Ard Louis

Abstract:

The assignment of infectious potency to test articles of adenovirus has been conducted mainly using classical end-point dilution methods, which rely on virus induced cytopathology to reveal the presence of infectious virus. These assays suffer the disadvantages of labor intensity, duration, throughput restriction and variability. In the course of our development of an Ad5 based HIV vaccine for clinical evaluation, we sought a facile method for the assignment of potency to the numerous test articles generated during the development of bioprocesses for bulk manufacture, downstream purification and formulation. In this paper we describe a quantitative PCR based potency assay (QPA) which uses QPCR to quantitate adenovirus genomes replicated 24h after the inoculation of a test article on 293 cell monolayers, and then relates that mass to potency by interpolation to a standard curve of replicated adenovirus genomes constructed with a reference adenovirus standard to which infectious potency has been previously assigned in the classical end-point dilution assay. The QPA assay for adenovirus is simple and rapid, with a throughput capacity adequate to the potency assay demands of bioprocess development, and with a precision expressed as a root variability of 16.8% R.S.D., allowing for close discriminations of the products of alternative process configurations. The adenovirus QPA principle can be applied to the quantitation of infectious potency of both RNA and DNA viruses and we report briefly on the development of QPA assays for measles and mumps. QPA assays owing to their simplicity and easy automation, rapidity, capacity and precision hold promise to become widely practiced methods for the quantitation of the potency of live virus vaccines and other recombinant virus vectors.

Abstract:

The preparation of polymeric particles and capsules by means of spontaneous droplet formation and subsequent polymer precipitation or synthesis is well-known. However, spontaneous emulsification is a phenomenon that has often been erroneously interpreted. This Minireview provides new insights into the preparation of metastable liquid dispersions by homogeneous liquid-liquid nucleation, and is based primarily on a recent study by Vitale and Katz (Langmuir, 2003, 19, 4105-4110). This spontaneous emulsification, which they named the Ouzo effect, occurs upon pouring, into water, a mixture of a totally water-miscible solvent and a hydrophobic oil--and optionally some water--thus generating long-lived small droplets, which are formed even though no surfactant is present. Herein, we review and reinterpret the most relevant publications on the synthesis of a variety of dispersions (pseudolatexes, silicone emulsions, biodegradable polymeric nanocapsules, etc.), which we believe have actually been synthesized using the Ouzo effect. The Ouzo effect may also become a substitute for high-shear techniques, which, to date have only been of limited utility on industrial scales.