Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma
Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, o...
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LLC Center of Pharmaceutical Analytics (LLC «CPHA»)
2024-03-01
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| Series: | Разработка и регистрация лекарственных средств |
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| Online Access: | https://www.pharmjournal.ru/jour/article/view/1757 |
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| author | A. N. Afanaseva V. B. Saparova I. E. Makarenko T. A. Selmenskikh D. V. Kurkin A. L. Hohlov R. V. Drai |
| author_facet | A. N. Afanaseva V. B. Saparova I. E. Makarenko T. A. Selmenskikh D. V. Kurkin A. L. Hohlov R. V. Drai |
| author_sort | A. N. Afanaseva |
| collection | DOAJ |
| description | Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies. |
| format | Article |
| id | doaj-art-b275653dfe8748d792b2e41f4c5157b3 |
| institution | DOAJ |
| issn | 2305-2066 2658-5049 |
| language | Russian |
| publishDate | 2024-03-01 |
| publisher | LLC Center of Pharmaceutical Analytics (LLC «CPHA») |
| record_format | Article |
| series | Разработка и регистрация лекарственных средств |
| spelling | doaj-art-b275653dfe8748d792b2e41f4c5157b32025-08-20T02:54:16ZrusLLC Center of Pharmaceutical Analytics (LLC «CPHA»)Разработка и регистрация лекарственных средств2305-20662658-50492024-03-0113124725510.33380/2305-2066-2024-13-1-15311225Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human PlasmaA. N. Afanaseva0V. B. Saparova1I. E. Makarenko2T. A. Selmenskikh3D. V. Kurkin4A. L. Hohlov5R. V. Drai6Closed Joint Stock Company "Pharm-Holding"Closed Joint Stock Company "Pharm-Holding"; Federal State Budgetary Educational Institution of Higher Education "A. I. Yevdokimov Moscow State University of Medicine and Dentistry" of the Ministry of Healthcare of the Russion FederationClosed Joint Stock Company "Pharm-Holding"; Federal State Budgetary Educational Institution of Higher Education "A. I. Yevdokimov Moscow State University of Medicine and Dentistry" of the Ministry of Healthcare of the Russion FederationClosed Joint Stock Company "Pharm-Holding"Federal State Budgetary Educational Institution of Higher Education "A. I. Yevdokimov Moscow State University of Medicine and Dentistry" of the Ministry of Healthcare of the Russion FederationFederal State Budgetary Educational Institution of Higher Education "Yaroslavl State Medical University" of the Ministry of Healthcare of the Russian FederationClosed Joint Stock Company "Pharm-Holding"Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies.https://www.pharmjournal.ru/jour/article/view/1757anti-drug antibodiesbiosimilarneutralizing antibody studiesclinical studies of immunogenicityvalidationcell viability determinationantibodies to romiplostim |
| spellingShingle | A. N. Afanaseva V. B. Saparova I. E. Makarenko T. A. Selmenskikh D. V. Kurkin A. L. Hohlov R. V. Drai Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma Разработка и регистрация лекарственных средств anti-drug antibodies biosimilar neutralizing antibody studies clinical studies of immunogenicity validation cell viability determination antibodies to romiplostim |
| title | Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma |
| title_full | Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma |
| title_fullStr | Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma |
| title_full_unstemmed | Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma |
| title_short | Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma |
| title_sort | experimental validation a method for assessing neutralizing antibodies of romiplostim in human plasma |
| topic | anti-drug antibodies biosimilar neutralizing antibody studies clinical studies of immunogenicity validation cell viability determination antibodies to romiplostim |
| url | https://www.pharmjournal.ru/jour/article/view/1757 |
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