title: Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.
abstract: BACKGROUND
  Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency.
  
  
  OBJECTIVE
  To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification.
  
  
  RESULTS
  The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product.
  
  
  CONCLUSIONS
  Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin.
authors: Octapharma AB, Elersvägen 40, SE-112 75 Stockholm, Sweden. Electronic address: stefan.winge@octapharma.se.
authors: Stefan
authors: S
authors: Winge
authors: Octapharma AB, Elersvägen 40, SE-112 75 Stockholm, Sweden.
authors: Louise
authors: L
authors: Yderland
authors: Octapharma Molecular Biochemistry, Walther-Nernst-Str. 3, 12489 Berlin, Germany.
authors: Christoph
authors: C
authors: Kannicht
authors: BAC BV - Thermo Fisher Scientific, 28 Huizerstraatweg, 1411 GP Naarden, The Netherlands.
authors: Pim
authors: P
authors: Hermans
authors: BAC BV - Thermo Fisher Scientific, 28 Huizerstraatweg, 1411 GP Naarden, The Netherlands.
authors: Simon
authors: S
authors: Adema
authors: Octapharma GmbH, Altenhöferallee 3, D-60438 Frankfurt, Germany.
authors: Torben
authors: T
authors: Schmidt
authors: Octapharma AB, Elersvägen 40, SE-112 75 Stockholm, Sweden.
authors: Gustav
authors: G
authors: Gilljam
authors: Octapharma AB, Elersvägen 40, SE-112 75 Stockholm, Sweden.
authors: Martin
authors: M
authors: Linhult
authors: Octapharma GmbH, Im Neuenheimer Feld 590, 69120 Heidelberg, Germany.
authors: Maya
authors: M
authors: Tiemeyer
authors: Octapharma AG, Seidenstrasse 2, 8853 Lachen, Switzerland.
authors: Larisa
authors: L
authors: Belyanskaya
authors: Octapharma AG, Seidenstrasse 2, 8853 Lachen, Switzerland.
authors: Olaf
authors: O
authors: Walter
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII
keywords: Haemophilia A; Human-cl rhFVIII; Nuwiq®; Pathogen safety; Purification process; Recombinant factor VIII