Large-Scale Identification and Quantification of Covalent Modifications in Therapeutic Proteins

Covalent modifications on therapeutic proteins are traditionally monitored by chromatographic techniques, which quantify limited number of protein modifications at a time. In this report, computer algorithms for automated analyses of liquid chromatography/tandem mass spectrometry (LC/MS/MS) data for large-scale identification and quantification of known and unknown modifications are described. Peptide identification is achieved by comparing the experimental fragmentation spectrum to the predicted spectrum of each native or modified peptide. Peak areas of related peptide ions under their selected-ion chromatograms (SIC) are used for relative quantification of modified peptides. A matched window function is used to generate SIC for more reliable quantification. In an LC/MS/MS analysis of a tryptic digestion of an IgG2 monoclonal antibody, 1712 peptide ions were identified with a false-discovery rate of ∼0.4%, and 227 modifications were identified and quantified. The accuracy of the mass spectrometry-based quantification is evaluated by comparing the abundance of different glycoforms determined by mass spectrometry to that determined by a fluorescence-based chromatography method. This large-scale method may potentially replace many chromatographic methods for assessing the quality attributes of therapeutic proteins.