Разработка методики пептидного картирования для оценки подлинности рекомбинантного интерферона бета-1b

Goloshchapova, E. O.; Minero, A. S.; Рунова, О.; Ustinnikova, O. B.

doi:10.30906/2073-8099-2021-13-1-21-26

Cited by 3 publications

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“…Хроматографирование проводили с использованием хроматографической колонки YMC-Pack ODS-A (100×4,6 мм, октадецилсилил (С18) модифицированный силикагель с диаметром частиц 5 мкм, размер пор 300 Å, YMC, Япония) при температуре 30 °С с применением спектрофотометрического детектора (длина волны 214 нм) и градиентного режима с использованием двух подвижных фаз: подвижная фаза 1: 0,1% три фторуксусная кислота в воде; подвижная фаза 2: 0,1% трифторуксусная кислота в смеси ацетонитрил-вода, 9:1 при скорости потока 1 мл/ мин [8] В качестве материала для получения кандидата в ФСО был выбран полупродукт рчИФН бета-1b производства АО «ГЕНЕРИУМ», отобранный на финальной стадии очистки белка до добавления стабилизатора -человеческого сывороточного альбумина (ЧСА). Для обеспечения стабильности молекулы рчИФН бета-1b без белкового стабилизатора полупродукт быстро разбавляли в глициновом буфере (pH 3,0), содержащем трегалозу (50 мг/мл) и полисорбат 80 (0,05 мг/мл).…”

Section: методы метод пептидного картирования с использованием обраще...unclassified

“…Далее для аттестации ФСО рчИФН бета-1b применяли методику пептидного картирования, разработанную на предыдущем этапе работы [8]. Анализ профилей пептидных карт кандидата в ФСО, полученных в процессе валидации вышеуказанной методики, позволил выбрать 7 характеристических пиков, отличающихся достаточной интенсивностью и стабильным На основании двадцати результатов, полученных в условиях воспроизводимости в соответствии с ГОСТ Р ИСО 5725-2-2002 (работу выполнили четыре специалиста в двух лабораториях с использованием четырех хроматографов), были рассчитаны диапазоны абсолютного и относительных времен удерживания характеристических пиков (табл.…”

Section: методы метод пептидного картирования с использованием обраще...unclassified

“…Монография в Европейской фармакопее и сооветствующий стандартный образец рчИФН бета-1b для подтверждения подлинности первичной структуры отсутствуют. Ранее нами была разработана методика пептидного картирования для рчИФН бета-1b, учитывающая стабильность белка при кислых значениях рН [8,9].…”

Section: Introductionunclassified

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Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Goloshchapova¹,

Rounova²,

Minero³

et al. 2022

Biological Products. Prevention, Diagnosis, Treatment

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Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification.The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping.Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation.Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

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Section: методы метод пептидного картирования с использованием обраще...unclassified

Section: Introductionunclassified

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Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Goloshchapova¹,

Rounova²,

Minero³

et al. 2022

Biological Products. Prevention, Diagnosis, Treatment

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“…При пептидном картировании исследуемые белки или стандартный и испытуемый образцы специфически расщепляются химически (например, с использованием BrCN) или ферментативно с помощью протеазы, чаще всего трипсина. После этого хроматографически или масс-спектрометрически регистрируются сигналы, соответствующие ожидаемым пептидам [3,4]. Надежность анализа обеспечивается одновременной регистрацией нескольких десятков соответствующих характеристических сигналов.…”

Section: Introductionunclassified

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

Konstantinov,

Zhdanov,

Toropygin

2024

Biological Products. Prevention, Diagnosis, Treatment

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SCIENTIFIC RELEVANCE. In the quality control of proteolytic enzyme components of medicinal products, the activity of proteases is determined by spectrophotometry, which involves measuring the amidase or esterase activity using a synthetic substrate and the proteolytic activity using the Anson method. These methods require special substrates and have low sensitivity; their specificity may be insufficient, which may lead to serious errors. Quantitative mass spectrometry is an alternative approach to protease activity assays, which involves adding an isotope-labelled peptide to hydrolysates of the test enzyme. This approach allows determining the activity of proteases, notably, by the hydrolysis of specific peptide bonds, while simultaneously confirming the identity and specificity of the test sample. Quantitative mass spectrometry has high sensitivity and does not require special substrates.AIM. This study aimed to investigate the possibility of enzymatic activity assay and enzyme identification by quantitative mass spectrometry with 18O labelling through an example of trypsin with casein.MATERIALS AND METHODS. The study used trypsin, casein, and H218O (Izotop, Russia). Peptide separation was performed using an Agilent 1100 HPLC system; mass spectra were obtained using a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer. Quantitative mass spectrometry was performed using a standard peptide, which was obtained from casein by tryptic digestion and HPLC purification. For 18O labelling, the authors dried the peptide and incubated it in H218О water. The quantitative analysis of the product was carried out using MALDI-TOF mass spectrometry. The authors used quantitative mass spectrometry with 18O labelling to determine enzymatic activity and calculate the Michaelis constant (KM).RESULTS. Following the tryptic digestion of casein, the authors identified the fragments corresponding to casein chains. The authors produced the isotope-labelled standard peptide and calculated its concentration using mass spectrometry. The authors determined the rate of casein digestion by trypsin and calculated the KM for trypsin, which was 13.65±0.60 μM. The standard deviation for repeated measurements showed that the mass-spectrometric method had a lower error of measurement than the spectrophotometric method. The sensitivity threshold for the mass-spectrometric method was 0.50±0.08 μM.CONCLUSIONS. The results obtained with trypsin confirm the possibility of enzymatic activity determination by the proposed method of quantitative mass spectrometry with 18O labelling. According to the sensitivity evaluation results, this method can be used for the simultaneous determination of enzyme activity, identity, and specificity. The proposed mass spectrometry approach is universal, it does not require expensive materials and reagents, and it can be easily adapted to determine the activity of virtually any protease.

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Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor

Zubareva,

Degterev,

Neronova

et al. 2023

Biological Products. Prevention, Diagnosis, Treatment

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Peptide mapping is a key method for studying the primary structure of proteins. With its sensitivity to the slightest changes in the covalent structure of a protein, this method is applicable both to medicinal product identification at the control stage and to production process stability monitoring.The aim of the study was to develop and validate a peptide-mapping procedure for the identification of a novel highly glycosylated recombinant C1 esterase inhibitor.Materials and methods. The authors studied recombinant human C1 esterase inhibitor products and trypsin. The study involved peptide mapping using reverse-phase high-performance liquid chromatography and high-resolution mass spectrometry. The following statistics were calculated to evaluate the results: mean, standard deviation, and coefficient of variation. The validation parameters included specificity, precision, and robustness.Results. The authors tested several variants of sample preparation for tryptic digests, including additional N-glycanase treatment and complete deglycosylation, and established the optimal conditions for sample preparation and chromatographic separation of C1 esterase inhibitor peptides to obtain consistent chromatographic profiles (peptide maps). The authors identified characteristic peaks and the corresponding relative retention time and area ranges. The absolute retention time of the second (characteristic) peak was approximately 16.5–16.9 minutes. The relative retention times were 2.14–2.21 for peak 9, 2.55–2.64 for peak 12, 2.97–3.14 for peak 14, 3.11–3.29 for peak 15, and 6.20–6.63 for peak 28.Conclusions. The authors developed a peptide-mapping procedure for C1 esterase inhibitors and optimised the conditions to achieve an over 18-hour reduction in sample preparation time. This procedure met the established acceptance criteria for specificity, precision, and robustness.

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Разработка методики пептидного картирования для оценки подлинности рекомбинантного интерферона бета-1b

Cited by 3 publications

References 0 publications

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Quantitative mass spectrometry with ¹⁸O labelling as an alternative approach for determining protease activity: an example of trypsin

Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor

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