Defining ‘full‐length’ recombinant factor VIII: a comparative structural analysis

Abstract

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.

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2‐D DIGE

“…LC-B is an extended LC that stems from a single cleavage of FVIII at position 1313. The extended LC is present in both commercial full-length rFVIII products (Advate and Kogenate FS) and in plasma-derived products such as Hemophil M, albeit to a varying extent [5]. BDD-rFVIII was isolated from an rFVIII-expressing CHO cell line, which has secreted rFVIII that consistently contains HC fragments with varying portions of the B domain including one that entirely lacks the B domain.…”

“…Neuraminidase-treated, buffer-treated and untreated rFVIII were labeled with distinct fluorescent dyes, mixed and separated on the same gel. In the first dimension, the sample was focused for 10 500 Vh on an immobilized non-linear pH gradient (3)(4)(5)(6)(7)(8)(9)(10)(11). After a reducing and alkylating step the sample was separated in the second dimension by SDS-PAGE on a 3-8% PA gradient gel (Tris-acetate).…”

“…Although useful, these methods either focus on a certain aspect of FVIII or do not resolve more subtle differences between FVIII samples. Combining techniques like tryptic mapping or size exclusion-HPLC with modern MS facilitated a more detailed structural characterization [5,17,19]. Nevertheless, the results of those approaches are rather complex and direct comparative analysis of various FVIII samples remains especially difficult.…”

“…Current substitution therapy uses plasma-derived and recombinant factor VIII (rFVIII) products, the latter comprising full-length and B-domain-deleted (BDD) variants [4][5][6]. BDD-rFVIII was developed because the B domain of FVIII is apparently not required for the catalytic activity of FVIIIa.…”

BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

“…Region 2 also contains active rFVIII that has been partially cleaved in the C-terminal region of the heavy chain B domain. Proteolytic processing of the inactive B domain is known to occur in all FVIII products, including plasma-derived products (primarily at Arg 1313 ) [20]. All cleaved fragments of the molecule that contain the highly glycosylated B domain elute within this main peak.…”