by SPR. Besides, a small number of clones was tested in functional assays by the impairment of cytotoxicity of hTNFα over L929 cells and the inhibition of ELAM-1 expression by HUVEC. The long term stability testing allowed to finally select 3 top clones, not inferior to adalimumab reference by the above criteria

Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coli and preliminary purification process

Submitted by Repositório Arca ([email protected]) on 2019-03-07T16:42:55Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) F98-1472-6750-14-1.pdf: 622348 bytes, checksum: f71c7f31ec9d0d84421d03dcc53f2068 (MD5)Approved for entry into archive by monique santos ([email protected]) on 2019-03-18T12:21:32Z (GMT) No. of bitstreams: 2 F98-1472-6750-14-1.pdf: 622348 bytes, checksum: f71c7f31ec9d0d84421d03dcc53f2068 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-03-18T12:21:32Z (GMT). No. of bitstreams: 2 F98-1472-6750-14-1.pdf: 622348 bytes, checksum: f71c7f31ec9d0d84421d03dcc53f2068 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Programa de Engenharia Química - COPPE. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Programa de Engenharia Química - COPPE. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Centro de Estudos de Saúde do Trabalhador e Ecologia Humana. Rio de Janeiro, RJ, Brasil.BACKGROUND: Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections. RESULTS: We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin. CONCLUSIONS: This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity