A new recombinant factor VIII: from genetics to clinical use

Figures & data

Table 1 Licensed recombinant factor VIII products

Generation	Product (manufacturer)	FVIII	Cell line	Culture medium	Stabilizer	Purification/viral inactivation
First	Recombinate^® (Baxter BioScience)	Full-length	CHO	Bovine serum albumin	Human albumin	IAC/IEC
Second	Kogenate^® FS (Bayer Healthcare)	Full-length	BHK	Human plasma protein solution	Sucrose	IAC/IEC/SD/UF
Second	Helixate^® FS (CSL Behring)	Full-length	BHK	Human plasma protein solution	Sucrose	IAC/IEC/SD/UF
Third	Advate^® (Baxter Healthcare)	Full-length	CHO	None	Trehalose	IAC/IEC/SD
Third	Xintha/Refacto^® AF (Pfizer)	B-domai-deleted	CHO	None	Sucrose	IAC/IEC/SD/NF
Third	Turoctocog alfa^® (Novo Nordisk)	B-domain truncated	CHO	None	Sucrose	IAC/IEC/SD/NF/SE

Note: Data from.^5–15

Abbreviations: BHK, baby hamster kidney; CHO, Chinese hamster ovary; FVIII, factor VIII; IAC, immunoaffinity chromatography; IEC, ion exchange chromatography; NF, nanofiltration; SD, solvent/detergent treatment; SE, size exclusion; UF, ultrafiltration.

Figure 1 Protein structures and post-translational modifications reported for factor VIII and turoctocog alfa, respectively.

Notes: Both are characterized by the same heavy chain and light chain and differ in the B-domain. Domains are indicated with capital letters and subdomains with lower case letters. Glycosylation sites are denoted by triangles, disulfide bonds are denoted by arches, reduced cysteine residues are denoted by orange vertical lines, and “S” inside a circle indicates sulfated tyrosine residues. Brackets mark the areas of interaction with corresponding clotting factors, phospholipids (Pl), von Willebrand factor (VWF), calcium (Ca²⁺), and copper (Cu⁺) ions.
Abbreviation: SP, signal peptide.

Table 2 Function specificity of sulfated tyrosine residues in factor VIII

Tyrosine residues	Significance and activity for FVIII
346 and 1664	Increase of affinity for thrombin interaction and thereby the rate of FVIII activation by thrombin
718, 719, and 723	Increase of specific procoagulant activity of FVIIIa in the complex with FIXa and FX bound to the phospholipid membrane
1680	Prerequisite for complex formation with VWF, influencing half-life of FVIII in the circulation

Note: Data from Michnick et al²⁶ and Kaufman.²⁷

Abbreviations: FVIII, factor VIII; FIXa, activated factor IX; FX, factor X; VWF, von Willebrand factor.

Table 3 Levels of nonsulfated tyrosine in rFVIII

Product	Origin	Nonsulfated tyrosine 1680 (%)
		LC-MS/MS^Citation36	LC-MS/MS^Citation35	MS-FT^Citation66
Turoctocog alfa	CHO	–	–	Below detection limit
Full-length, third-generation rFVIII	CHO	2.6–16.7	5.0–8.0	>9.0
Full-length, second-generation rFVIII	BHK	1–6.5	1.5	–
BDD third-generation rFVIII	CHO	4.5–13.9	4.0–5.0	–

Notes: Levels of nonsulfated tyrosine in rFVIII included Advate^® (full-length third-generation rFVIII), Kogenate FS^® (full-length second-generation rFVIII), and Xintha/Refacto AF^® (B-domain-deleted third-generation rFVIII). The sulfated form dominated for all proteins, with the proportion of nonsulfated thyrosine 1680 being highest for some third-generation rFVIII products. A small peak for the nonsulfated isoform was also observed for second-generation rFVIII, while no nonsulfated species was detected for turoctocog alfa.^35,36,66 Below detection limit means negligible signal, <1%, or <0.5%, trace.

Abbreviations: BDD, B-domain-deleted; LC-MS/MS, liquid chromatography tandem mass spectrometry; rFVIII, recombinant factor VIII; MS-FT, mass spectrometry high resolution Fourier transform scan; BHK, baby hamster kidney; CHO, Chinese hamster ovary.

NovoEight (antihemophilic factor [recombinant], prescribing information) Novo Nordisk Bagsværd, Denmark 2011 Available from: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR__Product_Information/human/002719/WC500157553.pdf Accessed June 30, 2014

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