Title: Efficient clone selection and enhancement of recombinant protein yield in CHO cells using FACS-based strategy and UCOE
Abstract:
In the biopharmaceutical industry, the quest for approaches to accelerate the generation of high-producing cell lines has always been challenging and crucial. In this regard, exploring novel techniques and combining them can assist in achieving this objective. One of the most challenging problems in this field is the random integration of vectors in the host genome. So, applying genetic regulatory elements such as ubiquitous chromatin-opening element (UCOE) can reduce the influence of vectors’ heterochromatin insertion and gene silencing effects. Also, utilizing a high-throughput fluorescent-activated cell sorting technique and EGFP as a reporter gene can facilitate and speed up the high-producing cell selection. In this sense, we applied UCOE to prevent the expression cassette random integration effect and performed FACS to isolate high-producing cells. Then, the impact of UCOE, FACS, and the combination of these two technologies was assessed.
To this end, two expression cassettes, pOptiVEC and UCOE-containing plasmid, CET1019HD, entailing DPO-LoxP-IRES-EGFP-LoxP-IRES-DHFR, were engineered. The LoxP sequences were applied to excise IRES-EGFP fragment after high-yield cell line development. The cloning process was confirmed by enzymatic digestion and PCR, followed by Sanger sequencing. Afterward, for the purpose of stable cell line development, both cassettes were linearized and transfected to the CHO DG44 cells through the FreeStyleTM Max reagent. The DHFR was used as a selection marker, and transfected cells were screened by changing the medium to an HT-deficient one. Subsequently, the integration of expression vectors in the host genome was assessed by PCR-Sanger sequencing. Then, the EGFP was utilized as an enrichment indicator in both populations to isolate high-producing cells through FACS. Ultimately, to evaluate the impact of UCOE and FACS on developing efficient cell lines, the DPO and EGFP expression levels were evaluated by qRT-PCR, western blotting, and ELISA.
The present research data demonstrated that UCOE highly influences on targeted protein transcription levels and protein synthesis. Isolating high-producing cells by FACS led to obtaining a cell pool with about 1.5-fold improvement of desired protein production. Combining these two approaches resulted in a population with approximately 9-fold increases in Darbepoetin alfa expression level.
Our results highlighted the potency of an EGFP-FACS-based approach for isolating high-producing cells in the shortest possible time, explored the straight relation between the gene of interest and EGFP expression level, which makes this fluorescent protein a valuable enrichment reporter, and underscored the UCOE’s substantial effect on productivity. This dual strategy, which simplifies the clonal selection process and boosts the desired protein yield, promises the strength of this method for industrial biotechnology applications.
Keywords: Cell line development, High-producing cell line, FACS, Cell sorting, UCOE
Audience Take Away Notes:
- The audience will gain the insights into the key challenges of recombinant protein production, particularly in cell line development and gene expression stability
- This dual approach offers a scalable, practical solution for enhancing target protein production and high-yield cell selection, which can be directly applied to the biopharmaceutical industries
