Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein

Abstract

Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

Keywords: haemophilia B; human cell line; manufacturing; rFIXFc; recombinant; viral clearance.

Figure 1

Overview of the recombinant factor IX Fc fusion protein (rFIXFc) manufacturing process.

Figure 2

SDS PAGE analysis of rFIXFc from a validation batch (Batch No. LP5-10-FIX-009) for identification and purity determination under non-reducing conditions. Non-reducing SDS PAGE was conducted on a 4–12% polyacrylamide gel in Bis-Tris buffer. Samples were denatured with SDS in the presence of 15 mM N-ethylmaleimide for 15 min at 25°C. The gel was stained with Colloidal Coomassie. Lane numbers: (1) MW markers; (2) rFIXFc assay sensitivity standard, 0.018 μg mass loading; (4) rFIXFc Reference standard, 6 μg mass loading; (6) rFIXFc product (Batch No. LP5-10-FIX-009), 6 μg mass loading; (3, 5) Blank lanes. MW, molecular weight; rFIXFc, recombinant factor IX Fc fusion protein; SDS PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Figure 3

Size exclusion chromatography (SEC) chromatogram of rFIXFc DS Batch No. LP5-11-FIX-001. SEC analysis was conducted on a TSK_gelG3000SW_XL column (Tosoh Bioscience, King of Prussia, PA, USA) using a mobile phase of 0.1 M sodium phosphate/0.1 M sodium chloride pH 6.5. Elution was monitored based on UV absorbance at 214 nm. rFIXFc, recombinant factor IX Fc fusion protein; DS, drug substance.