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1.
Sci Rep ; 12(1): 7239, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610229

RESUMO

Chinese hamster ovary (CHO) cells are widely used for manufacturing antibody drugs. We attempted to clone a novel high-expression promoter for producing monoclonal antibodies (mAbs) based on transcriptome analysis to enhance the transcriptional abundance of mAb genes. The efficacy of conventional promoters such as CMV and hEF1α decrease in the latter phase of fed-batch cell culture. To overcome this, we screened genes whose expression was maintained or increased throughout the culture period. Since CHO cells have diverse genetic expression depending on the selected clone and culture medium, transcriptome analysis was performed on multiple clones and culture media anticipated to be used in mAb manufacturing. We thus acquired the Hspa5 promoter as a novel high-expression promoter, which uniquely enables mAb productivity per cell to improve late in the culture period. Productivity also improved for various IgG subclasses under Hspa5 promoter control, indicating this promoter's potential universal value for mAb production. Finally, it was suggested that mAb production with this promoter is correlated with the transcription levels of endoplasmic reticulum stress-related genes. Therefore, mAb production utilizing the Hspa5 promoter might be a new method for maintaining protein homeostasis and achieving stable expression of introduced mAb genes during fed-batch culture.


Assuntos
Formação de Anticorpos , Técnicas de Cultura Celular por Lotes , Animais , Anticorpos Monoclonais/genética , Células CHO , Cricetinae , Cricetulus , Meios de Cultura
2.
AAPS PharmSciTech ; 16(5): 993-1001, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26288941

RESUMO

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.


Assuntos
Produtos Biológicos/síntese química , Equipamentos Descartáveis , Contaminação de Medicamentos/prevenção & controle , Indústria Farmacêutica , Gestão de Riscos , Tecnologia Farmacêutica/instrumentação , Produtos Biológicos/efeitos adversos , Produtos Biológicos/normas , Produtos Biológicos/provisão & distribuição , Qualidade de Produtos para o Consumidor , Equipamentos Descartáveis/normas , Indústria Farmacêutica/normas , Humanos , Segurança do Paciente , Controle de Qualidade , Medição de Risco , Fatores de Risco , Gestão de Riscos/normas , Tecnologia Farmacêutica/normas
3.
J Med Chem ; 56(5): 2155-9, 2013 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-23419007

RESUMO

Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in recent years. We introduce SITE (single-injection thermal extinction), a novel thermodynamic methodology that selects high-quality hits early in FBDD. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly fewer resources. We describe the principles of SITE and identify a novel family of fragment inhibitors of the enzyme ketosteroid isomerase displaying high values of enthalpic efficiency.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/síntese química , Bibliotecas de Moléculas Pequenas , Esteroide Isomerases/antagonistas & inibidores , Termodinâmica , Calorimetria/métodos , Inibidores Enzimáticos/isolamento & purificação , Ressonância de Plasmônio de Superfície
4.
Nat Methods ; 3(11): 923-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17060916

RESUMO

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.


Assuntos
Calmodulina/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Fenilalanina/análogos & derivados , Sistema Livre de Células , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Estrutura Molecular , Fenilalanina/síntese química , Fenilalanina/química , Conformação Proteica , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , Relação Estrutura-Atividade
6.
Protein Eng Des Sel ; 18(6): 273-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15928004

RESUMO

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Mutação/genética , RNA de Transferência/genética , Tirosina/genética , Sistema Livre de Células , Códon/genética , Escherichia coli/genética , Biblioteca Gênica , Proteínas de Fluorescência Verde/metabolismo , Mutagênese , Fenilalanina/análogos & derivados , Fenilalanina/genética , Fenilalanina/metabolismo , Engenharia de Proteínas , Tirosina/química
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