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Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli.
Selas Castiñeiras, Tania; Williams, Steven G; Hitchcock, Antony; Cole, Jeffrey A; Smith, Daniel C; Overton, Tim W.
Afiliação
  • Selas Castiñeiras T; Cobra Biologics, Stephenson Building, The Science Park, Keele ST5 5SP, UK.
  • Williams SG; School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
  • Hitchcock A; Institute of Microbiology & Infection, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
  • Cole JA; Cobra Biologics, Stephenson Building, The Science Park, Keele ST5 5SP, UK.
  • Smith DC; Cobra Biologics, Stephenson Building, The Science Park, Keele ST5 5SP, UK.
  • Overton TW; Institute of Microbiology & Infection, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Microbiology (Reading) ; 164(4): 440-452, 2018 04.
Article em En | MEDLINE | ID: mdl-29458685
ABSTRACT
As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fator de Necrose Tumoral alfa / Escherichia coli / Técnicas de Cultura Celular por Lotes Idioma: En Ano de publicação: 2018 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fator de Necrose Tumoral alfa / Escherichia coli / Técnicas de Cultura Celular por Lotes Idioma: En Ano de publicação: 2018 Tipo de documento: Article