Effects of selectable markers on the generation of high monoclonal antibody-producing cell lines.
Lee, Eric Yong Jing.
Date of Issue2009
The production of therapeutic monoclonal antibody is a time-consuming and costly process. With the increasing global demand for these recombinant proteins for therapeutic and research purposes, there is thus a need to improve this long and expensive production process. We have constructed several recombinant expression vectors to investigate the effects of selectable markers on the generation of high monoclonal antibody producing cell lines. The selectable markers investigated include neomycin-phosphotransferase (NPT) and its mutant variant which had reduced activity, dihydrofolate reductase (DHFR) and glutamine synthetase (GS). We stably selected for the cells and identified potential high-producers through surface fluorescent staining followed by fluorescent activated cell sorting. Due to time constraint, we could only assess the effect of wild-type NPT and its mutant variant. We observed that there were more potentially high-producing cells when the mutant NPT was used as compared to the wild-type NPT. This observation suggested that the mutation of the selectable marker sequence leading to its reduced activity had enhanced the stringency. As such, more high-titer cell clones were generated. Other selectable markers could be altered in a similar fashion to generate high-producing cell lines.
Final Year Project (FYP)
Nanyang Technological University