Detection and Quantification of Protein Aggregates by SEC–MALS

Proteins have a tendency to aggregate over time and the risk for drugs in the biopharmaceutical industry is that the presence of aggregates will stimulate an immune response. Size-exclusion chromatography (SEC) is a powerful tool that is commonly used to look at the aggregation of proteins.

SEC separates proteins by size, and is commonly used to measure molecular weight and characterize aggregation. By adding a light scattering detector to the system, the molecular weight of the protein monomer, oligomers, and aggregates in a sample can be measured independent of their elution volume. In addition, multi-angle light scattering (MALS) can also be used to measure the radius of gyration (Rg) of large aggregates that scatter light anisotropically.

Materials and Methods

Pepsin was characterized using a Viscotek TDAmax system connected to a Viscotek SEC-MALS 20 detector. Two Viscotek protein columns were coupled together for the separation. The detectors and columns were all held at 30 °C to ensure a good separation and to maximize baseline stability of the detectors.

Table 1: Measured molecular weights of the different peaks of the pepsin sample.

Results

Figure 1 shows that the pepsin sample contains 2 main components. The molecular weight of the second peak (18.5 mL) is measured at 34.7 kDa, which is very close to the known molecular weight of pepsin (35 kDa). The larger light scattering peak at 11 mL has a much higher and more variable molecular weight, clearly identifying it as some disordered aggregates, which are unlikely to be active. The MALS plot in Figure 2 shows that these aggregates are strongly anisotropic scatterers, and therefore have a large size compared to the protein.

Figure 1: Chromatogram of pepsin showing the refractive index (red) and SEC–MALS (90°) (orange) detector signals.

As pepsin is a digestive enzyme, the broad peak at 20.9 mL is most likely to be digestion products.

Figure 2: SEC-MALS plot showing different angular response for pepsin monomer and aggregates.

Conclusion

The molecular weight and aggregate content of pepsin was successfully measured using the Viscotek SEC-MALS 20 system, and their amounts quantified. Where the aggregates are large enough, their size (Rg) can also be measured.

Reference

(1) Measuring protein aggregation with the Viscotek SEC-MALS 20, Malvern Instruments application note, www.malvern.com/MRK1927

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