Validation of in vitro potency assays for tetanus immunoglobulin.

Abstract

The European Pharmacopoeia (Ph. Eur.) monograph Human tetanus immunoglobulin (0398) gives a clear outline of the in vivo assay to be performed to determine the potency of human tetanus immunoglobulins during their development. Furthermore, it states that an in vitro method shall be validated for the potency estimation. Since no further guidance is given on the in vitro assay, every control laboratory concerned is free to design and validate an in-house method. At the moment there is no agreed method available. The aim of this study was to validate and compare 2 alternative in vitro assays, i.e. an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA). The potency of 2 tetanus immunoglobulin preparations (Product 1, Product 2) was estimated against the WHO International Standard for tetanus immunoglobulin, using the tetanus EIA and TIA. The coefficient of variation (CV) to characterise the assay precision was 3.2% (EIA) and 3.6% (TIA), and the corresponding CV for intra-assay variation was 4.7% (EIA) and 5.5% (TIA). Using a spiking procedure, the 2nd part of the experiment investigated recovery of a known anti-tetanus potency. The recovery of samples spiked with defined amounts of reference preparation ranged from 104 112% (EIA) and 114 125% (TIA) respectively, resulting in a mean bias of 2.2 IU/ml (95% confidence interval (CI): -1.1-5.4 IU/ml, EIA) and 5.8 IU/ml (95% CI: 1.4 10.2 IU/ml, TIA). Good agreement was observed between the in vivo and in vitro assay results: the relative potency results of the EIA and TIA as compared to those of the in vivo assay performed by the manufacturers of the 2 tetanus immunoglobulins were for the EIA in the range of 104+/-10% for Product 1 and 100+/-6% for Product 2, and for the TIA in the range of 107+/-6% for Product 1 and 100+/-7% for Product 2. Tetanus EIA and TIA are suitable quality control methods for polyclonal tetanus immunoglobulin, which can be standardised in a quality control laboratory using a quality assurance system. In a collaborative study it will now be evaluated whether the validated methods can be proposed as common in vitro batch potency assays for replacement of the in vivo mouse assay.