Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293

Abstract

Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.

Keywords: CHO; HEK293; cell-penetrating peptides; mAb production; protein expression; protein production; transfection; transient transfection.

Figure 1

The total expressed reporter levels in adherent cells do not correlate with therapeutic protein production efficacy. (a) Transfection efficacy is assessed by quantitation of luminescence from cell lysate 24 h post-transfection of CHO-K1 adherent cells with pLuc. The top performers are highlighted in green. N = 5. (b) The production of Trastuzumab mAb in CHO 1H7 suspension cell culture in serum-free defined media 7 days after the transient transfection of pDNA. The complexes with PEI were formed at N/P ratio 20, and the complexes with CPPs were formed at CPP/pDNA charge ratio 2.5:1. They were assessed by SDS-PAAG 10%, with Coomassie blue staining. (c) List of reagents included in the further study. (d) A scheme depicting general workflow.

Figure 2

Suspension culturing improves the predictability of protein production. (a) Adherent cells were transfected with 0.2 µg of pLuc per 96-well plate well. CPP/pDNA complexes were formed at CR3:1, and PEI complexes were formed at N/P20. The total reporter levels were determined 24 h post-transfection. N = 10 for treatment groups. (b) The suspension cells were transfected with 0.1 µg of pLuc per 96-well plate well. CPP/pDNA complexes were formed at CR2:1. Total reporter levels were determined 48 h post-transfection. N = 10 for treatment groups. (c) Adherent cells were transfected in serum-free media with 0.5 µg of green fluorescent protein encoding plasmid (pGFP) per 24-well plate well. 4 h post-transfection media was changed to serum-containing media. CPP/pDNA complexes were formed at CR3:1, PEI N/P20. Analysis was performed 24 h post-transfection. N ≥ 5. (d) Suspension cells were transfected in serum-free media with 0.75 µg of pGFP per 24-well plate well. CPP/pDNA complexes formed at CR2:1. For PEI N/P20 was used in case of the HEK293FT cells and N/P60 in case of CHO-K1 cells. 4 h post-transfection 500 µL of fresh media was added to each well. 48 h post-transfection the cells were analyzed by flow cytometry. N ≥ 6 for treatment groups.

Figure 3

Using a suspension cell culture is a prerequisite for the prediction of protein production. (a) The trastuzumab mAb production in a suspension cell culture. 2 µg of pDNA was used per 6-well plate well. For CHO 1E9 cells assessment by SDS-PAAG 10%, Coomassie staining was performed 8 days post-transfection. For HEK293ALL analysis was performed 5 days post-transfection. (b–d) CHO-K1 or HEK293FT were cells transfected in serum-free media with 0.75 µg of pDNA. CPP/pDNA complexes were formed at CR2:1. For PEI N/P20 (HEK293FT) or N/P60 (CHO-K1) was used. 4 h post-transfection fresh media was added to the wells. Analysis was performed 48 h post-transfection. (b) CHO-K1 and (c) HEK293FT cells were transfected with pGFP. The fluorescent positive cell population was detected from the live cell population. The populations of fluorescent positive cells were divided into fractions of cells with low signal (+), medium signal (++), and strong signal (+++). (d) Cells transfected with pSEAP. RLU from SEAP-linked chemiluminescent assay were detected from collected media. N ≥ 3 for results shown in (b–d).

Figure 4

Cell viability post-transfection was assessed by the live/dead assay. Adherent (a) CHO-K1 cells and (b) HEK293 cells were transfected with 0.5 µg of pLuc per 24-well plate well. CPP/pDNA complexes were formed at CR3:1, and PEI complexes at N/P20. Analysis was done 24 h post-transfection. Calcein AM (live) and PI (dead) were added to detect live and dead cells. (c) The correlation graph depicting correlations between luciferase expression levels against viability of cells in adherent cell experiments. (d) The representative confocal images of cells 24 h post-treatment with pDNA, and cells transfected using jetOPTIMUS (jetOptimus) or NF55 (NF55). The red bar corresponds to 200 µm. Suspension (e) CHO K1 and (f) HEK293FT cells were transfected in serum-free media with 0.75 µg of pSEAP. CPP/pDNA complexes were formed at CR2:1, and PEI/pDNA at N/P20 (HEK293FT) or N/P60 (CHO-K1). 48 h post-transfection cells were collected, and Calcein AM and PI were added to detect live and dead cells from the whole cell population. N ≥ 3.

Figure 5

The protein production yield correlates with short-term assays of the secreted reporter protein and transfection positive population in CHO suspension culture. A scatterplot that highlights the correlations between the protein production yields and the assays of transfection-positive cell population (r = 0.86, p < 0.05) and SEAP protein secretion (r = 0.88, p < 0.05) in CHO suspension cells. The full correlation matrix with the Pearson coefficients is presented in Supplementary Table S3.