Kinetics of the interaction between anti-FVIII antibodies and FVIII from therapeutic concentrates, with and without von Willebrand factor, assessed by surface plasmon resonance.
The presence of VWF in plasma-derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti-FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full-length rFVIII (preincubated or not with purified VWF), B domain-deleted (BDD)-rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture-based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti-FVIII antibodies. Concentration ranges (nm) of FVIII products tested were 9-0.03 (rFVIII) and 6-0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3-5 min, whereas dissociation of the complex was followed for 5-20-240 min. A strong interaction of rFVIII and BDD-rFVIII with patient's IgG was detected with the K (D) values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm, respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K (D) was still in the picomolar range (4.1 ± 1.9 pm) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti-FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates.
R&D Area, Instituto Grifols S.A, Barcelona, Spain. salvador.grancha@grifols.com
S
S
Grancha
A M
AM
Ortiz
C
C
Marañón
K
K
Hampel
A
A
Moret
B
B
Zimmermann
J I
JI
Jorquera
J A
JA
Aznar
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D006467:Hemophilia A / Q000473:pathology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D051379:Mice; D012720:Severity of Illness Index; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D000936:Antigen-Antibody Complex; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; C577506:recombinant factor VIII N8; D014841:von Willebrand Factor; C427184:recombinant factor VIII SQ; D005169:Factor VIII