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Abstract Background Escherichia coli is one of the most important hosts for production of recombinant proteins in biopharmaceutical industry. However, when selecting a suitable production strain, it is often not considered that a lot of different sub-species exist, which can differ in their genotypes and phenotypes. Another important development step is the scale-up of bioprocesses with the particular challenge that heterogeneities and gradients occur at production scale. These in turn can affect the production organism and can have negative impact on the process and the product quality. Therefore, researchers developed scale-down reactors, which are used to mimic manufacturing conditions in laboratory scale. The main objectives of this study were to determine the extent to which scale-related process inhomogeneities affect the misincorporation of non-canonical amino acids into the recombinant target protein, which is an important quality attribute, and whether strain specific properties may have an impact. Results We investigated two industrially relevant E. coli strains, BL21(DE3) and HMS174(DE3), which produced an antigen binding fragment (Fab). The cells were cultivated in high cell density fed-batch mode at laboratory scale and under scale-down conditions. We demonstrated that the two host strains differ significantly with respect to norleucine misincorporation into the target protein, especially under heterogeneous cultivation conditions in the scale-down reactor. No norleucine misincorporation was observed in E. coli BL21(DE3) for either cultivation condition. In contrast, norleucine incorporation into HMS174(DE3) was already detectable in the reference process and increased dramatically in scale-down experiments. Norleucine incorporation was not random and certain positions were preferred over others, even though only a single codon exists. Differences in biomass and Fab production between the strains during scale-down cultivations could be observed as well. Conclusions This study has shown that E. coli BL21(DE3) is much more robust to scale-up effects in terms of norleucine misincorporation than the K12 strain tested. In this respect, BL21(DE3) enables better transferability of results at different scales, simplifies process implementation at production scale, and helps to meet regulatory quality guidelines defined for biopharmaceutical manufacturing.