N-glycosylation site occupancy heterogeneity reflects R27T size and pI differences. (A) SDS-PAGE and IEF analysis of glycoproteins. Lanes 2−4 show the main target peaks from R27T purification, and lanes 5−6 show intermediate peaks comprising mainly non- or partially glycosylated protein. Lane 7 is the singly glycosylated Rebif reference. (B) Microchip analysis (C) cIEF analysis