Development of a generic transient transfection process at 100 L scale

Abstract

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L(-1) IgG.

Fig. 1

Influence of conditioned medium on transient protein expression. Cells were seeded in culture medium at a density of 0.5 × 10⁶ cells mL⁻¹ with five different ratios of 72-h-old conditioned medium. The experiment was performed in 30 mL shaked cultures with pCEP4-AmCyan as expression vector. AmCyan expression was measured 24, 48 and 72 h post transfection using the signal-to-noise assay. Data (RFU) of one representative experiment are presented (░24 h, ▒48 h, □72 h)

Fig. 2

Influence of the time of transfection on transient protein expression. Cells were seeded in Wave bags at a density of 0,5 × 10⁶ cells mL⁻¹. Samples of 30 mL were taken almost every hour, transferred to a shaker or spinner flask and immediately transfected with a freshly prepared transfection complex using pCEP4-AmCyan. AmCyan expression was followed with the signal-to-noise assay for 3 days. Data of one representative experiment (Wave—shake flasks) are presented (░48 h, ▒72 h)

Fig. 3

Transfection of a 10 L HEK293-EBNA cells culture in a Wave Bioreactor. In two experiments (open symbols experiment 1, closed symbols experiment 2) cells were seeded in Wave bags at a density of 1 × 10⁶ cells mL⁻¹. Two hours later the cultures were transfected and after an additional 4 h incubation, the cultures were fed to the final culture volume. IgG concentration (○●) was determined by ELISA and the viable cell concentration (△▲) and viability (□■) were assessed using a Cedex automatic cell counter

Fig. 4

Transfection of 50 L HEK293-EBNA cells cultured in a 50 L (a) and 100 L (b) stirred tank bioreactor. In two experiments (open symbols experiment 1, closed symbols experiment 2) cells were seeded from a seeding bioreactor at a density of 1 × 10⁶ cells mL⁻¹. Two hours later the cultures were transfected and after an additional 4 h the cultures were fed to the final culture volume. IgG concentrations (○●) were determined by ELISA and the viable cell concentration (△▲) and viability (□■) were assessed using a Cedex automatic cell counter. (c) Shows the glucose (□■), lactate (△▲) and glutamine (○●) concentration monitored for the two 100 L cultures

Fig. 5

Comparison of the average cell specific productivities at 10, 50 and 100 L production scale. The average cell specific productivities were calculated over the whole production process as ((ΔIgG Δtime⁻¹ ΔCv⁻¹) × 24 h). The error bars show the standard deviation

Fig. 6

Transient transfection working scheme for transfections in 50 and 100 L bioreactors. The key-events from cell thawing to the final product formulation are indicated