Monoclonal antibodies (mAb) are glycosylated on asparagine residue 297 in CH<sub>2</sub> domain of each heavy chain. The N-linked glycan produced by CHO cells is a complex-type biantennary glycan that contains either no, one, or two galactose residues; terminal sialic acid residues are present in small amounts. Galactose, along with other residues in the N-linked glycan, interact with amino acids predominantly through CH-π interactions to stabilize the polypeptide in a conformation that facilitates antibody binding to Fcγ receptors, the complement component C1q, and FcRn receptors. Galactosylation thus has a role in modulating the effector functions of IgG, in particular by attenuating inflammation and enhancing complement dependent cytotoxicity. mAbs produced by CHO cells can have low levels of galactosylation. We have found a commercially available medium supplement that enhances galactosylation of our mAb. In addition, a supplement containing two precursors of UDP-galactose and a cofactor of the enzyme responsible for its synthesis also increases mAb galactosylation in a dose-dependent manner. The quantity of the G2F glycan was enhanced 43 and 76% when the commercial supplement was added on day 8 of culture versus day 3. A supplement containing galactose, uridine, and magnesium increased the quantity of the G2F glycan up to 47% higher than the control. There were smaller increases in the amounts of the glycans containing only one galactose residue—glycans G1F(1-6) and G1F(1-3). With the increase in galactosylated glycans there was a concomitant decrease in the non-galactosylated glycan G0F. It was interesting that a second medium supplement marketed to increase galactosylation actually caused a 50% decrease in the amount of G2F glycan and smaller decreases in the amounts of the mono-galactosylated glycans. These findings emphasize that the efficacy of a supplement cannot be predicted but must be evaluated in scaled-down cultures that closely resemble those that will be encountered in manufacturing

Enhanced galactosylation of a monoclonal antibody

253rd National Meeting (2017)

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