Abstract
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.
Highlights
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the generation of maternal alloantibodies as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father [1,2]
Human plasma remains an important source of established or novel therapeutic products, including polyvalent and hyper-immune immunoglobulins like anti-D Ig [19], which are on the WHO model list of essential medicines [30]
Since HPA-1a plasma from alloimmunized women will likely be available in limited volumes, not compatible with the current scale of the traditional ethanol plasma fractionation industry [18], a dedicated purification method is needed both for clinical proof-of-concept and future production
Summary
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the generation of maternal alloantibodies as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father [1,2]. FNAIT occurs in about 40 per 100’000 pregnancies, and the most feared complication, intracranial bleeding (ICH) in fetuses and newborns, in 3 or 4 children per 100’000 [3,4]. ICH may result in severe neurologic sequelae, miscarriage, and neonatal death [5]. No screening for FNAIT is performed and mothers with affected children are likely to be treated with intravenous immunoglobulin (IVIG), with or without steroids, in any subsequent pregnancies [7,8]. Intrauterin platelet transfusions are not recommended due to high risk of bleeding complications and the efficacy of this invasive fetal platelet transfusion has not been well studied [9]
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