Objective:
Baxter has developed BAX 855, a PEGylated form of Baxter’s recombinant full length FVIII (rFVIII) product based on the ADVATETM manufacturing process. Here we describe it ́s manufacturing and structural and functional characterization.
Methods:
A rFVIII intermediate of the ADVATETM manufacturing process is the starting material for the conjugation process for preparing BAX 855 by proprietary PEGylation technology from Nektar Therapeutics. Similar technology has been successfully employed for marketed and licensed PEGylated drug products and drugs in clinical use. The manufacturing process for BAX 855 comprises several steps, including controlled PEGylation followed by cation exchange chromatography. Final formulation uses the same excipients as ADVATETM. BAX 855 was characterized by a number of analytical methods, focusing on the elucidation of the primary structure, posttranslational modifications, PEGylation site distribution and three- dimensional structure. The overall hemostatic potency of BAX 855 in FVIII-deficient plasma was assessed by conventional FVIII 1-stage clotting and chromogenic assays and with a thrombin generation assay. The tenase cofactor activity of FVIII was determined by measuring the kinetics of FXa generation. Binding of BAX 855 in comparison to ADVATETM was determined to its ligands VWF and low-density lipoprotein-receptor-related protein (LRP).
Summary and Conclusions:
BAX 855 is a full-length rFVIII with extended half-life. PK studies in different animal species and humans with hemophilia A display longer survival of BAX 855 compared to ADVATETM. The product is based on the original, native FVIII protein utilized in the production of ADVATETM. Our analyses show that BAX 855 can be manufactured reproducibly without changes to the protein structure characteristic for a fully functional full-length rFVIII molecule. The process is suited to manufacture BAX 855 in large scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 855 has a specific activity similar to that of rFVIII in ADVATETM and PEGylation degrees in the range of 2 to 3 mol PEG / mol rFVIII. SDS-PAGE and Western blot analysis of BAX 855 confirm PEGylation and demonstrate an increase in the molecular weight of the various FVIII domains.
In comparison to ADVATETM the functional properties of BAX 855 were fully retained except for binding to LRP, indicating that PEGylation did not have an impact on the functional properties of rFVIII. The latter might explain why BAX 855 shows prolonged survival in the circulation of hemophilic species and patients with hemophilia A than ADVATETM.