Introduction: Human-cl rhFVIII (Nuwiq (R)), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. Aims: To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. Methods and 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 similar to 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. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license.