New Application Note Demonstrates Ability to Monitor Protein Aggregation States in Different Buffers Using Wyatt Technology’s Light Scattering Detectors

Wyatt Technology, the world leader in absolute macromolecular characterization, announces its instruments have been chosen by the Ridgefield Research and Development Division of Boehringer-Ingelheim Pharmaceutical, Inc. to monitor protein aggregation states in different buffers. A new application note details Wyatt’s innovative Multi-Angle Light Scattering (MALS) instruments were able to track changes in the proteins as the salt concentrations and buffer solutions were modified, thus giving greater insight into the behavior and predictability of the proteins.

Protein aggregation, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering since it eliminates or reduces the proteins’ biological function.

Diseases where protein aggregation is causal or an associated symptom include Down's syndrome, Alzheimer's and prion diseases such as BSE and CJD. Protein aggregation is encountered routinely during purification, refolding, sterilization, shipping and storage processes because of the presence of chemical, physical and thermal stresses. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention.

For this particular application, Wyatt’s 18 angle DAWN HELEOS instrument was coupled to a UV detector and Wyatt’s Optilab rEX refractive index detector. A TSKgel G3000SW column from Tosoh Bioscience was used while the analysis was performed within a 30 minute run time at a 1ml/min flow rate. A PBS isocratic mobile phase was employed. Prior to loading onto the HPLC column, the protein was thoroughly dialyzed into various buffers containing different concentrations of salt spanning 150mM to 500mM as well as different buffer systems with pH values ranging from 5 to 8.5.

The different states of the protein were separated into multiple peaks through SEC-HPLC and the molecular weights for these peaks were simultaneously determined by MALS. The molecular weights detected by MALS correlated very well with those expected for monomer, dimer, trimer and higher oligomers of the protein. As expected, in most of the buffer systems, the protein behaved similarly as the untreated control. However, MALS detection revealed that in a couple of buffers the molecular weights were lower or higher than those expected, suggesting salt/pH dependent dissociation or association of the protein subunits.

The results clearly demonstrated that several buffers significantly change the aggregation states of the protein. Moreover, in-line molecular weight determination by MALS will assist researchers in accurate interpretation of the SEC results. A copy of the new application note, entitled “Protein Aggregation States”, is available to download free of charge by visiting the article webpage.