Defining 'full-length' recombinant factor VIII: a comparative structural analysis.
Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.
Jankowski
M A
MA
Wyeth BioPharma, Andover, Massachusetts 01951, USA. MJankowski@wyeth.com
Patel
H
H
Rouse
J C
JC
Marzilli
L A
LA
Weston
S B
SB
Sharpe
P J
PJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000627:therapeutic use; D006467:Hemophilia A / Q000188:drug therapy; D006801:Humans; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
C078147:F8 protein, human; D005169:Factor VIII