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Updated: Jun 5, 2026
Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization
Published on: December 23, 2015
Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction
Mille Petersen Kolind1, Peder Lisby Nørby, Martin Werner Berchtold
1Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark. Mille_petersen@hotmail.com
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.
Insights
Adding von Willebrand factor (vWF) to serum-free media releases active recombinant clotting Factor VIII (rFVIII) from cell membranes. This strategy, along with using ortho-phospho-L-serine (OPLS), significantly boosts rFVIII production yield.
Area of Science:
- Biotechnology
- Biochemistry
- Cell Biology
Background:
- Clotting Factor VIII (FVIII) circulates bound to von Willebrand factor (vWF), preventing its interaction with phosphatidylserine (PS).
- During serum-free recombinant FVIII (rFVIII) production, secreted rFVIII binds to PS on production cells due to the absence of vWF.
- Up to 90% of rFVIII can be cell-membrane bound under these conditions, limiting recovery.
Purpose of the Study:
- To investigate the effect of including vWF in serum-free medium on rFVIII recovery.
- To evaluate compounds that compete for FVIII-PS interactions to enhance rFVIII release and yield.
- To optimize serum-free production of therapeutic rFVIII.
Main Methods:
- Addition of vWF to serum-free culture medium for rFVIII-producing cells.
- Studying rFVIII attachment to cell membranes using competitive assays with annexin V and ortho-phospho-L-serine (OPLS).
- Quantifying membrane-bound rFVIII using iodinated rFVIII (¹²⁵I-rFVIII) and assessing yield increases in stable FVIII-expressing CHO cells with OPLS.
Main Results:
- Addition of vWF to serum-free medium released active membrane-bound rFVIII into the culture supernatant.
- Annexin V and OPLS reduced the membrane-bound fraction of rFVIII by 70% and 30%, respectively.
- Supplementation with OPLS increased rFVIII yield by 50% in stably transfected CHO cells.
Conclusions:
- vWF inclusion in serum-free media facilitates the release of active rFVIII.
- Competitive inhibition of FVIII-PS interactions, particularly with OPLS, significantly enhances rFVIII recovery.
- These findings offer a strategy to considerably increase rFVIII yield during serum-free production.
