Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production

Abstract

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 (q p ) 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 q p 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 q p 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. K E Y W O R D SCHO perfusion cultivation, mild hypothermia, productivity, recombinant factor VIII, valeric acid

Shake tube perfusion cell cultures are suitable tools for the prediction of limiting substrate, CSPR, bleeding strategy, growth and productivity behavior

“…Perfusion bleed is usually considered waste, meaning product is lost in this split stream. Consequently, various strategies have been investigated in the past to limit the growth, and thus bleed rates, of perfusion cultures by adding chemical substrates (including valeric acid or cell cycle inhibitors) or by environmental intervention with mild hypothermia to 31–33 °C 28‐30 . Here, we demonstrate another possibility to limit growth while maintaining high specific productivities by carefully limiting the CSPR.…”

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode

“…Although other cell lines such as CHO cells were reported to grow efficiently when cooling the inclined settler to temperatures lower than 22 • C (Choo et al, 2007;Pohlscheidt et al, 2013;Coronel et al, 2019b), the AGE1.CR.pIX cells seemed to be sensitive to low temperatures in the recirculation loop. A previous case study showed through orthogonal partial least square multivariate analysis that the temperature in the inclined settler is one of the most important factor for the productivity variability (Shimoni et al, 2018).…”

“…DO and pH were controlled by sparging of O 2 and CO 2 , respectively. The IS was operated at a recirculation rate of 35 mL/min, intermittent vibration (15 s on, 10 min off) and with 30 • angle, as described previously by Coronel et al (2019b). Water at different temperatures was recirculated in the heat exchanger (between 20 • C and 27 • C).…”

“…Initial cultivations were done using a thermostatic bath to cool the water in the heat exchanger, that is commonly used during IS operation in biopharmaceutical production using other cell lines (e.g., CHO cells; Choo et al, 2007;Pohlscheidt et al, 2013;Coronel et al, 2019b). In our case, AGE1.CR.pIX cells did not efficiently grow (t d > 48 h) with set-points varying between 20-22 • C (IS1, Figures 2A,B), so higher set-points were used during the cell growth phase for run IS2 (25-27 • C; Figures 2A,B).…”

Process intensification for the production of yellow fever virus‐like particles as potential recombinant vaccine antigen

“…However, in view of the low production cost that is desirable for a vaccine to be accessible also for the population of low‐income countries, we considered it more adequate to proceed investigations using an inclined lamella settler, which is a low‐cost cell retention device that does not use membranes or any other consumables and is intellectual‐property free. Although no previous reports of HEK293 perfusion processes using a settler was found in literature, this cell retention device has been shown to provide very high separation efficiencies with no product retention in previous works with other mammalian cell lines (Coronel et al, 2020a, 2020b). It has also been industrially used to produce recombinant Factor VIII by Bayer since 1993, enabling long perfusion processes to be carried out.…”