Proteins have a tendency to aggregate over time and the risk for drugs in the biopharmaceutical industry is that the presence
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
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
Materials and Methods
Pepsin was characterized using a Viscotek TDAmax system connected to a Viscotek SEC-MALS 20 detector. Two Viscotek protein
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.
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.
The molecular weight and aggregate content of pepsin was successfully measured using the Viscotek SEC-MALS 20 system, and
quantified. Where the aggregates are large enough, their size (Rg) can also be measured.
(1) Measuring protein aggregation with the Viscotek SEC-MALS 20, Malvern Instruments application note,
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