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-1 a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1 a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1 a 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-1 a Ig G. 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 Pak12and 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.

Original languageEnglish
Article numbere0162973
JournalPLoS One
Volume11
Issue number9
DOIs
Publication statusPublished - Sep 1 2016

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Human Platelet Antigens
Neonatal Alloimmune Thrombocytopenia
thrombocytopenia
immunoglobulin G
Immunoglobulin G
antigens
Viruses
Hepacivirus
Immunoglobulins
Mothers
Platelets
Chromatography
Detergents
Assays
Blood Platelets
Hepatitis C virus
Monoclonal Antibodies
Complement C4
Plasma (human)
Plasmas

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Anti-human platelet antigen-1α immunoglobulin G preparation intended to prevent fetal and neonatal alloimmune thrombocytopenia. / Weng, Ying Jan; Husebekk, Anne; Skogen, Björn; Kjaer, Mette; Lin, Liang Tzung; Burnouf, Thierry.

In: PLoS One, Vol. 11, No. 9, e0162973, 01.09.2016.

Research output: Contribution to journalArticle

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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-1 a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1 a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1 a 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-1 a Ig G. 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 Pak12and 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.",
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AU - Husebekk, Anne

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AU - Lin, Liang Tzung

AU - Burnouf, Thierry

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