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

Maria J. Burggraef, Adam Oxley, Naveed A. Zaidi, Pedro R. Cutillas, Piers R. J. Gaffney and Andrew G. Livingston ()
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Maria J. Burggraef: Queen Mary University of London
Adam Oxley: Queen Mary University of London
Naveed A. Zaidi: Queen Mary University of London
Pedro R. Cutillas: Queen Mary University of London
Piers R. J. Gaffney: Queen Mary University of London
Andrew G. Livingston: Queen Mary University of London

Nature Communications, 2024, vol. 15, issue 1, 1-12

Abstract: Abstract 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 (Mw), 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 Mw, dispersity-free mPEG active ester, and a commercially available disperse 5 kDa mPEG active ester. We demonstrate that the defined Mw 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.

Date: 2024
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DOI: 10.1038/s41467-024-54076-6

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