engleski

Monolith chromatography in Ad5 vector research

In recent years chromatography has gained attraction in adenovirus research mostly as an alternative to classical ultracentrifuge purification. There are two kinds of stationary phases available: monolithic and classical beads based columns. In virology, monoliths provide several advantages over classical columns. They are cast as a single block of material with highly interconnected channel network without dead ends and channel diameter of ~1.5 µm that can easily accommodate virus particles. The mass transfer between mobile and stationary phase is based on convection and not diffusion therefore resolution and dynamic binding capacity are flow independent properties. The design of the monolith columns as short disks provide large surface area and it enables a high flow rate that leads to much shorter time necessary to perform experiments. In our research we used ion-exchange chromatography on convective interaction media (CIM) strong anion exchanger (quaternary amine, QA) to investigate several Ad5 based vectors. First we compared chromatographic profiles of uninfected cell lysates with Ad5 wild type (replication-defective first-generation vector with early region 1 deleted) infected cell lysates. We found two differential peaks: 1. a protein peak containing mostly adenovirus hexon proteins and 2. adenovirus particle peak. There was no infective virus present in the protein peak and the majority of it was found in the virus peak. Minor amounts were also found in DNA peaks. Next, we investigated chromatographic behaviour of several structurally modified Ad5 based vectors. Retention time in ion-exchange chromatography is connected with the amount of salt necessary to elute a certain macromolecules and this depends on macromolecule’s net charge. The changes in virus charge can also be related to different biological properties, e.g. differential targeting, therefore monolith chromatography can be used as an analytical tool in preparation of such vectors. We confirmed that certain changes in the hexon protein charge have an impact on hexon protein and virus retention time. Changes in the length of the fibre and insertions of RGD in the knob domain have no influence on virus chromatographic behaviour while substitution of the entire Ad5 fibre with that of Ad3 led to significant shift in the virus retention time compared to the structurally wild type virus.

monolith; Ad5; IEX; hexon; fibre

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