Chinese hamster ovary (CHO) cells are preferred for the production of biopharmaceuticals, due to their ability to express a variety of biopharmaceuticals at high titers with proper post-translational modifications. Current technologies facilitate two major aspects in stable cell generation – integration of the gene of interest (GOI) into the chromosomal DNA, and amplification of GOI to increase product yield. Cells that have successfully integrated the GOI are selected using “selection markers” based on antibiotic selection (Neomycin, Puromycin, Hygromycin, etc.) or metabolic selection (dihydrofolate reductase (DHFR)/methotrexate (MTX) selection and glutamine synthetase (GS)/ methionine sulfoximine (MSX) selection). In addition to enabling cell-selection, MTX also promotes gene amplification. Current technologies suffer various disadvantages including: time taken to select cells with stably integrated GOI, continuous expression of selection marker proteins impact the overall titer, and limitation of cell line options due to a necessity for auxotrophic/knockout cell lines (i.e. DHFR-ve, GS-ve) and finally gene amplification causes production instability. Thus, an innovation is required to generate stable cell lines without the above disadvantages. We have developed a technique, PTSelect™ that uses post-transcriptional regulation to overcome the above disadvantages. Here, there are no “selection markers”. Instead we have engineered an siRNA that is not originally found in the CHO genome, within an intron upstream of the GOI. Upon transcription of the GOI, functional siRNAs are processed and released post-transcriptionally. After transfecting cells with the GOI/siRNA plasmid, the selection of cells with integrated GOI is performed by transfecting an mRNA designed to be regulated by the siRNA processed along with the GOI. By exploiting the variable expression of the mRNA product due to siRNA downregulation, we were able to successfully select the cells that have integrated the GOI and siRNA construct into the genome. Using this technique, we were able to create pool clones within 14 days after transfection. This study compares various steps in the making of stable cell lines, production and stability of clones generated by PTSelect™ with antibiotic selection. PTSelect overcomes the limitations of drug-resistance genes by eliminating the need for the continued production of an extra protein thereby increasing GOI production and by reducing the selection timeframe.

Comparative analysis: An innovative siRNA-based technology versus traditional antibiotic-selection in the creation of stable expression of proteins/antibodies in CHO-K1 cells

2019 Spring National Meeting

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