The transient expression of CHIKV VLP in large stirred tank bioreactors

Abstract

Transient gene expression (TGE) bioprocesses have been difficult to scale up in large stirred tank bioreactors with volumes of more than 1.5 L. Low production levels are often observed, but the causes have not been investigated (Gutierrez-Granados et al. in Crit Rev Biotechnol 38:918-940, 2018). Chikungunya Virus-like particle (VLP), expressed by DNA-PEI transient transfection, is a representative case study for these difficulties. Clinical materials were produced in shake flasks, but the process suffered when transferred to large stirred tank bioreactors. The resulting process was not operationally friendly nor cost effective. In this study, a systematic approach was used to investigate the root causes of the poor scale up performance. The transfection conditions were first screened in ambr® 15 high throughput mini bioreactors then examined in 3 L stirred-tank systems. The studies found that production level was negatively correlated with inoculum cell growth status (P < 0.05). The pH range, DNA and PEI levels, order of the reagent addition, and gas-sparging systems were also studied and found to affect process performance. Further hydromechanical characterizations (Re, energy dissipation rates, and P/V, etc.) of shake flasks, ambr® 15, and 3-L stirred tank systems were performed. Overall, the study discovered that the shear stress (caused by a microsparger) and PEI toxicity together were the root causes of scale-up failure. Once the microsparger was replaced by a macrosparger, the scale-up was successful.

Keywords: Chikungunya virus; DNA and PEI; Large stirred tank bioreactor; Scale-up challenge; Shear stress; Transient gene expression; Virus-like particle vaccine; ambr® 15 bioreactor; pH.

Fig. 1

The effect of pH, inoculum source, and DNA–PEI concentration on CHIKV VLP production in the first ambr® 15 bioreactor study. Product titer was normalized to that of Vessel 1 condition (DNA 20 mg/L–PEI 40 mg/L at pH 7.3 ± 0.2) on Day 4. Vessel 16 had a control issue during the study and its data was not included. E: early exponential; M: middle exponential; L: late exponential; S: stationary

Fig. 2

The effect of pH and DNA–PEI concentration on cell growth in the second ambr® 15 bioreactor study. The first cell counts were taken after the addition of 1.5× CDM4HEK293 production medium

Fig. 3

The effect of pH and DNA–PEI concentration on CHIKV VLP production in the second ambr® 15 bioreactor study. Product titer was normalized to that of the control condition DNA 20 mg/L–PEI 40 mg/L on Day 4

Fig. 4

The toxicity of DNA, PEI, and combination of DNA and PEI measured by cellular viability in the third ambr® 15 bioreactor study. Samples were taken 3 h after transfection. Inoculum cells had a viability of more than 95%

Fig. 5

The effect of DNA and PEI addition order on CHIKV VLP product titers in the third ambr® 15 bioreactor study. Titers were normalized to that of DNA 20 mg/L–PEI 40 mg/L condition on Day 4

Fig. 6

Cell growth and CHIKV VLP production in 3-L bioreactors using microspargers. Product titer was normalized to that of the control shake flask on Day 4. Condition lo–lo (90 RPM–0.0125VVM) was lost due to contamination

Fig. 7

Cell growth and CHIKV VLP production in 3-L bioreactors using open tubes. Product titer was normalized to that of the control shake flask on day 4