Molar-mass characterization of cationic polymers for gene delivery by aqueous size-exclusion chromatography

Purpose. This study was performed to develop a reliable aqueous size-exclusion chromatography (SEC) method to obtain the absolute molar masses and distributions Of Various cationic polymers used in gene delivery. Methods. Water-soluble cationic [2-(dimethylamino) ethyl methacrylate] polymers (PDEs) with different molar masses and low polydispersities were synthesized by living polymerization and these were used to optimize the SEC conditions. Online Coupled multiangle light scattering (MALS) detection was applied to obtain the absolute molar masses. Narrow fractions of high molar mass were obtained by semipreparative SEC. Results. It was found that 0.3 M NaAc (pH 4.4) is a suitable eluent in combination with Shodex OHpak SB columns for SEC analysis of PDEs and other cationic polymers, such as poly(L-lysine) and poly(ethylene imine). The absolute molar masses of different PDEs were determined directly using SEC-MALS. A calibration curve was established using narrow PDEs. Conclusions. A reliable routine method for molar-mass characterization of cationic polymers was established. Because standards of known molar masses with narrow distributions are not commercially available for most polymers used in pharmaceutics and biotechnology, the procedure described in this work can also be applied for molar-mass characterization of other water-soluble polymers.