Establishment of Stable Recombinant Protein Expression in CHO Cells Using a PiggyBac Transposon Platform
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The generation of high-producing cell lines is extremely desirable for producing recombinant antibodies. This study aimed to optimize the expression and screening processes for high- producing cell lines to enable rapid identification.
Chinese hamster ovary (CHO) cells exhibit various characteristics that make them promising candidates for producing recombinant proteins. The process of developing high-yield stable cell lines includes transfection, selection, adaptation, and screening. This is a very laborious and time- consuming method, so different approaches should be optimized to facilitate the process. Different transfection reagents, presence and absence of hyperactive piggyBac transposase (hyPBase), and construct designs were assessed for their effects on the protein yield and expression efficiency of the cell line. Flow cytometry was used to analyse the cell population and compare the screening process with and without Spylink technology.
Three transfection reagents ExpiFectamine CHO (cationic lipid-based transfection reagent), FectoPRO reagent (cationic polymer-based reagent) and ExpiFectamine 293 (cationic lipid-based transfection reagent) were evaluated for the expression of the antibodies 9E10 IgG and 11N11- IgG-SC3. ExpiFectamine CHO yielded the highest production levels, whereas ExpiFectamine 293 resulted in the lowest yields. The hyPBase increased the yield by approximately 3 times in the transfection with a plasmid DNA construct without the internal ribosome entry site (IRES) and Blasticidin resistance gene (BlsR). Whereas in the case of the plasmid DNA with IRES BlsR the yield dropped by its addition. Whereas, the transfection with plasmid DNA with IRES-BlsR resulted in almost double the yield as compared to the yield of the transfection with plasmid DNA without IRES-BlsR. After the antibiotic selection, the viability of the cell culture transfected with hyPBase first declined till the 7th day to 42% and then rose to 85% on the 14th day. Whereas, for the cell lines transfected without hyPBase, the viability increased to 89% on the 3rd day after antibiotic selection, and on the 14th day it declined to 13%. Flow cytometry analyses predicted that Spylink technology could be a promising candidate for adaptor-based screening of high- producing transient and stable integrated cell lines.