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Figure S1. Detection of oligomers on Native PAGE Gels by SERVA system: The same range of factor IX, Mononine® total protein (62.5–1000 ng) as demonstrated in Fig. 1, was subjected to Bis-Tris gel SERVAGel™ Native Gel electrophoresis system (15 mm Bis-Tris, 50 mm Tricine pH 7.0) and stained using α-human FIX antibody and goat α-mouse conjugated to HRP secondary antibody.

Figure S2. Detection of recombinant factor IX oligomers on Native PAGE Gels: Factor IX, BeneFIX® (1000 ng) was subjected to native Bis-Tris gel electrophoresis and stained using α-human FIX antibody and goat α-mouse conjugated to HRP secondary antibody.

Figure S3. Size exclusion chromatography of FIX: (a) Gel-filtration pattern of Mononine® (0.5 mg mL−1, 500 μg) on Superdex-200 10/300 GL column eluted with standard PBS buffer (pH 7.4). Twenty four (24) μL from Fraction 13.5, 14, 14.5 and 15.9 mL were subjected to Native PAGE electrophoresis gel and stained using anti-human FIX antibody and goat anti-mouse conjugated to HRP secondary antibody (right top insert panel). (b) Gel-filtration pattern of Mononine® on Superdex-200 10/300 GL column eluted with 50 mm Bis-Tris, 50 mm Tricine (pH 6.8).

Figure S4. Detection of recombinant activated factor IX oligomers on Native PAGE Gels and factor IXa activity: (a) Factor IXa (~850 ng) was subjected to native Bis-Tris gel electrophoresis and stained using α-human FIX antibody and goat α-mouse conjugated to HRP secondary antibody. Factor IX- Mononine® was loaded in the same gel as a control. (b) Activated factor IX (FIXa) activity was determined for the same samples used in Fig. 3, gel-filtration (Superdex-200 10/300 GL) elution of Mononine® with a standard PBS (monomer peak) and samples eluted with 50 mm Bis-Tris, 50 mm Tricine pH 6.8 (dimer peak), with a statistically significant difference (P = 0.0003) (Please note that the FIXa activities are very low).

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