Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system

Abstract

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.

Tailoring recombinant protein quality by rational media design

“…Manganese plays a fundamental role in the glycosylation pathway, as shown by many authors 23,52,56 . It determines the levels of various oligosaccharide species including high mannose, galactosylated, fucosylated and sialylated proteins.…”

“…Given its potential, protein quality tuning by media design gained much interest in the last few years. Crowell et al 23 described in 2007 how manganese supplementation modulated the glycosylation state of erythropoeitin (EPO) in a CHO cell culture. These findings are based on early experiments by Kaufman et al who observed the pivotal role of manganese in the secretory pathway of complex N-linked and O-linked oligosaccharides, thus suggesting the unique requirement for manganese 56 .…”

“…It determines the levels of various oligosaccharide species including high mannose, galactosylated, fucosylated and sialylated proteins. As cofactor of many enzymes, manganese modulates the glycosylation profile and it was noted that a lack of this transition metal inhibits O-linked glycosylation 23 . Its specific role in each glycosylation processing step is described more thoroughly in the following subsections.…”

“…Cultures performed at higher Mn 2+ concentrations (40 µM) exhibited increased galactosylation and higher sialylation of recombinant human erythropoietin (EPO), however, the product yield was greatly reduced 23 . A decrease of Man5 and hence higher abundance of galactosylated species were observed at increasing Mn 2+ concentration: 0.25-1.0 µM 63 .…”

“…The environment that surrounds the cell while in suspension, namely the cell culture medium, plays a key role in the cell metabolism, and for this reason, media design lends itself to alter the key quality attributes of the molecule Introduction within the potential of a given cell line [19][20][21][22] . The adjustment of the levels of a choice of medium components and the addition of novel compounds either in the medium or the in bolus feeds, influence the enzymatic reactions in the endoplasmic reticulum and in the Golgi apparatus, the substrate generation as well as their transport into the two organelles 23,24 . Further enhancements and technology advancements of high-throughput cell culture systems, including 96-deepwell plates and TubeSpin bioreactor tubes, allow to test a plethora of different supplements at various concentrations simultaneously [25][26][27] .…”