Protein Glycosylation: Implications for In Vivo Functions and Therapeutic Applications - Advances in Biochemical Engineering Biotechnology - page 181

Glycan processing pathways have been genetically manipulated in hosts like
S.cerevisiae and CHO cells [188]. In general, the natures of glycan structure in
the expressed glycoprotein are protein and host specific. The terminal sugars
and, to a lesser extent, the chain branching are the features that are most influ-
enced by the host, as observed from the study of tPA and EPO [212]. Table 3a
shows the nature of glycans produced in three different glycoproteins expressed
in CHO cells. It is obvious that these recombinant proteins differ from their
native counterparts with respect to biological activities and circulatory half-
lives, mainly due to conformational differences and the nature of glycosylation.
The extent to which the host cells dictate the glycosylation differences is illust-
rated in Table 3b. Therefore, it is important to study the host cells and their gly-
cosylation machinery before considering them for the expression of the thera-
peutic proteins. This view is further substantiated by the observations with
pituitary hTSH, which is having complex glycans terminating predominantly
with SO 4 -GalNAc. CHO cells do not express GalNAc-transferase and sulfotrans-
ferase, and thus hTSH expressed in these contain only SA-Gal terminal sequen-
ces and have longer plasma half-life and higher in vivo activity despite lower in
vitro activity compared to pituitary hTSH [217].
5.3
Factors Controlling Glycosylation in Cultured Cells
Once the host cell line has been selected, the cell culture conditions are opti-
mized to minimize glycoprotein heterogeneity and to prevent deterioration of
product quality [218]. Cell status, bioreactor configuration, and culture condi-
tions (e.g.pH,concentration of NH + ) and media components (e.g.serum levels,
glucose, amino acids), presence of nucleotide sugars cytidine and uridine, and
lipids such as dolichol alone or in combination with lipoprotein carriers, have
been found to affect glycosylation in several systems [162]. Large scale cultured
tPA exhibited a higher clearance than tPA produced on a small scale, indicating
variability in glycosylation pattern [219]. Product degradation due to various
glycosidase activities has been measured in CHO cell lysates and culture super-
natants. Amongst these, sialidase is the most active enzyme at neutral pH [220]
and is found to degrade glycans from recombinant products [221]. Mouse cell
lines such as NSO myelomas and hybridomas display much lower sialidase
activity than CHO cell at neutral pH.
6
Biopharmaceutical Properties
Recombinant glycoproteins often have altered biological properties and func-
tions, increased immunogenicity and protease sensitivity compared to their
native counterparts.Cell lines that produce proteins containing glycans with the
blood group antigens or with terminal Gn residues are unsuitable as hosts
because of the presence of natural antibodies against blood group antigens and