Laust Bruun Johnsen

Purification and characterization of a new recombinant factor VIII (N8)

Summary.  A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B‐domain and a light chain of 79 kDa. The two chains are held together by non‐covalent interactions. The four‐step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800–9800 IU mg−1. Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N‐terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B‐domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N‐linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N‐linked glycans are sialylated bi‐antennary structures. An O‐glycosylation site is present in the B‐domain linker region. This site was glycosylated with a doubly sialylated GalNAc‐Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well‐characterized FVIII product with biochemical properties that equal other FVIII products.

The B-domain of Factor VIII reduces cell membrane attachment to host cells under serum free conditions

Factor VIII (FVIII) is an important protein in the blood coagulation cascade and dysfunction or deficiency of FVIII causes haemophilia A. Replacement therapy with exogenous recombinant FVIII (rFVIII) works as a substitute for the missing or non-functioning FVIII. The rFVIII protein has been engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N-linked glycosylations within the B-domain have no influence on either total expression level or membrane attachment properties.

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction.

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.