(5g) Uncovering the Mechanisms of Methotrexate-Mediated DHFR Amplification In Chinese Hamster Ovary Cells Through Transcriptome Analysis

In the past decade, the specific productivity of recombinant mammalian cells has increased to a level rivaling that of professional secretors in vivo. The development of such high protein-producing cell lines is generally aided by gene amplification, a process that leads to the creation of several copies of the recombinant gene on the chromosome, often resulting in dramatic increases in productivity. However, the physiological mechanisms of the amplification process that can transform a non-secretory cell line to achieve the productivities rivaling professional secretors, remain largely unknown. To this end, we used transcriptome analysis to study the amplification process in CHO cells.  A parental CHO cell line deficient in dihydrofolate reductase (DHFR) activity was transfected with a vector expressing a DHFR gene, a hygromycin-resistant gene, as well as antibody product genes. Methotrexate treatment was applied following transfection and initial selection. Clones isolated following transfection showed significant variation in their product expression profiles, as well as varying degrees of gene amplification. As a control, the same cell line was transfected with another vector carrying only the DHFR gene, thus providing a baseline to assess the importance of the expressed product in the transcriptional response. Following amplification, the clones were further subcloned, adapted to suspension and scaled up to fedbatch processes. Transcriptome analysis was performed on clones during the amplification process as well as on the resulting high and low producer subclones in fedbatch cultures. This study provides physiological insights into the mechanisms involved in gene amplification and clonal selection, and allows the design of a screening strategy for the selection of viable production cell line candidates.