SEC-MS for the Analysis of Aggregation in Protein Mixtures

Library Number:
Part Number:
Himanshu Gadgil;Da Ren;Paul Rainville;Reb Russell;Jeff Mazzeo [Waters]
WCBP, January 6-9 Washington D.C.
Content Type:
Content Subtype:
BSA; BSA Dimer; ß lactoglobulin ; Cytochrome c;
BioSuite 250 HR 5 µm SEC Column 7.8 mm 300 mm
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Coupling of chromatography to mass spectrometry allows rapid, efficient analysis of proteins, peptides and other biomolecules. Reversed phase chromatography utilizes a volatile mobile phase and is the most preferred separation mode for interfacing with a mass spectrometer. Size exclusion chromatography, ion exchange chromatography and affinity chromatography can also be coupled to mass spectrometry by modifying the mobile phase. We have presented earlier a method for the coupling of size exclusion chromatography to mass spectrometry. The method utilizes MS compatible mobile phase composed of 50 mM ammonium formate for the separation. Here we present the application of SEC-MS in the analysis of aggregates in a protein mixture. A mixture of beta lactoglobulin, cytochrome c and bovine serum albumin (BSA) was analyzed using SEC-MS. SEC resolves these proteins based on their hydrodynamic volume and online MS allows identification based on their molecular weights. We were able to confirm that the aggregate peak induced in the protein mixture by heat treatment, was composed exclusively of BSA based upon the molecular weight of individual components in the aggregate peak. This property of analyzing individual components within protein aggregate peaks is unique to SEC-MS. SEC-MS when used in conjunction with UV and light scattering detection should be a valuable tool for the analysis of aggregation in biotherapeutic products.

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