vedomostiregmedРегуляторные исследования и экспертиза лекарственных средствRegulatory Research and Medicine Evaluation3034-30623034-3453Federal State Budgetary Institution ‘Scientific Centre for Expert Evaluation of Medicinal Products’ of the Ministry of Health of the Russian Federation (FSBI ‘SCEEMP’)10.30895/1991-2919-2019-9-2-131-139vedomostiregmed-231Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESВалидация иммуноферментной тест-системы для доклинических исследований фармакокинетики ритуксимабаValidation of Enzyme Immunoassay for Preclinical Pharmacokinetic Trials of Rituximabhttps://orcid.org/0000-0003-3212-4369ПисаревВ. В.PisarevV. V

Писарев Владимир Викторович - кандидат химических  наук, генеральный директор.

Ленинские Горы, д. 1, стр. 75В, Москва, 119992

Vladimir V. Pisarev - Cand. Sci. (Chem.).

1/75B Leninskie Gory, Moscow 119992

info@probiotech.ruhttps://orcid.org/0000-0002-3837-4759УляшоваМ. М.UlyashovaMaria M

Уляшова Мария Морисовна - кандидат химических наук, руководитель лаборатории фармакологии лекарственных средств.

Ленинские Горы, д. 1, стр. 75В, Москва, 119992

Maria M. Ulyashova - Cand. Sci. (Chem.).

1/75B Leninskie Gory, Moscow 119992

info@probiotech.ruhttps://orcid.org/0000-0002-2537-2850ГильдееваГ. Н.GildeevaGelia N

Гильдеева Гэлия Нязыфовна - доктор фармацевтических наук, профссор кафедры организации и управления в сфере обращения лекарственных средств.

Трубецкая ул., д. 8, стр. 2, Москва, 119991

Gelia N. Gildeeva - Dr. Sci. (Pharm.), Professor.

8/2 Trubetskaya St., Moscow 119991

info@probiotech.ruОбщество с ограниченной ответственностью «Научно-производственный центр Пробиотек»РоссияScientific and Production Center Probiotech LLCRussian FederationФедеральное государственное автономное образовательное учреждение высшего образования «Первый Московский государственный медицинский университет им. И.М. Сеченова» Министерства здравоохранения Российской ФедерацииРоссияI. M. Sechenov First Moscow State Medical UniversityRussian Federation20190506201992131139Copyright © Писарев В.В., Уляшова М.М., Гильдеева Г.Н., 20192019Писарев В.В., Уляшова М.М., Гильдеева Г.Н.Pisarev V.V., Ulyashova M.M., Gildeeva G.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.vedomostincesmp.ru/jour/article/view/231

Неотъемлемой частью доклинических фармакокинетических исследований является разработка биоаналитической методики определения лекарственного средства в биологической жидкости.

Цель работы

Цель работы: оценка пригодности тест-системы на основе твердофазного иммуноферментного анализа (ИФА) (ООО «Научно-производственный центр Пробиотек») для количественного определения ритуксимаба в сыворотке крови лабораторных животных после внутривенного введения ритуксимаба в количестве, соответствующем терапевтической дозе у человека.

Материалы и методы

Материалы и методы: определение ритуксимаба проводили по «сэндвич»-схеме твердофазного ИФА в две стадии с последующей детекцией на основе пероксидазы хрена. Регистрация результатов ИФА проводилась с помощью микропланшетного фотометра при длине волны 450 нм.

Результаты

Результаты: в ходе работы установлены предел обнаружения (0,24 нг/мл) и нижний предел количественного определения (1,00 нг/мл) ритуксимаба в сыворотке крови кроликов, доказана высокая селективность определения аналита в многокомпонентной биологической матрице. Найденные средние значения концентрации ритуксимаба не отклонялись от номинальных значений более чем на 14 % во всем диапазоне применения методики, внутри- и межсерийная прецизионность тест-системы не превышала 7,4 %, общая ошибка метода — 20,1 %. Продемонстрированная линейность разведения позволяет применять данную тест-систему для анализа биологических образцов в широком диапазоне концентраций ритуксимаба. Стабильность аналита в сыворотке крови кроликов подтверждена при хранении образцов в течение 6 ч при комнатной температуре, 50 дней при —35 °С и после 3 циклов замораживания-оттаивания. Валидированная тест-система была успешно апробирована в ходе определения концентрации ритук-симаба в биологических образцах, полученных в рамках исследования фармакокинетики ритуксимаба у кроликов, во всем интервале определяемых концентраций подтверждена достоверность результатов анализа, продемонстрирован параллелизм между калибровочным графиком и результатами анализа серийно разведенных образцов сыворотки крови кроликов с максимальной концентрацией ритуксимаба.

Выводы

Выводы: изученная иммуноферментная тест-система пригодна для количественного определения ритуксимаба в сыворотке крови лабораторных животных, поскольку по всем валидационным параметрам соответствует критериям приемлемости, предъявляемым международными стандартами по валидации биоаналитических методов.

An integral part of preclinical pharmacokinetic studies is the development of a bioanalytical method for determination of the drug in a biological fluid.

The aim of the research was to assess the suitability of the test system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of rituximab in the blood serum of laboratory animals after intravenous administration of rituximab at a dose corresponding to the therapeutic dose in humans. Th test system was developed by the Scientific and Production Center Probiotech.

Materials and methods

Materials and methods: the determination of rituximab in biological samples was carried out using a two-stage sandwich-type ELISA, followed by detection based on horseradish peroxidase. The ELISA results were recorded using a microplate photometer at a wavelength of 450 nm.

Results

Results: the experiments helped to establish the detection limit (0.24 ng/mL) and the lower limit of quantitation (1.00 ng/mL) of rituximab in rabbit blood serum, they also demonstrated high selectivity of analyte determination in a multicomponent biological matrix. The mean rituximab concentration was within 14 % of the nominal value in the entire working range of the method. The within-run and between-run precision of the assay did not exceed 7.4 %, the total error of the method did not exceed 20.1 %. The linearity of dilution makes it possible to use the assay for the analysis of biological samples with a wide range of rituximab concentrations. The stability of the analyte in the rabbit blood serum was confirmed by storing samples for 6 hours at room temperature, for 50 days at —35 °C, and after 3 freeze-thaw cycles. The validated immunoassay was successfully used to determine the rituximab concentration in biological samples obtained in the rituximab pharmacokinetic trial in rabbits. The accuracy of the results was confirmed for the entire range of the determined concentrations; parallelism was demonstrated between the calibration curve and the results of analysis of serially diluted rabbit serum samples with the maximum concentration of rituximab.

Conclusions

Conclusions: the proposed enzyme immunoassay test system can be used for quantitative determination of rituximab in the blood serum of laboratory animals, as it meets acceptance criteria for all validation parameters described in the international guidelines on validation of bioanalytical methods.

ритуксимаббиоаналогфармакокинетикатвердофазный иммуноферментный анализвалидациядоклинические исследованияrituximabbiosimilarpharmacokineticsenzyme-linked immunosorbent assayvalidationpreclinical trialsReferencesPescovitz MD. Rituximab, an anti-CD20 monoclonal antibody: history and mechanism of action. Am J Transplant. 2006;6(5 Pt 1):859—66. https://doi.org/10.1111/j.1600-6143.2006.01288.xPescovitz MD. Rituximab, an anti-CD20 monoclonal antibody: history and mechanism of action. Am J Transplant. 2006; 6(5): 859-66.Vital EM, Kay J, Emery P. Rituximab biosimilars. Expert Opin Biol Ther. 2013;13(7):1049-62. https://doi.org/10.1517/14712598.2013.787064Vital EM, Kay J, Emery P. Rituximab biosimilars. Expert. Opin. Biol. Ther. 2013; 13(7): 1049-62.Seyfizadeh N, Seyfizadeh N, Hasenkamp J, Huerta-Yepez S. A molecular perspective on rituximab: A monoclonal antibody for B cell non Hodgkin lymphoma and other affections. Crit Rev Oncol Hematol. 2016;97:275-90. https://doi.org/10.1016/j.critrev-onc.2015.09.001Seyfizadeh N, Seyfizadeh N, Hasenkamp J, Huerta-Yepez S. A molecular perspective on rituximab: A monoclonal antibody for B cell non Hodgkin lymphoma and other affections. Crit. Rev. Oncol. Hematol. 2016; 97: 275-90.Roshique KK, Ravindran V. Efficacy and safety of a biosimilar rituximab in biologic naive patients with active rheumatoid arthritis. Clin Rheumatol. 2015;34(7):1289-92. https://doi.org/10.1007/s10067-015-2980-4Roshique KK, Ravindran V. Efficacy and safety of a biosimilar rituximab in biologic naïve patients with active rheumatoid arthritis. Clin. Rheumatol. 2015; 34(7): 1289-92.Gurcan HM, Keskin DB, Stern JN, Nitzberg MA, Shekhani H, Ahmed AR. A review of the current use of rituximab in autoimmune diseases. IntImmunopharmacol. 2009;9(1):10—25. https://doi.org/10.1016/j.intimp.2008.10.004Gürcan HM, Keskin DB, Stern JN, Nitzberg MA, Shekhani H, Ahmed AR. A review of the current use of rituximab in autoimmune diseases. Int. Immunopharmacol. 2009; 9(1):10-25.Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010;47(2):115-23. https://doi.org/10.1053/j.seminhematol.2010.01.011Weiner GJ. Rituximab: mechanism of action. Semin. Hematol. 2010; 47(2): 115-23.Blasco H, Lalmanach G, Godat E, Maurel MC, Canepa S, Belghazi M, et al. Evaluation of a peptide ELISA for the detection of rituximab in serum. J Immunol Methods. 2007;325(1-2):127-39. https://doi.org/10.1016/j.jim.2007.06.011Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues (EMEA/CHMP/BMWP/42832/2005 Rev1). European Medicines Agency. Available from: https://www.ema.europa.eu/documents/scientific-guideline/guideline-similar-biological-medicinal-products-containing-biotechnology-derived-proteins-active_en-2.pdfBeum PV, Kennedy AD, Taylor RP. Three new assays for rituximab based on its immunological activity or antigenic properties: Analyses of sera and plasmas of RTX-treated patients with chronic lymphocytic leukemia and other B cell lymphomas. J Immunol Methods. 2004;289(1-2):97-109. https://doi.org/10.1016/jjim.2004.03.012Guideline on similar biological medicinal products containing monoclonal antibodies – non-clinical and clinical issues (EMA/CHMP/BMWP/403543/2010). European Medicines Agency. Available from: https://www.ema.europa.eu/documents/scientific-guideline/guideline-similar-biological-medicinal-products-containing-monoclonal-antibodies-non-clinical_en.pdf.Berinstein NL, Grillo-Lopez, White CA, Bence-Bruckler I, Maloney D, Czuczman M, et al. Association of serum Rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin's lymphoma. Ann Oncol. 1998;(9):995-1001. https://doi.org/10.1023/A:1008416911099Guidelines on evaluation of similar biotherapeutic products (SBPs). World Health Organization. Available from: https://www.who.int/biologicals/areas/biological_ therapeutics/BIOTHERAPEUTICS_FOR_WEB_22APRIL2010.pdfHong K, Presta LG, Lu Y, Penn A, Adams C, Chuntharapai A, et al. Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20. J Immunol Methods. 2004;294(1-2):189-97. https://doi.org/10.1016/j.jim.2004.09.003Blasco H., Lalmanach G., Godat E., Maurel M. C., Canepa S., Belghazi M., et al. Evaluation of a peptide ELISA for the detection of rituximab in serum. J. Immunol. Methods 2007; 325(1-2): 127-39.Cragg MS, Bayne MB, Tutt AL, French RR, Beers S, Glennie MJ, et al. A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells. Blood. 2004;104(8):2540-2. https://doi.org/10.1182/blood-2004-05-1733Beum P V., Kennedy AD, Taylor RP. Three new assays for rituximab based on its immunological activity or antigenic properties: Analyses of sera and plasmas of RTX-treated patients with chronic lymphocytic leukemia and other B cell lymphomas. J. Immunol. Methods 2004; 289 (1-2): 97-109.Hampson G, Ward TH, Cummings J, Bayne M, Tutt AL, Cragg MS, et al. Validation of an ELISA for the determination of rituximab pharmacokinetics in clinical trials subjects. J Immunol Methods. 2010;360(1-2):30-8. https://doi.org/10.1016/jjim.2010.05.009Berinstein N.L., White C., Maloney D., Czuczman M., Green D., Rosenberg J., et al. Association of serum Rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin’s lymphoma. Ann. Oncol. 1998; 9: 995-1001.Schoergenhofer C, Schwameis M, Firbas C, Bartko J, Derhaschnig U, Mader RM, et al. Single, very low rituximab doses in healthy volunteers — a pilot and a randomized trial: Implications for dosing and biosimilarity testing. Sci Rep. 2018;8(1):124. https://doi.org/10.1038/s41598-017-17934-6Hong K., Presta L.G., Lu Y., Penn A., Adams C., Chuntharapai A., et al. Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20. J. Immunol. Methods 2004; 294 (1-2): 189-97.Crowther JR. The ELISA guidebook. Methods Mol Biol. 2000;149:1-413. PMID: 11028258Cragg M.S., Bayne M.B., Tutt A.L., French R.R., Beers S., Glennie M.J., et al. A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells. Blood 2004; 104(8): 2540-2.Cartron G, Blasco H, Paintaud G, Watier H, Le Guellec C. Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit Rev Oncol Hematol. 2007;62(1):43-52. https://doi.org/10.1016/j.critrevonc.2006.09.004Hampson G., Ward T.H., Cummings J., Bayne M., Tutt A.L., Cragg M.S., et al. Validation of an ELISA for the determination of rituximab pharmacokinetics in clinical trials subjects. J. Immunol. Methods 2010; 360(1-2): 30-8.Yoo DH, Suh C-H, Shim SC, Jeka S, Cons-Molina FF, Hrycaj P, et al. A multicentre randomised controlled trial to compare the pharmacokinetics, efficacy and safety of CT-P10 and innovator rituximab in patients with rheumatoid arthritis. Ann Rheum Dis. 2017;76(3):566-70. https://doi.org/10.1136/annrheumdis-2016-209540Schoergenhofer C., Schwameis M., Firbas C., Bartko J., Derhaschnig U., Mader R.M., et al. Single, very low rituximab doses in healthy volunteers - A pilot and a randomized trial: Implications for dosing and biosimilarity testing. Sci. Rep. 2018; 8(1): 6-13.Ryan AM, Sokolowski SA, Ng C, Shirai N, Collinge M, Shen AC, et al. Comparative nonclinical assessments of the proposed biosimilar PF-05280586 and Rituximab (MabThera"). Toxicol Pathol. 2014;42(7):1069-81. https://doi.org/10.1177/0192623313520351Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2**). European Medicines Agency. Available from: https://www.ema.europa.eu/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdfda Silva A, Kronthaler U, Koppenburg V, Fink M, Meyer I, Papandriko-poulou A, et al. Target-directed development and preclinical characterization of the proposed biosimilar rituximab GP2013. Leuk Lymphoma. 2014;55(7):1609-17. https://doi.org/10.3109/10428194.2013.843090Rules for conducting bioequivalence studies of drugs in the framework of the Eurasian Economic Union. Appendix №6. Council of the Eurasian Economic Commission (2016). Available from: http://www.consultant.ru/document/cons_doc_LAW_207405/Cartron G., Blasco H., Paintaud G., Watier H., Le Guellec C. Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit. Rev. Oncol. Hematol. 2007; 62(1): 43-52.Cartron G., Blasco H., Paintaud G., Watier H., Le Guellec C. Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit. Rev. Oncol. Hematol. 2007; 62(1): 43-52.Yoo D.H., Suh C-H., Shim S.C., Jeka S., Cons-Molina F.F., Hrycaj P., et al. A multicentre randomised controlled trial to compare the pharmacokinetics, efficacy and safety of CT-P10 and innovator rituximab in patients with rheumatoid arthritis. Ann. Rheum. Dis. 2017; 76(3): 566-70.Yoo D.H., Suh C-H., Shim S.C., Jeka S., Cons-Molina F.F., Hrycaj P., et al. A multicentre randomised controlled trial to compare the pharmacokinetics, efficacy and safety of CT-P10 and innovator rituximab in patients with rheumatoid arthritis. Ann. Rheum. Dis. 2017; 76(3): 566-70.Ryan AM, Sokolowski SA, Ng C., Shirai N. Comparative Nonclinical Assessments of the Proposed Biosimilar PF-05280586 and Rituximab (MabThera). Toxicol. Pathol. 2014; 42(7): 1069-81.Ryan AM, Sokolowski SA, Ng C., Shirai N. Comparative Nonclinical Assessments of the Proposed Biosimilar PF-05280586 and Rituximab (MabThera). Toxicol. Pathol. 2014; 42(7): 1069-81.Silva A., Kronthaler U., Koppenburg V., Fink M., Meyer I., Papandrikopoulou A., et al. Target-Directed Development And Preclinical Characterization Of The Proposed Biosimilar Rituximab Gp2013. Leuk Lymphoma 2014; 55 (7): 1609-17.Silva A., Kronthaler U., Koppenburg V., Fink M., Meyer I., Papandrikopoulou A., et al. Target-Directed Development And Preclinical Characterization Of The Proposed Biosimilar Rituximab Gp2013. Leuk Lymphoma 2014; 55 (7): 1609-17.

The authors declare that there are no conflicts of interest present.