Zinc supplementation modulates intracellular metal uptake and oxidative stress defense mechanisms in CHO cell cultures

Abstract

Trace metal variation in culture media has the potential to affect CHO cell culture performance in an unpredictable manner. Previously, zinc variation in CHO cultures were shown to impact cell growth, viability, specific activity and apoptosis [1]. In mammalian cells, zinc deficiency is frequently tied to oxidative stress. Although recombinantly produced protein drugs are affected by oxidative stress, the relationship between cellular metal uptake and key oxidative stress defense mechanisms remains unclear in CHO cells. Here, we developed a strategy to assess the intracellular zinc and iron content of cultured CHO cells. Next, we supplemented a CHO cell line producing β-glucuronidase (GUS) with 0, 50, 100, or 150 μM zinc sulfate cultured in batch mode in spinner flasks and in fed-batch mode in parallel bioreactors. Zinc supplementation impact on GUS production was marginal (≤ 6%), but, impacted glycan fucosylation by decreasing up to ∼15 % compared to control cultures. Zinc depletion in the supplemented culture medium was as much as ∼20 μM under intense fed-batch cultures in parallel bioreactors. However, in less intense spinner flasks batch cultures zinc depletion was less profound at ∼2−5 μM over the course of the run. Intracellular zinc analysis showed that most zinc consumption occurs primarily during lag phase and minimal consumption thereafter. Harvest day analyses showed a five-fold increase in intracellular iron, higher peroxidase activity and lowered total superoxide dismutase activity in zinc-deficient control cells indicating enhanced oxidative stress defense activity correlated to zinc deficiency. Comparatively smaller impact on CHO culture performance in spinner flask cultures indicated a stronger need for zinc supplementation under intense cell culture conditions.