biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2026-727biopreparat-727Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESТерапевтические гликопротеины: способ пробоподготовки для профилирования N-гликанов моноклональных антителTherapeutic glycoproteins: sample preparation for N-glycan profiling of monoclonal antibodieshttps://orcid.org/0000-0001-6029-4563НероноваМ. Ю.NeronovaM. Yu.

Неронова Мария Юрьевна

ул. Заводская, стр. 273, пос. Вольгинский, Владимирская область, 601125

Maria Yu. Neronova

273 Zavodskaya St., Volginsky, Vladimir Region, 601125

https://orcid.org/0009-0004-6049-9322КаргополовИ. А.KargopolovI. A.

Каргополов Иван Андреевич

ул. Заводская, стр. 273, пос. Вольгинский, Владимирская область, 601125

Ivan A. Kargopolov

273 Zavodskaya St., Volginsky, Vladimir Region, 601125

kargopolov@generium.ruhttps://orcid.org/0009-0009-1768-8992МериновА. А.MerinovA. A.

Меринов Артем Андреевич

ул. Заводская, стр. 273, пос. Вольгинский, Владимирская область, 601125

Artem A. Merinov

273 Zavodskaya St., Volginsky, Vladimir Region, 601125

https://orcid.org/0000-0002-9452-7879КалмыковаА. А.KalmykovaA. A.

Калмыкова Алена Александровна

ул. Заводская, стр. 273, пос. Вольгинский, Владимирская область, 601125

Alena A. Kalmykova

273 Zavodskaya St., Volginsky, Vladimir Region, 601125

https://orcid.org/0000-0002-6178-0050ЗубареваЕ. В.ZubarevaE. V.

Зубарева Екатерина Валерьевна

ул. Заводская, стр. 273, пос. Вольгинский, Владимирская область, 601125

Ekaterina V. Zubareva

273 Zavodskaya St., Volginsky, Vladimir Region, 601125

Акционерное общество «ГЕНЕРИУМ»РоссияGENERIUM JSCRussian Federation20221004202600Принято в печать (Online First)727Copyright © Неронова М.Ю., Каргополов И.А., Меринов А.А., Калмыкова А.А., Зубарева Е.В., 20222022Неронова М.Ю., Каргополов И.А., Меринов А.А., Калмыкова А.А., Зубарева Е.В.Neronova M.Y., Kargopolov I.A., Merinov A.A., Kalmykova A.A., Zubareva E.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.biopreparations.ru/jour/article/view/727ВВЕДЕНИЕ

ВВЕДЕНИЕ. Гликановый профиль — критический параметр качества лекарственных средств (ЛС) на основе моноклональных антител (МкАТ), контроль которого обязателен при разработке и выпуске каждой серии ЛС. Актуальность работы обусловлена необходимостью разработки воспроизводимого протокола пробоподготовки для анализа гликанового профиля, адаптированного к условиям стандартной физико-химической лаборатории и не требующего применения коммерческих наборов ограниченной доступности.

ЦЕЛЬ

ЦЕЛЬ. Разработать альтернативный способ пробоподготовки для количественной характеризации гликанового профиля терапевтических антител без использования коммерческих наборов.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. Объекты исследования — лекарственные средства на основе МкАТ: омализумаб, устекинумаб, канакинумаб, тоцилизумаб, натализумаб и иммуноглобулин класса G2, специфичный к рецептору PD-1. N-гликаны отщепляли от МкАТ пептид-N-гликозидазой (ПНГаза F), затем присоединяли к ним флуоресцентные метки — 2-аминобензамид (2-АВ) или 2-аминобензойную кислоту (2-АА) или InstantAB и очищали пробы от посторонних примесей. Для анализа смесей гликанов использовали высокоэффективную жидкостную хроматографию гидрофильного взаимодействия с детектированием флуоресценции (HILIC-FLD) на оборудовании Alliance e2695 и Acquity Arc Bio с детектором флуоресценции FLR 2475. Гликановый профиль оценивали по содержанию функциональных групп гликанов.

РЕЗУЛЬТАТЫ

РЕЗУЛЬТАТЫ. В работе подобраны следующие условия пробоподготовки N-гликанов терапевтических МкАТ: инкубация с 2 мЕД ПНГазой F на 100 мкг белка в 10 мМ трис-HCl (pH 8,0) в течение 3 ч при температуре 37 °C без предварительной денатурации белка; окрашивание гликанов 2-АА в течение 1,5 ч при 65 °C; удаление избытка красителя путем экстракции ацетонитрилом. Осаждение ацетонитрилом меченых 2-АА гликанов пригодно для очистки и концентрирования проб. Применение метода HILIC-FLD позволило определять в пробах минорные гликаны с содержанием до 0,1–0,2%. Сравнение наших данных с данными, полученными при использовании коммерческого набора, указало на приемлемую сопоставимость результатов хроматографического анализа. При пробоподготовке важно учитывать структурные особенности конкретных МкАТ, например для высокосиалированных МкАТ обоснован контроль десиалирования в процессе окрашивания.

ВЫВОДЫ

ВЫВОДЫ. Разработанный способ пробоподготовки пригоден для анализа омализумаба, канакинумаба и натализумаба. Предложенные аналитические подходы к пробоподготовке будут также полезны при разработке методики анализа гликанового профиля других гликопротеинов с использованием метода HILIC-FLD.

INTRODUCTION

INTRODUCTION. Glycan profile is a critical quality parameter for therapeutic monoclonal antibodies (mAbs) that is consistently estimated during development and release of each drug batch. The need to develop a reproducible sample preparation protocol for glycan profiling brings about the relevance of the study. The profile should be adapted to the conditions of a standard physicochemical laboratory, so that it avoids using commercial preparation kits that are currently in short supply.

AIM

AIM. This study aimed to develop an alternative sample preparation procedure for quantitation of glycans in therapeutic monoclonal antibodies without using commercial kits.

MATERIALS AND METHODS

MATERIALS AND METHODS. Omalizumab, ustekinumab, canakinumab, tocilizumab, natalizumab, and human anti-PD IgG2 were used as the study objects. N-glycans were released from mAbs by peptide-N-glycosidase (PNGase F) and labeled with fluorescent tags 2-aminobenzamide (2-АВ) or 2-aminobenzoic acid (2-АА) or InstantAB. Subsequently, glycan samples were cleaned from impurities. Glycan compounds were analyzed using hydrophilic interaction liquid chromatography (HILIC-FLD) on Alliance e2695 and Acquity Arc Bio chromatographic systems equipped with FLR 2475 fluorescence detector. The glycan profile was assessed by the content of functional glycan groups.

RESULTS

RESULTS. Conditions for preparing therapeutic mAbs N-glycans were chosen as follows: incubation with 2 mEU PNGase F per 100 µg protein in 10 mM Tris-HCl (pH 8.0) at 37 °C for 3 h without protein denaturation; derivatization of glycans with 2-AA at 65 °C for 1.5 h; extraction of 2-AA excess with acetonitrile. Centrifugation of labeled glycans with acetonitrile is suitable for purification and concentration of samples. Minor glycans with a content not more than 0.1–0.2% were determined using HILIC-FLD. Comparison with data obtained using a commercial kit for sample preparation indicated acceptable comparability of the results. When preparing the samples, we took into account structural features of specific mAbs, for instance, desialylation control during the staining was found necessary for high-sialylated mAbs.

CONCLUSIONS

CONCLUSIONS. The developed sample preparation procedure is suitable for the analysis of N-glycans of omalizumab, canakinumab and natalizumab and may be used to develop the analysis methods of the glycan profile of other glycoproteins using HILIC-FLD mode.

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