Key result
While F2A-mediated tricistronic vectors yielded higher monoclonal antibody expression levels than IRES-mediated vectors in CHO cells, over 40% of the F2A products were aggregates and improperly processed.
IRES-mediated tricistronic vectors, particularly with the light chain as the first cistron, are superior to F2A-mediated vectors for producing high-quality, properly processed monoclonal antibodies in CHO cells.
IRES vectors may improve mAb quality in CHO production; leaves open optimal vector selection for therapeutic manufacturing.
Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.
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Ho et al. (2013) studied this question. Furin-2A (F2A) mediated tricistronic vectors vs. Internal Ribosome Entry Site (IRES) mediated tricistronic vectors was evaluated on Monoclonal antibody expression level and quality. While F2A-mediated tricistronic vectors yielded higher monoclonal antibody expression levels than IRES-mediated vectors in CHO cells, over 40% of the F2A products were aggregates and improperly processed.
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