Paraytec expands UV detection to new application areas

The latest results have been condensed into two application notes and describe how the active pixel UV area imaging detector can be used to size proteins as small as 1nm as well as used to save time and reagents when conducting substrate specificity experiments on enzymes such as protein kinases.

Paraytec made a big impression at Pittcon 2007, with its ActiPix D100 capillary-based UV/Vis detector snatching the Silver Pittcon Editor's Award from under the noses of industry giants just days after its release. The company has since built on this success by winning an R&D100 award.

Last December, the company won a £1m (€1.26m) grant from UK's TSB (Technology Strategy Board) and the EPSRC (Engineering and Physical Sciences Research Council) to lead a multidisciplinary consortium with partners Avecia, Intertek ASG, Lilly, Lonza and the University of Bradford with the aim of developing instruments for quantification of biopharmaceutical aggregation for use in R&D/QC and on-line in bioprocessing.

The sizing application works by measuring a phenomenon known as Taylor dispersion, which provides information about the size of a molecule from the broadening of an injected sample zone driven by pressure through a capillary

When a liquid flows through a capillary, liquid flows faster at the centre than at the walls, causing species that are present in the liquid to be dispersed along the tube axis. Acting counter to this is diffusion transverse to the tube axis.

Since small molecules diffuse faster than proteins or aggregates, small molecules give narrower bands than proteins, whilst aggregated proteins and particles give even wider bands.

As long as it contains a chromophore, the size of any species can be determined by measuring the change in the width of its UV band as it flows through a capillary.

According to Professor David Goodall, Chief Scientific Officer at Paraytec, the technique can be used to generate highly precise and accurate sizing data about molecules that are too small to be determined using light scattering techniques.

Light scattering techniques can only determine the size of particles, however by changing the UV wavelength filter the ActiPix detector can also distinguish between different materials in successive experiments.

This is proving to be particularly useful for sizing membrane proteins that typically have to be studied in detergent solutions, as the detergent forms micelles that tend to dominate the results gained using light scattering techniques.

However, because the micelles are not UV active, the ActiPix can study the proteins themselves, unhindered by the presence of the micelles.

While initial studies involved sizing proteins, the firm has also demonstrated sizing of nanoparticles – an area that is currently receiving much interest due to concerns about the toxicological effects of nanoparticles.

Working in collaboration with Paraytec during a PhD at the University of York, Pawel Urban developed a new technique that uses the ActiPix area detector to study enzyme activity and kinetics.

The continuous engagement electrophoretically mediated microanalysis (EMMA) assay offers an alternative to enzyme assays that involve the separate steps of incubation, sampling at a number of time points, separation and detection.

“Using EMMA assays will enable drug companies to quickly study the activity of enzymes such as kinases against putative substrates and inhibitors as well as quickly analysing the kinetics of the reaction,” ​said Prof. Goodall.

A key advantage of the new technique is that it permits lower concentrations of enzyme to be used than other assay methods, as the interaction between enzymes and substrates can occur throughout the entire run. This is particularly useful when working with expensive, or very rare enzymes.

The technique works by injecting a plug of putative substrates into a fused silica capillary containing the dissolved enzyme.

During the initial part of the run the components are separated, avoiding the potential competitive binding and inhibition problems that can occur in standard enzyme assays.

The UV peaks are measured at multiple time points along the looped capillary, with those components that have decreasing peak areas over time being good substrates and the multiple analysis points enabling kinetic profiles for each reaction to be obtained.

“The technique should enable cross-reactivity studies to be conducted far more efficiently than ever before and will no doubt prove to be of immense use to pharmaceutical and biotechnology companies in the future,”​ said Prof Goodall.