Lactate metabolism variations are frequently encountered in mammalian cell culture processes, especially during process scale-up. In this study, we took a multipronged approach to investigate the impact of pH, pCO₂ , osmolality, base addition, and mixing conditions on the observed lactate variations in a Chinese Hamster Ovary (CHO) fed-batch process at 2,000 L scale. Two cultivating methods, CO₂ -controlled and pH-controlled, were used to decouple the individual and synergistic effects from those factors. The individual effects from pH, pCO₂ , and osmolality on lactate consumption/reproduction in the stationary phase were insignificant in the ranges studied though the initial lactate production rates varied. In contrast, lactate metabolism was found to be impacted by an interaction between mixing conditions and CO₂ accumulation. High CO₂ accumulation and poor mixing led to lactate reproduction, whereas either low CO₂ or improved mixing were sufficient to result in lactate consumption. Base addition was not required for pH control in the low CO₂ conditions, and therefore lactate reproduction was correlated with base addition under poor mixing conditions. Under good mixing conditions, CO₂ -triggered base addition did not significantly impact lactate reproduction. It is reasonable to postulate that increased mixing times further promoted lactate production during base addition. As lactate reproduction results in more base addition to maintain pH, a cycle could be formed between lactate production and base addition. As a remediation, we showed that such lactate reproduction could be eliminated by improving CO₂ removal at 2,000 L scale. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:756-766, 2018.

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