HPLC-based quantification of haemagglutinin in the production of egg- and MDCK cell-derived influenza virus seasonal and pandemic vaccines.

The haemagglutinin (HA) content is an important specification of influenza vaccines. Recently, a reversed-phase high performance liquid chromatography (RP-HPLC) method for quantification of HA in PER.C6 cell culture-based whole virus vaccines has been reported, having a high sensitivity, precision, broad range, and high sample throughput [Kapteyn JC, Drissi Saidi M, Dijkstra R, Kars C, Tjon CMS-K, Weverling GJ et al. Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 cells: a novel RP-HPLC method. Vaccine 2006;24:3137-44]. This RP-HPLC assay is based on measuring the peak area of HA1, the hydrophilic subunit of HA, which turned out to be proportional to the amount of HA analyzed. Here, we present data demonstrating that this RP-HPLC method is also highly suitable for HA quantification of active and BPL- or formaldehyde-inactivated egg-based and MDCK cell-based whole virus samples, including egg allantoic harvest, and in final (monovalent) subunit vaccines, including those for pandemic H5N1 strains and for virosomal vaccines. In addition, the RP-HPLC assay was demonstrated to be a very powerful tool in the early stages of seasonal influenza vaccine production, when homologous serial radial immunodiffusion (SRID) reagents are not yet available, enabling fast and reliable viral growth studies in eggs in order to select the best growing virus strains or reassortants for the production of the seasonal trivalent influenza vaccine. Because of its high sensitivity, the RP-HPLC assay has shown its enormous value in supporting small scale MDCK-based (H5N1) influenza virus production models. Finally, the observed differences between HA1 molecules from various HA subtypes in UV absorbance, FLD response, and in the actual retention times in RP-HPLC are discussed in relation to the primary structure of the HA1 molecules studied.

Aquatic toxicity testing of sparingly soluble, volatile, and unstable substances and interpretation and use of data. Task Force of the European Centre for Ecotoxicology and Toxicology of Chemicals.

Aquatic toxicity tests were originally designed for individual compounds that are soluble and stable in water. For sparingly soluble substances that are not toxic at the solubility limit, the issue is whether tests should be performed with insoluble test substance present. Based on a literature evaluation of the physiology of uptake, it was concluded that only the dissolved fraction is available for uptake and that the insoluble test substance may introduce artifacts that aggravate data interpretation. Therefore, toxicity tests should be conducted only up to the solubility limit. Testing of volatile, unstable, or adsorptive substances is complicated by the ability to keep exposure concentrations relatively constant. For these, appropriate test protocols including adequate design of the dosing systems must be employed. For test medium preparation, physical methods and, where necessary, use of low concentrations of certain solvents are recommended to support handling and speed of dissolution. However, recommendation is made against the use of dispersants. Water-accommodated fractions are recommended as one approach for dosing multicomponent substances. Interpretation of observed effects depends on appropriate test medium preparation, correct measurement and expression of exposure levels, and differentiation of true toxicity from indirect physical effects of the substance, or the toxicity of impurities.

Bioaccumulation and lack of toxicity of octachlorodibenzofuran (OCDF) and octachlorodibenzo-p-dioxin (OCDD) to early-life stages of zebra fish (Brachydanio rerio).

Previous studies with octachlorodibenzo-p-dioxin (OCDD) and octachlorodibenzofuran (OCDF) in juvenile or adult fish exposed via water revealed no toxicity, despite significant bioaccumulation. With 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), the fish early-life stage study has been shown to be the most sensitive test system. Therefore, the effects of OCDD and OCDF on the early-life stages of zebra fish (Brachydanio rerio) were determined during a flow-through test based on a column generator method. No statistically significant effect of OCDD and OCDF on the survival and hatching time of the eggs was found. Furthermore, no effects on survival, weight, general appearance or behaviour of the larvae were observed at the end of the exposure period of 32 days. GC-MS analysis of test solution samples revealed geometric mean measured concentrations of 32 (OCDD) and 34 ng/l (OCDF), respectively. Concentrations in surviving larvae at the end of the study were 61 (OCDD) and 94 (OCDF) micrograms/kg, respectively. These concentrations in zebra fish larvae were several orders of magnitude higher than concentrations in fish collected from the wild. In a review of the available laboratory fish experiments, we found a lack of biomagnification of OCDD and OCDF. We do not expect to find adverse effects of these compounds on the aquatic environment.