Stéphanie Tissot

Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

Large‐scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33 °C after DNA delivery. With this approach, transient recombinant antibody yields of 60–80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady‐state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector‐dependent, but the presence of the woodchuck hepatitis virus post‐transcriptional regulatory element in the 3′ untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3‐fold at 31 °C as compared to expression at 37 °C. The yields achieved by the low‐temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).

Surface wave dynamics in orbital shaken cylindrical containers

Be it to aerate a glass of wine before tasting, to accelerate a chemical reaction, or to cultivate cells in suspension, the “swirling” (or orbital shaking) of a container ensures good mixing and gas exchange in an efficient and simple way. Despite being used in a large range of applications this intuitive motion is far from being understood and presents a richness of patterns and behaviors which has not yet been reported. The present research charts the evolution of the waves with the operating parameters identifying a large variety of patterns, ranging from single and multiple crested waves to breaking waves. Free surface and velocity fields measurements are compared to a potential sloshing model, highlighting the existence of various flow regimes. Our research assesses the importance of the modal response of the shaken liquids, laying the foundations for a rigorous mixing optimization of the orbital agitation in its applications.

Efficient and reproducible mammalian cell bioprocesses without probes and controllers

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here.

k(L)a as a predictor for successful probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation.

Hydrodynamic stress in orbitally shaken bioreactors.

Orbitally shaken bioreactors (OSRs) of nominal volume from 50 mL to 2’000 L have been developed for the cultivation of suspension-adapted mammalian cells. Here we study the hydrodynamics of OSRs for mammalian cells. The results are expected to allow the determination of key parameters for cell cultivation conditions and will facilitate the scale-up of OSRs.

Engineering Principles and Cell Culture Performance of Orbitally Shaken Cylindrical Bioreactors

We are investigating the use of orbitally shaken cylindrical containers for the cultivation of suspension-adapted mammalian cells at working volumes from a few mLs to 1000 L. Here we present data on mixing, oxygen transfer, and cell culture performance in these bioreactors. kLa values for volumes up to 1000 L and mixing times for volumes up to 400 L were measured at shaking speeds suitable for mammalian cell culture. Without pH or oxygen control, cell densities equivalent to fully controlled bioreactors were obtained at scales up to 100 L in a 200-L bioreactor. A 500-L culture run in a 2000-L orbitally shaken bioreactor (non-optimized prototype) reached 85% of the maximal cell density and 65% of the recombinant protein concentration compared to 10-mL cultures in 50-mL Tubespins. Only air was used as the oxygen source at all the scales, demonstrating that oxygen transfer through the free surface in orbitally shaken bioreactor is sufficient for mammalian cell cultures. Overall, our data indicate that orbitally shaken bioreactors are an interesting alternative to conventional stirred-tank bioreactors.

kLa as a predictor for probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors

Background Orbitally shaken flasks are commonly used at an early stage of bioprocess development with mammalian cells. In contrast to large-scale stirred-tank bioreactors, shaken flasks are usually operated in probe-independent bioprocesses, i.e. without strictly controlling the pH or dissolved oxygen concentration (DO). As a consequence, gas transfer issues are thought to limit the effectiveness of orbitally shaken flasks and bioreactors (OSRs). To define optimal operating conditions for probe-independent bioprocesses in OSRs, we tested the effects of the mass transfer coefficient of oxygen (kLa) on mammalian cell growth, recombinant protein production, and environmental conditions of the culture (pH, DO).

Transient Gene Expression in Chinese Hamster Ovary Cells at Low Temperature – The Effects of Cold-Induced Proteins and an mRNA Regulatory Element

Exposure of transfected Chinese hamster ovary (CHO) cells to low temperature increased transient recombinant protein yield up to 18-fold. This phenomenon was not plasmid specific, but the enhancement of protein production under hypothermic conditions varied from 2- to 18-fold depending both on the reporter gene and the presence or absence of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). When located in the 3′-untranslated region (UTR) of the transgene it increased protein production 3-fold at 31°C compared to expression from the isogenic plasmid without the element. Cold shock proteins are also known to act in a post-transcriptional manner, but the overexpression of the cold-induced RNA binding protein (CIRBP) and the RNA binding motif 3 (Rbm3) had no effect on the enhancement of transient recombinant protein production in CHO cells.