Peptide Mapping of a Monoclonal Antibody using a Microfluidic-based HPLC-Chip coupled to an Agilent Accurate-Mass Q-TOF LC/MS
The document details a method for sequence characterization of monoclonal antibodies (mAbs) at low nanogram levels. It utilizes an Agilent HPLC-Chip system coupled with an Agilent Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS instrument. The high performance and mass accuracy of the platform, combined with MassHunter and BioConfirm software, enable complete sequence coverage and mass confirmation of peptides.
| HPLC-Chip Column | 5 ym, ZORBAX 300SB-C18 (300A), 40 nL enrichment column and a 75 mm x 43 mm analytical column |
|---|---|
| Flow rate (enrichment) | 3 L/min |
| Flow rate (analytical) | 600 nL/min |
| Sample injected | 50 ng |
| Mass accuracy | 4 ppm |
| Sequence coverage (trypsin) | 95% heavy chains, 85% light chains |
Data was processed using Agilent MassHunter Qualitative Analysis software, Agilent MassHunter BioConfirm and Agilent Spectrum Mill MS Proteomics Workbench software packages.
The flow rate from the Agilent 1200 Series Capillary Pump to the enrichment column is 3 L/min.