Quantification methods for carbohydrate compounds in biologicals: a review

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

Comparability assessment of the results of thiomersal quantification in adsorbed immunobiological medicinal products by colourimetry and by cold vapor atomic absorption spectrometry

To ensure the quality of immunobiologicals, it is required to quantify the thiomersal preservative present in a number of them. The authors have previously developed an analytical procedure for thiomersal quantification in non-adsorbed immunobiological medicinal products, which is based on cold vapor atomic absorption spectrometry (CV AAS). The aim of the study was to analyse the possibility of using the CV AAS procedure for thiomersal content determination in adsorbed immunobiologicals and evaluate the comparability of thiomersal quantification results obtained by colourimetry and CV AAS. Materials and methods: the study used the national reference standard of mercury ions content and the pharmacopoeial reference standard of thiomersal content in adsorbed medicinal products (PhRS 3.1.00427), as well as samples of immunobiologicals by different manufacturers: a DTP vaccine, anatoxins, hepatitis B and influenza vaccines, and combined vaccines. The study involved CV AAS and the colourimetric reaction between mercury and dithizone. Results: the specificity of the CV AAS procedure is demonstrated by the coefficient of variation (3.95%) and the coefficient of correlation between the test sample volume and thiomersal content (0.9956). The regression analysis and the Fisher’s test value of 0.16 indicate the absence of bias. The trueness of the method is satisfactory, as the percent recovery differs from the total spiked amount by less than 10%. For the sensitivity of the CV AAS procedure, its quantification and detection limits are 6.9×10-3 μg/ mL and 2.3×10-3  μg/ mL, respectively. The Fisher’s test value obtained in the comparability assessment of the results of thiomersal quantification by colourimetry and CV AAS (1.29) is lower than the conventional tabulated one (3.96). Conclusions: according to the study, it is possible to use the CV AAS procedure for thiomersal quantification in adsorbed immunobiologicals. The established detection limit allows evaluating residual amounts of thiomersal in in-process intermediates during the production of preservative-free immunobilogical dosage forms. The comparability assessment of the results of thiomersal quantification by colourimetry and CV AAS, carried out using oneway ANOVA and Fisher’s test, showed the possibility of using PhRS 3.1.00427 to control the consistency of operation when reproducing the CV AAS procedure

Prospects for ion chromatography in quality assessment of biologicals

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

DEVELOPMENT OF A QUALIFICATION PROCEDURE FOR METHIONINE FORM OF INTERFERON ALFA-2b STANDARD TO CONFIRM ITS AUTHENTICITY BY MEANS OF A PEPTIDE MAPPING METHOD

Authenticity evaluation of proteins obtained with recombinant DNA technology is an important step in confirming efficacy and safety of the drugs based on them. One of the main ways to assess the authenticity is to compare molecular structure of the test and standard samples using the peptide mapping method with chromatographic separation of the products obtained by enzymatic degradation. Proper selection of a standard reference sample is essential in order to achieve reliable results. A standard sample of Interferon (CRS, Chemical Reference Substances) recommended by the European Agency for the Quality of Medicines for interferon alpha-2b substances containing N-terminal methionine is inappropriate, since the Interferon CRS sample doesn’t contain methionine. We present the results of development of qualification procedure for methionine form of Interferon alfa-2b industrial standard sample (ISS). The range of use for this ISS is authenticity confirmation for the methionine form of interferon alpha-2b substance using peptide mapping method with reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The quality assessment was performed for all the parameters specified by the manufacturer of this candidate substance at the initial stage of qualification procedure, due to changed application area, and changed package size. Further, 30 peptide cards of the ISS candidate substance were obtained after pre-trypsinolysis of the protein followed by validated HPLC method with proven repeatability.It was shown that the hydrolysis conditions, i.e., the choice of trypsin preparations, may significantly affect the peptide map profile. Therefore, a reference to specific manufacturer and the catalog number of the product should be provided in description of application conditions for the ISS proposed.A set of eight reference peaks (peaks of comparison) has been justified, as based on evaluation of peptide maps and results of high-resolution mass spectrometry. The peak with maximally stable yield and intensity was selected as the main peak with an established absolute retention time. Two peaks with relative retention times were chosen as essential peaks for evaluation, i.e., the 1st peak containing N-terminal methionine, and the 2nd peak of highest molecular weight with an established amino acid sequence covering 11% of the studied interferon molecule.We have also qualified ISS parameters expressed as absolute (minimum for one reference peak), and relative (for the remaining reference peaks) retention time periods. Authenticity of the ISS candidate was further confirmed by the peptide mapping method, as compared with interferon CRS reference standard. Their peak patterns proved to be near-similar, except of a peak with eluted peptide containing N-terminal methionine as revealed by high-resolution mass spectrometr

Development and certification of reference standards for phenolic content in biologicals, based on comparison of results obtained by GLC, HPLC, spectrophotometric, and colorimetric methods

Phenol is used as a preservative in a number of biological products. Methods that are used for quantitative determination of phenol differ a lot. Current requirements for accredited laboratories include continuous internal quality control. Reference standards with a certified content of the analyte are an effective metrological tool for ensuring such control. The aim of the study was to develop and certify reference standards for phenolic content in biological products, based on comparison of results obtained by GLC, HPLC, spectrophotometric, and colorimetric methods. Materials and methods: diluent for allergens by (candidate reference standard), 2.5 and 5 mg/mL phenol solutions, and 2.5 mg/mL 2-phenoxyethanol solution were used in the study. The experiments were performed using spectrophotometric, colorimetric, HPLC, and GLC procedures. The statistical analysis of results included calculation of the arithmetic mean, standard deviation, coefficient of variation, and analysis of variance with Student’s t-test and Fisher’s F-test. Results: the results of phenolic content determination by the spectrophotometric, colorimetric, and HPLC methods were statistically comparable. The F value obtained for equal sample sizes (n = 40) was F = 0.9343, given the critical value Fcrit = 3.96. A reference standard certified by one of these methods can be used to control the consistency of phenol determination by a relevant method. The results of phenolic content determination by the GLC method showed statistically significantly differences: F = 17.47, given Fcrit = 3.96, which demonstrated the need for certification of another reference standard. Conclusions: two reference standards were certified in the study: reference standard 42-28-449 with the certified phenolic content of 2.56‒3.32 mg/mL, to be used with the spectrophotometric, colorimetric, and HPLC methods; and reference standard 42-28-451 with the certified phenolic content of 2.92‒3.28 mg/mL, to be used with the GLC method

Обзор методов определения соединений углеводной природы в биологических лекарственных препаратах

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.Соединения углеводной природы широко используются в качестве наполнителей и стабилизаторов в биологических лекарственных препаратах (БЛП). Наличие данных соединений в составе лекарственного препарата гарантирует стабильность действующего вещества в процессе производства, транспортирования и хранения. При этом нормирование содержания вспомогательных веществ и их количественное определение является фармакопейным требованием к оценке качества БЛП.Цель работы — выявление перспективных методов для разработки методик количественного определения соединений углеводной природы в биологических лекарственных препаратах.Проведен анализ нормативных документов зарегистрированных в Российской Федерации БЛП. Показано, что наиболее часто в качестве вспомогательных веществ используются полиолы (сорбитол и маннитол), моносахариды (глюкоза), дисахариды (трегалоза, сахароза, лактоза, мальтоза) как по отдельности, так и в смесях различного состава. На основании данных научной литературы рассмотрены методы количественного определения полиолов, моно- и дисахаридов, применяемые при оценке качества БЛП. Для количественного определения стабилизаторов углеводной природы применяют титриметрические, спектрофотометрические, ферментативные, хроматографические методы. Представлен анализ достоинств и недостатков данных методов. Показаны преимущества метода ионообменной высокоэффективной жидкостной хроматографии (ВЭЖХ) с амперометрическим детектированием и метода гидрофильной ВЭЖХ с рефрактометрическим детектированием и испарительным детектором светорассеяния, обладающих достаточной селективностью и способностью идентификации исходных веществ без дериватизации. Сделан вывод о перспективности разработки методик определения стабилизаторов углеводной природы на основе методов ионообменной и гидрофильной ВЭЖХ

Рекомендации к составлению нормативной документации на иммунобиологические лекарственные препараты в части физико-химических показателей качества

These recommendations present the specifics of laboratory testing and examination of regulatory documents. The recommendations include information about the basic principles of presentation of the quality assessment methods. The paper demonstrates the following: a general presentation scheme of regulatory documents sections with a detailed description of subsections structure, the peculiarities of presentation of the methods for evaluation of physicochemical quality characteristics of immunobiologicals and the typical mistakes made in the process of drafting regulatory documents.В настоящих рекомендациях отражены особенности лабораторной экспертизы и экспертизы нормативной документации. Рекомендации содержат информацию об основных принципах изложения методик оценки качества. Приведена общая схема изложения разделов нормативной документации с подробным описанием структуры подразделов, рассмотрены особенности изложения методик оценки физико-химических показателей качества иммунобиологических лекарственных препаратов, указаны типичные ошибки, допускаемые при составлении нормативной документации

Recommendations on the certification of reference standards for structure identification of recombinant therapeutic proteins

Reference standards for structure identification of recombinant therapeutic proteins are essential for quality assessment of recombinant protein-based biotechnological medicinal products. The development and certification of such reference standards hold special relevance because of, firstly, the absence of international, national or compendial reference standards for a number of new or recently approved proteins and, secondly, the disruption of supply chains providing the biopharmaceutical industry of the Russian Federation with international reference standards. Moreover, international and national regulatory documents contain only general requirements for the procedure of reference standards certification but not the considerations specific to the standards for biotechnologicals’ structure identification, which vary with the production technologies for each individual active moiety. The aim of this work was to provide recommendations on the procedure for the development and certification of reference standards used to identify the structure of recombinant therapeutic proteins. These recommendations define 4 main stages of the procedure: stage 1 covers the development of requirements for the reference standard, including the justification of material and formulation choices, the elaboration of quality specifications, and the assessment of quality; stage 2 comprises the selection of analytical procedures and the establishment of the values for the certified parameters; stage 3 includes stability studies and shelf-life setting; and stage 4 involves the development of documentation for the reference standard. The paper dwells upon the scope of the stages, taking into account the specific considerations for recombinant therapeutic proteins and the use of reference standards. The recommendations are based upon the extensive experience in biotechnologicals testing and standardisation of the employees of the Scientific Centre for Expert Evaluation of Medicinal Products. These recommendations can provide a base for the establishment of protein-specific certification programmes for reference standards used in structure identification. This approach will allow for systematisation of the process for standards development and ensure the traceability of information and the validity of results. The reference standards certified in accordance with these recommendations can be considered primary standards, if necessary

Определение мертиолята в несорбированных ИЛП методом атомно-абсорбционной спектроскопии холодного пара (ААС-ХП) по ионам ртути. Часть 1: отработка методики и оценка статистической значимости различий результатов определения ионов ртути колориметрическим методом и методом ААС-ХПО

The article describes development of methods of quantitative determination of thimerosal in non-adsorbed immunobiological preparations by cold vapor atomic absorption spectroscopy as well as an estimation of the statistical significance of differences in the results determination of thimerosal conventional colorimetric method in combination with dithizone and CV AAS using one-way analysis of variance with Fisher’s exact test (F-test).В статье представлены материалы по отработке условий количественного определения содержания мертиолята в несорбированных иммунобиологических препаратах (ИЛП) методом атомно-абсорбционной спектроскопии холодного пара (ААС-ХП), а также проведена оценка статистической значимости различий результатов определения мертиолята традиционным колориметрическим методом в комплексе с дитизоном и методом ААС-ХП: использован однофакторный дисперсионный анализ с применением критерия Фишера (F-критерия)

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

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