The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that beta-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn(2+)), a cofactor for beta-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn(2+) addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn(2+) addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn(2+) only had this effect late in the culture process. These data indicate that the addition of Mn(2+) has complex effects on stressed batch fed cultures.

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Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.