Modifications in native and glycoPEGylated N9. (A) Separation of Gla species in rFIX and N9 by anion exchange HPLC using a linear ammonium acetate gradient. The fractional content of indicated Gla species is given in parentheses and was calculated by relating the area of the corresponding peak to the total peak area. (B) Domain structure of N9 and location of identified N- and O-linked (●) carbohydrates. The 2 N-linked glycans in the activation peptide (AP) were predominantly core-fucosylated tri- and tetra-antennary complex structures composed of fucose (◁), N-acetylglucosamine (□), mannose (○), galactose (⊙), and sialic acid (♢). Enzymatic transfer of 40k-PEG-sialic acid to desialylated N9, and sialylation of remaining exposed galactoses are indicated by arrows. The relative distribution of PEG between the 2 N-glycans is given in parentheses. (C) N9-GP (lane 2) separated into 2 bands by SDS-PAGE corresponding to mono- and di-PEGylated (1x and 2xPEG, respectively) forms. Incubation with 2.5nM FXIa for 6 hours at pH 7.4 and 25°C converted 500nM N9-GP (lane 4 left panel) into heavy and light chains with the same mobility as activated N9 (lane 3 left panel) concomitant with the release of mono and di-PEGylated activation peptides (1x and 2xPEG-AP, respectively) evident by barium iodide (PEG) staining (lane 4 right panel). (D) The distribution of PEGylated species was determined by rpHPLC using a linear acetonitrile gradient which resolved N9-GP into unmodified (1%), mono-PEGylated (95%), and di-PEGylated (4%) forms. In comparison, N9 eluted as a single peak.