ABSTRACT
Polysorbates are complex mixtures of over a thousand components with a wide range of hydrophobicity. This paper describes a methodology for characterization of heterogeneity and stability of polysorbates in therapeutic protein formulation. The method utilizes on-line coupling of a hydrophobic interaction chromatography (HIC) column with reverse phase liquid chromatography (RPLC) and charged aerosol detection (CAD)/mass spectrometer (MS). The addition of a low concentration of formic acid and organic solvent (e.g. 0.05% formic acid and 3% acetonitrile) in the mobile phase enables the use of the HIC column to separate small molecule excipients (including major components of polysorbates) and the large protein molecules by a mixed mechanism of size exclusion chromatography (SEC) and RPLC. The protein and the charged excipients, which elute early from the HIC column, are directed by a switching valve to waste. The polysorbates and other neutral excipients, which elute later from the HIC column, are directed to the RPLC column for further separation. The separated polysorbate components are detected by CAD, and characterized by MS. This method has been used to characterize the degradation of polysorbate 80 (PS80) in placebo and protein formulations. Our studies have revealed different degradation behavior of PS80 in placebo vs. protein formulation.
