Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells

Abstract

Trace element composition of culture medium can be altered to modulate glycoform of recombinant glycoproteins. In this study, we show that Zn²⁺ supplementation at or above 100 μM decreases galactosylation of recombinant IgG expressed in Chinese Hamster Ovary cells. This decrease in galactosylation is not due to reduced galactosyltransferase expression. This effect persists upon supplementation of galactose and uridine to the culture, indicating that it may not be due to reduced UDP-Gal availability. Measurements of galactosyltransferase activity in the cell lysate show that activity decreases with increasing Zn²⁺/Mn²⁺ ratio. This suggests that one possible explanation of the effect of Zn²⁺ may be reduced intracellular galactosyltransferase activity due to increase in Zn²⁺/Mn²⁺ ratio. Consistent with this, the decrease in galactosylation of IgG could be reversed by supplementation of Mn²⁺ (a cofactor of galactosyltransferase) which increases intracellular Mn²⁺ content. Measurement of total intracellular Zn²⁺ content, however, indicates no significant upregulation of total intracellular Zn²⁺ content and no significant downregulation of intracellular Mn²⁺ content with Zn²⁺ supplementation. One possible explanation could be that cellular detoxification response to higher extracellular Zn²⁺ concentration might lead to changes in intracellular distribution of Mn²⁺. In this case, Zn²⁺ supplementation would be expected to interfere with other known effects of Mn²⁺. Indeed, the previously reported increase in high mannose glycans upon Mn²⁺ supplementation in the absence of glucose is reversed by Zn²⁺ supplementation. This study also suggests the use of Mn²⁺ supplementation as a strategy to overcome the effect of lot-to-lot variability in trace element concentrations on galactosylation.