Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

Polysorbate 80 (PS80) is a widely used surfactant in therapeutic monoclonal antibodies (mAbs) because it prevents mAb aggregation and stabilizes the drug product. Various analytical methods have been published and are available for measuring PS80, each with its own limitations. In this study, an HPLC method employing an evaporative light scattering detector (ELSD) was developed for multiproduct analysis of PS80. Method validation was conducted across multiple laboratories using different types of instruments and ELSD detectors. The method was validated using various immunoglobulin G (IgG) mAb molecules and different sources of PS80, with the results showing that it can accurately measure PS80 concentrations ranging from 0.05 to 0.5 mg/mL. Robustness tests demonstrated that the method tolerates up to 160 mg/mL mAb interference, through either sample dilution or protein precipitation. This method can be recommended as a release/quality control method for PS80 concentration determination in therapeutic mAbs.