Аттестация стандартного образца мутности бактерийных взвесей

The article presents the results of research carried out FSBI "SCEMAP" of the Ministry of Health of the Russian Federation in order to bring qualification procedures for opacity standard samples of bacterial suspensions in line with current requirements for certified reference materials. The studies include: the justification requirements for the certification of opacity standard samples of bacterial suspensions, the development of the program and procedure of its evaluation, validation of the measurement procedure for the certified characteristics, statistical analysis of test results and calculation of metrological characteristics.В работе приведены результаты исследований, выполненных ФГБУ «НЦЭСМП» Минздрава России с целью приведения процедуры аттестации стандартного образца (СО) мутности в соответствие с современными требованиями. Исследования включают: обоснование требований к аттестации СО мутности бактерийных взвесей, разработку программы и методики его аттестации, валидацию методики измерений значений аттестуемой характеристики СО, статистическую обработку результатов испытаний и расчет метрологических характеристик СО

The component composition of integrated foreign language competence of future specialists of State Fire Service of the Emergency Ministry of Russia

The authors of the article consider one of the areas of modern vocational education as the formation of integrated foreign language communicative competence of future specialists. It is analyzed the reasons of integration lack of professional and foreign language background in the foreign language qualification in high school. They actualize the education content as a process of integration of vocational and language experience in the field of activity of the future specialist.Рассматривается одна из проблемных областей современного профессионального образования – формирование интегрированной иноязычной коммуникативной компетентности будущих специалистов. Анализируются причины отсутствия единства освоения профессионального и языкового опыта в иноязычной подготовке вуза. Актуализируются вопросы содержания образования как процесс интеграции профессионального и языкового опыта в сфере деятельности будущего специалиста

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

The use of small volumes of test samples in subvisible particle testing by the light obscuration method

The light obscuration method described in the State Pharmacopoeia of the Russian Federation for subvisible particle testing, provides for preparation of a pooled sample with a minimum volume of 25 mL to be used in four measurements, each with 5.0 mL of the test sample. In the case of, for example, ready-to-use 0.2–0.3 mL pre-filled syringes, the method requires pooling the contents of a large number of products, which is economically costly. The use of small volumes of test samples in measurements by the light obscuration method is especially relevant for expensive medicines. Current particle counters allow for testing of 0.1 mL samples, but this requires assessment of the procedure’s accuracy. The aim of the study was to assess the accuracy of subvisible particle testing by the light obscuration method for small volumes of test samples. Materials and methods: we used an HIAC 9703+ liquid particle counter; particle count reference standards containing 0.998×106 particles/mL and 3.800 particles/mL; suspensions of standard latex particles with a known size (20 μm). Results: the study assessed the accuracy of subvisible particle determination by the light obscuration method for small test samples of 0.1‒0.5 mL: trueness was 96–100%; repeatability was 0.8–1.8%; linear correlation coefficients for the calculated versus theoretical number of particles were more than 0.999. The use of 0.1 mL test samples is impractical because of insufficient accuracy of the test results. The relative standard deviation of subvisible particle measurements obtained with 0.2–5.0 mL test samples did not exceed the measurement error of the instrument. The use of small test samples (0.2–1.0 mL) requires the use of a 1 mL sampling syringe. The study demonstrated the need for determination of the pre-run volume (not less than 0.1 mL). Comparative testing of standard (5.0 mL) and small (0.5 mL) samples of protein-based biological products showed comparable results. Conclusions: the study demonstrated that the light obscuration method could be used for small volumes of test samples

Influence of the material of weighing bottles on loss-on-drying reproducibility

One of the factors influencing the uncertainty of residual moisture measurements in biological medicinal products is the accumulation of electrostatic charge on the surfaces of weighing bottles and laboratory balances, which results in poor weighing reproducibility. The authors believe that the simplest and most economical solution to this problem is to use weighing bottles made of a conductive material, e.g. metal. The aim of the work was to evaluate the influence of the material of weighing bottles on the reproducibility of loss-on-drying (LOD) methods. Materials and methods: Model samples for the study were prepared from a sucrose-gelatin medium by lyophilisation and subsequent moisture sorption to achieve a certain residual moisture content. The authors assessed the samples’ mass uniformity using Shewhart’s X-charts, and analysed their residual moisture content using a loss-on-drying procedure with glass and metal weighing bottles. Statistical processing of the results was carried out by calculating the main statistical indicators: Student’s t-test and Fisher’s F-test. Results: Four batches of model samples were prepared and standardised in terms of average mass using Shewhart’s charts. The effect of weighing bottle materials was most pronounced at low residual moisture contents (less than 0.5%), with the relative standard deviation (RSD) values for the results obtained with glass and metal weighing bottles reaching 76% and 35%, respectively. For the samples with a higher residual moisture content (2–5%), the minimum RSDs with glass and metal weighing bottles were 15% and 6%, respectively. Conclusions: The study allowed for evaluating the influence of the material of weighing bottles on the results of LOD measurements and demonstrated a higher reproducibility with metal weighing bottles. This confirms the possibility of using metal weighing bottles in quality assessment of biological medicinal products for human use with LOD methods

Standardisation of the method for prekallikrein activator determination in human immunoglobulin and albumin products

Assessment of prekallikrein content is essential for safety control of human immunoglobulin and albumin products. The inherent variability of human prekallikrein reagents and chromogenic substrates indicates the need for standardisation of the chromogenic assay, using the components of a reference standard (RS) not only for construction of calibration curves, but also for confirmation of validity, consistency, and reproducibility of results within different established ranges. The aim of the study was to improve the quality control of human plasma products in terms of prekallikrein activator content. Materials and methods: prekallikrein activator content was determined by the chromogenic assay according to the procedure described in General Monograph 1.8.2.0013.18 of the Russian Pharmacopoeia, using various prekallikrein reagents. An RS was developed in a spiking test, using human albumin solution and Hageman factor beta-fragment reagent. Shewhart control charts were prepared based on the results of determination of prekallikrein activator content in the RS control component.Results: a two-component RS for prekallikrein activator content with an assigned Hageman factor beta fragment content was developed using the spiking test. The authors substantiated the necessity of using a Russian-produced prekallikrein reagent as the RS component. The in-house reference standard IRS 42-28-445 was certified using all available human prekallikrein reagents, and the IRS 42-28-446 was certified using the prekallikrein reagent included in the kit. The certified prekallikrein activator content is: 51 IU in the batches 1 of IRS components intended for prekallikrein determination; 8.3–11.9 IU/mL in the IRS 42-28-445 control component, after reconstitution in 1.0 mL of purified water, and 5.4–6.6 IU/mL after reconstitution in 2.0 mL of purified water; and 9.1–11.1 IU/mL and 5.6–6.4 IU/mL in the IRS 42-28-446 control component after reconstitution in 1.0 mL and 2.0 mL of purified water, respectively. The IRS component intended for prekallikrein determination is designed for calibration curve construction, while the IRS control component is designed for assessing the validity of test results and preparation of control charts. The analysis of the control charts for the control component made it possible to evaluate the consistency of the analytical process. Conclusions: the components of the developed RSs in combination with Shewhart control charts allow for both determination of prekallikrein activator content, and control of the analytical process, as well as assessment of changes related to the replacement of the reagent batch. The RS control component allows for assessment of analytical process consistency and ensures the standardisation of the test procedure

Lyophilisation of bacterial test strains in a manifold-type apparatus: Effects of freezing and drying parameters, ampoule fill volume, and cotton filter density

Scientific relevance. Lyophilisation is the preferred method at the National Collection of Pathogenic Microorganisms (NCPM) of the Scientific Centre for Expert Evaluation of Medicinal Products of the Ministry of Health of the Russian Federation. Lyophilisation is used to provide for high standards of test-strain deposition, storage, and transportation and to ensure that test strains maintain their properties. Successful lyophilisation requires conducting experiments to establish the key parameters and critical conditions of the process.Aim. The study aimed to evaluate the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of NCPM indicator microorganisms lyophilised in a manifold-type apparatus.Materials and methods. Pseudomonas aeruginosa NCTC 12924, Staphylococcus aureus NCTC 10788, and Salmonella Abony NCTC 6017 were freeze-dried using a manifold-type apparatus (M. S. R. 18, Usifroid). The authors used a low-temperature freezer at –70±2 °C for slow freezing and a mixture of dry ice and alcohol for quick freezing. The statistical analysis was performed using Microsoft Excel and Statistica 10.Results. The minimum time needed for freezing the samples in a low-temperature freezer at –70±2 °C was 4 hours. Further storage at this temperature for up to 1 month was shown possible without compromising the quality of the final product. The time needed for freezing the samples in a mixture of dry ice and alcohol was under 1 minute. No differences in quality parameters were observed between the lyophilised samples frozen slowly or quickly, except for the cake appearance. Quick freezing resulted in cakes that were non-uniform, crumbled, and pulled away from the ampoule walls, which is considered undesirable. The primary drying stage for ampoules with a fill volume of 0.2 mL took 6–8 hours. The secondary drying stage of 11, 18, 35, and 59 hours resulted in comparable lyophilisate quality: the authors observed no statistically significant differences in viable cell counts (CFU/mL) at the end of lyophilisation and at the end of stress testing. The residual moisture content after 59-hour secondary drying was less than 2%. The cotton filter density had a critical influence on the lyophilisate quality. Therefore, the authors recommend using cotton filters weighing 50 mg or less.Conclusions. The authors analysed the main stages of the lyophilisation process used for NCPM test strains and considered the effects that the speed and time of freezing, the time of drying, the fill volume of ampoules, and the density of cotton filters have on the quality of the final lyophilised product. The NCPM has implemented the results of this study in its work

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

Использование малых объемов аналитических проб при определении невидимых механических включений счетно-фотометрическим методом

The light obscuration method described in the State Pharmacopoeia of the Russian Federation for subvisible particle testing, provides for preparation of a pooled sample with a minimum volume of 25 mL to be used in four measurements, each with 5.0 mL of the test sample. In the case of, for example, ready-to-use 0.2–0.3 mL pre-filled syringes, the method requires pooling the contents of a large number of products, which is economically costly. The use of small volumes of test samples in measurements by the light obscuration method is especially relevant for expensive medicines. Current particle counters allow for testing of 0.1 mL samples, but this requires assessment of the procedure’s accuracy. The aim of the study was to assess the accuracy of subvisible particle testing by the light obscuration method for small volumes of test samples. Materials and methods: we used an HIAC 9703+ liquid particle counter; particle count reference standards containing 0.998×106 particles/mL and 3.800 particles/mL; suspensions of standard latex particles with a known size (20 μm). Results: the study assessed the accuracy of subvisible particle determination by the light obscuration method for small test samples of 0.1‒0.5 mL: trueness was 96–100%; repeatability was 0.8–1.8%; linear correlation coefficients for the calculated versus theoretical number of particles were more than 0.999. The use of 0.1 mL test samples is impractical because of insufficient accuracy of the test results. The relative standard deviation of subvisible particle measurements obtained with 0.2–5.0 mL test samples did not exceed the measurement error of the instrument. The use of small test samples (0.2–1.0 mL) requires the use of a 1 mL sampling syringe. The study demonstrated the need for determination of the pre-run volume (not less than 0.1 mL). Comparative testing of standard (5.0 mL) and small (0.5 mL) samples of protein-based biological products showed comparable results. Conclusions: the study demonstrated that the light obscuration method could be used for small volumes of test samples.Счетно-фотометрический метод определения невидимых механических включений, описанный в Государственной фармакопее Российской Федерации, предусматривает формирование из образцов лекарственных препаратов пробы объемом не менее 25 мл, необходимой для проведения четырех измерений, каждое объемом 5 мл. Для препаратов, выпускаемых, например, в готовых к использованию преднаполненных шприцах объемом 0,2–0,3 мл, метод требует объединения большого количества первичных упаковок, что экономически затратно. Для дорогостоящих препаратов актуальным является использование малых объемов аналитических проб при проведении испытаний счетно-фотометрическим методом. Применяющиеся на практике счетчики частиц позволяют проводить анализ лекарственных препаратов в объемах от 0,1 мл, но это требует оценки точности методики. Цель работы: оценить точность определения невидимых механических включений счетно-фотометрическим методом с использованием малых объемов аналитических проб. Материалы и методы: в работе использовали счетчик частиц HIAC 9703+; стандартные образцы счетной концентрации, содержащие 0,998×106 частиц/мл и 3800 частиц/мл; суспензии стандартных латексных частиц с заданным размером (20 мкм). Результаты: оценена точность методики определения количества невидимых частиц счетно-фотометрическим методом при использовании малых аналитических проб объемом от 0,1 мл до 5,0 мл: правильность составила 96–100%; повторяемость – 0,8–1,8%; коэффициенты корреляции линейной зависимости расчетного количества частиц от теоретического значения – более 0,999. Проведение измерений с аналитической пробой объемом 0,1 мл нецелесообразно из-за недостаточной точности результатов. Относительное стандартное отклонение результатов измерений количества невидимых частиц, получаемых при использовании аналитических проб объемом от 0,2 до 5,0 мл, не превышает относительной погрешности результатов измерений счетчика частиц. При проведении испытания с использованием малых аналитических проб (0,2–1,0 мл) рекомендуется использовать шприц-пробоотборник объемом 1 мл. Показана необходимость предварительной установки объема преаналитической пробы (не менее 0,1 мл). Сравнительные испытания биологических лекарственных препаратов белковой природы (7 наименований) при использовании стандартной (5,0 мл) и малой (0,5 мл) аналитических проб продемонстрировали сопоставимые результаты. Выводы: анализ результатов проведенных исследований свидетельствует о возможности использования счетно-фотометрического метода с малыми объемами аналитических проб

Оценка неопределенности результатов измерений при определении потери в массе при высушивании биологических лекарственных препаратов

Scientific relevance. GOST ISO/IEC 17025-2019 requires testing laboratories to evaluate the measurement uncertainty of their results. Estimating the uncertainty of analytical methods intended for biologicals is a challenging task that requires time, effort, and a special approach. Measurement uncertainty estimation is of particular interest in the case of measuring loss on drying (LOD) for biologicals, since LOD testing procedures involve analysing measurements of    a physical value, i.e. mass.Aim. This study aimed to estimate the measurement uncertainty of LOD determination in biological medicinal products.Materials and methods. The study examined a powdered active substance intended for a Bifidobacterium product (test sample). The authors conducted the LOD test in accordance with the State Pharmacopoeia of the Russian Federation (OFS.1.2.1.0010.15). Statistical processing of the results was performed using Microsoft Excel. To estimate the measurement uncertainty, the authors employed the bottom-up approach or used the standard deviation from testing results.Results. The authors identified the uncertainty components that affected the LOD determination results. When calculated using the bottom-up approach, the expanded uncertainty was 0.34% (coverage factor, k=2; approximate confidence level, 95%). In particular, the largest contributor to the expanded uncertainty was the uncertainty of measuring the mass of weighing bottles containing dried test samples (0.147%), whereas the smallest contributor was the uncertainty of weighing empty bottles (0.003%). When calculated using the standard deviation, the uncertainty of two parallel measurements amounted to 0.32%.Conclusions. Both approaches to calculating LOD measurement uncertainty yield comparable results. According to the uncertainty budget analysis, the uncertainty of measuring the mass of weighing bottles with dried test samples is the major  contributor to  the test result. For  this reason, the conditions of sample preparation should be carefully controlled. The study results confirm that the LOD measurement uncertainty can be calculated using the standard deviation. Testing laboratory teams may benefit from the methods for identifying the factors influencing LOD test results and the methods for calculating the uncertainty of measurement described in this study.Актуальность. Исследование неопределенности результатов измерений, проводимых испытательными лабораториями, предусмотрено ГОСТ ISO/IEC 17025-2019. Оценивание неопределенности методик испытаний биологических лекарственных препаратов представляет собой сложную задачу,  требующую особого подхода и  значительных  временных и трудовых ресурсов. Актуальной является оценка неопределенности измерений на примере методики определения потери в массе при высушивании биологических лекарственных препаратов, поскольку в процессе ее реализации анализируется именно результат измерения (взвешивания) физической величины — массы.Цель. Оценить неопределенность результатов измерений при  определении потери в  массе при высушивании биологических лекарственных препаратов.Материалы и методы. Субстанция-порошок для изготовления бифидосодержащего препарата (исследуемый образец). Испытание проводили в соответствии с требованиями Государственной фармакопеи Российской Федерации ОФС.1.2.1.0010.15. Статистическую обработку результатов выполняли с использованием программы Microsoft Exсel. Расчет неопределенности проводили  двумя   способами:   при   помощи   подхода   «снизу   вверх» и с использованием доверительного интервала.Результаты. Идентифицированы составляющие неопределенности, влияющие  на  результат измерения потери в массе при высушивании. Расширенная неопределенность, рассчитанная с использованием подхода «снизу вверх»,  составила  0,34%  (коэффициент  охвата k=2, уровень доверия приблизительно 95%), при этом наибольший вклад вносит неопределенность измерения массы бюкса после высушивания образца — 0,147%; наименьший вклад — неопределенность измерения массы пустого бюкса — 0,003%. Неопределенность двух параллельных измерений, рассчитанная с помощью доверительного интервала, составила 0,32%.Выводы. Два подхода к расчету неопределенности результатов  измерений  потери  в  массе при высушивании дают сопоставимые результаты. Анализ бюджета неопределенности выявил, что наибольший вклад в результат испытания вносит неопределенность  измерения массы бюкса после высушивания образца, что требует тщательного контроля условий пробоподготовки. Для оценки неопределенности может  быть использован способ  расчета с помощью доверительного интервала. Методология анализа  методики  потери  в  массе при  высушивании с  точки зрения  выявления факторов, влияющих на  результат  испытания, и представленные способы расчета неопределенности могут быть полезны специалистам испытательных лабораторий