Using Design of Experiments to Increase Protein Yield in HEK293 6E cells

HEK293 6E cells (which we have on license from the National Research Council (NRC) in Canada) are a well-established mammalian expression system used at Peak Proteins for expressing recombinant proteins. In this case study Sarah Beck describes the experiments that Calum Wilson our summer intern student performed under her guidance, that aimed to improve our standard HEK cell culture protocols. Calum carried out a Design of Experiments (DoE) study to investigate transfection conditions and feeding regimes in HEK293 6E cells with the overall aim being to optimise yields of recombinant protein.

Which variables were investigated?

Cell density at transfection, DNA concentration, DNA:PEI ratio, and feeding regimes were the variable studied. Two of these were categorical factors so we were unable to use a definitive screening design. Instead, a custom design was created in the JMP software that still allowed us to investigate 2-way factor interactions in two blocks of 12 runs, with each block including a control with our current culture conditions. In total twenty-four different 50ml cultures of HEK293 6E cells were performed with cell viability and mCherry protein expression levels compared as the output for each condition tested. The data obtained was statistically analysed with the JMP software.

Interpretation of DoE data

Analysis of DoE data for mCherry yield showed the interaction of DNA:PEI ratio and DNA concentration together had more of a significant effect (P value < 0.05) than individual variables on their own (Figure 1). It was proven to be a good model with R-squared value of 0.91, suggesting that 91% of the observed variation could be explained by the model generated by the software. Using the prediction profiler tool in JMP showed the optimal settings for each of the factors for highest mCherry yield. These conditions were matched by the analysis of the cell viability output (data not shown).
e optimal conditions for highest mCherry yield.

Figure 1: Logworth analysis of DoE data for mCherry yield

Confirmation Run: Testing the predicted optimal conditions in HEK cell transfections

A confirmation run was then performed with the new predicted optimal settings from the DoE for each of the factors. mCherry transfections in HEK cells were tested and compared to standard (original) conditions (Figure 2). It was found that the new optimal conditions produced a higher, 80% improved, mCherry yield and these cells also had a higher percentage cell viability at harvest compared to our standard conditions.

Figure 2: mCherry yield at harvest for HEK cells transfected in standard conditions versus optimal conditions.

Conclusion

This Design of Experiments (DoE) study was a rapid way to investigate four variables to increase protein yield in our HEK293 6E mammalian expression system. Optimal conditions were identified which successfully improved mCherry yield by 80%. These also included using a lower cell density and DNA concentration at transfection which is advantageous in terms of consumption of materials. Design of experiments is a hugely powerful tool and will be extended to optimise other cell culture protocols in use at Peak Proteins.

Applying DoE at Peak Proteins for your projects

As mentioned above, we can apply DoE variable testing to optimise cell growth. We have also use this to optimise a client protein purification protocol. If you have a protein expression and purification project that you think would benefit from a DoE approach, then please don’t hesitate to get in touch with us at info@peakproteins.com.