Подобраны условия ферментативного гидролиза с применением эндопротеиназы Glu-C, рабочая область рН которой соответствовала рН-диапазону стабильности белка. Экспериментально подтверждено отсутствие аутогидролиза фермента. Анализ полученных пептидных карт, основанный на оценке стабильности времен удерживания и интенсивности 7 основных пиков, позволил выбрать их в качестве реперных. Масс-спектрометрический анализ состава фракций, соответствующих данным пикам, позволил идентифицировать 81 аминокислотный остаток, что соответствует 50 % аминокислотного покрытия первичной последовательности исходной молекулы. Разработана методика пептидного картирования с последующим разделением продуктов ферментативного гидролиза методом ВЭЖХ. Полученные результаты свидетельствуют о специфичности и стабильности полученных пептидных карт. Методика может быть использована при подтверждении подлинности структуры рекомбинантного интерферона бета-1b, не содержащего вспомогательных веществ белковой природы.

Carbohydrate compounds are widely used as fillers and stabilisers in biological products. When present, these compounds guarantee that the active pharmaceutical ingredient will remain stable during production, transportation, and storage. At the same time, pharmacopoeias standardise the excipient content and require that excipients should be quantified for assessing the quality of biological products.The aim of the study was to identify promising methods for the development of quantification procedures for carbohydrate compounds in biological products.The authors analysed regulatory documents for biological products approved in the Russian Federation. The most widely used excipients, both individually and in combinations, are polyols (sorbitol and mannitol), monosaccarides (glucose), and disaccharides (trehalose, sucrose, lactose, and maltose). Using literature data, the authors reviewed the methods used for quantifying polyols, monosaccharides, and disaccharides to assess the quality of biological products. Quantitative determination of carbohydrate stabilisers employs titrimetric, spectrophotometric, enzymatic, and chromatographic methods. This review presents an analysis of the advantages and disadvantages of these methods. It highlights the advantages of ionic HPLC with amperometric detection and hydrophilic HPLC with refractometric and evaporative light scattering detection, which are sufficiently selective and can identify substances without prior derivatisation. In conclusion, ionic and hydrophilic HPLC methods are a promising base for the development of quantification procedures for carbohydrate stabilisers.

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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Quantitative characterisation of excipients in biologicals is an important part of the quality assurance process both at the level of finished products and intermediates, as well as active pharmaceutical ingredients. Ion chromatography with amperometric and conductometric detection of separation products has a number of advantages. The main of the advantages is the possibility of direct determination of semivolatile compounds that have neither chromophoric groups, nor intrinsic fluorescence. The aim of this study was to compare ion chromatography with alternative methods in order to identify promising areas for its use in assessing the quality of biologicals. The authors analysed regulatory documents and literature and summarised the methods applied for quantitative determination of ionic excipients in biological medicinal products. The authors investigated the possibility of using ion chromatography for determination of the main active pharmaceutical ingredient in polysaccharide vaccines and excipients in biologicals. The study demonstrated the feasibility of ion chromatography for simultaneous quantitation of cations (ammonium, calcium, magnesium) and anions (chlorides, sulfates, nitrates) in reconstitution solvents for lyophilised biologicals; quality assessment of active pharmaceutical ingredients in biologicals (quantitative analysis of polysaccharides in polysaccharide vaccines, profiling of glycosylated proteins, etc.); and determination of several carbohydrate stabilisers in biologicals with the same analytical procedure. According to the conclusions, ion-exchange chromatography with conductometric and amperometric detection, aimed at quality assessment of biological products, can shortly take a leading position in quantitation of ionic excipients, carbohydrate stabilisers, and main active ingredients (polysaccharides) in polysaccharide vaccines, including the vaccines in the immunisation schedule.