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.

High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human Embryonic Kidney 293-Epstein-Barr Virus Nuclear Antigen Cells

“…Cells were allowed to adapt for 2 h before transfection. Transfection mixture (0.5 liter total) was prepared with 2 mg of plasmid DNA and 5 mg of PEI (Tuvesson et al, 2008) and transferred to the culture. Four hours after transfection 5 liters of supplemented DHI medium was added to the culture.…”

“…Large-Scale Transient Transfection Suspension Cultures. The cultures with MRP2 and BCRP were performed according to the largescale protocol published earlier (Tuvesson et al, 2008). The transfection was carried out at a cell density of 1.0 ϫ 10 6 cells/ml in half of the final volume for several hours, after which the remaining medium was added.…”

“…The HEK293-EBNA cell line was chosen because it exhibits a low background of both uptake and efflux transporters (Ahlin et al, 2008(Ahlin et al, , 2009) and has been used widely for expression of various recombinant proteins (Wurm, 2004). Moreover, this cell line is amenable to large-scale polyethylenimine (PEI)-mediated transient transfection for large-scale expression of recombinant protein (Tuvesson et al, 2008). By using this method, high-activity membrane vesicles containing P-gp, MRP2, or BCRP were produced in large quantities from transiently transfected HEK293-EBNA cells.…”

Rapid, Large-Scale Production of Full-Length, Human-Like Monoclonal Antibodies

“…In addition, these experiments observed the use of 100% conditioned media during transfection resulted in a reduced titer. This observation is also consistent with reported studies in literature Tuvesson et al This study also found that 50% conditioned media did not significantly reduce protein levels. Tuvesson et al (2008) found that the use of more than 25% conditioned media during transfections impairs transfections.…”

“…High titer processes, however, utilize high density cultures with cell concentration and medium exchange steps prior to transfection. All such protocols published to date, utilize external batch centrifugation, using lab-scale centrifuges, for media exchange and cell concentration Tuvesson et al 2008). This method is typically limited in scale to 20 liters or less, as cells are typically processed in batches of 6-L or less (i.e, 6 x 1-L bottles).…”