Quantitation and characterization of process impurities and extractables in protein-containing solutions using proton NMR as a general tool.

The ability to detect and quantitate a variety of components in solution has become increasingly important in carrying out efficient and rigorous validation studies for biopharmaceutical manufacturing processes. Here, we demonstrate the general applicability of NMR spectroscopy for the identification and quantitation of leachables and other impurities in protein-based drugs, at low levels previously unattainable in protein-containing solutions. With improved NMR technology (i.e., CryoProbes) and the application of a Carr-Purcell-Meiboom-Gill pulse sequence (CPMG) to attenuate protein signals, we have been able to use NMR to quantify impurities in a protein-based biopharmaceutical product at ~1 μg mL(-1) . The data indicate that NMR spectra can be used to quantitate a range of impurities, from small molecule components to higher molecular weight leachables, without removing protein from solution. Furthermore, quantitation of impurities by NMR is reliable and accurate enough for biopharmaceutical process validation, even for high molecular weight extractables whose structures are not precisely known.