title: Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells.
abstract: 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.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Steven C L
authors.initials: SC
authors.lastname: Ho
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Jia Juan
authors.initials: JJ
authors.lastname: Lee
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
authors.forename: Yen Wah
authors.initials: YW
authors.lastname: Tong
authors.forename: Lin-Tang
authors.initials: LT
authors.lastname: Goh
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D007074:Immunoglobulin G / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D012270:Ribosomes / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D045683:Furin; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D018719:Erb-b2 Receptor Tyrosine Kinases; D014409:Tumor Necrosis Factor-alpha; D042461:Vascular Endothelial Growth Factor A