Hydrophobized PEG for unexposed aminoacid conjugation.

PEGylation has been largely applied to ameliorate the pharmacokinetic, immunological and physicochemical properties of protein therapeutics (1). However, the therapeutic performance of the bioconjugates is strictly related to the PEG molecular weight and architecture, the extent of polymer conjugation and the conjugation site. Aimed at generating derivatives with defined structural properties and high biological activity, few single site directed conjugation strategies have been set up (1, 2). Typically, site specific bioconjugates are obtained by terminal NH2 derivatization. Alternatively, PEG can be attached to infrequent aminoacids, namely Cys, which can also be properly introduced into the protein sequence by altering the DNA sequence encoding for the protein. However, Cys is usually located inside deep hydrophobic pockets that prevent the penetration of large hydrated molecules like PEG making difficult its modification. Though PEGylation is possible under controlled protein denaturation, the unfolding/re-folding process and the insertion of a hydrophilic macromolecule into a hydrophobic pocket can induce conformational protein alteration, which may compromise dramatically its activity and stability. In order to conjugate low accessible protein aminoacids under native conditions, we prepared a new 20 kDa PEG derivative end functionalised with an hydrophobic linear chain made of 18 C. The new polymer PEG-C18-Mal [PEG-NHCO-(CH2)11-NHCO(CH2)5-Mal] was found to possess the same reactivity towards the thiol groups as the commercial PEG-maleimide. The ability to modify unexposed aminoacids was investigated by using rh-GCSF which contains a Cys17 that can not be modified by commercial PEG-maleimide or PEG-vinylsulfone. Under native conditions, the PEGylation reaction was found to proceed slowly to yield over 55% Cys conjugation in about 140 hours. The higher efficacy in Cys bioconjugation can be ascribed to a better thermodynamic interaction between PEG-C18-Mal and the hydrophobic aminoacids surrounding the protein pocket, and to extended stability of the PEG maleimide group by micelles formation. Circular dichroism analysis showed that the PEGylation did not induce significant alteration of tertiary structure of the native protein. A stability study carried out by protein incubation at 37°C showed that the polymer conjugation yielded a derivative with lower stability (aggregation half life 60 hrs) as compared to the native protein (aggregation half life higher than 100 hrs). However, rh-GCSF-PEG conjugate obtained with the new polymer under native conditions was much more stable than the counterpart obtained under denaturant conditions either with the new or the commercial PEG-maleimide (aggregation half life 30-90 min). The present study shows that end hydrophobized PEGs can be successfully used for modification of inaccessible aminoacids, which are located into deep hydrophobic pockets of the protein. The modification can be performed under physiological conditions thus avoiding protein unfolding which may be detrimental for the protein activity and stability