A Rapid Desalting Procedure for the Mass Spectrometric Analysis of Intact Proteins

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Himanshu S. Gadgil , Da Ren, Paul Rainville, Reb J. Russell II and Jeff R. Mazzeo [Waters]
WCBP, January 6-9 Washington D.C.
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IgG; Intact IgG and glycoforms
BioSuite Prototype 5 µm Desalting Column 2.1 mm 20 mm
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Reversed phase chromatography, of intact proteins coupled to online mass spectrometric analysis has enabled detection of structural difference in proteins. Reversed phase chromatography is the most preferred method for coupling to mass spectrometer as it utilizes a volatile mobile phase. Biotherapeutics and other proteins are usually stored in salt containing buffer solutions. Non volatile salts such as sodium phosphate, sodium chloride, etc. can cause adducts with proteins and also suppress ionization during MALD-MS and ESI-MS analysis. Hence mass spectrometric analysis of proteins in salt solution can be challenging. We have developed a rapid online desalting procedure for the analysis of proteins by ESI-MS. The method has a cycle time of 15 min and could be configured in a parallel mode for high throughput analysis of proteins. We evaluated several reversed phase chemistries for protein desalting. The LC-MS conditions such as the concentration of acid modifier were studied for intact protein analysis. We show application of online desalting to the characterization of an intact IgG. We were able to detect several glycoforms in intact IgG using this method. This method should be effective in rapid analysis of biotherapeutic formulations.

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