Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

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.

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics

“…host cell line. 22,23 With the use of a low amount of 25 μM MSX on CHO-K1SV GS −/cells, the bulk culture productivity was doubled when compared to parental CHO-K1SV cells. The efficiency of finding high-producing clones (>2g/L) was increased sixfold.…”

“…The stability of the GS selection system in CHO cells has previously been evaluated. 23,25,35,36 Evaluation of high-producing GS-selected clones demonstrated that instability stems from promoter silencing, antibody gene copy loss, and generation of secondary poor-producing populations. 25,36 Gene copy loss could be due to unstable chromosomal structures, as shown previously in GS-selected CHO cells.…”

“…16,[18][19][20] However, selection in such CHO cell lines may result in the survival of a large number of poor producers, as some CHO cells are able to survive up to 5 mM MSX. 21 To improve selection stringency for the GS system, a CHO GS knockout host cell line was generated, 22,23 and enhanced productivity has been observed. To further improve selection stringency and eliminate the use of MSX, the SV40E promoter driving the GS gene expression was weakened to reduce the GS level.…”

“…It has been observed that a significant percentage of GS-selected CHO production cell lines lost more than 20-30% of their original productivity. 23,25 Thus, the process of trying to obtain a high-producing, and yet stable, clone can be time-consuming and laborious.…”

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine‐replete conditions and can be inhibited by serine

“…Given the interconnected nature of transport of amino acids, such an effect of serine may be observed to varying effects in different medium formulations. For the commercially important GS‐CHO and GS‐NS0 cell lines which are cultivated in the absence of glutamine, this is an important observation in the context of the design of culture medium and feed. Supplementation of glutamine for culture of GS‐CHO to control culture pH and reduce lactate generation is also reported and the results reported in this study will be relevant in such cases.…”