Online Protein A Affinity-MS for the Analysis of Therapeutic Antibodies

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Himanshu Gadgil;Da Ren;Paul Rainville;Reb Russell;Jeff Mazzeo [Waters]
WCBP, January 6-9 Washington D.C.
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Albumin; IgG 1; IgG 2
The field of therapeutic antibodies has been growing exponentially in the past few years. Antibodies and their derivatives are thought to constitute twenty five percent of therapeutics in development. Mass spectrometric analysis of antibodies and other intact proteins can provide valuable information about the global structure of the molecule. Reversed phase chromatography, size exclusion chromatography, and ion exchange chromatography have all been successfully coupled to mass spectrometry for the analysis of intact proteins. Protein A affinity chromatography is the most efficient method for the purification of immunoglobulins. We have developed an online Protein A affinity-MS method for the analysis of antibodies in crude protein extract. The coupling of Protein A affinity column to a mass spectrometer was achieved by using a modified mass spectrometry compatible mobile phase. The binding and washing buffer consisted of 50 mM ammonium formate buffer. The elution of purified Antibody was brought about by using 1 % formic acid. The eluent was then analyzed online with a QTOF instrument after post column addition of acetonitrile. We show the application of this method to the analysis of glycosylated and non-glycosylated IgG1 from crude ascites fluids. This method should be a valuable tool for the global analysis of monoclonal antibodies during large scale fermentation.

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