N-glycan modification of the anti–HPA-1a mAb SZ21. Coomassie blue staining (CBS) was used to analyze the general structure modifications of SZ21 after its treatment with endo F. Two bands with apparent molecular weights of <50 kDa (∼47 kDa) and 25 kDa representing the heavy and light chains of mAb SZ21 and NGM-SZ21 were visible under reducing conditions (upper left panel). N-glycan was then detected using LCA (upper right panel). A strong signal was solely detected at a molecular weight of ∼50 kDa of the lane loaded with SZ21 (lane 1). No signal was detected by testing endo F–treated NGM-SZ21 (lane 2), indicating the removal of the N-glycan. The specificity of deglycosylation was analyzed by MALDI-TOF mass spectroscopy (MS). As shown in the lower panels, the only difference between the heavy chain of mAb SZ21 (left panel) and the heavy chain of NGM-SZ21 (right panel) was in fragment 1506 corresponding to the N-glycan that is linked to the asparagine at position 297. No additional modifications were observed in the heavy chain of the NGM-SZ21.