title: Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.
abstract: Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.
authors.affiliation: aNovo Nordisk A/S, Biopharmaceutical Research Unit, Mål⊘v bNovo Nordisk A/S, CMC Supply, Gentofte, Denmark.
authors.forename: Haleh
authors.initials: H
authors.lastname: Ahmadian
authors.forename: Ernst B
authors.initials: EB
authors.lastname: Hansen
authors.forename: Johan H
authors.initials: JH
authors.lastname: Faber
authors.forename: Lars
authors.initials: L
authors.lastname: Sejergaard
authors.forename: Johan
authors.initials: J
authors.lastname: Karlsson
authors.forename: Gert
authors.initials: G
authors.lastname: Bolt
authors.forename: Jens J
authors.initials: JJ
authors.lastname: Hansen
authors.forename: Lars
authors.initials: L
authors.lastname: Thim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003412:Cricetulus; D015195:Drug Design; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion
chemical_list: D011994:Recombinant Proteins; C577506:recombinant factor VIII N8; D005169:Factor VIII