(T1130-08-43) Peptide Mapping of Monoclonal Antibody (mAb) Using an Integrated Online Protein Digest LCMS Platform

Purpose: For recombinant protein pharmaceuticals, such as monoclonal antibodies (mAbs), peptide mapping is widely used for proof of identity, primary structural characterization, and quality assurance (QA). A bottom-up strategy using enzymes to digest the therapeutic protein into its constituent peptides is usually used to generate a peptide map. Typical peptide-mapping procedures, especially for protein digestion, consist of many laborious steps, which can vary due to the differences among protocols or operators.  As such they are susceptible to changes that affect reproducibility, reduce assay sensitivity, and significantly increase analysis times. An automated, new method is needed to overcome these drawbacks and provide a simple, efficient, and reliable alternative. We utilized Perfinity workstation – an automated on-column protein digestion platform – directly coupled with a LCMS-QTOF mass spectrometer for high throughput and high sequence coverage peptide mapping.

Methods: In this study, peptide mapping experiments were performed using the NISTmAb standard (reference material 8671). Three types of enzymatic columns – trypsin column, non-reduction/alkylation (NoRA) trypsin column, and Lys-C column – were used for protein digestion on Perfinity. A C18 desalt column was used to clean up the samples after digestion and a Restek Ultra C18 (2.7μm, 150mm x 2.1mm) column was used for reserved phase separation. All data was obtained on a Q-TOF mass spectrometer, LCMS-9030. Data dependent acquisition (DDA) with three dependent events was used for data acquisition. For one set of experiments, NISTmAb was diluted to 1 mg/mL with water then directly injected into Perfintiy for a 6-minute on-column protein digestion. For another set of experiments, NISTmAb was first reduced by adding 50 mM dithiothreitol (DTT), then alkylated with 500 mM iodoacetamide prior to the injection into Perfinity. Protein Metrics software was used for comprehensive peptide identification of the digested protein.

Results: For the NISTmAb sample injected directly into Perfinity without reduction and alkylation, 30.2% sequence coverage of the heavy chain and 44.1% sequence coverage of the light chain was obtained with 6-min trypsin column digestion at 50 °C. 37.3% sequence coverage of the heavy chain and 50.2% sequence coverage of the light chain was obtained with 6-min NoRA trypsin column digestion at 70 °C. For the NISTmAb sample with pre-reduction and alkylation steps, 98.7% sequence coverage of the heavy chain and 100% sequence coverage of the light chain was obtained with 6-min trypsin column digestion at 50 °C. 100% sequence coverages of both the heavy and light chains were obtained with 6-min NoRA trypsin column digestion at 70 °C. 94.4% sequence coverage of the heavy chain and 95.3% sequence coverage of the light chain was obtained with 6-min Lys-C column digestion at 50°C. A set of quality attributes post translational modifications (PTMs), such as oxidation, glycosylation, and deamidation, was identified and quantified by relative abundance. The results indicate sample preparation-induced PTMs were insignificantly low.

Conclusion: The combination of the Perfinity workstation coupled with a Q-TOF mass spectrometer provides a simple and rapid protein digestion protocol for peptide mapping analysis, which is more efficient and reproducible than the traditional benchtop digestion methods. For NISTmAb, reduction and alkylation prior to digestion is necessary for achieving high sequence coverage. Even with pre-reduction and alkylation, a comprehensive peptide mapping result can be obtained within 2 hours. The most commonly targeted modifications were successfully identified and relatively quantified.