Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology

Abstract

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO-S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO-S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO-S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO-S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression.