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Comprehensive characterization of dihydrofolate reductase‐mediated gene amplification for the establishment of recombinant human embryonic kidney 293 cells producing monoclonal antibodies
Author(s) -
Lee Sang Yoon,
Baek Minhye,
Lee Gyun Min
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000351
Subject(s) - hek 293 cells , dihydrofolate reductase , monoclonal antibody , microbiology and biotechnology , biology , cell culture , recombinant dna , transfection , embryonic stem cell , gene , chinese hamster ovary cell , antibody , biochemistry , genetics
Human embryonic kidney 293 (HEK293) cells with glycosylation machinery have emerged as an alternative host cell line for stable expression of therapeutic glycoproteins. To characterize dihydrofolate reductase/methotrexate (DHFR/MTX)‐mediated gene amplification in HEK293 cells, an expression vector containing dhfr and monoclonal antibody (mAb) gene was transfected into dhfr ‐deficient HEK293 cells generated by knocking out dhfr and dhfrl1 in HEK293E cells. Due to the improved selection stringency, mAb‐producing parental cell pools could be generated in the absence of MTX. When subjected to stepwise selection for increasing MTX concentrations such as 1, 10, and 100 nM, there was an increase in the specific mAb productivity ( q mAb ) of the parental cell pool upon DHFR/MTX‐mediated gene amplification. High producing (HP) clones with a q mAb of more than 2‐fold of the corresponding cell pool could be obtained using the limiting dilution method. The q mAb of most HP clones obtained from cell pools at elevated MTX concentrations significantly decreased during long‐term culture (3 months) in the absence of selection pressure. However, some HP clones could maintain high q mAb during long‐term culture. Taken together, a stable HP recombinant HEK293 cell line can be established using DHFR/MTX‐mediated gene amplification together with dhfr − HEK293 host cells.