Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation

Objectives Addition of the antimicrobial preservative benzyl alcohol to reconstitution buffer promotes the formation of undesirable aggregates in multidose protein formulations. Herein we investigated the efficiency of PEGylation (attachment of poly(ethylene glycol)) to prevent benzyl alcohol-induced aggregation of the model protein alpha-chymotrypsinogen A (aCTgn). Methods Various PEG-aCTgn conjugates were prepared using PEG with a molecular weight of either 700 or 5000 Da by varying the PEG-to-protein ratio during synthesis and the formation of insoluble aggregates was studied. The effect of benzyl alcohol on the thermodynamic stability and tertiary structure of aCTgn was also examined. Key findings When the model protein was reconstituted in buffer containing 0.9% benzyl alcohol, copious amounts of buffer-insoluble aggregates formed within 24 h (> 10%). Benzyl alcohol-induced aggregation was completely prevented when two or five molecules of PEG with a molecular weight of 5000 Da were attached to the protein, whereas two or four molecules of bound 700 Da PEG were completely inefficient in preventing aggregation. Mechanistic investigations excluded prevention of structural perturbations or increased thermodynamic stability by PEGylation from being responsible for the prevention of aggregation. Simple addition of PEG to the buffer was also inefficient and PEG had to be covalently linked to the protein to be efficient. Conclusions The most likely explanation for the protective effect of the 5000 Da PEG is shielding of exposed hydrophobic protein surface area and prevention of protein-protein contacts (molecular spacer effect).