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 BufferPBS
Flow Rate [mL/min] 0.500
Column SetSuperdex200
Column Temp (°C)30.0
Injection volume μL30.0
Mw Number Average [Da]38,303
Mw Weight Average [Da]38,320
Sample Rh [nm]5.30
Sample IV [dL/g]0.247
Chromatogram of PEGylated protein
Chromatogram of PEGylated protein. Refractive index RI, ultraviolett absorption at 280nm UV, Viscometer, and right angle light scattering RALS signal versus elution volume.

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.


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