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1.
Protein J ; 40(5): 786-798, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34023982

RESUMO

With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.In this study, non-glycosylated anti-TNF-α Fab' moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.


Assuntos
Biomassa , Certolizumab Pegol , Citoplasma , Escherichia coli , Certolizumab Pegol/biossíntese , Certolizumab Pegol/genética , Citoplasma/genética , Citoplasma/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes
2.
Cytokine ; 101: 56-63, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27567553

RESUMO

Tumor necrosis factor (TNF)-α is a potent pro-inflammatory and pathological cytokines in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti-TNF-α therapy has been established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal anti-TNF-α full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab' fragment of anti-TNF-α antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1-Fc fusion protein etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibodies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in inflammatory bowel diseases. Besides the common effect of anti-TNF-α agents on neutralization of soluble TNF-α, each anti-TNF-α agent has its own distinctive pharmacological properties which cause the difference in clinical efficacies. Here we focus on the distinctions of action of anti-TNF-α agents especially in following points; (1) blocking ability against ligands, transmembrane TNF-α and lymphotoxin, (2) effects toward transmembrane TNF-α-expressing cells, (3) effects toward Fcγ receptor-expressing cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea how to modify anti-TNF-α agents to enhance the clinical efficacy in inflammatory diseases.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/genética , Artrite Reumatoide/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab/efeitos adversos , Adalimumab/genética , Adalimumab/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/administração & dosagem , Antirreumáticos/efeitos adversos , Antirreumáticos/uso terapêutico , Artrite Reumatoide/imunologia , Certolizumab Pegol/efeitos adversos , Certolizumab Pegol/genética , Certolizumab Pegol/uso terapêutico , Modelos Animais de Doenças , Etanercepte/efeitos adversos , Etanercepte/uso terapêutico , Humanos , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Imunoglobulina G/efeitos adversos , Imunoglobulina G/genética , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/genética , Fatores Imunológicos/uso terapêutico , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Doenças Inflamatórias Intestinais/imunologia , Infliximab/efeitos adversos , Infliximab/genética , Infliximab/uso terapêutico , Camundongos , Polietilenoglicóis/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia
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