Human Immunoglobulin Preparations Research Articles

Evaluation of the Stability of Performance of the Analytical Test Method Used for Determination of Anticomplementary Activity of Human Immunoglobulin Preparations

The evaluation of anticomplementary activity, being an obligatory component of the quality control ofhuman immunoglobulin preparations, requires the use of a number of reagents of biological origin that are difficult to standardise. In order to standardise the quality control method used for determination of antiсomplementary activity it is advisable to use a reference standard which demonstrates whether obtained results comply with the acceptance criteria, and helps to assess the stability of analytical performance. The aim of the study was to assess the stability of analytical performance of the test procedure used for determination of antiсomplementary activity of human immunoglobulin preparations using human immunoglobulin reference standards of various grades. Materials and methods: anticomplementary activity was determined by the complement fixation test in accordance with the general monograph of the State Pharmacopoeia of the Russian Federation (14th ed.) OFS.1.8.2.0007.15 using different batches of guinea pig complement and sheep red blood cells. The test procedure was standardised using the OSO 42-28-430-2018 human immunoglobulin reference standard and the human immunoglobulin BRP, batch 1 (cat. No. Y0001504). The obtained results of anticomplementary activity of the reference standards were used to construct Shewhart control charts, using the certified values as control limits. Results: the analysis of the constructed Shewhart charts helped to assess trends, and estimate the influence of different batches of guinea pig complement and sheep red blood cells on the stability of analytical performance of the test procedure used for determination of antiсomplementary activity of human immunoglobulin preparations. Conclusions: the use of the OSO 42-28-430-2018 reference standard in combination with Shewhart control charts for the quality control of human immunoglobulin preparations in terms of Anticomplementary activity makes it possible to control the testing process, and assess any of its changes associated with the replacement of the batch of the reagent. At the same time, the reference standard of the European Pharmacopoeia, which has a wide range of permissible values, can only be used to confirm the acceptance of the results. Further studies to determine the mean value of the anticomplementary activity of control samples, as well as the standard deviation, may increase the possibilities of using this reference standard for assessment of analytical performance stability.

Method Development for Determining Prekallikrein Activator in Medicinal Preparations of Human Immunoglobulins and Albumin

Method development for determining prekallikrein activator in medicinal preparations of human immunoglobulins and albumin found that the adequacy of chromogenic determination of prekallikrein activator depends on the activity of the used prekallikrein reagent, the quantitative component ratio in the reaction mixture and its incubation time, and the concentration of employed chromogenic reagent. Acceptance criteria for commercial prekallikrein reagents are justified. The main method parameters and validation test results confirming the linearity in the range 0 – 35 IU/mL, accuracy, and intralaboratory precision are presented and correspond to those for biological analytical methods. The elaborated method can be used for quality assessment of prekallikrein activator content in medicinal preparations of human immunoglobulins and albumin.

Effects of Euglena gracilis EOD-1 Ingestion on Salivary IgA Reactivity and Health-Related Quality of Life in Humans.

Euglena gracilis EOD-1, a microalgal strain known for high yields of the β-1, 3-glucan paramylon, is suggested to function as a dietary fiber and enhance immunity. Here, we aimed to investigate the effects of E. gracilis EOD-1 biomass (EOD1BM) ingestion on immunoglobulin A (IgA) antibody titers in saliva, its reactivity, and the health-related quality of life (QOL) in humans. Reacting human immunoglobulin preparations and saliva with paramylon granules revealed the presence of anti-paramylon antibodies in the blood and saliva. We conducted a placebo-controlled, double-blind, crossover study involving 13 healthy subjects who ingested the placebo or EOD1BM for 4 weeks. Saliva was collected from each subject before and after ingestion, and IgA titers and E. gracilis EOD-1 paramylon (EOD1PM) reactivity were compared. In the EOD1BM Ingestion group, the anti-EOD1PM IgA content and titer increased after EOD1BM ingestion. No such change was observed in the Placebo group. Furthermore, the health-related QOL, especially mental health, increased in the EOD1BM Ingestion group. Thus, EOD1BM ingestion led to the production of paramylon (PM)-specific IgA antibody and increased salivary IgA antibody titers. We demonstrate that EOD1BM ingestion enhanced the immunity in the mucosal surface, evoked an antigen-specific response, and increased the health-related QOL, thereby contributing to health improvement.

Human antibodies eluted from ligand-free Sepharose capable of binding bacterial polysaccharides and sulfated glycans

Sepharose matrix without immobilized ligands binds antibodies from human blood serum or immunoglobulin preparations. The eluted antibodies bind bacterial polysaccharides having no structural similarity to agarose (Sepharose is a cross-linked polysaccharide agarose) with a high affinity. It is concluded that the identified antibodies are capable of recognizing spatial rather than linear epitopes of bacterial polysaccharides. This side activity of Sepharose matrix should be taken into account in isolating target antibodies and other proteins from human blood.

Technological Aspects of Ensuring the Specific Safety of Human Immunoglobulin and Albumin Preparations

Modern technological approaches to ensuring the specific safety of human immunoglobulin and albumin preparations are analyzed. Introduction into human immunoglobulin preparation technology of donor screening for group- and rhesus-affiliations and chromatographic purification steps to reduce the contents of anti-A and anti-B hemagglutinins and anti-D antibodies are shown to be promising. Technological capabilities for reducing the anticomplementary activity and thrombogenic potential of immunoglobulin preparations and decreasing the contents of prekallikrein activator in human i.v. immunoglobulin and albumin preparations are discussed.

Технологические особенности обеспечения специфической безопасности препаратов иммуноглобулинов и альбумина человека

Представлен анализ современных технологических подходов к обеспечению специфической безопасности препаратов иммуноглобулинов и альбумина человека. Показана перспективность включения в технологию препаратов иммуноглобулинов человека скрининга доноров по групповой и резус-принадлежности, а также этапов хроматографической очистки с целью снижения содержания в них анти-А и анти-В гемагглютининов, анти-D антител. Рассмотрены технологические возможности снижения антикомплементарной активности и тромбогенного потенциала препаратов иммуноглобулинов, а также снижения содержания активатора прекалликреина как в препаратах иммуноглобулинов человека для внутривенного введения, так и в препаратах альбумина человека.

Standardization of the Method for Determining Hemagglutinin Content in Human Immunoglobulin Preparations in Russia

Documentation for standardization of the method for determining hemagglutinins (HAs) in human immunoglobulin preparations used in Russia is presented. The proposed general pharmacopoeial monograph “Determination of anti-A and anti-B hemagglutinins in medicinal preparations of human immunoglobulin” allows quality control of human immunoglobulin preparations for HAcontent using indirect hemagglutination under standard conditions. The positive and negative components of the domestic standard sample (SS) of HAs contain 1:32 anti-A and 1:16 anti-B, respectively; less than 1:2 anti-A and -B; and 1:64 components of the HA content limit. Introduction of the SS for contents of anti-A and anti-B HAs into pharmacopoeial practice would ensure that results obtained in various ranges are accurate and enable the quantitative contents of the corresponding HAs in human immunoglobulin preparations to be determined.

Стандартизация метода определения содержания гемагглютининов в препаратах иммуноглобулинов человека в России

Представлены материалы по стандартизации метода определения содержания гемагглютининов в препаратах иммуноглобулинов человека в России. Показано, что разработанная общая фармакопейная статья «Определение анти-A и анти-B гемагглютининов в лекарственных препаратах иммуноглобулинов» позволяет контролировать качество препаратов иммуноглобулинов человека по содержанию гемагглютининов методом непрямой гемагглютинации в стандартных условиях. Положительный и отрицательный компоненты отечественного стандартного образца содержания гемагглютининов содержат, соответственно, 1:32 анти-A гемагглютининов, 1:16 анти-B гемагглютининов и менее 1:2 анти-A и анти-B гемагглютининов, а компоненты лимита содержания — 1:64. Внедрение стандартного образца содержания анти-A и анти-B гемагглютининов в фармацевтический анализ позволит обеспечить достоверность полученных результатов в различных диапазонах значений, а также определить количественное содержание соответствующих гемагглютининов в препаратах иммуноглобулинов человека.

Production of pure fractions of immunoglobulin G subclass autoantibodies against tumor necrosis factor.

Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal. Our protocol purified IgG subclass autoantibodies directed against TNF from a human immunoglobulin preparation. The isolation of these anti-TNF autoantibodies will enable evaluation of the effect of TNF-specific antibodies on TNF biological activity. Our newly developed technique for purifying subclasses of anti-TNF autoantibodies may be important for both basic research on the functional activity of these autoantibodies and for clinical immunology.

Development of the suppository form of human immunoglobulin preparation with high titers of antibodies to herpes simplex virus types 1 and 2 for the treatment of chronic forms of herpetic disease

In spite of the vast arsenal of therapeutic agents, therapy of herpes virus infection (HVI) is very difficult, particularly in pregnant women, newborns and children in the first years of life, as well as in patients with immune deficiency. In this regard, possibility of using immunoglobulins for the treatment of HVI is currently attracting the attention of doctors. The aim of this work was to develop a suppository form of the drug containing donor immunoglobulins with high levels of neutralizing antibodies to herpes simplex virus types 1 and 2 for the treatment of chronic forms of herpetic disease. The study included the following steps: 1) selection of gamma-globulins with high antibody titer for HSV-1 and HSV-2 ELISA test; 2) determination of the level of neutralizing antibodies in the selected series of gamma-globulins in tests in tissue cultures and animals; 3) lyophilization of immunoglobulins; 4) development of the suppository form of the preparation containing gamma-globulin donors with high levels of neutralizing antibodies to HSV-1 and HSV-2; 5) study of the safety of the activity of neutralizing antibodies to HSV-1 and HSV-2 in the suppository form of the drug with hyaluronic acid used as immunomodulator. As the result of this work, immunoglobulin preparation in the suppository form was developed. The developed preparation meets the requirements for safety and efficacy. It is not toxic or pyrogenic. The problems of clinical use of this drug as a method of HVI therapy are discussed.

Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens.

Naturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG), representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule.

Update on the use of immunoglobulin in human disease: A review of evidence

Human immunoglobulin preparations for intravenous or subcutaneous administration are the cornerstone of treatment in patients with primary immunodeficiency diseases affecting the humoral immune system. Intravenous preparations have a number of important uses in the treatment of other diseases in humans as well, some for which acceptable treatment alternatives do not exist. We provide an update of the evidence-based guideline on immunoglobulin therapy, last published in 2006. Given the potential risks and inherent scarcity of human immunoglobulin, careful consideration of its indications and administration is warranted.

Аттестация стандартного образца иммуноглобулина человека для определения антикомплементарной активности

Представлены материалы по аттестации и изучению стабильности впервые разработанного отечественного стандартного образца иммуноглобулина человека для определения антикомплементарной активности, применяемого для оценки стабильности и приемлемости результатов испытания. По результатам проведенных исследований аттестованные значения антикомплементарной активности отрицательного контроля стандартного образца находятся в интервале от 37,9 до 45,1 %, положительного контроля — в интервале от 73,6 до 82,8 % (с доверительной вероятностью 0,95). На основании материалов по изучению стабильности обоснован срок годности 1,5 года. Внедрение аттестованного стандартного образца в фармацевтический анализ лекарственных препаратов иммуноглобулинов человека для внутривенного введения при проведении экспертизы и контроля качества в рамках государственных заданий, а также с целью подтверждения соответствия требованиям нормативной документации позволит обеспечить достоверность определения антикомплементарных свойств.

Method Validation for Anticomplementary Activity of Human Immunoglobulin Drugs

Amethod for determining anticomplementary activity of human immunoglobulin (Ig) preparations for i.v. administration by complement fixation test was validated for accuracy, precision (repeatability, intralaboratory precision), limit of quantitation (LOQ), linearity, and analytical domain. The method could be used for quality assessment using the “anticomplementary activity” as part of the pharmaceutical quality examination of human Ig preparations.

Валидация методики определения антикомплементарной активности лекарственных препаратов иммуноглобулинов человека

Валидирована методика определения антикомплементарной активности лекарственных препаратов иммуноглобулинов (Ig) человека для внутривенного введения методом реакции связывания комплемента по показателям правильность, прецизионность (повторяемость, внутрилабораторная прецизионность), предел количественного определения, линейность и аналитическая область. Методика может быть использована для оценки качества по показателю «Антикомплементарная активность» в рамках проведения фармацевтической экспертизы качества лекарственных препаратов Ig человека.

Development of Pharmacopoeial Methods for Determining the Anticomplementary Activity of Human Immunoglobulin Preparations

The development of a pharmacopoeial method for determining the anticomplementary activity of human immunoglobulin preparations was reviewed. It was shown that the methods for determining anticomplementary activity that are used by domestic manufacturers of human immunoglobulin preparations did not comply with current international requirements, primarily because a standard sample was not used. The general pharmacopoeial monograph “Determination of anticomplementary activity of human immunoglobulin preparations for intravenous administration” that was published in the Russian Federation State Pharmacopoeia, XIIIth Edition, was formulated for the first time as a result of optimizing the conditions for determining anticomplementary activity and harmonization of the method with major foreign pharmacopoeias.

Разработка фармакопейной методики определения антикомплементарной активности препаратов иммуноглобулинов человека

Представлен обзор исследований по разработке фармакопейной методики определения антикомплементарной активности препаратов иммуноглобулинов человека. Показано несоответствие методик определения антикомплементарной активности, применяемых отечественными производителями препаратов иммуноглобулинов человека, современным международным требованиям, в первую очередь, по причине отсутствия использования стандартного образца. В результате проведенных исследований по оптимизации условий определения антикомплементарной активности и гармонизации методики с ведущими зарубежными фармакопеями впервые разработана общая фармакопейная статья «Определение антикомплементарной активности лекарственных препаратов иммуноглобулинов человека для внутривенного введения», опубликованная в Государственной фармакопее Российской Федерации XIII издания.

Neuronal low-density lipoprotein receptor-related protein 1 (LRP1) enhances the anti-apoptotic effect of intravenous immunoglobulin (IVIg) in ischemic stroke

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional and multi-ligand endocytic receptor abundantly expressed in neurons. Intravenous immunoglobulin (IVIg) is a purified preparation of plasma-derived human immunoglobulin used for the treatment of several neurological inflammatory disorders, and proposed for the treatment of stroke for its potent neuroprotective effects. LRP1 has been shown to be involved in the transcytosis of IVIg, and IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation, which may contribute to the anti-inflammatory effects of IVIg. However, the question remains whether IVIg could induce its neuroprotective effects via LRP1 in neurons under ischemic stroke conditions. In cultured neurons and in a transient ischemic mouse model, ischemia decrease LRP1 levels and phosphorylation, and IVIg blocks these effects. In ischemic neurons, LRP1 antagonism by receptor associated protein (RAP) enhances the activation of pro-death signaling pathways such as nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and caspase-3, and IVIg reduces these effects. When applied to ischemic neuronal cultures, RAP induces a dramatic drop in Akt activation, and IVIg reverses this effect, as it does with the decrease in Bcl-2 levels caused by ischemic injury in the presence of RAP. Altogether, these results show evidence of LRP1 expression and activity modulation by IVIg, and support the role of LRP1 as a partner of IVIg in the execution of its neuroprotective effects.

Optimization of the Conditions for Assay of the Anti-Complement Activity of Human Immunoglobulin Preparations for Intravenous Administration

Anti-complement activity is a measure of the specific safety of human immunoglobulins characterizing one of the unwanted effect - spontaneous activation of the complement system. In compliance with the specifications of the European Pharmacopeia and WHO recommendations, the anti-complement activity of human immunoglobulin preparations must not be greater than 50%, which corresponds to a maximum of 1 CH50/mg of immunoglobulin. The measurement method was based on the ability of human immunoglobulins to produce “nonspecific” (in the absence of an antigen-antibody complex) binding of complement, preventing lysis of sensitized sheep red blood cells. We present here the results of analysis of the critical parameters of the method for measuring anti-complement activity, along with assessment of existing methodological approaches to its optimization. We report experimental studies of the effects of components of the hemolytic system and complement in guinea pigs on the results of measurements of anti-complement activity. The optimum conditions for the method of measuring the anti-complement activity of human immunoglobulins are based in evidence and the anti-complement properties of human immunoglobulin preparations for intravenous use of Russian origin are evaluated.

Development of a Reference Standard for the Determination of Human Immunoglobulin Anticomplementary Activity

Results of studies devoted to determination of the anticomplementary activity of human immunoglobulin preparations in various formulations were presented. The optimal conditions for developing a positive control, i.e., a reference standard of human immunoglobulin that can be used for determining the anticomplementary activity, were defined. The first domestic reference sample of human immunoglobulin was developed. This standard is intended to be used for quality assessment of human immunoglobulin preparations for intravenous administration in terms of an anticomplementary activity parameter. Use of the proposed reference standard of human immunoglobulin for determination of anticomplementary activity will improve the evaluation accuracy of commercial human immunoglobulin preparations for intravenous administration.