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AAPS J. Jun 2007; 9(2): E251–E259.
Published online Jun 29, 2007. doi:  10.1208/aapsj0902028
PMCID: PMC2573386
NIHMSID: NIHMS74123
O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII
Razvan D. Miclea,1 Vivek S. Purohit,2 and Sathy V. Balu-Iyercorresponding author1,2
1Department of Molecular & Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, NY
2Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, 14260 Buffalo, NY
Sathy V. Balu-Iyer, Phone: (716) 645-2842, Fax: (716) 645-3693, svb/at/acsu.buffalo.edu.
corresponding authorCorresponding author.
Received September 5, 2006; Accepted May 24, 2007.
Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, ~15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid bindinding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free-vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDRFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.
Keywords: B domain deleted recombinant factor VIII, O-phospho-L-serine, protein formulation, excipient, physical stability, immunogenicity, inhibitor development
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