Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster ovary cells

摘要: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet rising demand novel production with high productivity. Destabilization dihydrofolate reductase (dhfr) selection marker by addition AU-rich elements murine ornithine decarboxylase PEST region was previously shown improve specific productivities recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated combinations engineered motifs further attenuation alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem promote protein degradation, internal ribosome entry site (IRES) mutations impede translation initiation, codon-deoptimized dhfr reduce efficiency. After a 2-step methotrexate (MTX) amplification 50 nM that took less than 3 months, expression IRES point mutation dhfr-PEST gave maximum titer 1.05 g/l top producer pool. Further MTX 300 1.15 g/l. Relative transcript copy numbers pools were also analysed demonstrate transcription rhA1AT genes correlated due linkage, strategies attenuating use mutated PEST, but not codon deoptimization, effective reducing levels suspension serum free culture. Novel studied result highest reported our knowledge shake flask batch culture stable mammalian at 1.15 g/l, highlighting applicability optimization generating producing cells essential therapeutics Our results suggest usage should be considered applications may involve gene culture, since overall thus general regulation host proteins affected surviving