The Engineering of Chinese Hamster Ovary Cells to Achieve More Efficient Gene Amplification for Improving Biopharmaceutical Development

This dissertation addresses the issue of the long development times of obtaining Chinese hamster ovary (CHO) cells capable of producing high quantities of therapeutic proteins. It addresses the specific time bottlenecks associated with developing high producing CHO cell lines and reviews various methods that are employed to alleviate these bottlenecks. The specific time consuming process of gene amplification is the focus of this work. Gene amplification is the process of selecting CHO cells which have been genetically modified to contain many copies of a therapeutic transgene, and therefore has the ability to produce a high amount of therapeutic protein. Two separate projects are described which decrease the time necessary to obtain a high producing cell. The first project describes a novel process developed which can measure and quantify the amplification rate of a transgene in CHO cells. This process was used to successfully isolate a CHO cell clone with the capability of amplifying a transgene targeted to a specific location in the genome and thus produce higher quantities of protein in a shorter time period. Site-specific recombination (SSR) technology was utilized to target the transgene to this location which was deemed capable of amplifying a transgene at a high rate. The second project also utilizes SSR technology to integrate many copies of a transgene into many recombination sites in the CHO genome. A cell line containing several thousand integration sites was isolated, however only about twenty of these sites successfully integrated a transgene after optimizing cell transfection conditions. Efforts towards engineering an improved recombinase for this purpose has led to the result that DNA sequences flanking recombination sites have the ability to greatly improve this integration process. Potential future experiments are described which may isolate such sequences and ultimately increase the number of transgenes integrated into the CHO cell genome. Overall, these improvements to CHO cells have the ability to ultimately isolate a higher producing cell line faster, thus decreasing the time to get a potential drug candidate to market.