Collaborative study for establishment of the European Pharmacopoeia BRP batch 1 for diphtheria toxin.

A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.

Are potency tests for tetanus vaccines really necessary?

There is no simple relationship connecting the antigen content to the immunogenic potency of tetanus vaccines and the need for potency tests therefore remains inescapable. However, the quantal response methods of assay presently used for the measurement of potency appear to use unnecessarily large numbers of animals and substantial reductions could be made, particularly in the control of combined vaccines, if toxic challenge were replaced by in vitro titration of antitoxin.

United States standard diphtheria toxin for the Schick text and the erythema potency assay for the Schick text dose.

A diphtheria toxin shown to have high toxicity and avidity and to combine with antitoxin in multiple proportions was selected for the U.S. standard diphtheria toxin for the Schick test and freeze-dried. Assayed values per vial were 1.09 L(f), 1.09 L(r), 1,090 Schick test doses (STD), 33 LD(80), 38 LD(50), and 43,000 minimum skin reactive doses. One STD (L(r)/1000) is slightly more toxic than one unit of the international standard (L(f)/1,000). Experiments showed that the potency assay of Schick test toxin by guinea pig erythema toxicity, determined relative to the toxicity of the standard, was highly reproducible and significantly more reproducible than the lethal (minimum lethal dose) test and that the STD, defined as one L(r)/1000, was equivalent to approximately 1/50 minimum lethal dose. The erythema potency assay was prescribed in the U.S. standards for Schick test toxin effective in 1969.