Characterizing Protein Aggregates Using Combined Technologies

As a dynamic and static light scattering (DLS & SLS) system, the Zetasizer ìV can measure molecular size and molecular weight in a batch cuvette, as well as in combination with a size-exclusion chromatography (SEC) system equipped with an OmniFACE connection box (Figure 1).

With superior signal-to-noise ratio, 90° detection angle, and a variable power laser and attenuator, the Zetasizer ìV provides a wide dynamic range pertaining to the light scattering count rate.

The Zetasizer µV and OmniFACE A/D converter box.

Figure 1. The Zetasizer µV and OmniFACE A/D converter box.

In addition, with a very small light scattering volume, the Zetasizer ìV can measure very small volumes of samples. With a batch cuvette, this system can make measurements in as small as 2ìl, while the volume is mere 8ìl with a flow cell. It also takes only seconds to change between batch and chromatography modes.

The Zetasizer ìV is primarily used in protein characterization, and this article demonstrates the application of the Zetasizer ìV to measure two proteins in batch and chromatographic modes.

Experimental Results

Batch DLS Measurements

At first, a BSA protein is measured and its batch DLS measurement is shown in Figure 2A. The presence of a peak reveals that the protein is a very clean sample, free from any aggregated material.

The peak’s Z-average radius is 3.7 nm, a value slightly more than for a pure BSA monomer. Additionally, the polydispersity index (PDI) determined is 0.16, revealing that the population is slightly polydisperse.

However, Cuvette-based batch DLS measurements are not able to resolve the individual oligomers, thus providing a single peak with a higher polydispersity.

BSA Sample Measurements in Chromatographic Mode

The same BSA sample is measured in chromatographic mode using the Zetasizer ìV equipped with third party ultraviolet (UV; purple) and refractive index (RI; red) detectors (Figure 2C).

The molecular separation in SEC is carried out in such a manner with the smallest ones eluting last. The molecular weights of the peaks reveal that the main peak is the monomer, while the secondary peak is the dimer and the shoulder on the leading edge of the dimer peak is the trimmer (Figure 2B).

Figure 2C depicts the measured radii of the three peaks are 3.5, 4.5 and 5.4 nm, respectively. The SEC’s resolution allows measuring each of the individual populations. Based on the stability of the molecular weight and size within the monomer and dimer peaks, each peak represents a single form of the protein (Figure 2B).

The co-elution of higher order oligomers in small quantities is represented by the slightly upward shoulder representing the trimer tails. The proportion of dimer and trimer in the protein sample is slightly below 10%. The contribution of these two populations elucidates the slightly higher Z-average value obtained in the batch DLS measurement when compared to a pure monomer.

A. Batch DLS measurement of BSA. B. Derived chromatogram of BSA with molecular weight overlaid. C. Raw chromatogram of BSA.

Figure 2. A. Batch DLS measurement of BSA. B. Derived chromatogram of BSA with molecular weight overlaid. C. Raw chromatogram of BSA.

â-lactoglobulin is the second protein measured and its batch and chromatographic measurements are presented in Figures 3 and 4.

Figure 3A shows the batch DLS measurements made before filtration through a 0.02ìm filter. A consistent population with 2.8nm radius is observed across three repeat measurements, clearly revealing that the population is â-lactoglobulin.

The extra peaks ranging from 30 nm up to 1ìm in size observed in the measurements indicate the presence of aggregated material in the sample. The Z-average of roughly 100 nm and PDI of 0.08 of the sample represent the contribution of the aggregated material. The filtered sample has a Z-average of 2.8 nm and PDI of 0.07, revealing the elimination of the aggregated material through the sample filtration.

 A. Batch measurement of unfiltered â-lactoglobulin. B. Batch measurement of filtered â-lactoglobulin. C. Derived chromatogram with molecular weight overlaid.

 A. Batch measurement of unfiltered â-lactoglobulin. B. Batch measurement of filtered â-lactoglobulin. C. Derived chromatogram with molecular weight overlaid.

Figure 3. A. Batch measurement of unfiltered â-lactoglobulin. B. Batch measurement of filtered â-lactoglobulin. C. Derived chromatogram with molecular weight overlaid.

A SEC measurement of the unfiltered sample is presented in Figure 4, clearly showing two peaks. The molecular weight of the peak eluting after 18 ml determines this peak as the â-lactoglobulin.

The low polydispersity of the molecular weight and size reveal that this is the stable form of the protein. The deviation of the size (Figure 3C) and molecular weight (Figure 4) within the peak indicates the high instability expected with protein aggregates when compared with oligomers.

Raw chromatogram with DLS radius overlaid.

Figure 4. Raw chromatogram with DLS radius overlaid.

Conclusions

The Zetasizer ìV can measure molecular size and weight in both batch and chromatographic modes. DLS is simple yet very quick, providing non-invasive size measurement of proteins in small sample volumes. Its sensitivity to larger species makes it suitable to determine the presence of aggregated material within a sample.

Individual populations within a polydisperse sample can be separated using SEC. The combination of SLS and DLS enables measurement of both the size and molecular weight of the populations, thereby facilitating the characterization of the state of oligomerization.

Furthermore, it is possible to quantify the amount of aggregated material determined in the batch DLS measurement.

This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.

For more information on this source, please visit Malvern Panalytical.

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