The Chromatogram Series: Fab
Fragment antigen binding (Fab) proteins are fragments of monoclonal antibodies (mAb) which can be obtained by enzymatically digesting mAb with papain. About a third in molecular weight, Fabs have been used to treat macular degeneration, cardiovascular diseases, and snake bites, where the faster tissue penetration is an advantage after the administration of anti-venom.
In scientific research, Fabs have been of interest in structural biology to for example help understand antibody-antigen interactions, or obtain molecular structures through Fab-mediated protein crystallization.
This is an example of an Antibody fragment. The run conditions and some results are detailed in the table below.
| Run Buffer | PBS |
| Flow Rate [mL/min] | 0.800 |
| Column Set | P2500 + P3000 |
| Column Temp (°C) | 30.0 |
| Injection volume μL | 100.0 |
| Concentration [mg/mL] | 6.28 |
| Mw Weight Average [Da] | 46,183 |
| Sample dn/dc [mL/g] | 0.185 |
| Sample IV [dL/g] | 0.0284 |
This IgG antibody has undergone degradation to Fab fragments which have been partially purified. Three peaks are identified as Fab monomer and dimer and undegraded IgG with molecular weights of 46, 97 and 159 kDa respectively. Molecular weights are stable across the peaks showing monodisperse populations are eluting under these separation conditions. Data were acquired on a Malvern Viscotek TDAmax system.
Both the 7º (LALS) and 90º (RALS) light scattering measurements overlay demonstrating that at these molecular sizes, only one angle is necessary for highly accurate and reproducible molecular weight measurements. Typically for small molecules, the RALS signal is the best choice, due to the lower influence of noise on the signal baseline.
Previously
- Where can I find dn/dc?
- Biophysical Characterization: Questions and Answers
- 5 Steps to improve SEC-LS data