TY - JOUR
T1 - Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction
AU - Kolind, Mille Petersen
AU - Nørby, Peder Lisby
AU - Berchtold, Martin Werner
AU - Johnsen, Laust Bruun
N1 - Copyright © 2011 Elsevier B.V. All rights reserved.
PY - 2011/2/20
Y1 - 2011/2/20
N2 - 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.
AB - 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.
KW - Animals
KW - Annexin A5
KW - CHO Cells
KW - Cell Culture Techniques
KW - Cell Membrane
KW - Cricetinae
KW - Cricetulus
KW - Culture Media, Serum-Free
KW - Factor VIII
KW - Genetic Vectors
KW - HEK293 Cells
KW - Humans
KW - Protein Binding
KW - Serine
KW - Thrombin
KW - Transfection
KW - von Willebrand Factor
U2 - 10.1016/j.jbiotec.2010.12.019
DO - 10.1016/j.jbiotec.2010.12.019
M3 - Journal article
C2 - 21219947
SN - 0168-1656
VL - 151
SP - 357
EP - 362
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 4
ER -