Regulation of recombinant protein expression during CHO pool selection enhances high producer frequency

To address the needs of the growing antibody market, it is crucial to develop robust and efficient fed-batch cell culture processes that can ensure high product yields and quality. In this work, we have investigated the combined effect of both process and metabolic engineering strategies on the N-glycosylation state of an antibody. To this end, we have used two CHO cell clones exhibiting distinct phenotypes and performed fed-batch cultures employing two concentrated feed formulations, allowing us to infer the relative impact of these changes on antibody productivity and product quality. The comparative study was done with a parental CHO cell line and one clonal derivative stably expressing the PYC2 gene and characterized by a significantly altered lactate metabolism. We have also assessed these strategies in combination with or without manganese and galactose addition, two culture supplements commonly employed to modulate the glycosylation profile of antibodies. A comparative study of the resulting glycan distribution profiles revealed that the degrees of galactosylation and sialylation were largely similar between the two cell lines, despite their significantly different lactate metabolism. However, higher product yields were consistently achieved with PYC2-expressing cells and their faster production kinetics enable an early harvest that can circumvent the adverse effects of increased culture duration on antibody glycosylation. Our study also shows that galactose and manganese supplementation can help to mitigate the negative impacts of culture longevity on antibody glycosylation.