Selection of high‐producing CHO cells using NPT selection marker with reduced enzyme activity

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Abstract
We developed an expression system that aimed to increase the proportion of high producers in a transfected cell population in order to reduce the effort in clone screening. The principle is based on the impairment of the selection marker. Twelve single‐point mutations in more or less conserved domains of the resistance marker gene neomycin‐phosphotransferase (NPT) resulted in different degrees of reduced enzyme activity, depending on the amino acid conservation and the kind of amino acid exchange. In all transfected, mutant‐NPT bearing CHO‐DG44 cell pools surviving the selection with G418, the ratio of high‐producing cells to total cell number was higher than in pools selected with wildtype‐NPT. Furthermore, these pools showed, in comparison to wildtype‐NPT selected pools, not only higher NPT‐RNA levels but also increased specific productivities and higher titers of a coexpressed biopharmaceutically relevant product. Elevated productivity could be ascribed to higher gene copy numbers, integration into chromatin regions with higher transcriptional activity, or a combination of both effects. Thus, the use of NPT‐mutants as selection markers is suitable for the enrichment of high producers in a transfected CHO‐DG44 cell population, since cell survival is achieved only if the enzymatic impairment of the cointegrated resistance marker is compensated by a higher expression level.