Abstract
To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R2 ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.
Highlights
For large-scale production of therapeutic proteins including monoclonal antibodies, recombinant Chinese hamster ovary cells are established using dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection or glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection[1]
CHO-K1 and GS KO host cell lines were transfected with a vector containing gs and monoclonal antibody (mAb) genes as described earlier
Due to the short timeline of the cell line generation, a GS-based MSX-selection system has been increasingly used for the establishment of recombinant Chinese hamster ovary (rCHO) cell lines
Summary
For large-scale production of therapeutic proteins including monoclonal antibodies (mAbs), recombinant Chinese hamster ovary (rCHO) cells are established using dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection or glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection[1]. The DHFR-based system, which has been extensively characterized with respect to gene amplification and production stability, has been the most widely used system because it results in high levels of gene amplification and expression[2,3,4,5,6,7,8] It requires a multi-round of MTX selection for stepwise gene amplification, resulting in a longer timeline for cell line generation[9]. Despite the increasing use of the GS-based system, rCHO cell line generation using the GS-based system has not been fully characterized with respect to gene amplification and production stability. Using two different host cell lines (CHO-K1 and GS-knockout CHO (GS KO)), mAb producing rCHO cell clones were generated by a single round of selection at various MSX concentrations. Clones generated by a single round of selection were subjected to a higher MSX concentration to evaluate the potential of GS-mediated gene amplification
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