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.
Kolind
Mille Petersen
MP
Novo Nordisk A/S, Maaloev Park, 2760 Maaloev, Denmark.
Nørby
Peder Lisby
PL
Flintegaard
Thomas Veje
TV
Berchtold
Martin Werner
MW
Johnsen
Laust Bruun
LB
D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012995:Solubility / Q000187:drug effects; D013329:Structure-Activity Relationship; D014162:Transfection
D016895:Culture Media, Serum-Free; D050505:Mutant Proteins; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011134:Polysaccharides; D005169:Factor VIII