Methotrexate gene amplification for development of erythropoietin with 2 additional N-link producing cell lines

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As a workhorse for manufacturing recombinant proteins, ChineseHamster Ovary(CHO)cells are becoming an extremely valuable cell for producing recombinant proteins having glycosylation patterns like in human system. Development of cell line, basically, focused on achieving the maximum amount of active proteins by screening the cell line to select higher producing clone. Currently, one of mammalian cell line development technologies used by most biopharmaceutical companies is based on gene amplification technology with methotrexate (MTX) to increase protein expression. With that in mind, the aim of this study is part of an effort to develop cell line capable of producing erythropoietin with 2 additional N-links. Gene amplification was performed on CHO-DG44 cell pools expressing EPO through addition of different concentrations of MTX to medium. The cells were cultured using orbital shaker at the speed of 130 rpm at 37°C and 5% CO2 condition. Stepwise increasing MTX concentrations from 200 and 300nM to 4000 nM were carried out. To measure the EPO produced from the culture, ELISA analysis was performed. The whole process of this amplification took approximately 60 days. From this study it was found that the use of 4000 nM of MTX gave the highest titers of EPO which was approximately 170 mg/liter. This data shows that stepwise increasing MTX concentration is very powerful step for gene amplication and subsequently for cell line development as a whole.