extract was utilized for synthesis of the selenoprotein. Such in vitro selenium bioavailability was consistent with our previous results of in vivo assessment in mice. Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab. Nakamura HOda-Ueda N Ueda TOhkuri TBiochem. Biophys. Res. Commun., Vol. 503 (2),pp. 752-756201830 DDOI: 10.1016/j.bbrc.2018.06.071. The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development.
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