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.
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.
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.Reference standards (RS) play an important role in immunological drug (ID) quality assurance systems according to an analysis of pharmacopoeial requirements imposed on ID quality control methods. RS are widely used in practice as indicators of the certified compositions and properties of the samples [1, 2].Currently, the anticomplementary activity (ACA) of human immunoglobulin (IG) preparations is determined in international practice using RS of human immunoglobulin (RS IG) [Human Immunoglobulin (ACA and molecular size), BRP batch 1] with certified ACA values of a negative control in the range 10 -40% and of a positive control in the range 60 -100% [3,4]. The positive and negative controls confirm the reliability of the results in the different ACA ranges. The WHO recommends strongly the use of national RS, the application of which is more economically advantageous for manufacturing and quality assessment [5].The goal of the present work was to develop a RS for determining the ACA of human IG preparations.
EXPERIMENTAL PARTVarious IG formulations with protein contents >45 mg/mL in addition to samples of human IG preparations that were heat treated on a water bath (56 ± 1°C) were used to develop the RS. Ampuls were treated for 5 -7 min; vials, 15 min.
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