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Scientific relevance. Currently, only the rabbit pyrogen test is used to test the Vianvac® typhoid Vi polysaccharide vaccine for pyrogenicity. As part of the product specification file, the gel-clot test for bacterial endotoxins (BE) will improve the reliability of quality control, as well as harmonise the requirements for the vaccine with the requirements outlined for this group of medicinal products by leading world pharmacopoeias.Aim. This study aimed at an experimental assessment of the applicability of the gel-clot test to the quantification of BE in the typhoid Vi polysaccharide vaccine.Materials and methods. This study used samples from 5 batches of the typhoid Vi polysaccharide vaccine (0.5 mL/dose, solution for subcutaneous injection), LAL and TAL reagents. The analysis included the gel-clot test and the pyrogenicity test according to the State Pharmacopoeia of the Russian Federation (OFS.1.2.4.0006.15 and OFS.1.2.4.0005.15, respectively).Results. According to calculations, the BE limit for the tested vaccine was 96 EU/mL, and the maximum valid dilution (MVD) was 3200. The authors determined the regulatory requirements for typhoid Vi polysaccharide vaccine quality in terms of BE (not more than 48 EU/dose). The in vitro BE tests were positive at vaccine dilutions of 1/16 to 1/32 and negative at 1/64 to 1/256. The authors selected and validated a working vaccine dilution of 1/128. The BE content measured in the tested samples ranged from 0.24 to 0.48 EU/dose. The in vivo pyrogen tests were positive at dilutions of 1/16 to 1/128 and negative at 1/256 in all experiments with samples from 5 vaccine batches at dilutions ranging from 1/16 to 1/256.Conclusions. This study has experimentally proven that the gel-clot test can quantify BE in the typhoid Vi polysaccharide vaccine. The authors have recommended introducing the gel-clot BE test in the monograph of the State Pharmacopoeia of the Russian Federation on the typhoid Vi polysaccharide vaccine (FS.3.3.1.0012.15).

Relevance. The presence of pyrogenic impurities in finished dosage forms of concentrated cultural anti-rabies vaccines produced in the Russian Federation is determined using pyrogenicity tests on rabbits (in vivo). In accordance with the decision of the Board of the Eurasian Economic Commission dated September 7, 2018 N 151 «On the approval of guidelines for drawing up a regulatory document on the quality of a medicinal product», one of the requirements for parenteral drugs is the determination of bacterial endotoxins. This document indicates that the regulatory documentation should include a test and an admissibility criterion for bacterial endotoxins (BE) using the horseshoe crab amoebocyte lysate technique.Aims. Experimental evaluation of the possibility of using the LAL-test to determine bacterial endotoxins in national vaccines for the prevention of rabies.Materials and methods. The research of the drug «Cultural antirabies vaccine concentrated inactivated purified» of national production was carried out in accordance with the National Pharmacopoeia of the Russian Federation, General Pharmacopoeia Monograph OFS.1.2.4.0006.15 in three modifications: gel-clot test: methods A, B; turbidimetric kinetic test: method C; chromogenic kinetic test: method D.Results. Was investigated 6 series of the national vaccines for the prevention of rabies from two national manufacturers (using three pharmacopoeial methods). LAL reagent produced by two companies (Charles River Endosafe® and Lonza). In order to confirm the reproducibility of the method, the gel-clot test was carried out at different time intervals by one or two operators. During the research was determined the possibility of using photometric methods (method C and D).Conclusions. The research proved the possibility of determining bacterial endotoxins by methods: gel-clot test (method A), turbidimetric kinetic test (method C) and chromogenic kinetic test (method D). Method B is recommended for quantitative analysis of vaccine without instrumental methods. During the research all national vaccines for the prevention of rabies was free from bacterial endotoxins (no more 25 EU/ml).

Objective(s): Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized. Methods: GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD. Results: It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes. Conclusions: Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs. [ABSTRACT FROM AUTHOR]

Various test methods are used to detect and quantify bacterial endotoxins: a gel-clot test, which is arbitral and is based on the formation of a gel, and photometric (instrumental) tests that determine the intensity of the color or turbidity in the test solutions. In this article we consider the features of modern methods for detecting bacterial endotoxins, their advantages and disadvantages. Based on information analysis and practical experience, the main criteria for choosing a method for determining pyrogenic impurities are established. The gel-clot test and photometric determinations of bacterial endotoxins are sufficiently reliable and reproducible. The choice is left for the analyst when all necessary equipment and reagents are available.Instrumental methods are used in rare case due to their high sensitivity, which allows to avoid the influence of interfering factors in the LAL-test.