Date Published: June 6, 2019
Publisher: Public Library of Science
Author(s): Maren Bleckmann, Margitta Schürig, Michelle Endres, Anke Samuels, Daniela Gebauer, Nadine Konisch, Joop van den Heuvel, Saw-See Hong.
Virus-free, transient gene expression (TGE) in High Five cells was recently presented as an efficient protein production method. However, published TGE protocols have not been standardized to a general protocol. Therefore, reproducibility and implementation of the method in other labs remains difficult. The aim of this study is to analyse the parameters determining the reproducibility of the TGE in insect cells. Here, we identified that using linear 40 kDa PEI instead of 25 kDa PEI was one of the most important aspects to improve TGE. Furthermore, DNA amount, DNA:PEI ratio, growth phase of the cells before transfection, passage number, the origin of the High-Five cell isolates and the type of cultivation medium were considered. Interestingly, a correlation of the passage number to the DNA content of single cells (ploidy) and to the transfection efficacy could be shown. The optimal conditions for critical parameters were used to establish a robust TGE method. Finally, we compared the achieved product yields in High Five cells using our improved TGE method with both the baculoviral expression system and TGE in the mammalian HEK293-6E cell line. In conclusion, the presented robust TGE protocol in High Five cells is easy to establish and produces ample amounts of high-quality recombinant protein, bridging the gap in expression level of this method to the well-established mammalian TGE in HEK293 cells as well as to the baculoviral expression vector system (BEVS).
Protein expression in insect cells has serval advantages. Insect cell lines offer the majority of posttranslational modifications e. g. phosphorylation of serine , tyrosine sulfation  or palmitoylation , while being easier to handle than mammalian cells. In comparison to mammalian cells, insect cells can be cultivated without additional CO2 aeration at 27°C in serum-free media, thereby decreasing costs for the experimental set-up and reducing the risk of contamination with human pathogens . However, compared to mammalian cells the glycosylation pattern in insect cells mainly consists of truncated N-glycans of the paucimannose type . This glycosylation pattern is a relevant difference for secreted glycoprotein from insect cells, as mammalian glycosylation might be required for pharmaceutical applications and/or full biologic activity. To circumvent this limitation, both insect cell lines and baculovirus expression system have been adapted to achieve authentic mammalian-like glycosylation [6–9]. On the other side, the paucimannose glycosylation type is more homogenous and less flexible, which presents an advantage for 3 D structural analysis by crystallization. The high number of eukaryotic membrane protein structures deposited in the Protein Database (PDB) reflects this advantage of proteins produced in insect cells .
In this study we tested our plasmid-based method published before  in regard of reproducibility and feasible implementation in different laboratories. The replacement of the linear 25 kDa PEI by linear 40 kDa PEI was the most important factor to circumvent the observed batch-to-batch variability in performance of different 25 kDa PEI Lots. So far, we could confirm that tested 40 kDa PEI Lots do not show relevant batch-to-batch variability for independently purchased lots. In addition, 40 kDa PEI performance was not influenced by different storage conditions and time of storage. Furthermore, we could successfully transfer and implement the described method to different laboratories by cultivating the commercially available High Five cell line (Thermo Fisher) over ~80 passages in EX-CELL 405 medium. The high passage number resulted in mainly tetraploid cells likely, explaining the increase in recombinant protein production. The cell density of the pre-culture was not influencing the reproducibility of the method, as long as exponential growth was maintained and viability was above 95%. In contrast, the choice of culture medium is important for high levels of transfection efficiency and protein production. Cells cultivated in EX-CELL 405 medium showed the best performance in TGE.