(2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Abbreviations: CHO, Chinese hamster ovary; HC, heavy chain; LC, light chain; mAb, monoclonal antibody; F2A, 2A peptide derived from the foot-and-mouth disease virus; E2A, 2A peptide derived from the equine rhinitis virus; P2A, 2A peptide derived from the porcine teschovirus-1; T2A, 2A peptide derived from the Thosea asigna virus; GF2A, F2A with the GSG linker; GE2A, E2A with the GSG linker; GP2A, P2A with the GSG linker; GT2A, T2A with the GSG linker; IgG, immunoglobulin G; IRES, internal ribosome entry site; G, glycine; P, proline; K, lysine; MTX, methotrexate; SEC, size exclusion chromatography; MS, mass spectrometry; PFM, protein-free medium; HT, hypoxanthine and thymine; GFP, green fluorescence protein; PVDF, polyvinylidene difluorideLinking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

Many important therapeutic proteins are produced in recombinant mammalian cells. Upon the introduction of the product gene, the isolated clones typically exhibit a wide range of productivity and high producers are subsequently selected for use in production. Using DNA microarray, two-dimensional gel electrophoresis (2DE), and iTRAQ as global surveying tools, we examined the transcriptome and proteome profiles of 11 lines of NS0 cells producing the same antibody molecule. Genes that are significantly differentially expressed between high and low producer groups statistically fall into a number of functional classes. Their distribution among the functional classes differs somewhat between transcriptomic and proteomic results. Overall, a high degree of consistency between transcriptome and proteome analysis are seen, although some genes exhibiting inconsistent trends between transcript and protein levels were observed as expected. In a novel approach, functional gene networks were retrieved using computational pathway analysis tools and their association with productivity was tested by physiological comprehension of the possible pathways involved in high recombinant protein production. Network analysis indicates that protein synthesis pathways were altered in high producers at both transcriptome and proteome levels, whereas the effect on cell growth/death pathways was more prominent only at the transcript level. The results suggest a common mechanism entailing the alteration of protein synthesis and cell growth control networks leading to high productivity. However, alternate routes with different sets of genes may be invoked to give rise to the same mechanistic outcomes. Such systematic approaches, combining transcriptomic and proteomic tools to examine high and low producers of recombinant mammalian cells will greatly enhance our capability to rationally design high producer cells. This work is a first step towards shedding a new light on the global physiological landscape of hyper productivity of recombinant cells.

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