Investigations of PEGylated Recombinant Adenovirus, Using Fluorescein-Labeled Polyethylene Glycol

Abstract

As with certain successful protein drug treatments, the attachment of polyethylene glycol (PEG) molecules to recombinant adenovirus (rAd) can augment their therapeutic potential. Unlike these proteins, the rAd particle has thousands of target sites for PEG conjugation. The reliable measurement of the average number of PEG molecules attached to the virion, or the degree of PEGylation (DP), is crucial not only for the characterization of PEGylated virus but also for optimization of the PEGylation reaction. Using a fluorescein-labeled PEG-SPA linker (SPA, succinimidyl ester of PEG propionic acid) with a 5-kDa linear PEG moiety, multiple preparations of fluoro-PEG-rAds were produced under various reaction conditions, purified, and analyzed by size-exclusion high-performance liquid chromatography (HPLC) with fluorescence quantification of the virus peak. The DP was strongly dependent on the percent linker concentration in the reaction. For example, under one set of conditions, fluoro-PEG-rAd samples prepared at 1.3, 2.5, 5.0, 7.4, and 10.0% linker concentration had DPs of approximately 540, 1,000, 1,590, 1,990, and 2,170, respectively. The fluoro-PEG-rAds were compared with a set of nonfluorescent PEG-rAds. Analytical ultracentrifugation in CsCl density gradients showed distinct peaks at decreased buoyant density corresponding to the increased DP of the rAd samples; sodium dodecyl sulfate-polyacrylamide gel electrophoresis/scanning densitometry showed decreased hexon monomer and penton base. Both techniques were used to estimate the DP of nonfluorescent PEG-rAds versus fluoro-PEG-rAds, and anion-exchange HPLC revealed the different surface chemistries of the two vector types. In summary, these studies should provide investigators with the ability to reproducibly prepare and characterize PEGylated rAds or other large viral or nonviral particles for further in vivo studies.