Establishment of the European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) for hepatitis A vaccine type B (Aventis Pasteur) batch 2.

A collaborative study was organised by the European Directorate for the Quality of Medicines with the goal to calibrate a candidate European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) for hepatitis A vaccine (adsorbed, inactivated) type B (Aventis Pasteur) batch 2 against the International Standard using the antigen content- and immunogenicity assay. Nine laboratories participated in the study; seven carried out both assays. Three samples were to be assayed, sample A, the 1st International Standard for hepatitis A vaccine (inactivated), sample B, the candidate BRP and sample C, the hepatitis A vaccine BRP type B (Aventis Pasteur) batch 1. Samples B and C represent the currently marketed vaccine and contain an antigen that is formalin inactivated and alum adjuvanted. For samples B and C, desorption was necessary prior to the antigen content assay; a proprietary desorption procedure had to be used. Due to an insufficient number of samples, sample C could only be tested in the antigen content assay. Samples A and B had to be tested in 3 independent experiments in the immunogenicity assay; an assay had to be used that produced a dose response curve. All three samples had to be tested also in three independent experiments in the antigen content assay using a standard method. The results from the immunogenicity assays were submitted to probit analyses, the results from the antigen content assays to parallel line analyses on log transformed responses. Based on the results of the collaborative study, the candidate BRP batch 2 appeared suitable as reference preparation for the immunogenicity assay of adsorbed, inactivated hepatitis A vaccines from Aventis Pasteur. A potency value of 262 IU/ml was assigned based on the calibration against the 1st International Standard. The attempt to reproduce the in vitro potency of the BRP batch 1 in the current study failed; only about 50% of the previously established potency was found. It was hypothesised that this loss in potency might be due to ageing of the antigen which changed adsorption behaviour, and subsequently would lead to reduced desorption and lower in vitro potency. Additional experiments were performed to investigate this hypothesis. For older vaccines a much lower degree of desorption and thus a much lower in vitro potency was found, when using the proprietary desorption method. With more vigorous desorption procedures, this problem could only partly be overcome. Thus the results obtained in the antigen content assay in the collaborative study and in the additional experiments did not allow proposing the candidate reference preparation for use in the antigen content assay for hepatitis A vaccine from Aventis Pasteur. The candidate BRP was adopted by the European Pharmacopoeia Commission at its session in March 2002 as Ph. Eur. BRP for hepatitis A vaccine (adsorbed, inactivated) type B (Aventis Pasteur) batch 2 to be used for the immunogenicity assay.