Capillary electrophoresis-mass spectrometry methods for tryptic peptide mapping of therapeutic antibodies.
Tryptic peptide mapping is routinely used in the biotech industry to confirm primary sequence, cell line stability, and to analyze posttranslational modifications. Peptide analysis is generally done by reverse phase liquid chromatography with UV or mass spectrometric detection. This method provides excellent resolution and sequence coverage. However, traditional methods are slow, and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. In this work, complementary CE-MS peptide analysis methods have been developed. The analyses are performed on a traditional CE-MS instrument with a sheath interface, and also on a novel sheathless interface that promises improved resolution and limit of detection. The methods were performed on a tryptic digest of a therapeutic monoclonal antibody for which LC-MS detects 97% sequence coverage. The 3% not covered consists of 11 peptides containing three amino acids or fewer, including two in the critical complementarity binding domain. Without further processing, the same tryptic digest was analyzed by CE-MS. Separation and detection of the 11 small peptides was achieved on CE-MS systems with both interfaces. The sheathless system produced better peak capacity and gave mass spectra with significantly less noise, while the sheath system proved to have better repeatability.
Protein Analytical Chemistry, Genentech, Inc, South San Francisco, CA, USA.
Colin D
CD
Whitmore
Lynn A
LA
Gennaro
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014357:Trypsin