William M. Hurni

Analysis of a vaccine purification process by capillary electrophoresis

Abstract Free-zone capillary electrophoresis (CZE) was applied to the analysis of samples from the individual purification steps in the production of a licensed vaccine product. With this technique, real-time analysis can be performed to ensure that purification parameters are being met before proceeding to the next step. In addition to monitoring the product peak, a unique pattern of associated host cell contaminants also is displayed. This unique “fingerprint” at each step of the purification process may have practical utility for production in that it demonstrates lot-to-lot consistency in the manufacturing process of the vaccine.

Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system.

Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.

A robot for performing radioiodinations

Abstract A robotic system for radioiodination of proteins has been developed by Merck, Sharp & Dohme Research Laboratories and Zymark Corporation. The robot radioiodinates the protein of interest, separates the free from the bound 125 I, makes appropriate dilutions so that the free and bound peaks can be located using a γ counter and performs a complete clean-up after the procedure. The robot runs unattended during the iodination procedure so that operator exposure is minimized. Operator time and attention is also reduced.

Viral Enhancement and Interference Induced in Cell Culture by Hepatitis A Virus: Application to Quantitative Assays for Hepatitis A Virus

Abstract Hepatitis A virus (HAV) growing in human diploid lung fibroblast (MRC5) monolayers can either interfere with or enhance the cytopathic effect of Newcastle Disease virus (NDV) challenge. Enhancement of NDV occurred if HAV-infected monolayers were challenged with a low multiplicity of infection of NDV and incubated at 35°C. Interference occurred if HAV-infected monolayers were given a high NDV multiplicity of infection and incubated at 32°C. These phenomena were applied to assays for quantifying HAV and may be useful in providing new insights into viral interference and enhancement.

Automated cytopathic effect (CPE) assays

An automated CPE procedure has been developed that increases the precision and ease of performing titrations of measles, mumps and rubella viruses in vaccine materials. By this procedure, additions of cell suspensions and reagents and the dilution of samples are performed automatically by a modified Dynatiter instrument, using 96-well microtitre plates. Cell monolayers are stained with carbolfuchsin dye to eliminate the need for microscopic examination. Finally, the trays are read in an optical scanner and the end points calculated automatically by a programmable calculator. The increased accuracy and precision attained by performing greater numbers of replicate assays at reasonable cost will be of particular value to vaccine manufacturers.