Human anti-D immunoglobulin preparations: potency standardisation milestones

Human anti-D immunoglobulin preparations derived from human immune plasma are much needed and highly effective for specific anti-D prevention of perinatal complications and treatment of primary immune thrombocytopenia. The effectiveness of immune suppression is a direct function of the active ingredient dose received with the medicinal product. To improve the accuracy of anti-D antibody quantification, it is recommended to use certified reference materials with values assigned in international units (IUs). The aim of this study was to analyse the main stages in the development of the international standards (ISs) for human anti-D immunoglobulin potency testing and to substantiate the need for a national standard for anti-Rho(anti-D) antibody quantification. The article describes the creation of the first and subsequent ISs, the procedure for establishing the IU equivalent for the anti-Rho(anti-D) antibody concentration, the characteristics of the raw materials and preparations used, and the anti-Rho(anti-D) antibody assay methods applied to certify the ISs. According to the study conclusions, it is necessary to develop and certify a national standard for the content of anti-Rho(anti-D) antibodies that will meet the requirements of the corresponding Russian regulations

Blood preparations of humans and animals in terms of their quality, efficacy and safety

The problems of quality and safety products derived from human blood plasma and hyperimmune animal sera as well as recombinant blood products resolved strict government regulation of their production processes. The risk of implications is minimized by plasma fractionation and purification of a specific drugs from various impurities (immunoglobulin aggregates, protease, plasmin, plasminogen, prekallikrein activator, IgA and IgM etc.). Viral safety is achieved by multi-step manufacturing process that includes at least two independent methods (treatment with solvent/detergent + incubation at low pH or pasteurization, combined with polyethylene glycol processing). It was justified that for today the technological process of the development of plasma preparations and hyperimmune animal sera has reached its limit. Their further development is the most likely to refer to specific improvements. The improvements will relate to increasing the efficiency of manufacturing technologies and methods of clinical use (preparations for subcutaneous administration, combinations of different immunoglobulin preparations, etc.), viral safety, ways to eliminate component, that were previously not considered to be able to influence the outcome of clinical use (soluble molecules CD4, CD8, HLA, thrombin, trace amounts of blood clotting factors VIII, IX, X, XI, XII etc.). At the same time new genetic engineered preparations with well-characterized molecular composition and a high selectivity for target impact are expected to appear on the market because of these unsolved issues. These are recombinant blood factors with altered properties; cocktails of recombinant antibodies and Fab-fragments of IgG, highly affine for toxin epitopes, etc. Therefore, in the upcoming years it is necessary to create in Russia a new system for assessing the quality, efficacy and safety of blood products, taking into account the future course of their development

Этапы стандартизации препаратов антирезусного иммуноглобулина человека по показателю качества «Специфическая активность»

Human anti-D immunoglobulin preparations derived from human immune plasma are much needed and highly effective for specific anti-D prevention of perinatal complications and treatment of primary immune thrombocytopenia. The effectiveness of immune suppression is a direct function of the active ingredient dose received with the medicinal product. To improve the accuracy of anti-D antibody quantification, it is recommended to use certified reference materials with values assigned in international units (IUs). The aim of this study was to analyse the main stages in the development of the international standards (ISs) for human anti-D immunoglobulin potency testing and to substantiate the need for a national standard for anti-Rho(anti-D) antibody quantification. The article describes the creation of the first and subsequent ISs, the procedure for establishing the IU equivalent for the anti-Rho(anti-D) antibody concentration, the characteristics of the raw materials and preparations used, and the anti-Rho(anti-D) antibody assay methods applied to certify the ISs. According to the study conclusions, it is necessary to develop and certify a national standard for the content of anti-Rho(anti-D) antibodies that will meet the requirements of the corresponding Russian regulations.Препараты антирезусного иммуноглобулина человека, получаемые из иммунной плазмы крови человека, являются востребованными и высокоэффективными средствами специфической анти-D профилактики перинатальных осложнений и при лечении первичной иммунной тромбоцитопении. Эффективность предотвращения иммунного ответа напрямую зависит от дозы введенного действующего вещества препарата. Для повышения точности количественного определения анти-D антител рекомендовано использование стандартных образцов (СО), аттестованных в международных единицах. Цель работы — анализ основных этапов разработки международного стандартного образца (МСО) для определения специфической активности антирезусного иммуноглобулина человека и обоснование необходимости создания отечественного СО содержания антирезус Rho(D) антител. В статье описаны процесс создания первого и последующих МСО, процедура установления эквивалента международных единиц для выражения концентрации антирезус Rho(D) антител, приведены характеристики сырья и продуктов, а также методы определения антирезусных антител, используемые при аттестации МСО. Сделан вывод о необходимости разработки и аттестации отечественного СО содержания антирезус Rho(D) антител, соответствующего требованиям нормативных актов Российской Федерации в данной области

Разработка критериев приемлемости оценки результатов количественного определения анти-D-антител IgG в препаратах иммуноглобулина человека антирезус Rhо(D) иммуноферментным методом

The competitive enzyme-linked immunosorbent assay (ELISA) used for the quantification of specific antibodies, anti-D (Rho) IgGs, in human anti-D (Rho) immunoglobulin preparations relies upon the competition between anti-D antibodies in the preparation and anti-D monoclonal antibodies (mAbs) for immunochemical binding to D-antigen epitopes of the immunosorbent, human erythrocytes of the ccDEE phenotype immobilised on a solid support. The immunosorbent is prepared in-house under laboratory conditions. The certification of the International Standard (IS) for anti-D immunoglobulin included attempts to use the CCDee phenotype, which is more common (16.01%) than the ccDEE phenotype (2.16%). It is possible to use the CCDee phenotype, as long as the user adheres to the acceptance criteria for the results, developed during method validation. The aim of this study was to develop the acceptance criteria for the results of anti-D IgG quantification in human anti-D immunoglobulin preparations by the ELISA method that would allow using CCDee erythrocytes to prepare the immunosorbent should there be no erythrocytes of the ccDEE phenotype available. Materials and methods: the study used human anti-D immunoglobulin preparations; anti-D IgG–spiked samples; the IS for  anti-D immunoglobulin; the reference standard (RS) for anti-D IgG–free immunoglobulin; anti-D mAbs; and erythrocytes of the CCDee, ccDEE, and ccddee phenotypes. The authors quantified anti-D IgG antibodies by the competitive ELISA. The statistical analysis used parametric and nonpar ametric tests. Results: the study demonstrated the possibility of using CCDee  erythrocytes for competitive immunochemical binding with anti-D mAbs and anti-D IgG of various human  anti-D immunoglobulin preparations. The suitability of the analytical procedure with the CCDee phenotype was confirmed by validation parameters: trueness, intermediate precision, linearity, selectivity, specificity, and robustness of the method and comparability of the results obtained when using the CCDee and ccDEE phenotypes. The authors developed the acceptance criteria: the relative values of anti-D IgG content in the test sample should range within ±20% of the nominal value; the coefficient of determination (R2) should be at least 0.9; the relative standard deviation (RSD, %) of three absorbance values for each concentration should not exceed 20%. Conclusions: these acceptance criteria guarantee the reliability of assay results of anti-D IgG quantification by the ELISA, which allows them to be used by specialists of control and analytical laboratories to assess the quality of human anti-D immunoglobulin preparations.Метод конкурентного иммуноферментного анализа (ИФА) для количественного определения специфических антител (Ат) — анти-D-Ат IgG, в препаратах иммуноглобулина человека антирезус Rho(D) (ИГЧА) основан на конкурентном иммунохимическом связывании между анти-D-Ат в препарате ИГЧА и моноклональными анти-D-Ат к эпитопу D-антигенов иммуносорбента — эритроцитов человека фенотипа ccDEE, иммобилизированных на твердой фазе. Приготовление иммуносорбента проводится в условиях лаборатории самостоятельно. При аттестации Международного стандартного образца (МСО) анти-D иммуноглобулина предприняты попытки использования и фенотипа ССDee как наиболее часто встречаемого (16,01%) в сравнении с фенотипом ccDEE (2,16%). Использование фенотипа CCDee возможно при условии соблюдения критериев приемлемости оценки результатов, разработанных при валидации методики. Цель работы: разработать критерии приемлемости оценки результатов количественного определения анти-D-Ат IgG в препаратах ИГЧА методом ИФА, в котором для приготовления иммуносорбента, в случае отсутствия эритроцитов фенотипа ccDEE, могут быть использованы эритроциты фенотипа CCDee. Материалы и методы: препараты ИГЧА, модельные образцы с известным содержанием анти-D-Ат IgG, МСО анти-D иммуноглобулина, стандартный образец (СО) иммуноглобулина, не содержащий анти-D-Ат IgG, моноклональные анти-D-Ат, эритроциты фенотипов CCDee, ccDEE и ccddee. Определение анти-D-Ат IgG проводили методом конкурентного ИФА. Применяли параметрические и непараметрические методы статистики. Результаты: показано, что применение эритроцитов фенотипа CCDee позволяет использовать их для конкурентного иммунохимического связывания с моноклональными анти-D-Ат и антиD-Ат IgG различных препаратов ИГЧА. Подтверждена пригодность методики с применением фенотипа CCDee по валидационным характеристикам: правильность, промежуточная прецизионность, линейность, селективность, специфичность, устойчивость методики и сопоставимость результатов при использовании эритроцитов фенотипов CCDee и ccDEE. Разработаны критерии приемлемости: относительные значения содержания анти-D-Ат IgG в испытуемом образце должны быть в интервале ±20% от номинального значения; коэффициент детерминации (R2) должен быть не менее 0,9; относительное стандартное отклонение (RSD, %) трех значений оптической плотности для каждой концентрации не должно превышать 20%. Выводы: разработанные критерии приемлемости гарантируют достоверность получаемых результатов количественного определения анти-D-Ат IgG методом ИФА, что позволяет их использовать специалистами контрольно-аналитических лабораторий для оценки качества препаратов ИГЧА

Особенности разработки, аттестации и применения фармакопейного стандартного образца содержания иммуноглобулинов класса А в препаратах иммуноглобулинов человека для парентерального применения

Scientific relevance. The immunoglobulin A (IgA) impurity content in parenteral human immunoglobulins should be determined in accordance with the State Pharmacopoeia of the Russian Federation by kinetic nephelometry, radial immunodiffusion, or enzyme immunoassay (ELISA) with a reference standard. The International Standard (IS) for the content of IgA is certified using gravimetry and radial immunodiffusion. However, neither of the existing standards for  the content of IgA in human immunoglobulins is currently certified using all three compendial methods. This prevents analysts from comparing test results obtained by different methods  and may lead to an underestimation of the IgA content in human immunoglobulins.Aim. This study aimed to determine the procedure for the development, certification, and use of a pharmacopoeial reference standard (RS) for the content of IgA in human immunoglobulins.Materials and methods. The authors studied candidate RSs for the IgA content derived from human plasma for fractionation. The IgA content determination involved kinetic nephelometry, radial immunodiffusion, and ELISA, as well as commercial test kits and the IS. The authors quantified the IgA impurity in samples of commercial human immunoglobulins from various manufacturers. The data analysis involved descriptive statistics and variance analysis using Microsoft Excel and Statistica 10.Results. The  authors  established  a  pharmacopoeial  standard  with  a  certified  IgA  content  of 1.98 mg/mL (expanded uncertainty, 0.44 mg/mL; coverage coefficient, k=2; confidence level, 95%) for IgA impurity quantification in human immunoglobulins by radial immunodiffusion and ELISA and that of 1.31–2.64 mg/mL (expanded uncertainty, 0.67 mg/mL; coverage ratio, k=3; confidence level, 99%) for intralaboratory quality control of IgA impurity quantification by kinetic nephelometry, radial immunodiffusion, and ELISA.Conclusions.  The  pharmacopoeial  standard  developed   in   the   study   has   been   included in the register of standards of the State Pharmacopoeia of the Russian Federation as the Reference Standard for the Content of Immunoglobulin Class  A  (IgA) (Registry  No.  3.1.00454). The pharmacopoeial standard is intended for the standardisation of analytical methods for the-determination of the IgA impurity content in parenteral human immunoglobulins.Актуальность. Оценку содержания примеси иммуноглобулинов класса А (IgA) в парентеральных лекарственных формах препаратов иммуноглобулинов человека необходимо проводить в соответствии с требованиями Государственной фармакопеи Российской Федерации  (ГФ  РФ)  методами  кинетической  нефелометрии,  радиальной  иммунодиффузии и иммуноферментного анализа (ИФА) с использованием стандартного образца. Международный стандартный образец (МСО) содержания IgA аттестован с использованием весового метода и метода радиальной иммунодиффузии. В настоящее время стандартный образец содержания IgA в препаратах иммуноглобулинов человека, аттестованный с использованием трех фармакопейных методов, отсутствует, что не позволяет сопоставить результаты, полученные разными методами, и может стать причиной  некорректной оценки содержания нежелательной примеси IgA.Цель. Определить порядок разработки, аттестации и применения фармакопейного стандартного образца (ФСО) содержания IgA в лекарственных препаратах иммуноглобулинов человека.Материалы и методы. Исследуемые образцы кандидата в ФСО содержания IgA изготавливали с использованием субстанции «Плазма человека для фракционирования». Определение содержания IgA проводили методами кинетической нефелометрии, радиальной иммунодиффузии  и  ИФА  с  использованием  коммерчески  доступных  наборов  реагентов  и МСО. Определение содержания примеси IgA проводили в образцах коммерчески доступных препаратов иммуноглобулинов человека различных производителей. Использовали методы описательной статистики и дисперсионного анализа с применением программ Microsoft Excel и Statistica 10.0.Результаты. Разработан   ФСО   с    аттестованной    характеристикой    содержания    IgA: 1,98 мг/мл (расширенная неопределенность 0,44 мг/мл; коэффициент охвата k=2; уровень доверия 95%) — для количественного определения  содержания  примеси  IgA  в  препаратах иммуноглобулинов человека методами радиальной иммунодиффузии и  ИФА;  от  1,31  до 2,64 мг/мл (расширенная неопределенность 0,67 мг/мл; коэффициент охвата k=3; уровень доверия 99%) — для  внутрилабораторного  контроля  качества  аналитических  работ по оценке содержания примеси IgA методами кинетической нефелометрии, радиальной иммунодиффузии и ИФА.Выводы. Разработанный ФСО содержания IgА включен в реестр ФСО ГФ РФ под названием «Стандартный образец содержания иммуноглобулинов класса А (IgA)» (реестровый номер ФСО 3.1.00454) и предназначен для стандартизации методов оценки содержания примеси IgA в парентеральных лекарственных препаратах иммуноглобулинов человека

Development of acceptance criteria for the results of anti-D IgG quantification in human anti-D (Rh_о) immunoglobulin preparations by enzyme immunoassay

The competitive enzyme-linked immunosorbent assay (ELISA) used for the quantification of specific antibodies, anti-D (Rho) IgGs, in human anti-D (Rho) immunoglobulin preparations relies upon the competition between anti-D antibodies in the preparation and anti-D monoclonal antibodies (mAbs) for immunochemical binding to D-antigen epitopes of the immunosorbent, human erythrocytes of the ccDEE phenotype immobilised on a solid support. The immunosorbent is prepared in-house under laboratory conditions. The certification of the International Standard (IS) for anti-D immunoglobulin included attempts to use the CCDee phenotype, which is more common (16.01%) than the ccDEE phenotype (2.16%). It is possible to use the CCDee phenotype, as long as the user adheres to the acceptance criteria for the results, developed during method validation. The aim of this study was to develop the acceptance criteria for the results of anti-D IgG quantification in human anti-D immunoglobulin preparations by the ELISA method that would allow using CCDee erythrocytes to prepare the immunosorbent should there be no erythrocytes of the ccDEE phenotype available. Materials and methods: the study used human anti-D immunoglobulin preparations; anti-D IgG–spiked samples; the IS for  anti-D immunoglobulin; the reference standard (RS) for anti-D IgG–free immunoglobulin; anti-D mAbs; and erythrocytes of the CCDee, ccDEE, and ccddee phenotypes. The authors quantified anti-D IgG antibodies by the competitive ELISA. The statistical analysis used parametric and nonpar ametric tests. Results: the study demonstrated the possibility of using CCDee  erythrocytes for competitive immunochemical binding with anti-D mAbs and anti-D IgG of various human  anti-D immunoglobulin preparations. The suitability of the analytical procedure with the CCDee phenotype was confirmed by validation parameters: trueness, intermediate precision, linearity, selectivity, specificity, and robustness of the method and comparability of the results obtained when using the CCDee and ccDEE phenotypes. The authors developed the acceptance criteria: the relative values of anti-D IgG content in the test sample should range within ±20% of the nominal value; the coefficient of determination (R2) should be at least 0.9; the relative standard deviation (RSD, %) of three absorbance values for each concentration should not exceed 20%. Conclusions: these acceptance criteria guarantee the reliability of assay results of anti-D IgG quantification by the ELISA, which allows them to be used by specialists of control and analytical laboratories to assess the quality of human anti-D immunoglobulin preparations

HAEMAGGLUTINATION TECHNIQUES TO EVALUATE SPECIFIC SAFETY OF HUMAN INTRAVENOUS IMMUNOGLOBULINS

The safety issues of human intravenous immunoglobulin preparations are particularly important in modern pharmacotherapy for immunodeficiencies, hematologic and neurologic diseases, like as at transplant centers. Upon massive infusions of these media some complications are detected that are associated with spontaneous activation of complement system accompanied by production of anaphylatoxins, as well as activation of kallikrein/kinin, plasmin, and blood coagulation systems, changed blood rheology, initiation of intravascular hemolysis. For distinct groups of patients, these complications may be due to presence of some anti-erythrocyte antibodies (e.g., anti-A and anti-B haemagglutinins, anti-D antibodies) in the intravenous human immunoglobulin preparations. In the present review article, we show development of current quality standards for human intravenous immunoglobulins based on determination of antibody contents. Antibodies to erythrocytes represent a special safety index aiming to minimize risk of possible adverse effects connected with transfusions of human blood preparations. Different haemagglutination tests were compared to assess contents of anti-A, anti-B haemagglutinins and anti-D antibodies for specific safety of human intravenous immunoglobulins. Analysis of haemagglutination techniques for evaluation of human intravenous immunoglobulin preparations revealed their relative advantages and disadvantages. Various modifications of the methods are discussed, thus allowing to optimize process of quality control for these preparations based on detection of haemagglutinins and anti-D antibodies. We demonstrate a necessity to adjust regulations and to improve evaluation techniques for haemagglutinin determination in human immunoglobulin preparations at amounts of 100 mg/ml of protein. Special features of Russian national quality standards for human immunoglobulin preparations are considered with respect to assessment of haemagglutinins and anti-D contents. One may conclude that haemagglutination methods present the most informative and economically substantiated approach when assessing specific safety of human intravenous immunoglobulins by measuring contents of anti-A, anti-B haemagglutinins, and anti-D antibodies

METHODOLOGICAL APPROACHES TO EXPERT EVALUATION OF PRECLINICAL AND CLINICAL TRIALS OF HUMAN IMMUNOGLOBULIN PRODUCTS

The article considers the experience of Russian and leading foreign regulatory agencies in organisation and conduction of preclinical and clinical trials of human immunoglobulin products. The authors suggest a classification of human immunoglobulins and provide updated information on authorization of these products in Russia. The article summarizes methodological approaches, basic scientific principles and criteria relating to expert evaluation of preclinical and clinical trials of blood products. The authors further define the expert body’s requirements for data on preclinical and clinical trials of human normal immuniglobulins and human specific immunoglobulins for the prevention and/or treatment of infectious and non-infectious diseases which are submitted as part of applications for marketing authorization or marketing authorization variation. The article suggests programs of preclinical and clinical trials for human normal immunoglobulins and human specific immunoglobulins for the prevention and/or treatment of infectious and non-infectious diseases that are aligned with the Russian legislation and Eurasian Economic Union’s regulations on medicines circulation, and have been elaborated with respect to the guidelines of the European Medicines Agency

Certification of the branch standard sample of anti-alpha-staphylolysin content, and some aspects of its use

The article summarises materials on the certification of a candidate branch standard sample (BSS) of anti-alpha-staphylolysin content - OSO 42-28-342-2017 FSBI «SCEEMP» of the Ministry of Health of Russia, batch No. 34, production date April 1, 2017, expiry date April 1, 2018. According to the Certification programme the reaction of neutralization of staphylococcal toxin hemolytic properties was carried out to establish the range of the certified property of the candidate BSS against the WHO international standard (21±2 IU/ml). The BSS of anti-alpha-staphylolysin content can be used for quantitative determination of antibodies to staphylococcal exotoxin (alpha-staphylolysin) in the reaction of neutralization of staphylococcal toxin (alpha-toxin) hemolytic properties by specific antibodies present in human blood plasma and in a number of biologicals (normal and specific human immunoglobulins, purified and adsorbed staphylococcal toxins, and diagnostic staphylococcal toxin)

Certification and potential future use of the reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis

The article gives an account of certification performed for a new batch of a Reference standard to be used with the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis -industry reference standard (IRS) 42-28-77 - in accordance with existing requirements. A candidate reference standard was represented by a batch of normal human serum intended for diagnostic purposes (more than 500 donors) and a batch of immunoelectrophoresis serum against human serum proteins (antiserum against human serum proteins). In accordance with the certification programme, the fractional (antigenic) composition of the reference standard was determined by immunoelectrophoresis using «KliniTest-EF» buffer and 0.05 M borate buffer solution and the modes stipulated in the general monograph 1.8.2.0002.15 Agar gel immunoelectrophoresis. The reaction between the antiserum against human serum proteins and the normal human serum components of the reference standard produced at least 15 precipitation lines. The study demonstrated that the bromphenol blue dye could be used together with «KliniTest-EF» buffer to assess albumin migration, and the pyronin B dye could be used together with «KliniTest-EF» buffer and 0.05 M borate buffer solution to assess immunoglobulin migration. The study confirmed the applicability of the certified reference standard designed for the test system for determination of the fractional (antigenic) composition of human serum products by immunoelectrophoresis, and set the criteria for its use in the context of human serum products identification (species specificity) testing not only by immunoelectrophoresis, but also by agar-gel immunodiffusion