Transient Transfection of HEK293 Cells in Suspension : Process Characterization and Optimization by Applying Invasive Nucleotide and Non-invasive Electronic Nose Technology

Abstract

The increasing demand for milligrams of recombinant proteins (r-proteins) to be used for therapeutics and structural studies justifies the need for a rapid and scalable expression system. Transient transfection of mammalian cells is a powerful technology for the fast production of large amounts of these r-proteins. This system depends on a multitude of different factors including the cell line, its physiological state, the type of expression vector and the medium formulation. Moreover, monitoring bioprocess parameters, and control of the bioprocess at its optimal state, enable the reduction of production costs, increase the yield and maintain the quality of the desired product. Off-line and on-line monitoring methods have been used to characterize and to optimize the transient transfection process using HEK293 cells and polyethylenimine as transfection mediator. As an off-line method the intracellular nucleotide pool was used, which has been considered as a reliable tool reflecting the metabolic status, the growth potential, and the overall physiological condition of the cell. As an on-line and non-invasive method a completely new technology, the electronic nose, was applied for bioprocess monitoring and the identification of characteristic process states. HEK293 cells were cultured in suspension (HEK293s) using a completely new medium, totally free of proteins or animal-origin components, which facilitates the transfection process by allowing growth and transfection of the cells without medium exchange. This is a central pre-requisite for scaling the technology up to large volume cultivation processes. A special bicistronic plasmid was constructed under control of the cytomegalovirus promoter. The plasmid DNA was combined with polyethylenimine (PEI) using an efficient minimum amount of DNA at a DNA:PEI ratio of 0.50:1.50 (µg:µg) showing 70-80 % of GFP positive cells under serum-containing as well as serum-free culture conditions. Specific cells taken from an early (from 40 to 77) and a late (from 89 to 150) passage number were compared as hosts for transient transfection. The combined NucleotideTriPhosphate ratio expressed as NTP/U = [ATP+GTP] [UDP-GNAc] / [UTP+CTP] [UTP] was calculated in parallel during cultivation and transient transfection under serum-containing and serum-free culture conditions for process monitoring and characterization. The results showed that nucleotide ratios can be used to distinguish between: HEK293s cells from early and late passage numbers; Cells cultured under serum-containing conditions from those under protein/serum-free medium conditions; Transfected and untransfected cells in culture; DNA:PEI complex uptake (transfection procedure) from PEI uptake alone. The BioNose was connected to the off-gas line of the bioreactor cultivation. The data were collected using the NST Senstool software and evaluated applying multivariate methods such as Principal Component Analysis (PCA). The results showed, that the BioNose can generate characteristic pattern specific for a particular process being an easily accessible tool to monitor cell cultivations as well as to distinguish between different culture conditions.