During virus development processes, it is vital to discern the virus concentration at each stage of the procedure in order to ensure optimise the clone used and facilitate maximum production yields.
For example, instances such as clone screening, multiplicity of infection (MoI) optimisation and adaptations of cell culture methods are instances when virus concentration, or virus titre as it is also commonly called, is of interest.
Dynamic light scattering (DLS) can be used in virus development to measure drug substance or in a screening function to separate good and stable samples from those with contaminants or aggregates.
With the new multi-angle DLS (MADLS) based particle concentration measurement available on the Zetasizer
Ultra, it is now possible to get both size and size distribution, as well as particle concentration per population, within a few minutes.
In this application note, Allergan are kindly sharing data from their evaluation of the Zetasizer Ultra, examples of three adeno-associated virus (AAV) samples are shown.
The concentration results are compared to results from capsid ELISA-based virus titre assays.
Method
Samples were measured using the low-volume quartz cuvette (ZEN2112).
The measurements were performed on a Zetasizer Ultra, using the particle concentration measurement, which gives both the MADLS size distribution and the concentration per peak.
When this measurement is used, the following parameters need to be provided by the user:
Sample material; the measurements were done using protein as a material, as the virus capsid is made from a protein shell.
Dispersant material; the dispersant was initially set to water, but the viscosity was later corrected to obtain the correct size and concentration by editing the particle concentration result.
The accuracy of the measured size is critical to the particle concentration calculation. The buffer viscosity was measured using bead lacing, where we recommend using a 60nm-200nm polystyrene latex bead.
Note that larger beads scatter more and therefore it is not necessary to add as much to the sample.
Buffer scattering count rate (at backscatter angle); the buffer scattering intensity was measured in the same cuvette used for the measurement (ZEN2112), by simply setting up a single angle measurement using the backscatter angle and setting the measurement position to the centre of the cuvette.
Run as a normal measurement, the derived count rate value is used in the ‘Buffer Scattering’ field for the particle concentration measurements.
The run duration for three repeat measurements is less than four minutes.
Please visit the Malvern Panalytical website to view the complete application note.
