chemicallytechFine Chemical TechnologiesТонкие химические технологии2410-65932686-7575MIREA – Russian Technological University (RTU MIREA).10.32362/2410-6593-2026-21-2-165-178SNRUWUchemicallytech-2385Research ArticleCHEMISTRY AND TECHNOLOGY OF MEDICINAL COMPOUNDS AND BIOLOGICALLY ACTIVE SUBSTANCESХИМИЯ И ТЕХНОЛОГИЯ ЛЕКАРСТВЕННЫХ ПРЕПАРАТОВ И БИОЛОГИЧЕСКИ АКТИВНЫХ СОЕДИНЕНИЙDevelopment, characterization, and stability assessment of a lyophilized Eculizumab formulation for use as a reference materialРазработка, характеризация и оценка стабильности лиофилизированной формы экулизумаба для использования в качестве стандартного образцаhttps://orcid.org/0000-0002-5542-982XЗыбинД. И.ZybinD. I.

Зыбин Дмитрий Игоревич, к.х.н., начальник лаборатории по разработке аналитических методик

Scopus Author ID 57189868539

Researcher ID P-8049-2016

117246, Москва, Научный пр-д, д. 8, корп. 1

 

Dmitry I. Zybin, Cand. Sci. (Chem.), Head of the Research Laboratory

Scopus Author ID 57189868539, Researcher ID P-8049-2016

8, b. 1, Nauchnyi proezd, Moscow, 117246

mithtchem@gmail.comКлишинА. А.KlishinA. A.

Клишин Анатолий Анатольевич, начальник сектора контроля качества

117246, Москва, Научный пр-д, д. 8, корп. 1

Anatoly A. Klishin, Head of the Research Laboratory

8, b. 1, Nauchnyi proezd, Moscow, 117246

klishin@pharmapark.ruhttps://orcid.org/0000-0003-4161-5880ОрловаН. В.OrlovaN. V.

Орлова Наталья Владимировна, к.б.н., заместитель директора по науке

117246, Москва, Научный пр-д, д. 8, корп. 1

Natalya V. Orlova, Cand. Sci. (Biol.), Deputy Director for Science

8, b. 1, Nauchnyi proezd, Moscow, 117246

orlova.chemist@gmail.comСорокинаТ. С.SorokinaT. S.

Сорокина Татьяна Сергеевна, директор по науке

117246, Москва, Научный пр-д, д. 8, корп. 1

Tatiana S. Sorokina, Director of Science

8, b. 1, Nauchnyi proezd, Moscow, 117246

sorokina@pharmapark.ruhttps://orcid.org/0000-0002-5485-9297КапустинД. В.KapustinD. V.

Капустин Дмитрий Валерьевич, д.х.н., старший научный сотрудник, Лаборатория полимеров для биологии

Scopus Author ID 6602903079,

Researcher ID B-5773-2014

117997, Москва, ул. Миклухо-Маклая, д. 16/10

Dmitry V. Kapustin, Dr. Sci. (Chem.), Senior Researcher, Laboratory of Polymers for Biology

Scopus Author ID 6602903079, Researcher ID B-5773-2014

16/10, Miklukho-Maklaya ul., Moscow, 117997

kapustin@ibch.ruООО ФАРМАПАРКРоссияPHARMAPARKRussian FederationИнститут биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наукРоссияM.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of SciencesRussian Federation202606052026212165–178Copyright © Zybin D.I., Klishin A.A., Orlova N.V., Sorokina T.S., Kapustin D.V., 20262026Зыбин Д.И., Клишин А.А., Орлова Н.В., Сорокина Т.С., Капустин Д.В.Zybin D.I., Klishin A.A., Orlova N.V., Sorokina T.S., Kapustin D.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.finechem-mirea.ru/jour/article/view/2385Objectives

Objectives. The study set out to develop a stable lyophilized formulation of the monoclonal antibody Eculizumab, comprehensively characterize the resulting material, and assess its stability for qualifying it as a reference material. This involved developing a matching placebo formulation, determining the optimal lyophilization conditions, and conducting a rigorous stability study.

Methods

Methods. In the development of the formulation and lyophilization conditions for Eculizumab, we tested various buffer systems and cryoprotectants. The residual moisture content in the resulting lyophilized samples was determined by Karl Fischer titration. Peptide mapping was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) following enzymatic hydrolysis with trypsin. The structural, physicochemical, and biological properties were analyzed using various analytical methods, including RP-HPLC, high-performance liquid chromatography mass spectrometry, capillary sodium dodecyl sulfate electrophoresis, size-exclusion high-performance liquid chromatography, and enzyme-linked immunosorbent assay.

Results

Results. A placebo solution for lyophilization of Eculizumab was selected with the following composition: 20 mM sodium phosphate, 4% trehalose, 0.2% polysorbate 80, pH 7.0. The results demonstrated a high degree of similarity between the candidate reference material and Eculizumab EU. Stability studies under storage conditions at 2–8°C demonstrated the material’s stability for one year, with control points at 3, 6, 9, and 12 months.

Conclusions

Conclusions. The absence of any effect of the drying process on the primary and spatial structure, post-translational modifications, content of related impurities, composition of isoforms, and specific activity was confirmed. Furthermore, stability studies demonstrated no significant changes in protein quality during storage at 2–8°C for at least 12 months, which represents the entire available data period at the time of manuscript preparation. The results indicate that the developed lyophilized material is a viable candidate for an international reference material, although its official qualification would require additional collaborative trials and long-term stability data.

Цели

Цели. Целью данного исследования была разработка стабильной лиофилизированной формы моноклонального антитела экулизумаб, характеризация полученного материала и оценка его стабильности для аттестации в качестве стандартного образца. Для достижения поставленных целей требовались разработка соответствующего состава плацебо, определение оптимальных условий лиофилизации и проведение исследования стабильности.

Методы

Методы. В ходе разработки состава плацебо и условий лиофилизации экулизумаба были протестированы различные буферные системы и криопротекторы. Содержание остаточной воды в лиофилизированных образцах определяли методом титрования по Карлу Фишеру. Пептидное картирование проводили методом обращенно-фазовой высокоэффективной жидкостной хроматографии (ВЭЖХ) после ферментативного гидролиза трипсином. Структурные, физико-химические и биологические свойства анализировали с использованием широкого ряда аналитических методов, включая обращенно-фазовую ВЭЖХ, жидкостную хромато-масс-спектрометрию, капиллярный гель-электрофорез, эксклюзионную ВЭЖХ и иммуноферментный анализ.

Результаты

Результаты. Для лиофилизации экулизумаба был выбран буферный раствор следующего состава: 20 мМ фосфат натрия, 4% трегалозы, 0.2% полисорбата 80, pH 7.0. Полученные результаты продемонстрировали высокую степень сходства между кандидатом на стандартный образец и референсным образцом экулизумаба. Исследования стабильности при хранении при 2–8°C показали стабильность материала в течение одного года с контрольными точками через 3, 6, 9 и 12 месяцев.

Выводы

Выводы. Подтверждено отсутствие влияния процесса лиофилизации на первичную и пространственную структуру, посттрансляционные модификации, содержание родственных примесей, состав изоформ и специфическую активность. Кроме того, исследования стабильности показали отсутствие значимых изменений качества белка при хранении при 2–8°C в течение по меньшей мере 12 месяцев, что соответствует всему доступному объему данных на момент подготовки рукописи. Полученные результаты свидетельствуют о том, что разработанный лиофилизированный материал является перспективным кандидатом на роль международного стандартного образца, однако его официальная аттестация потребует проведения дополнительных межлабораторных исследований и получения данных по долгосрочной стабильности.

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