Developing a lactate sensing and controlling system in CHO cells

Chinese Hamster Ovary (CHO) cells are the preferred host for the biomanufacturing of monoclonal antibodies (mAb), which represent more than 50% of the FDA approved biopharmaceuticals. Cultured CHO cells show enhanced glycolysis during exponential cell growth, and lactate is formed as a by-product. Acidification of the culture media by the accumulation of lactate is typically alleviated by the addition of a base. Both increased lactate concentration and addition of a base decrease cell viability and show impaired mAb production. Unsurprisingly, lactate has been identified as the single most important process parameter in CHO cell cultures, and lower lactate has been shown to correlate with increased final mAb titre in industrially-relevant cultures. Current solutions to mitigate lactate accumulation in the biotechnology industry are based on media and process optimisation. However, they have not been established in large-scale mammalian cGMP cultures. An alternative approach is based on cell line engineering strategies, such as rewiring the fate of cytosolic pyruvate away from lactate production. However, these approaches usually rely on the constitutive expression of transgenes, which cannot dynamically respond to changes throughout cell culture. Inducible systems that can pair the expression of transgenes with culture phases have shown higher transgene expression than 'constitutive' expression systems. The overarching goal of this Thesis is to establish a genetic 'sense-and-control' system in CHO cells, in which an effector gene is placed downstream of a lactate-inducible regulatory module, aiming to mitigate lactate build-up. In this configuration, the effector is expressed only when lactate accumulates in the cell environment (an autonomous response), and its expression level can vary as a function of lactate concentration (a dynamic response), thus limiting lactate accumulation and improve mAb production.