Internal ribosome entry site (IRES) elements, which have a length of several hundred base pairs, allow expression of multiple genes in one mRNA. When vectors get fragmented, the mRNA unit would be incomplete and no genes would be expressed. Tricistronic vectors that express LC, HC, and selection marker genes in one mRNA are able to alleviate the above problems of traditional vectors. Having HC in excess can cause ER stress and proteasome overloading, creating a burden to the cell machinery and can inhibit cell proliferation. The ratio of LC:HC expression can also affect mAb qualities such as aggregation and glycosylation. It has been demonstrated that expression of LC in excess was beneficial for mAb expression –. LC is required to facilitate the folding and release of HC from BiP to form a complete IgG monomer. The other disadvantage is the lack of control over the ratio of LC:HC expression. One disadvantage of such designs is that vector fragmentation could result in non-expressing clones surviving drug selection. Each gene is under the control of its own promoter and transcribed separately. CHO DG44 cells are commonly used due to their compatibility with dihydrofolate reductase (DHFR), an amplifiable selection marker. mAbs are commonly produced by stable transfection of Chinese hamster ovary (CHO) cells with the HC, LC and selection marker on either one or two separate vectors –. Most mAbs in the market are immunoglobulin G (IgG) consisting of two identical heavy chain (HC) and two identical light chain (LC) polypeptides assembled via disulfide bridges. Monoclonal antibodies (mAbs) are currently the fastest growing class of biotherapeutic molecules. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by the Biomedical Research Council/Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. Received: NovemAccepted: ApPublished: May 21, 2013Ĭopyright: © 2013 Ho et al. Jude Children's Hospital, United States of America (2013) Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.Ĭitation: Ho SCL, Bardor M, Li B, Lee JJ, Song Z, Tong YW, et al. 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. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. 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. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. 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. LC and HC genes are arranged as either the first or the second cistron. 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.
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