Detection and Quantification of Protein Aggregates by SEC–MALS - - Chromatography Online
Detection and Quantification of Protein Aggregates by SEC–MALS

The Application Notebook
pp. 385

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


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

Results


Figure 1: Chromatogram of pepsin showing the refractive index (red) and SEC–MALS (90) (orange) detector signals.
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 2: SEC-MALS plot showing different angular response for pepsin monomer and aggregates.
As pepsin is a digestive enzyme, the broad peak at 20.9 mL is most likely to be digestion products.

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, http://www.malvern.com/MRK1927


Malvern Instruments Ltd.
Enigma Business Park, Grovewood Road, Malvern, UK
Tel: +44 (0) 1684 892456
E-mail:

Website: http://www.malvern.com/

ADVERTISEMENT

blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:




 

LCGC COLUMNISTS 2014

Sample Prep Perspectives | Ronald E. Majors: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments.
LATEST: UV Detector Problems


Perspectives in Modern HPLC | Michael W. Dong: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Superficially Porous Particles: Perspectives, Practices, and Trends


MS — The Practical Art | Kate Yu: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis


LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: Problems with Large-Molecule Separations


More LCGC Chromatography-Related Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>


Source: The Application Notebook,
Click here