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.

The Development of Cell-Penetrating Peptides for Efficient and Selective In Vivo Expression of mRNA in Spleen Tissue

“…Although both the NF55 and PF14 were able to transfect almost all cells with mRNA ( Figure S2 b), PF14 did not achieve as high as the total reporter levels. We have previously shown that for the pDNA delivery, both CPPs show similar efficacies and effects on cell viability [ 17 ]. Nevertheless, the CPPs may affect other cellular processes, including protein translation or exhibit other unknown undesired side-effects.…”

“…The best performing CPP was NF55, which induced high expression of luciferase with both mRNA and pDNA. Interestingly, the CPP NF71, which was designed for siRNA delivery [ 11 ], also achieved efficient miRNA delivery [ 12 ] as well as efficient pDNA delivery [ 17 ] ( Figure 1 a) and negligible reporter expression with mRNA. This could be due to the less optimal complexation–release of NF71/mRNA complexes compared to shorter RNA or larger pDNA.…”

Approaches for evaluation of novel CPP-based cargo delivery systems

“…The level at which the delivery system should be tested depends on the application. For example, the application of CPPs as plasmid delivery vectors for recombinant protein production in mammalian cell cultures was assessed on CHO and HEK293 suspension cell cultures (Porosk et al, 2022). In contrast, the evaluation of the CPP-cargo for therapeutic purposes must include preclinical assessment.…”

“…The cell cycle consists of several phases and check points, which can be reflected with specific assays. BrdU-based assay, which reflects the integration of the nucleoside analog into the new strands of DNA synthesized during DNA replication, has been used to assess the effect of CPPs and other transfection reagents (Porosk et al, 2022). Ki-67 has been used as a proliferation marker for human tumor, although it reflects other additional processes (Sun and Kaufman, 2018).…”

“…Apoptotic cells can be detected by flow cytometry accompanied by specific staining, with TUNEL assay (DNA fragmentation) or Annexin V-FITC/PI double staining assay. Live/dead assay, which reflects both the viable cell population and cells with damaged membranes, is compatible with both flow cytometry and microscopy (Porosk et al, 2022). Caspase 3 assay is also useful when assessing pro-apoptotic effect of anticancer treatment.…”