This application note describes a method for analyzing antibody samples using an Agilent 1200 series HPLC coupled with a 6210 TOF MS. The results indicate that this method can quickly and accurately determine the molecular weight (MW) of antibodies and can resolve the various antibody subpopulations present in a sample.
Monoclonal antibodies (mAbs) are complex structures requiring accurate characterization, particularly for determining molecular weight. Traditional methods like gel analysis or MALDI-TOF often suffer from imprecision or lengthy processing times.
This study evaluates the combination of Agilent Technologies Poroshell column, 1200 HPLC system, and 6210 Time-of-Flight mass spectrometer for analyzing intact IgG4 antibodies. The method achieves a short retention time of 2.78 minutes with good chromatographic peak shape. Mass spectral analysis successfully resolved various antibody subpopulations, demonstrating routine mass accuracy better than 25 ppm.
| Retention time for intact IgG4 | 2.78 min |
|---|---|
| Routine mass accuracy | better than 25 ppm |
| Total analysis time | 9-minute |
| HPLC flow rate | ~500 l/min |
| Column oven temperature | 90°C |
| Capillary voltage (TOF MS) | 5500 V |
| Drying gas temperature | 300°C |
The routine mass accuracy demonstrated by the TOF MS was better than 25 ppm.
The Poroshell C8 column was used, resulting in very good chromatographic peak shape.