Certification of a pharmacopoeial reference standard for potency testing of adsorbed tetanus toxoid in guinea pigs

INTRODUCTION. Currently, the national pharmacopoeial reference standard (RS) FSO.3.2.00247 for the potency of adsorbed tetanus toxoid (TT) is certified only in mice, whereas the WHO International Standard (IS) for TT is certified in two animal species — mice and guinea pigs. Given the development of the Eurasian Economic Union (EAEU) regulatory framework and the harmonization of the EAEU Pharmacopoeia with the European Pharmacopoeia, it is necessary to augment the certified characteristic of FSO.3.2.00247 with the potency value determined by the lethal challenge assay in guinea pigs.

AIM. This study aimed to determine the potency of the pharmacopoeial reference standard for tetanus toxoid adsorbed using the lethal challenge assay in guinea pigs.

MATERIALS AND METHODS. The study used the 4th WHO International Standard for tetanus toxoid adsorbed and the FSO for potency testing of TT adsorbed (FSO.3.2.00247, series 011-210619). The potency of the pharmacopoeial RS was assessed relative to the IS using the lethal challenge method in guinea pigs, in accordance with the European Pharmacopoeia (monograph 2.7.8). A total of 352 outbred guinea pigs (250–350 g) were used. Animals were evenly distributed into experimental groups (8–10 animals per group) for immunization: four groups for the IS and four for the pharmacopoeial RS. After 28–30 days, the animals were injected with tetanus toxin (50 LD₅₀). Results were evaluated over five days, with clinical signs of tetanus intoxication monitoring in accordance with the international scoring system. To control tetanus toxin activity, a group of non-immunized animals received injections of 2, 1, 0.5, and 0.25 LD₅₀ of the toxin. The potency of the pharmacopoeial RS and the LD₅₀ of the toxin were calculated using Kerber’s formula.

RESULTS. A certification program for FSO.3.2.00247 (series 011-210619) was developed. The pharmacopoeial RS was certified using the lethal challenge assay in guinea pigs. Four experimental studies were shown that the potency of FSO.3.2.00247 was determined to be 220 IU/ampoule. Specification of FSO.3.2.00247 (series 011-210619) may be augmented with the potency value 220 IU/ampoule for guinea pigs based on the findings obtained. The results demonstrate that pharmacopoeial RS may be used not only in the lethal challenge assay but also in alternative methods for quality control, including enzyme-linked immunosorbent assay.

CONCLUSIONS. The certified characteristic of the pharmacopoeial reference standard for potency testing of tetanus toxoid adsorbed may be augmented with the potency value determined by the lethal challenge assay in guinea pigs — 220 IU/ampoule.

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