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Defining ‘full‐length’ recombinant factor VIII: a comparative structural analysis
Author(s) -
JANKOWSKI M. A.,
PATEL H.,
ROUSE J. C.,
MARZILLI L. A.,
WESTON S. B.,
SHARPE P. J.
Publication year - 2007
Publication title -
haemophilia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.213
H-Index - 92
eISSN - 1365-2516
pISSN - 1351-8216
DOI - 10.1111/j.1365-2516.2006.01388.x
Subject(s) - recombinant dna , complementary dna , immunoglobulin light chain , glycoprotein , computational biology , coagulation , heavy chain , homogeneous , microbiology and biotechnology , biology , antibody , genetics , gene , medicine , physics , psychiatry , thermodynamics
Summary. 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.