RESUMO

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.

Assuntos

Fator VIII/biossíntese , Ácidos Pentanoicos/metabolismo , Perfusão , Temperatura , Animais , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Células CHO , Células Cultivadas , Cricetulus , Fator VIII/química , Humanos , Ácidos Pentanoicos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química

RESUMO

ABSTRACT Valeriana wallichii DC., Caprifoliaceae, is used to have anti-ulcer, anti-spasmodic, anti-epileptic, memory enhancer, anti-anxiety, anti-rheumatic, sedative, anti-asthmatic and diuretic activities. V. wallichii is reported to contain valpotriates, valeric acid, valerenic acid, valechlorine, valerianine, resins and alkaloids. Valeric acid, found in V. wallichii appears similar in structure to the neurotransmitter GABA. Valeric acid also acts as an NMDA-receptor antagonist. The aim of present study was to investigate the neuroprotective effect of V. wallichii containing valeric acid and its possible mechanism of action in amelioration of intracerebroventricular streptozotocin induced neurodegeneration in Wistar rats. The rhizomes of V. wallichii were powdered coarsely and extracted by percolation method using dichloromethane. Wistar rats (220–250 g) of either sex were divided into 5 groups, comprising 6 animals each. Valeric acid was isolated from plant extract and characterized using FT-IR. Picrotoxin (2 mg/kg) was used as GABA-A antagonist. Intracerebroventricular streptozotocin administration caused significant (p < 0.05) increase in escape latency, retention transfer latency on morris water maze on 17th, 18th, 19th and 20th day and elevated plus maze on 19th and 20th day respectively, as compared to normal untreated rats. Treatment with V. wallichii extract 100 and 200 mg/kg and valeric acid 20 and 40 mg/kg significantly decreased the escape latency and retention transfer latency, as compared to intracerebroventricular-streptozotocin group. Plant extract and valeric acid also decreased the level of lipid peroxidation and restored glutathione level in rat brains. Administration of picrotoxin significantly reversed the effects produced by plant extract and valeric acid in intracerebroventricular-streptozotocin treated rats. The findings may conclude that valeric acid present in V. wallichii has significant GABAergic effect in amelioration of experimental dementia.