(474a) Invited Talk: Eliminating the “Essential” Warburg Effect and Other Metabolic Liabilities in Mammalian Cell Biomanufacturing

100 years ago, Otto Warburg reported a striking feature of cancer cells, wherein they consume excess glucose and secrete large amounts of lactic acid. However, this metabolic phenotype extends to many other normal and pathological settings. In particular, the Warburg effect has posed a constant challenge in mammalian bioprocessing since the field began. Indeed, the predisposition of mammalian cells to secrete large quantities of lactic acid through the Warburg effect leads to premature cell death, reduced product yields, and often lower quality products. Efforts to suppress it have only led to the emergence of more metabolic liabilities, such as ammonia. Thus, over the past decades, numerous innovations in the mammalian cell culture field have focused on mitigating the secretion of lactate and ammonia, including through media optimization, feeding control, chemical inhibition, etc. Despite extensive efforts from many researchers, these toxic metabolites remain a problem. For example, several independent efforts to knock out lactate dehydrogenase (the enzyme responsible for producing lactic acid from pyruvate) have been unsuccessful, as it has proven essential for immortalized cell growth.

Here I present our work in which we discovered panels of genes involved in genetic feedback circuits that control lactic acid secretion (5-6 genes) or are key contributors to ammonia production (3 genes) in mammalian cells. Knocking out individual genes in serial was unsuccessful since LdhA and other targets are essential for CHO cell growth. However, we knocked out these genes simultaneously and overcame the “essentiality” of these genes, leading to the successful elimination of lactic acid secretion and suppression of ammonia production in mammalian bioproduction hosts.

Since many hypotheses have been proposed regarding the essentiality of the secretion of these metabolites for rapid cell proliferation in cancer, immune cell activation, and embryonic development, we were interested to study how the complete elimination of the Warburg effect and ammonia production impacts CHO cells. Surprisingly, the cells show improved metabolic and growth phenotypes, despite the elimination of this fundamental metabolic activity. To understand how immortalized mammalian cells can cope without this seemingly essential metabolic process, we conducted a comprehensive analysis of these cell lines using time-course RNA-Seq, metabolomics, C-13 fluxomics, etc. We further characterized the impact of cell engineering on recombinant drug production yields and quality. Thus, through a multiplex metabolic engineering effort and comprehensive systems biology analysis, we have been able to engineer out leading challenges in biotherapeutic manufacturing and begin to understand now a cell can survive without a seemingly essential process.