RESUMEN
Perfusion operation mode remains the preferred platform for production of labile biopharmaceuticals (e.g., blood factors) and is also being increasingly adopted for production of stable products (e.g., monoclonal antibodies). Regardless of the product, process development typically aims at maximizing production capacity. In this work, we investigated the impact of perfusion cultivation conditions on process productivity for production of human factor VIII (FVIII). Recombinant CHO cells were cultivated in bioreactors coupled to inclined settlers and the effects of reducing the temperature to 31°C with or without valeric acid (VA) supplementation were evaluated. Increases in cell specific productivity (qp ) up to 2.4-fold (FVIII concentration) and up to 3.0-fold (FVIII biological activity) were obtained at 31°C with VA compared to the control at 37°C. Biological activity is the most important quality attribute for FVIII and was positively affected by mild hypothermia in combination with the chemical inducer. The low temperature conditions resulted in enhanced product transcript levels, suggesting that the higher qp is related to the increased mRNA levels. Furthermore, a high-producer subclone was evaluated under the perfusion conditions optimized for the parental clone (31°C with VA), yielding increases in qp of 6-fold and 15-fold compared to the parental clone cultivated under the same condition and at 37°C, respectively. The proposed perfusion strategy enables increased product formation without increasing production costs, being potentially applicable to perfusion production of other CHO-derived biopharmaceuticals. To the best of our knowledge, this is the first report showing the benefits of perfusion combining mild hypothermia with VA supplementation.
Asunto(s)
Factor VIII/biosíntesis , Ácidos Pentanoicos/metabolismo , Perfusión , Temperatura , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Células CHO , Células Cultivadas , Cricetulus , Factor VIII/química , Humanos , Ácidos Pentanoicos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
Valepotriate content levels in samples of in vitro cultures of Valeriana edulis ssp. procera were compared with those of roots and rhizomes of wild plants in the reproductive stage. Rhizomes and roots of regenerated and wild plants showed a similar valepotriate content. The data obtained support the hypothesis that valepotriate production in V. edulis spp. procera is closely related to rhizome and root differentiation. The large-scale propagation of this endangered plant may offer an attractive alternative for its production for medicinal purposes.