ESA Biosciences has conducted a study to resolve Pegylated monoclonal antibody (PEG-MAb) and free PEG using size exclusion chromatography and the Corona charged aerosol detector.
Conjugation of polymer chains such as polyethylene glycol (PEG) with proteins, Pegylation, has been used as an approach to improve the pharmacokinetic properties of many therapeutic proteins.
Advantages of Pegylation are: 'masking' the bio-therapeutic from the host's immune and metabolic systems; increasing the hydrodynamic size (size in solution) of the agent, which prolongs its circulatory time by reducing renal clearance; and providing water solubility to hydrophobic molecules.
However, Pegylation typically results in product mixtures necessitating complex clean-up and characterisation protocols.
An application brief from ESA describes a method that uses size exclusion chromatography (SEC) and Corona charged aerosol detection to resolve both Pegylated monoclonal antibody (PEG-MAb) and free PEG.
The Corona detector is a sensitive, mass-based HPLC detector that can analyse other non-volatile and some semi-volatile analytes.
The analyte response is less dependent on chemical structure than other detectors.
In this study, the typical elution order expected for SEC is reversed with this mobile phase due to hydrophobic interactions causing the PEG-MAb to retain longer.
The limit of detection (LOD) for PEG was 16ng (on column).
The RSD percentage (n=5) for 400ng of PEG was 2.6 per cent.
This approach can readily detect 0.1 per cent PEG in the presence of the PEG-MAb product.
