Comprehensive characterization of dihydrofolate reductase‐mediated gene amplification for the establishment of recombinant human embryonic kidney 293 cells producing monoclonal antibodies

Abstract

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 longterm 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.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells

“…After 3 days, the limiting dilution method was performed in 96-well culture plates. Every single clone generated in the 96-well plates was split into two 96-well plates containing DMEM supplemented with 10% dFBS with or without 1× hypoxanthine-thymidine (HT; Thermo Fisher Scientific) to select for DHFR − CHO-K1 clones via HT-dependency screening ( Lee et al, 2021 ). To confirm the modifications of the genomic DHFR sequence, genomic DNA was extracted from the HT-auxotrophic clones using QuickExtract™ (Lucigen, Teddington, United Kingdom) and amplified using primers specific to the DHFR target region.…”

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

“…Process and media parameters, such as expression vector, growth media, transfection agent and culture conditions, have been optimized to improved recombinant protein yields from HEK293. Notable innovative approaches include 3D collagen microsphere culture system [ 41 ]; the establishment of a glutamine-ammonia ligase (GLUL) mediated gene selection system [ 42 ]; glutamine synthase (GS) mediated selection/amplification system, and a dihydrofolate reductase (DHFR) amplification system [ 43 , 44 ], synonymous with the CHO system [ 45 ]. Growth and media optimization have led to the development of several formulations of chemically defined growth media and animal-derived component free media additives tailored to HEK293 cell growth and protein expression [ 46 ].…”

Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells

“…Due to their ability to produce proteins including natural human products, the production of therapeutic proteins in human cell lines, particularly in human embryonic kidney 293 (HEK293) cells, is expanding [ 1 , 2 ]. HEK293 cells have been utilized as expression host cells for transient gene expression, but recent studies have established HEK293-based expression systems for stable and high-level production of therapeutic proteins [ 2 , 3 ]. To improve recombinant protein production, various approaches have been developed in industrially relevant mammalian cells such as CHO and HEK293 cell lines, among which expression under mild hypothermia (MH; 30–35 °C) is an effective method to extend culture longevity and improve productivity [ 4 , 5 , 6 ].…”