Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells.

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

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Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage ϕ6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of ϕ6. In addition, binding of ϕ6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 10 PFU/mg of protein and ~65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.

Imidazolium‐based ionic liquids are used as new solvents for the synthesis of statistical copolymers from methacrylates of strongly differing polarities and solubilities, namely of n‐butylmethacrylate and 3‐(N‐2‐[methacryloyloxyethyl]‐N,N dimethyl‐3‐ammonio)propane sulfonate. Though difficult to prepare in classical solvents, true copolymers of these monomers can be obtained in ionic liquids, providing zwitterionic amphiphilic polymers. The relative reactivity of the monomers and thus the copolymer composition is influenced by the ionic liquid used. Analysis of the copolymers using 1H NMR spectroscopy, elemental analysis, and asymmetrical flow field‐flow fractionation (AF4) shows the influence of both the length of the alkyl chain bound at the nitrogen atom of the imidazolium ring and the anion of the ionic liquid on the structure of the copolymers. Furthermore, the highly polar methacrylate with a sulfobetaine moiety is only slightly preferred in copolymerization if ionic liquids are used as solvents for copolymerization. The ratio of monomer segments in the copolymer can be influenced by the selection of the ionic liquid used as a solvent for polymer synthesis and by the variation of the monomer ratio selected for copolymerization if it is carried out in an ionic liquid. This opens new possibilities for a broad variation of amphiphilic copolymer structures obtained through free radical copolymerization of differently polar monomers. Copyright © 2008 John Wiley & Sons, Ltd.