Abstract
PEGylation (covalent attachment of poly(ethylene glycol) (PEG)) is an increasingly important process that extends the in vivo circulation half-lives of therapeutic proteins, thereby increasing their efficacy and reducing both the frequency and the level of dosage required for effective treatment. PEGylation is known to significantly increase the size of proteins, so size exclusion chromatography (SEC) is a logical candidate for analysis and purification. In this paper, the results of size exclusion studies are presented for several proteins, with molecular weights from 12.2 to 68 kDa, PEGylated with PEG's ranging from 2 to 40 kDa and having between 0 and 8 PEG groups attached. Results show that PEG maintains a constant surface area to volume ratio whether it is in free solution or attached to a protein surface, in the latter case doing so by adjusting the thickness of the PEG layer according to the native protein molecular size and the total amount of PEG attached. The thickness of the PEG layer, and therefore the size of the PEGylated protein, is shown to be independent of the number of PEG groups attached to the protein. It will be shown that the size of PEGylated proteins is quantitatively predictable from the native protein and total PEG molecular weights alone and that the elution volumes of PEGylated proteins in SEC can therefore be predicted. This allows not only the identification of PEGylated species by analytical SEC but quantitative design of production scale SEC for the separation of PEGylated proteins.
