title: Impact of gene vector design on the control of recombinant monoclonal antibody production by Chinese hamster ovary cells.
abstract: In this study, we systematically compare two vector design strategies for recombinant monoclonal antibody (Mab) synthesis by Chinese hamster ovary (CHO) cells; a dual open reading frame (ORF) expression vector utilizing separate cytomegalovirus (CMV) promoters to drive heavy chain (HC) and light chain (LC) expression independently, and a single ORF vector design employing a single CMV promoter to drive HC and LC polypeptide expression joined by a foot and mouth disease virus F2A polypeptide self-cleaving linker sequence. Initial analysis of stable transfectants showed that transfectants utilizing the single ORF vector designs exhibited significantly reduced Mab production. We employed an empirical modeling strategy to quantitatively describe the cellular constraints on recombinant Mab synthesis in all stable transfectants. In all transfectants, an intracellular molar excess of LC polypeptide over HC polypeptide was observed. For CHO cells transfected with the single ORF vectors, model-predicted, and empirical intracellular intermediate levels could only be reconciled by inclusion of nascent HC polypeptide degradation. Whilst a local sensitivity analysis showed that qMab of all transfectants was primarily constrained by recombinant mRNA translation rate, our data indicated that all single ORF transfectants exhibited a reduced level of recombinant gene transcription and that Mab folding and assembly reactions generically exerted greater control over qMab. We infer that the productivity of single ORF transfectants is limited by ER processing/degradation "capacity" which sets a limit on transcriptional input. We conclude that gene vector design for oligomeric recombinant proteins should be based on an understanding of protein-specific synthetic kinetics rather than polypeptide stoichiometry.
authors: Dept. of Chemical and Biological Engineering, University of Sheffield, Mappin St., Sheffield, U.K.
authors: Sarah L
authors: SL
authors: Davies
authors: Peter M
authors: PM
authors: O'Callaghan
authors: Jane
authors: J
authors: McLeod
authors: Leon P
authors: LP
authors: Pybus
authors: Yun Hee
authors: YH
authors: Sung
authors: James
authors: J
authors: Rance
authors: Stephen J
authors: SJ
authors: Wilkinson
authors: Andrew J
authors: AJ
authors: Racher
authors: Robert J
authors: RJ
authors: Young
authors: David C
authors: DC
authors: James
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism*; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins