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利用層析法純化與去病毒之抗血小板免疫球蛋白預防新生兒血小板減少症之研究
Other Title
A chromatographically-purified, virally-inactivated, plasma-derived anti-human platelet antigen-1a immunoglobulin G for preventing fetal and neonatal alloimmune thrombocytopenia
Type
thesis
Date Issued
2015-07-07
Author(s)
翁英然
Advisor
白台瑞
Subjects
系所名稱：生醫材料暨組織工程研究所
Description
學位別：碩士
語文別：英文
指導教授：白台瑞
共同指導教授：
口試委員：林良宗;Anne Husebekk
中文關鍵字：新生兒血小板減少症;層析法純化;病毒去活化
英文關鍵字：FNAIT;Anti-HPA-1a;Chromatographic purification;Viral inactivation
語文別：英文
指導教授：白台瑞
共同指導教授：
口試委員：林良宗;Anne Husebekk
中文關鍵字：新生兒血小板減少症;層析法純化;病毒去活化
英文關鍵字：FNAIT;Anti-HPA-1a;Chromatographic purification;Viral inactivation
Abstract
Background
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibody–mediated destruction of fetal or neonatal platelets. Maternal immunization due to an incompatibility of platelet-specific antigens between the fetus and the mother is the basis for FNAIT. In Caucasian population, fetal–maternal incompatibility for human platelet antigen-1a (HPA-1a) is the most common cause of this disorder. Platelet specific antibodies induce fetal thrombocytopenia that may cause intracranial bleeding and death. To date, there is no way to prevent such alloimmunization, nor is there any safe and effective treatment for FNAIT until delivery. The strong pathological similarities that exist between FNAIT and hemolytic disease of the newborn, which can be prevented by the injection of anti-rhesus immunoglobulin (IgG), supports the potential for a similar therapeutic approach against FNAIT based on antibody-mediated immune suppression (AMIS). Until now, no manufacturing process complying with the requirements of Good Manufacturing Practice has been developed to obtain clinical-grade prophylactic IgG for preventing FNAIT.
Aim
Develop a state-of-the-art scalable process for the purification and viral reduction of a human plasma-derived anti-HPA-1a IgG that could possibly be used for preventing maternal alloimmunization.
Methods
HPA-1a positive plasma was collected by apheresis from volunteer women who became alloimmunized in connection to pregnancy and gave birth to severely thrombocytopenic children. They provided informed consent. Plasma was cryoprecipitated at 2-4°C, and the supernatant was treated by 5% caprylic acid pH 5.5 at 25°C. The supernatant was then directly incubated with solvent/detergent [S/D; 1% tri-n-butyl phosphate (TnBP) / 1% Triton X-100, >1 hour, at 22 +/- 1°C] to inactivate lipid-enveloped viruses. The protein and S/D mixture was then purified on a S-HyperCel cation exchange column (Pall Life Sciences) to purify the IgG in binding/elution mode and remove the S/D agents and part of IgA and IgM. IgG polishing was achieved by HyperCel STAR AX anion-exchanger (Pall Life Sciences) used in a flow-through mode. The IgG-rich flow-through was subjected to 20 nm nanofiltration (Planova 20N, Asahi Kasei Medical) for viral removal, concentrated with centrifugal devices and then dialyzed against PBS buffer by dynamic dialysis device. Fractions were analyzed to determine protein content, protein profile by SDS-PAGE and zone electrophoresis, IgG, IgA, and IgM by ELISA, complement factor 3 (C3) and 4 (C4) by PEG enhanced immunoturbidimetry, and risk of thrombogenic activity by a thrombin generation assay (TGA). Pak12 assay (Immucor Inc.) and monoclonal platelet antigen capture assay (MAIPA) were used for follow-up of the anti-HPA-1a IgG along the purification process. The capacity of the nanofiltration step to remove viruses was evaluated by spiking experiments using hepatitis C virus (HCV) as a relevant virus model. Viral infectivity before and after nanofiltration was assessed on HuH-7.5 cell cultures and the extent of viral removal calculated by comparing HCV infectivity of the IgG before and after nanofiltration.
Results
The caprylic acid treatment precipitated most non-Ig plasma proteins yielding approximately 90% pure immunoglobulin supernatant. S-HyperCel and HyperCel STAR AX were found to ensure a high IgG recovery (>80% compared to caprylic acid supernatant) and purity (>99.5%), as well as efficient IgA and IgM removal (undetectable). Complement factor C3 and C4 were < 0.5 mg/dL and < 0.4 mg/dL, respectively. TGA showed low thrombin generation activity (<154 nM) in purified concentrated anti-HPA-1a IgG (154nM). Pak12 kit and MAIPA assay showed that the developed purification process could successfully be used to extract the anti-HPA-1a IgG with an HPA-1a antigen binding capacity up to 665 IU. Spiking experiments evidenced that the nanofiltration step of the HyperCel STAR AX breakthrough efficiently removed 3 log of HCV infectivity.
Conclusions
We demonstrate that it is technically achievable to fractionate plasma of HPA-1a-alloimmunized women, using a manufacturing process combining cryoprecipitation, caprylic acid precipitation, cation-exchange and anion-exchange chromatography that allows to obtain essentially pure anti-HPA-1a hyperimmune IgG. The process includes three steps expected to contribute to robust viral reduction: caprylic acid precipitation, S/D treatment, and 20-nm nanofiltration. The process could be readily scaled-up for producing batches for clinical evaluations.
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibody–mediated destruction of fetal or neonatal platelets. Maternal immunization due to an incompatibility of platelet-specific antigens between the fetus and the mother is the basis for FNAIT. In Caucasian population, fetal–maternal incompatibility for human platelet antigen-1a (HPA-1a) is the most common cause of this disorder. Platelet specific antibodies induce fetal thrombocytopenia that may cause intracranial bleeding and death. To date, there is no way to prevent such alloimmunization, nor is there any safe and effective treatment for FNAIT until delivery. The strong pathological similarities that exist between FNAIT and hemolytic disease of the newborn, which can be prevented by the injection of anti-rhesus immunoglobulin (IgG), supports the potential for a similar therapeutic approach against FNAIT based on antibody-mediated immune suppression (AMIS). Until now, no manufacturing process complying with the requirements of Good Manufacturing Practice has been developed to obtain clinical-grade prophylactic IgG for preventing FNAIT.
Aim
Develop a state-of-the-art scalable process for the purification and viral reduction of a human plasma-derived anti-HPA-1a IgG that could possibly be used for preventing maternal alloimmunization.
Methods
HPA-1a positive plasma was collected by apheresis from volunteer women who became alloimmunized in connection to pregnancy and gave birth to severely thrombocytopenic children. They provided informed consent. Plasma was cryoprecipitated at 2-4°C, and the supernatant was treated by 5% caprylic acid pH 5.5 at 25°C. The supernatant was then directly incubated with solvent/detergent [S/D; 1% tri-n-butyl phosphate (TnBP) / 1% Triton X-100, >1 hour, at 22 +/- 1°C] to inactivate lipid-enveloped viruses. The protein and S/D mixture was then purified on a S-HyperCel cation exchange column (Pall Life Sciences) to purify the IgG in binding/elution mode and remove the S/D agents and part of IgA and IgM. IgG polishing was achieved by HyperCel STAR AX anion-exchanger (Pall Life Sciences) used in a flow-through mode. The IgG-rich flow-through was subjected to 20 nm nanofiltration (Planova 20N, Asahi Kasei Medical) for viral removal, concentrated with centrifugal devices and then dialyzed against PBS buffer by dynamic dialysis device. Fractions were analyzed to determine protein content, protein profile by SDS-PAGE and zone electrophoresis, IgG, IgA, and IgM by ELISA, complement factor 3 (C3) and 4 (C4) by PEG enhanced immunoturbidimetry, and risk of thrombogenic activity by a thrombin generation assay (TGA). Pak12 assay (Immucor Inc.) and monoclonal platelet antigen capture assay (MAIPA) were used for follow-up of the anti-HPA-1a IgG along the purification process. The capacity of the nanofiltration step to remove viruses was evaluated by spiking experiments using hepatitis C virus (HCV) as a relevant virus model. Viral infectivity before and after nanofiltration was assessed on HuH-7.5 cell cultures and the extent of viral removal calculated by comparing HCV infectivity of the IgG before and after nanofiltration.
Results
The caprylic acid treatment precipitated most non-Ig plasma proteins yielding approximately 90% pure immunoglobulin supernatant. S-HyperCel and HyperCel STAR AX were found to ensure a high IgG recovery (>80% compared to caprylic acid supernatant) and purity (>99.5%), as well as efficient IgA and IgM removal (undetectable). Complement factor C3 and C4 were < 0.5 mg/dL and < 0.4 mg/dL, respectively. TGA showed low thrombin generation activity (<154 nM) in purified concentrated anti-HPA-1a IgG (154nM). Pak12 kit and MAIPA assay showed that the developed purification process could successfully be used to extract the anti-HPA-1a IgG with an HPA-1a antigen binding capacity up to 665 IU. Spiking experiments evidenced that the nanofiltration step of the HyperCel STAR AX breakthrough efficiently removed 3 log of HCV infectivity.
Conclusions
We demonstrate that it is technically achievable to fractionate plasma of HPA-1a-alloimmunized women, using a manufacturing process combining cryoprecipitation, caprylic acid precipitation, cation-exchange and anion-exchange chromatography that allows to obtain essentially pure anti-HPA-1a hyperimmune IgG. The process includes three steps expected to contribute to robust viral reduction: caprylic acid precipitation, S/D treatment, and 20-nm nanofiltration. The process could be readily scaled-up for producing batches for clinical evaluations.