LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics
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Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.
KeywordsBiopharmaceutical Glycoprotein Glycan Mass spectrometry Standards Reference material
The authors would like to thank Dr. Carlito Lebrilla for technical guidance regarding pronase digestion and Dr. Illarion Turko for assistance performing MALDI-MS.
Commercial equipment, instruments, and materials are identified throughout this paper to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by NIST nor does it imply that the equipment, instruments, or materials are necessarily the best available for the purpose.
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