In Situ Quantification of Polysorbate in Pharmaceutical Samples of Therapeutic Proteins by Hydrodynamic Profiling by NMR Spectroscopy.

Polysorbates are nonionic surfactants often used at variable levels in various formulations of protein therapeutics. Their quantification in pharmaceutical samples has posed an analytical challenge. Here we present an approach based on 1H NMR spectroscopy which can accurately estimate the concentration of polysorbate 80 (PS80) in intact pharmaceutical samples of an arbitrary formulation. The method, HAP-NMR (hydrodynamic profiling by NMR), is an extension of the protein fingerprint by line shape enhancement method (PROFILE) approach ( Poppe , L. ; Jordan , J. B. ; Lawson , K. ; Jerums , M. ; Apostol , I. ; Schnier , P. D. Anal. Chem. 2013 , 85 (20) , 9623 - 9629 ) and is based on the 1D 1H pulsed field gradient stimulated echo (PFGSTE) NMR experiment, which allows for the rectification of the 1D 1H NMR spectrum to a level suitable for a quantitative hydrodynamic analysis. Here we describe the methodology as applied to an antibody sample formulated in 9% (w/v) sucrose and with variable levels of PS80, ranging from 0.01% to 0.20% (w/v) sample concentrations. Equally important, we present evidence and propose a novel mechanism of how polysorbate stabilizes protein in pharmaceutical formulations.