Spectroscopic techniques such as circular dichroism (CD) and Fourier transform infrared spectroscopies (FTIR) are widely used to characterize the higher order structure (HOS) of proteins. Additionally, the use of nuclear magnetic resonance (NMR) �fingerprint spectra� has become a viable option to compare samples. Though the characterization of the HOS is important, the sensitivity of these techniques must be evaluated for comparability testing especially of large proteins, e.g. monoclonal antibodies.We assessed the relative sensitivity of CD, FTIR and NMR spectroscopies to other conventional analytical methods such as size-exclusion and ion-exchange chromatographic methods using a degraded panel of samples of monoclonal antibody. For FTIR, we found that samples with dramatic changes in the SEC or IEC chromatograms did not show appreciable shifts in the Amide I band of the FTIR spectra. Similarly, CD signal changes were largely within the instrument variability. As a comparison, one-dimensional 1H-NMR �fingerprinting� was also assessed.The spectra of CD, FTIR and NMR techniques provide information on the sample ensemble, which must be considered in comparability studies wherein degradant concentrations can be relatively small and signal changes are potentially within assay variability. With this in mind, we experimentally quantified the sensitivity of CD using an inline 2-cell cuvette experiment design (Cuvette #1: native condition, Cuvette #2: denaturing condition) and compared these changes to the assay variability. The protein sample required at least a 5% and 15% random coil component to distinguish a signal change in the Far- and Near-UV signal, respectively, compared to assay variability. This suggests that a broad panel of chromatographic assays may be more appropriate than relatively insensitive assays such as CD or FTIR to determine comparability of mAb samples. Additionally, the values and utilities of 1-D NMR fingerprinting method will be presented and discussed.

Assessment of higher order structure techniques (CD, FTIR and NMR) for characterization and comparability of monoclonal antibodies

248th National Meeting (2014)

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