Possibility of using mathematical analysis methods to evaluate the potency of antibodies to hepatitis B virus surface antigen in human immunoglobulin preparations

Scientific relevance. Anti-hepatitis B virus surface-antigen (HBsAg) immunoglobulins are used to prevent hepatitis B in adults and children after exposure and to treat mild to moderate acute viral hepatitis B. The clinical effectiveness of human immunoglobulin preparations is determined by their potency, which is assessed by the content of antibodies to hepatitis B virus surface antigen (anti-HBs antibodies). Currently, this assessment involves using immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).Aim. This study examined several mathematical methods for analysing the experimental data obtained in ELISA-based anti-HBs antibody potency assays of human immunoglobulin preparations.Materials and methods. This study used the international standard for human anti-HBs immunoglobulin, two immunoglobulin preparations, and an ELISA test kit for the detection and quantification of anti-HBs antibodies in serum or plasma samples.Results. Using sandwich ELISA, the authors ascertained that the measured anti-HBs antibody concentration depended on the choice of calibration curve calculation method (i.e. manual analysis, parallel-line analysis using PARALINE software, linear regression, and 4-parameter logistic regression). The measured anti-HBs antibody concentrations varied by ± 19 IU/mL. According to the study results, an incorrectly selected method of data analysis can lead to an erroneous calculation of the analyte potency (concentration) in the test sample.Conclusions. The study demonstrated the need for improved mathematical methods for the evaluation of experimental data used to determine the anti-HBs antibody concentration in human immunoglobulin preparations. It is essential to switch from manual to automated calculation (for example, using a 4-parameter logistic model), taking into account the requirements for bioanalytical methods and the capabilities of the available equipment

Current Challenges of Preventive Vaccination Against Rabies

The urgency of the rabies problem for all mankind and the search for new ways of eradicating the disease entailed the creation of a new global initiative for rabies elimination ‒ «United Against Rabies» which sets a highly ambitious goal of achieving zero rabies human deaths by 2030. The many years of international experience in elimination of street dogs, which account for 99 % of rabies cases, did not produce the desired results, therefore the focus was shifted to mass vaccination of dogs (minimum 70 % of dog population). The rabies problem is complex and global, it requires efforts from all the parties involved as well as hefty investment. The paper presents the results of a continuous long-term analysis of the rabies situation in Russia and across the world, as well as analysis of the current state of vaccination against rabies which plays an important, if not crucial, role in prevention of rabies in humans who got bitten by infected animals. The paper formulates the main currently existing ways of solving the rabies problem, namely: mass vaccination of dogs; improvement of dosing schedules and administration routes of medicines against rabies; analysis of immunity development mechanisms in immunocompromised patients; progressive implementation of vaccination of people who got bitten by infected animals, and alternative administration routes; development of an express method of the neutralising antibody titer determination; raising public awareness about disease hazards

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

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

Preventive Vaccination against Poliomyelitis: Modern View

Poliomyelitis is a typical anthroponosis, in natural conditions it infects only humans. The only effective strategy for combating the infection is preventive vaccination. The polio vaccine induces long-lasting humoral and local immunity. The article presents a brief history of polio vaccine development, and compares live and inactivated vaccines currently licensed and used in Russia. It also dwells upon the benefits and shortcomings of each of these vaccines. The results of analysis demonstrated that all foreign-made and domestically-produced polio vaccines currently used in Russia meet international requirements in terms of main quality characteristics and comply with the WHO recommendations. The article looks into some issues arising from the use of live polio vaccine, in particular the development of vaccine-associated paralytic polio, and the appearance of vaccine-derived polioviruses. It reviews the main approaches of the current WHO polio eradication initiative, and summarises the outcomes of the 30-year period since the adoption of the Global Polio Eradication Initiative. The article describes the transition from live attenuated oral polio vaccine (types 1, 2 and 3) to bivalent vaccine (live attenuated oral polio vaccine, types 1 and 3). It discusses the necessity of using polio vaccines (both live and inactivated) at the final stage of polio eradication. The article presents the new National Immunisation Schedule

Изучение возможности применения математических методов анализа для оценки специфической активности антител к поверхностному антигену вируса гепатита В в препаратах иммуноглобулинов человека

Scientific relevance. Anti-hepatitis B virus surface-antigen (HBsAg) immunoglobulins are used to prevent hepatitis B in adults and children after exposure and to treat mild to moderate acute viral hepatitis B. The clinical effectiveness of human immunoglobulin preparations is determined by their potency, which is assessed by the content of antibodies to hepatitis B virus surface antigen (anti-HBs antibodies). Currently, this assessment involves using immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).Aim. This study examined several mathematical methods for analysing the experimental data obtained in ELISA-based anti-HBs antibody potency assays of human immunoglobulin preparations.Materials and methods. This study used the international standard for human anti-HBs immunoglobulin, two immunoglobulin preparations, and an ELISA test kit for the detection and quantification of anti-HBs antibodies in serum or plasma samples.Results. Using sandwich ELISA, the authors ascertained that the measured anti-HBs antibody concentration depended on the choice of calibration curve calculation method (i.e. manual analysis, parallel-line analysis using PARALINE software, linear regression, and 4-parameter logistic regression). The measured anti-HBs antibody concentrations varied by ± 19 IU/mL. According to the study results, an incorrectly selected method of data analysis can lead to an erroneous calculation of the analyte potency (concentration) in the test sample.Conclusions. The study demonstrated the need for improved mathematical methods for the evaluation of experimental data used to determine the anti-HBs antibody concentration in human immunoglobulin preparations. It is essential to switch from manual to automated calculation (for example, using a 4-parameter logistic model), taking into account the requirements for bioanalytical methods and the capabilities of the available equipment.Актуальность. Препараты иммуноглобулинов человека, содержащие в составе антитела к поверхностному антигену вируса гепатита В (HBsAg), являются средством экстренной профилактики гепатита В у взрослых и детей и применяются для лечения легких и среднетяжелых форм острого вирусного гепатита В. Клиническая эффективность таких препаратов определяется специфической активностью, оцениваемой по содержанию антител к поверхностному антигену вируса гепатита В (anti-HBs антитела), которую в настоящее время определяют с использованием метода иммуноферментного анализа (ИФА).Цель. Анализ различных математических методов оценки экспериментальных данных, полученных с применением ИФА для определения активности антител к поверхностному антигену вируса гепатита В в препаратах иммуноглобулинов человека.Материалы и методы. В работе использовали международный стандартный образец иммуноглобулина человека, содержащий anti-HBs антитела; препараты иммуноглобулинов; набор реагентов для иммуноферментного качественного и количественного определения антител к HBsAg в сыворотке (плазме) крови.Результаты. С использованием сэндвич-варианта ИФА установлена зависимость получаемого результата определения концентрации anti-HBs антител от выбора способа построения калибровочного графика («ручной» подход, метод параллельных линий с использованием программы «PARALINE», метод линейной регрессии, 4-параметрическая логистическая модель). Разброс полученных значений концентрации anti-HBs антител составил ±19 МЕ/мл от среднего значения. Установлено, что неверно подобранный метод обработки данных может приводить к ошибочным расчетам значений активности (концентрации) анализируемого вещества в испытуемом образце.Выводы. Показана необходимость совершенствования математических методов оценки экспериментальных данных, используемых для определения концентрации anti-HBs антител в препаратах иммуноглобулинов человека, и важность перехода от «ручного» к автоматизированному обсчету получаемых результатов (например, с использованием 4-параметрической логистической модели) в соответствии с требованиями, предъявляемыми к биоаналитическим методам испытаний, и с учетом возможностей современного оборудования

Современные проблемы вакцинопрофилактики бешенства

The urgency of the rabies problem for all mankind and the search for new ways of eradicating the disease entailed the creation of a new global initiative for rabies elimination ‒ «United Against Rabies» which sets a highly ambitious goal of achieving zero rabies human deaths by 2030. The many years of international experience in elimination of street dogs, which account for 99 % of rabies cases, did not produce the desired results, therefore the focus was shifted to mass vaccination of dogs (minimum 70 % of dog population). The rabies problem is complex and global, it requires efforts from all the parties involved as well as hefty investment. The paper presents the results of a continuous long-term analysis of the rabies situation in Russia and across the world, as well as analysis of the current state of vaccination against rabies which plays an important, if not crucial, role in prevention of rabies in humans who got bitten by infected animals. The paper formulates the main currently existing ways of solving the rabies problem, namely: mass vaccination of dogs; improvement of dosing schedules and administration routes of medicines against rabies; analysis of immunity development mechanisms in immunocompromised patients; progressive implementation of vaccination of people who got bitten by infected animals, and alternative administration routes; development of an express method of the neutralising antibody titer determination; raising public awareness about disease hazards.Актуальность проблемы бешенства для всего человечества, поиск новых путей искоренения инфекции привели к созданию новой глобальной системы для ликвидации бешенства – «Объединенные против бешенства», поставившей перед собой чрезвычайно амбициозную задачу по достижению нулевого уровня смертности людей от гидрофобии во всем мире к 2030 году. Принимая во внимание предыдущий многолетний мировой опыт уничтожения безнадзорных собак, на долю которых приходится 99 % случаев гидрофобии, что не привело к желаемому результату, в настоящее время взят курс на массовую вакцинацию собак (не менее 70 % популяции животных). Проблема бешенства комплексная, без усилия всех заинтересованных служб, серьезных финансовых затрат вряд ли удастся решить такую глобальную задачу. Материалы данной статьи являются результатом постоянного многолетнего анализа ситуации по бешенству в мире и в Российской Федерации, анализа современного состояния проблемы вакцинопрофилактики бешенства, которая играет огромную, если не ведущую, роль в предупреждении развития гидрофобии у людей, пострадавших от укусов больных бешенством животных. Сформулированы основные направления решения проблемы заболевания бешенством на современном этапе, а именно: массовая вакцинация собак; совершенствование схем и методов применения антирабических препаратов; изучение закономерностей образования иммунного ответа у лиц с ослабленным иммунитетом; постепенное внедрение в практику вакцинации людей, пострадавших от укусов больных бешенством животных, альтернативных методов введения препаратов; разработка экспресс-системы определения титров вируснейтрализующих антител; повышение уровня информированности населения об опасности заболевания

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

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

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