Comprehensive N- and O‑Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines

Glycoproteomic
analysis of three Chinese hamster ovary (CHO) suspension
host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in
biopharmaceutical settings for recombinant protein production is reported.
Intracellular and secreted glycoproteins were examined. We utilized
an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins
or glycopeptides were first immobilized through reductive amination,
and the sialyl moieties were amidated for protection. The desired
N- or O-glycans and glycopeptides were released from the immobilization
resin by enzymatic or chemical digestion. Glycopeptides were studied
by Orbitrap Liquid chromatography–mass spectrometry (LC/MS),
and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time
of flight (MALDI-TOF). Differences were detected in the relative abundances
of N- and O-glycopeptide types, their resident and released glycans,
and their glycoprotein complexity. Ontogeny analysis revealed key
differences in features, such as general metabolic and biosynthetic
pathways, including glycosylation systems, as well as distributions
in cellular compartments. Host cell lines and subfraction differences
were observed in both N- and O-glycan and glycoprotein pools. Differences
were observed in sialyl and fucosyl glycan distributions. Key differences
were also observed among glycoproteins that are problematic contaminants
in recombinant antibody production. The differences revealed in this
study should inform the choice of cell lines best suited for a particular
bioproduction application.