Protein Aggregation

Combining technologies for the superior detection, separation and characterization of protein aggregates

Introduction

The Zetasizer μV is a dynamic and static light scattering (DLS & SLS) system capable of making measurements of molecular size and molecular weight in a batch cuvette and also when connected to a size-exclusion chromatography (SEC) system in conjunction with an OmniFACE connection box (figure 1).

The Zetasizer μV has a 90° detection angle with excellent signal-to-noise and a variable power laser and attenuator to help it achieve a wide dynamic range with respect to the light scattering count rate. Additionally, the light scattering volume, which is the intersection of the illuminating laser and detector field, is very small. This allows very small volumes of sample to be measured. In a batch cuvette, measurements can be made in as little as 2 μl while the flow cell used with the system is just 8 μl. Switching between batch and chromatography modes takes only seconds for the removal of the batch cuvette and insertion of the flow cuvette or vice versa.

The application arena for the Zetasizer μV is primarily the characterization of proteins. This application note describes the measurement of two proteins in batch and chromatographic modes.

Results

The first protein measured is bovine serum albumin (BSA). Figure 2A shows a batch DLS measurement of the protein. One peak is observed demonstrating that this is a very 'clean' sample with no aggregated material present. The Z-average radius of the peak is 3.7 nm which is slightly larger than a pure BSA monomer. Furthermore, the polydispersity index (PDI) is measured to be 0.16. This indicates that the population is slightly polydisperse. BSA is known to form oligomers such as dimers and trimmers in solution. Cuvette-based batch DLS measurements are unable to resolve the individual oligomers so the sample appears as a single peak with a higher polydispersity.