Bulaon C.J.I.Jaratsittisin J.Rattanapisit K.Suwanchaikasem P.Guo S.Boonha K.Pitaksajjakul P.Simsom N.Limprasutr V.Phoolcharoen W.Mahidol University2025-10-062025-10-062025-12-01Biotechnology Reports Vol.48 (2025)https://repository.li.mahidol.ac.th/handle/123456789/112472Plant systems offer scalable and cost-effective platforms for antibody production, but plant-specific glycans may affect pharmacokinetics and immunogenicity. To evaluate the impact of Fc glycosylation, four Pembrolizumab glycovariants were generated in Nicotiana benthamiana: wild-type glycosylation (Pembro-WT), high-mannose with SEKDEL (Pembro-KD), aglycosylated N297A mutant (Pembro-NG), and a core fucose/xylose-deficient variant (Pembro-XF). Glycoproteins were transiently expressed either in wild-type or ΔXF plants, purified, and characterized for glycan composition, in vitro binding, and in vivo pharmacokinetics. LC-MS confirmed distinct glycoform patterns, while PD-1 binding was retained across all variants. Pembro-XF showed the highest FcRn binding affinity and longest serum half-life (45.83 h) in mice, compared to Pembro-WT (26.7 h), Pembro-KD (32.95 h), Pembro-NG (34.27 h), and Keytruda® (33.26 h). As an initial efficacy evaluation, Pembro-WT demonstrated strong antitumor activity in a murine colon cancer model. These findings support plant glycoengineering as a strategy to enhance antibody pharmacokinetics and advance next generation antibody therapeutics.Biochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyArticleSCOPUS10.1016/j.btre.2025.e009272-s2.0-1050174468112215017X