Journal: International Journal of Molecular Sciences
Article Title: A Deletion Variant of Human Factor VIII Displaying Low Immunogenicity in a Murine Model of Hemophilia A
doi: 10.3390/ijms262412093
Figure Lengend Snippet: Purity, structural integrity analysis, and specific activity of the recombinant Factor VIII produced in our laboratory (rhFVIII-H6A) compared to reference commercial products. ( A ) Factor VIII light chain (LCh) Western blot analysis of the pool of samples derived from media conditioned by the H6A cell clone (well 2) (0.04 IU); after the anion exchange chromatography step (well 3) (0.07 IU); after the affinity chromatography step (well 4) (0.13 IU); retained fractions after the 100 kDa ultrafiltration step (well 5) (0.49 IU); rhFVIII-H6A final formulation (well 6) (0.5 IU); recombinant product Kogenate ® FS (well 7) (0.5 IU); and plasma derived Fanhdi ® (well 9) (0.5 IU). Water in well number 8. ( B ) Evaluation of the protein mixture complexity was carried out for the same samples described in ( A ) using 8% denaturing polyacrylamide gel electrophoresis, followed by staining with Coomassie blue G-250 colloidal using 2 IU of formulated rhH6A (well 6); a total of 2 IU of rhFVIII Kogenate ® FS (well 7) and 1 IU pdFVIII Fanhdi ® (well 9). Molecular weight ladder was applied in well number 1. ( C ) Factor VIII specific activity obtained for formulated rhFVIII-H6A. Specific activity was quantified based on the chromogenic in vitro Factor VIII activity assay and protein quantification using the colorimetric bicinchoninic acid assay. ** 0.01 < p < 0.05.
Article Snippet: Total protein was quantified using the colorimetric assay based on bicinchoninic acid, namely the BCA Protein Assay commercial kit (Lot CI48587) from Pierce (ThermoFisher Scientific, Rockford, IL, USA), following the manufacturer’s instructions.
Techniques: Activity Assay, Recombinant, Produced, Western Blot, Derivative Assay, Chromatography, Affinity Chromatography, Formulation, Clinical Proteomics, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, In Vitro, Acid Assay
