ANALYSIS OF GLYCOSYLATION HETEROGENEITY IN IFN-¥ PRODUCED BY CHO CELLS DURING BATCH AND CONTINUOUS CULTURE.

Abstract

The production of recombinant human interferon-x (IFN- X¥ ) by Chinese hamster ovary (CHO) cells in serum-free medium was heterogeneous with up to twelve molecular weight variants secreted. Using the enzyme N-glycanase to remove all the (N-linked) oligosaccharides or tunicamycin to inhibit glycosylation, it was found that the variation was due to both variable asparagine residue occupation (Asnyg and/or Asn- 100) by complex oligosaccharide and to truncation of the IFN-$ polypeptide due to proteolytic cleavage at the carboxy-terminal end. Using neuraminidase treatment, terminal sialic acid expression was found to be invariant. When the CHD cells were grown in two litre batch suspension culture in serum-free medium further heterogeneity was discovered, seen as a reproducible shift towards the secretion of underglycosylated IFN-Y with time. This phenomenon was independent of glucose concentration in the medium. The IFN- ¥ was produced during the exponential growth phase and declined in line with the cell growth rate and glucose uptake rates. In contrast, during glucose-limited chemostat culture at a constant dilution rate of 0.015h”l, the relative levels of each major glycosylation variant did not change significantly, although the proportion of fully glycosylated IFN-¥ was still less than that seen at the start of batch culture while non-glycosylated IEN- ¥ represented 15% of the total IFN- ¥ secreted.