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1.  O-Phospho-L-Serine, Multi-functional Excipient for B Domain Deleted Recombinant Factor VIII 
The AAPS journal  2007;9(2):E251-E259.
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 binding 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.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development
2.  O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII 
The AAPS Journal  2007;9(2):E251-E259.
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.
doi:10.1208/aapsj0902028
PMCID: PMC2573386  PMID: 17907766
B domain deleted recombinant factor VIII; O-phospho-L-serine; protein formulation; excipient; physical stability; immunogenicity; inhibitor development
3.  PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice 
The AAPS Journal  2011;14(1):35-42.
Hemophilia A is an X-linked bleeding disorder caused by the deficiency of factor VIII (FVIII). Exogenous FVIII is administered therapeutically, and due to a short half-life, frequent infusions are often required. Fifteen to thirty-five percent of severe hemophilia A patients develop inhibitory antibodies toward FVIII that complicate clinical management of the disease. Previously, we used phosphatidylinositol (PI) containing lipidic nanoparticles to improve the therapeutic efficacy of recombinant FVIII by reducing immunogenicity and prolonging the circulating half-life. The objective of this study is to investigate further improvements in the FVIII–PI formulation resulting from the addition of polyethylene glycol (PEG) to the particle. PEGylation was achieved by passive transfer of PEG conjugated lipid into the FVIII–PI complex. PEGylated FVIII–PI (FVIII–PI/PEG) was generated with high association efficiency. Reduced activity in vitro and improved retention of activity in the presence of antibodies suggested strong shielding of FVIII by the particle; thus, in vivo studies were conducted in hemophilia A mice. Following intravenous administration, the apparent terminal half-life was improved versus both free FVIII and FVIII–PI, but exposure determined by area under the curve was reduced. The formation of inhibitory antibodies after subcutaneous immunization with FVIII–PI/PEG was lower than free FVIII but resulted in a significant increase in inhibitors following intravenous administration. Passive transfer of PEG onto the FVIII–PI complex does not provide any therapeutic benefit.
doi:10.1208/s12248-011-9309-2
PMCID: PMC3291190  PMID: 22173945
factor VIII; hemophilia A; immunogenicity; inhibitor development; PEGylation
4.  Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice 
The AAPS Journal  2010;12(3):473-481.
Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH2-A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII–PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII–PI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A.
doi:10.1208/s12248-010-9207-z
PMCID: PMC2895449  PMID: 20517659
factor VIII; hemophilia A; inhibitor development; immunogenicity; phosphatidylinositol
5.  Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII 
The AAPS Journal  2009;11(3):424-431.
Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.
doi:10.1208/s12248-009-9119-y
PMCID: PMC2758112  PMID: 19499345
factor VIII; glycosylation; hemophilia A; immunogenicity; inhibitor development

Results 1-5 (5)