Characterization and improvement of interferon-[gamma] glycosylation in Chinese hamster ovary cell culture

Abstract

Glycoproteins representing an important category of therapeutics for human health care, generally exist as a set of glycosylated variants exhibiting heterogeity with respect to both the proportion of potential glycosylation sites that are occupied (i.e., macroheterogeneity) and the oligosaccharide structures observed at each glycosylation site (i.e., microheterogeneity). Therefore, it is essential to characterize and. if possible, optimize the glycosylation profiles of recombinant glycoproteins to ensure their quality and consistency as effective pharmaceuticals and to meet increasing regulatory demands. In this study, the two potential N-linked glycosylation sites (i.e., Asn25 and Asn97) of recombinant human interferon-[gamma]y (IFNY-[gamma]) derived from Chinese hamster ovary (CHO) cell culture were characterized by a sensitive (- 1 [mu]) and rapid (- 3 h) analytical methodology employing a variety of tandem chromatographic techniques, capillary electrophoresis and mass spectrometry. Although complex biantennary glycans were the predominant structures at both glycosylation sites, Asn25 -linked glycans possessed higher proportions of tri- and tetraantennary structures and were exclusively fucosylated compared to glycans at Asn97. Although the proportions of glycan antennary structures were found to be relatively invariable throughout typical CHO batch cultures, the oligosaccharide structures were selectively altered by the introduction of various inhibitors of intracellular glycosylation pathways. For example, the use of dexamethasone, an inhibitor of the enzyme responsible for oligosaccharide branching, was found to decrease the proportions of tri- and tetraantennary glycan structures and, thus, improve the glycosylation homogeneity of CHO-derived IFNY-[gamma]. A critical aspect of glycosylation is sialylation since the presence of sialic acid can dramatically extend a glycoprotein's circulatory lifetime. The sialylation patterns of CHO-derived IFNY-[gamma] were characterized by RP-HPLC separations of glycosylation site-specific tryptic glycopeptides. IFNY-[gamma] displayed both site- and branch-specific differences in sialic acid content as Asn25-linked glycans and the Man([alpha]1-3) branch of the predominant complex biantennary glycan structures at each site were preferentially sialylated. Both incomplete intracellular sialylation and extracellular desialylation resulting from release of sialidase during cell lysis were found to determine the sialic acid content of the final product. Primatone RL, an animal tissue hydrolysate commonly employed as a serum substitute to stimulate cell growth, was found to have an adverse impact on intracellular sialylation of CHO-derived IFNY-[gamma] in both batch and fed-batch cultures. However, incomplete intracellular sialylation was minimized through feeding of Nacetylmannosamine (ManNAc), a synthetic orecursor of sialic acid. Feeding of 20 mM ManNAc reduced the proportion of undersialylated glycan structures by 50%. Radiolabeled MarNAc was utilized to confirm that the supplemental ManNAc was incorporated into IFNY-[gamma] as sialic acid. The intracellular pool of CMP-sialic acid. the nucleotide sugar substrate for sialyltransferase, was also extracted and quantitated by RP-HPLC. Although feeding of 20 mM ManNAc increased the intracellular pool of CMP-sialic acid nearly 30-fold, in vitro incubation of isolated Golgi with radiolabeled CMP-sialic acid revealed a limitation for transport of the substrate to the site of sialylation.