Introduction: The PEGylated proteins, including peptide and antibody fragment conjugates, have been approved to date. In many cases, PEGylated protein was synthesized by the reaction between PEG possessing activated ester and amine(s) in the protein. This reaction has been recognized as the gold standard of PEGylation reactions since last several decades. However, basicity of amine (s) in a protein significantly reduced after the amide bond formation, resulting in the significant inactivation and structural changes of native proteins. Accordingly, alternative method for the protein PEGylataion which enables high biological activity is highly demanded. In this study, we described a novel chemistry for the protein PEGylation. Developed method can afford PEGylated protein possessing high biological activity under the mild reaction condition.
Results and Discussion: Synthesized PEG derivative (PEG-GALD) possesses a glutaraldehyde at the terminus and two chains of PEG, resulting in the branched form of conjugate after the PEGylation of protein. PEG-GALD affords the conjugate via alkylation reaction with primary amine(s) in the protein, that is, the basicity of amine is retained even after the conjugation. Accordingly, this method is useful for the conjugation in the case the basicity of amine is critical for the retention of biological activity and protein structure. Importantly, no small molecules liberate during the conjugation reaction, which facilitates purification process. The detail structure of the conjugate was analyzed by NMR and MS. It should be noted this alkylation reaction doesn’t require additional chemical reagents such as NaBH3CN which must be used in the conjugation reaction with mono-aldehyde-PEG.
Inactivation of protein by PEGylation is particularly serious problem for the small protein which acts on the large molecule. In this study, lysozyme was chosen a model protein because the size of this protein is small (14.4 kDa), and the substrate is large peptidoglycan on the bacterial cell wall. PEGylation of lysozyme was carried out in the phosphate buffer (50 mM, pH 7.0) at 4°C for 6 hours and purified by cation exchange chromatography to afford PEGylated lysozyme in which only one PEG chain was attached. This conjugate was found to be stable during the storage for several months. PEGylated protein prepared by the developed technology showed more than 20 times higher activity compared to the conventionally PEGylated protein via activated ester technique.
References:
[1] Y. Ikeda, J. Katamachi, H. Kawasaki, Y. Nagasaki, Novel protein PEGylation chemistry via glutalaldehyde functionalized PEG, Bioconjugate Chemistry, 24, 1824-1827(2013)