RESUMEN
OBJECTIVE: To compare in vitro and in vivo biological and biochemical properties of five liquid intravenous immunoglobulin (IVIg) preparations licensed for therapeutic use in Europe. METHODS: ClairYg(®) was compared in a blinded manner to four other liquid IVIg preparations licensed in Europe (Octagam(®) , Kiovig(®) , Gamunex(®) , Privigen(®) ). Three batches of each preparation were tested, except for the IgG repertoires and the animal model. RESULTS: Levels of anti-A and anti-B antibodies were lower in ClairYg(®) (0·11/0·11) relative to a positive EDQM standard and Octagam(®) (0·11/0·08) than in other preparations (0·33-0·69/0·42-0·46). IgG in ClairYg(®) recognized 365 and 416 protein spots in HEp-2 cell and Escherichia coli protein extracts vs. 230-330 and 402-842 protein spots, respectively, for IgG in other preparations. IgA content (301 vs. 165-820 ng/mg of IgG), Factor XI and Factor XII antigen (0·46 vs. 0·85-2·40 mU/mg of IgG and 7·8 vs. 20·0-46·2 lU/mg of IgG) C1q binding (0·42 vs. 0·67-1·89 arbitrary units) and C5a uptake (0·41 vs. 0·45-0·66% of activation) were lower in ClairYg(®) than in other preparations. Finally, intravenous infusion of ClairYg(®) , Gamunex(®) and Privigen(®) had no major effect on arterial blood pressure in spontaneously hypertensive rats. CONCLUSIONS: Our results evidence some differences in the biological and biochemical properties among licensed liquid IVIg preparations.
Asunto(s)
Inmunoglobulinas Intravenosas/administración & dosificación , Inmunoglobulinas Intravenosas/química , Animales , Presión Arterial/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Hipertensión/tratamiento farmacológico , Inmunoglobulinas Intravenosas/inmunología , Masculino , RatasRESUMEN
BACKGROUND: Since the early 1990 s the Committee for Proprietary Medicinal Products has set the mandatory requirement that all manufacturing processes for blood products include two virus removal/inactivation steps that are complementary in their action. OBJECTIVES: The objective was to develop a manufacturing process for factor VIII (FVIII) including two complementary steps of viral inactivation/elimination. METHODS: A 35-15 nm nanofiltration step was added to a former FVIII manufacturing process that included solvent/detergent (S/D) treatment to generate a new FVIII concentrate called Factane. The impact of nanofiltration on the structural and functional characteristics of FVIII, as well as virus/transmissible spongiform encephalopathy reduction factors were assessed. RESULTS: Using an innovative approach, FVIII was successfully nanofiltered at 35-15 nm, while the biological properties of the active substance were unmodified. FVIII coagulant and antigen content for Factane and previous S/D-treated FVIII (FVIII-LFB, commercialized as Facteur VIII-LFB) were comparable. The FVIII one-stage chromogenic and coagulant/antigen ratios confirmed that nanofiltered FVIII was not activated. After nanofiltration, the copurified von Willebrand factor (vWF) was reduced but vWF/FVIII binding properties were unaffected. Phospholipid binding and thrombin proteolysis studies displayed no differences between Factane and FVIII-LFB. The rate of factor Xa generation was slightly lower for Factane when compared to FVIII-LFB. Viral validation studies with different viruses showed no detectable virus in the filtrate. CONCLUSIONS: Nanofiltration of FVIII at 15 nm is feasible despite the large molecular weight of FVIII and vWF. Nanofiltration has been proven to be highly effective at removing infectious agents while preserving the structural and functional integrity of FVIII.
Asunto(s)
Factor VIII/aislamiento & purificación , Eliminación de Componentes Sanguíneos/métodos , Eliminación de Componentes Sanguíneos/normas , Calcio/metabolismo , Detergentes , Factor VIII/química , Factor VIII/metabolismo , Factor Xa/metabolismo , Filtración/métodos , Filtración/normas , Humanos , Técnicas In Vitro , Filtros Microporos , Nanotecnología , Fosfolípidos/metabolismo , Plasma/virología , Priones/sangre , Priones/aislamiento & purificación , Unión Proteica , Estructura Cuaternaria de Proteína , Seguridad , Solventes , Trombina , Virus/aislamiento & purificación , Factor de von Willebrand/química , Factor de von Willebrand/aislamiento & purificación , Factor de von Willebrand/metabolismoRESUMEN
SCID mice were grafted with human PBL (hu-PBL-SCID) from healthy or haemophilia A donors. Those containing human and no murine Ig in their plasma, were injected with 100 U VIII:Ag of a plasma derived (pd) FVIII or recombinant deleted Factor VIII (FVIII deltaII) and with 10 microg of tetanus toxoid as control immunogen. The frequency and the intensity of the humoral specific responses were measured in 253 mice humanized with PBL from 13 different donors. There was no significant difference in the frequency or intensity of the anti-FVIII immune responses to pd FVIII and FVIII deltaII. Neutralizing antibodies were only detected in the plasma of mice humanized with cells from haemophiliacs having FVIII inhibitors in their blood. The immune responses observed in hu-PBL-SCID mice correlated with the immune status of the corresponding human donor.
Asunto(s)
Factor VIII/inmunología , Adulto , Animales , Formación de Anticuerpos , Factor VIII/antagonistas & inhibidores , Femenino , Hemofilia A/sangre , Hemofilia A/terapia , Humanos , Inmunización , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Toxoide Tetánico/inmunología , Trasplante HeterólogoRESUMEN
Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VIII forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg. At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the 80-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor-VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin. In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45-50-70 kDa). The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.