The Chromatogram Series: PEGylated Protein
This is a screenshot of a mono-PEGylated protein, plot generated with the OmniSEC report designer. The run conditions are detailed in the table below.
| Run Buffer | PBS |
| Flow Rate [mL/min] | 0.500 |
| Column Set | Superdex200 |
| Column Temp (°C) | 30.0 |
| Injection volume μL | 30.0 |
| Mw Number Average [Da] | 38,303 |
| Mw Weight Average [Da] | 38,320 |
| Sample Rh [nm] | 5.30 |
| Sample IV [dL/g] | 0.247 |
This highly purified mono-PEGylated protein conjugate was measured by tetra-detection SEC. The UV signal measured the protein concentration while the RI signal measured the conjugate concentration. Due to the
mixed composition, the RI and UV detectors were both needed for accurate molecular weight measurement by light scattering. The RALS signal was used to measure the molecular weight at each slice across the
peak. This sample shows an extremely narrow polydispersity index PDI = Mw/Mn value of 1.0 – in other words it is very monodisperse. The viscometer allowed the hydrodynamic size Rh(visc) determiniation through the Einstein relation.
Update: we also have an application note on Interferon-α5 where the % PEGylation was determined. The individual protein and PEG molecules, as well as the PEGylated conjugates are characterized by SEC and their composition, molecular weight and intrinsic viscosity measured.
Previously
- The definite guide to Malvern GPC/SEC information – how to get an overview of all the chromatography details on malvern.com
- Biophysical Characterization: Frequently Asked Questions
- 5 Steps to improve SEC-LS data