Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

Abstract

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL(-1) 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These 'nucleotide ratios' changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.

Transient pentameric IgM fulfill biological function—Effect of expression host and transfection on IgM properties

“…Transient transfection poses an attractive method for fast and efficient IgM screening and a useful tool for fast IgM production. However, low reproducibility in transient transfections might contribute to high variabilities during transfections as a result of variabilities in transfectability of the host cell line [36]. Transient transfection may be accompanied by stress due to polyethyleneimine treatment and therefore result in less processed glycan forms.…”

Stable recombinant production of codon‐scrambled lubricin and mucin in human cells

“…The vector also contained a second cassette under control of an EF1α promoter for expression of the rtTA-M2 tetracycline transactivator and a bicistronic neomycinresistance gene for selection ( Figure 1e). To test the performance of the expression system, we cloned mCherry2 into the vector and transfected 293-F cells with cationic PEI condensates following standard protocols (Boussif et al, 1995;de los Milagros Bassani Molinas, Beer, Hesse, Wirth, & Wagner, 2014;Sonawane, Szoka Jr. & Verkman, 2003). Stable cell populations were isolated after 2 weeks of selection, and mCherry2 production was validated by flow cytometry.…”

Selection and Characterization of Single-Stranded DNA Aptamers Binding Human B-Cell Surface Protein CD20 by Cell-SELEX

“…A gradually increasing dose of hygromycin B (150, 250, 350 and 450 µg/mL) was employed to select the clones with the highest expression of CD20 for further experiments. The overexpression of the CD20 membrane protein was evaluated by flow cytometry (Sysmex Partec GmbH, Görlitz, Sax Germany) using FITC-conjugated anti-CD20 monoclonal antibody (The Dako Group, Glostrup, Denmark) [ 21 ].…”

“…Though, monoclonal antibodies act differently against each of the CD20 epitopes and there is also a great interest in the production of novel antibodies against CD20, but due to some of the disadvantages of classical antibodies such as thermal instability and immunogenicity, this makes limitation of using antibodies and leading to create alternative molecules resemble to them. Among these alternative molecules, the production of aptamers were considered [ 21 ]. Aptamers are desirable because of rapid detection methods, cheap, fast and reliable with no complexity.…”