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Characterizing Deamidation and Oxidation in Adalimumab with Low pH Peptide Mapping and Middle-Up Mass Spec Analysis

Part # PS297


Chris Hosfield1, Eric Largy2, Anicet Catrain2, Fabrice Cantais2, Géry Van Vyncht2, Michael Rosenblatt1, Sergei Saveliev1, Marjeta Urh1& Arnaud Delobel2
1Promega Corporation, Madison, WI, USA, 2Quality Assistance, Thuin, Belgium

Non-enzymatic chemical modifications such as deamidation, oxidation and disulfide bond scrambling can affect the stability and efficacy of biotherapeutic proteins. Peptide mapping, the method of choice for site-specific monitoring of these modifications, has a significant drawback which is that the sample preparation methods often induce the same modifications intended to be measured. A primary source of these sample preparation artifacts is the alkaline pH favored by trypsin and other commonly used proteases. To address this problem we have developed a sample preparation procedure in which all steps including proteolytic digestion are performed under acidic conditions. Although trypsin is inhibited at low pH, we have overcome this limitation by supplementing the digestion with a low pH resistant Lys-C protease. Here we describe the utility of this new sample preparation procedure for analysis of deamidation in Adalimumab (Humira). We show that observed deamidation sites appear to be artifacts induced during conventional sample preparation procedures. In addition, we have analyzed peroxide-induced oxidation in Adalimumab, both by peptide mapping and subunit-level analysis following digestion with IdeZ protease. An oxidation suppressant added to the peptide mapping protocol reduces oxidation when digestion occurs in the presence of peroxide.

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