SCIENTIFIC RELEVANCE. The complexity of standardising immunobiologicals, including bacillus Calmette–Guérin (BCG) vaccines, requires improving the approach to statistical analysis of consistency in their quality indicators. Quality analysis tools include Shewhart control charts, which can be used for real-time and retrospective identification and analysis of trends in the processes of interest.AIM. This study aimed to analyse quality indicators of a BCG vaccine for bladder cancer immuno­therapy using Shewhart control charts to monitor consistency in manufacturing processes and quality testing of vaccines.MATERIALS AND METHODS. The study involved a retrospective analysis of the bacterial concentration, dispersity index, loss-on-drying, and potency data obtained during the quality control of Imuron-vac, a BCG vaccine for bladder cancer immunotherapy, in the past 5 years. As part of the statistical data analysis, the authors calculated the means, investigated the corre­lation between the test results provided by the manufacturer and the quality control results obtained at the testing centre (TC), and checked the distributions for normality. Individuals and moving range control charts were plotted and analysed for each quality indicator.RESULTS. The correlation coefficients (r) ranged from 0.34 to 0.70 for the data submitted by the manufacturer and the data obtained at the TC. Pearson’s goodness-of-fit test showed that the distribution of the quality control results obtained at the TC was normal, with χ² = 14.03 for the dispersity index values and χ² = 4.93 for the loss-on-drying measurements. Shewhart control charts for loss-on-drying and potency data suggested that the production process and/or the testing process deviated from the state of statistical control. Consistency monitoring of the bacterial concentration and the dispersity index showed that the state of statistical control was achieved. The authors established control limits for further real-time monitoring of the dispersity index consistency.CONCLUSIONS. Shewhart control charts are applicable to the quality control of BCG vaccines for bladder cancer immunotherapy at the TC. Continuous monitoring of consistency in quality indicators is needed to improve the standardisation of vaccine production processes and testing methods, as this standardisation will result in high product quality.

Scientific relevance. Viable cell counting is an important microbiological test for the quality assessment of medicinal products containing live microorganisms. To optimise labour and material costs and enhance testing precision and reproducibility, it is practical to use partially automated instrumental methods, such as the spiral plating method.Aim. The aim was to conduct a validation study of the spiral plating method for assessing the potency of biologicals containing live bacterial cells, using a lactobacillus-containing probiotic medicinal product as a case study.Materials and methods. This study used a culture of Lactiplantibacillus plantarum isolated from a probiotic medicinal product. Spiral plating on agar-based biological culture media was performed using an automatic Eddy Jet 2 plating system. The study used an IUL Flash & Go colony counter for automatic reporting of the results. The validation study was conducted according to the general chapter on the validation of microbiological testing methods (OFS.1.1.0021.18) of the State Pharmacopoeia of the Russian Federation.Results. According to the validation results for the main parameters, the spiral plating method had the range of 104–105 CFU/mL; the limit of quantification of 102 CFU/mL; and the coefficient of linear determination, R2, of 0.99. The accuracy of the spiral plating method in determining the potency of the test sample was 93%; the repeatability was 4.9%.Conclusions. The study results confirm the similarity of the spiral plating method with an automatic colony counter to Koch’s plating method, which is currently used in accordance with the State Pharmacopoeia of the Russian Federation, in terms of the validated parameters. Therefore, spiral plating can be used to evaluate the potency of lactobacillus-containing probiotics. Spiral plating can help improve the cost-effectiveness and accuracy of testing.

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.

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.

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.

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.

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.

The State Pharmacopoeia of the Russian Federation, 14th edition provides for determination of sub-visible particles (less than 100 µm in size) in parenteral dosage forms using the Coulter method, in addition to the light obscuration particle count test and microscopy. However, the proposed 100 µm aperture tube does not enable assessment of the whole range of sub-visible particle sizes. Therefore, research is needed to find optimal test conditions for determination of sub-visible particulate matter by the Coulter method. The aim of the study: modification of the Coulter-based procedure using a 200 µm aperture tube, and performance of validation studies. Materials and methods: Multisizer 4e Coulter counter, suspensions of reference latex particles (10 µm, 20 µm, and 43 µm), and a particulate count reference standard containing 0.998 × 106 particles/mL were used in the study. The following parameters were assessed during validation: accuracy, repeatability, linearity. Results: the study confirmed the feasibility of using the modified Coulter-based procedure with a 200 µm aperture tube. The following values were obtained during validation of the modified test procedure: accuracy was 5.3% (deviation from the mean value) as compared to the particulate count reference standard, and 4.2% as compared to the light obscuration method. Repeatability was 1% (relative standard deviation) for the particle concentration of approximately 10000 per 1 mL, and 7.6% for the particle concentration of approximately 300 per 1 mL. The study demonstrated the linearity of the procedure, the linear correlation coefficient was more than 0.99. Conclusions: the studied validation parameters of the modified test procedure were shown to comply with the acceptance criteria. The modified test procedure will enable assessment of the whole range of sub-visible particle sizes when testing parenteral solutions for particulate contamination: sub-visible particles.

The article presents the results of the study of the possibility of using the method of electrosensitive zones (Coulter's method) to evaluate the indicator «Particulate matter (invisible)» in domestic medicinal human blood-derived products. The studies were carried out on suspensions of latex particles, the size and quantity of which corresponded to those normalized for parenteral preparations. Suspensions simulated human blood-derived products, which can presumably contain some invisible particles. Factors that can influence the reliability of test results conducted using the method of electrosensitive zones are determined: the electrical conductivity of the test sample, the volume of the analytical sample, the degree of dilution of the sample.

Water for injections is one of the most popular diluents used for preparation of parenteral dosage forms. The European  Pharmacopoeia recommends two methods for the determination of  subvisible particulate matter: Light Obscuration Particle Count Test  and Microscopic Particle Count Test. The Russian Pharmacopoeia,  13th ed., additionally allows for the use of the Coulter principle  (Electrical Sensing Zone method). Thus, a procedure had to be developed for subvisible particles determination in water for  injections based on the Coulter principle (hereinafter — procedure).  The article presents the results of development and validation of the  procedure, i.e. the characteristics of accuracy, dilutional linearity,  ruggedness in terms of the time factor, and repeatability for particles more than 10 μm in size. The results of subvisible particles  determination obtained with the help of the developed procedure based on the Coulter principle were compared to the  results obtained with the help of the light obscuration particle count  test. The accuracy of the developed procedure was supported by the  statistical insignificance of the differences between the obtained  results. The values of ruggedness in terms of the time factor (NMT  14 %) and repeatability (NMT 15 %) did not exceed the established  acceptance criterion which is equal to the acceptable limit of the  instrument error for particle count in the tested samples (20 %). The  dilutional linearity of the procedure was demonstrated  (coefficient of determination R2 = 0.999). The results obtained  during the validation studies support the possibility of using the  Coulter principle for the assessment of subvisible particles in water  for injections.

Water for injections is one of the most popular diluents used for preparation of parenteral dosage forms. The European Pharmacopoeia recommends two methods for the determination of subvisible particulate matter: Light Obscuration Particle Count Test and Microscopic Particle Count Test. The Russian Pharmacopoeia, 13th ed., additionally allows for the use of the Coulter principle (Electrical Sensing Zone method). Thus, a procedure had to be developed for subvisible particles determination in water for injections based on the Coulter principle (hereinafter — procedure). The article presents the results of development and validation of the procedure, i.e. the characteristics of accuracy, dilutional linearity, ruggedness in terms of the time factor, and repeatability for particles more than 10 μm in size. The results of subvisible particles determination obtained with the help of the developed procedure based on the Coulter principle were compared to the results obtained with the help of the light obscuration particle count test. The accuracy of the developed procedure was supported by the statistical insignificance of the differences between the obtained results. The values of ruggedness in terms of the time factor (NMT 14 %) and repeatability (NMT 15 %) did not exceed the established acceptance criterion which is equal to the acceptable limit of the instrument error for particle count in the tested samples (20 %). The dilutional linearity of the procedure was demonstrated (coefficient of determination R2 = 0.999). The results obtained during the validation studies support the possibility of using the Coulter principle for the assessment of subvisible particles in water for injections.

On the basis of WHO recommendations and the principles developed for the standardization of erythropoietins we selected one epoetin alfa pharmaceutical substances included in the State Register of Medicines as a candidate reference sample. The results of the manufacturer’s monitoring of this pharmaceutical substance complied with the requirements of the normative documentation and additional verification of identity by capillary zone electrophoresis demonstrated the presence of eight isoforms, which corresponded to the requirements of the European Pharmacopeia. Conditions for the preparation of an industrial reference sample in lyophilized form were developed. The industrial reference sample was attested in interlaboratory tests and the value of the attested property - specific activity - was 2400 IU/ampule, with confidence limits of 94.5% to 105.8%. Studies of the stability of the industrial reference sample developed here gave a shelf life of five years at a storage temperature of -20°C, with potential to extend this on the basis of stability monitoring results.

Основываясь на рекомендациях ВОЗ и разработанных принципах стандартизации эритропоэтинов, в качестве материала для кандидата в стандартный образец выбрана одна из фармацевтических субстанций эпоэтина альфа, внесенных в Государственный реестр лекарственных средств. По результатам контроля производителя выбранная фармацевтическая субстанция соответствовала требованиям нормативной документации, дополнительный контроль подлинности методом капиллярного зонного электрофореза показал присутствие 8 изоформ, что соответствует требованиям Европейской фармакопеи. Разработаны условия получения отраслевого стандартного образца в лиофилизированной форме. Аттестацию отраслевого стандартного образца провели в межлабораторных исследованиях, значение аттестованной характеристики — специфической активности — составило 2400 ME в 1 ампуле, доверительный интервал от 94,5 до 105,8 %. Изучение стабильности разработанного отраслевого стандартного образца позволило установить срок годности 5 лет при температуре хранения –20 °C с возможностью его продления по результатам мониторинга стабильности.