title: Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction.
abstract: In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-L-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.
authors.affiliation: Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark. Mille_petersen@hotmail.com
authors.forename: Mille Petersen
authors.initials: MP
authors.lastname: Kolind
authors.forename: Peder Lisby
authors.initials: PL
authors.lastname: Nørby
authors.forename: Martin Werner
authors.initials: MW
authors.lastname: Berchtold
authors.forename: Laust Bruun
authors.initials: LB
authors.lastname: Johnsen
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D016466:CHO Cells; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000737:chemistry / Q000378:metabolism*; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017304:Annexin A5; D016895:Culture Media, Serum-Free; D014841:von Willebrand Factor; D012694:Serine; D005169:Factor VIII; D013917:Thrombin