Exactly defined molecular weight poly(ethylene glycol) allows for facile identification of PEGylation sites on proteins.

PEGylation (the covalent attachment of one or more poly(ethylene glycol) (PEG) units to a therapeutic) is a well-established technique in the pharmaceutical industry to increase blood-residence time and decrease immunogenicity. A challenging aspect of PEGylation is the dispersity of PEGylation agents, which results in batch-to-batch variations and analytical limitations. Herein, we present an approach to overcome these limitations by manufacturing a defined molecular weight (dispersity-free) PEGylation agent. We synthesise a defined molecular weight (M), linear 5 kDa methoxy-PEG (mPEG) active ester in an efficient and scalable manner using an iterative liquid-phase approach based on Nanostar Sieving. We then perform a comparative study on the random PEGylation and subsequent characterisation of the protein bovine serum albumin (BSA), using both the defined M, dispersity-free mPEG active ester, and a commercially available disperse 5 kDa mPEG active ester. We demonstrate that the defined M PEG both allows for facile monitoring of chemical modification reactions during the synthesis of the PEGylation agents, and facilitates straightforward identification of the PEGylated fragments within a PEGylated protein via a simple peptide mapping approach using UPLC-MS.