Determining Spectroscopic Quantitation Limits for Misfolded Structures

Abstract Protein secondary structures are frequently assessed using infrared and circular dichroism spectroscopies during drug development (e.g. during product comparability and/or biosimilarity studies, reference standard characterization, etc.) However, there is little information on the lower limits of quantitation of structural misfolds and/or impurities for these methods. A model system employing a monoclonal antibody reference material was spiked at various levels with a protein that had a significantly different secondary structure to represent the presence of a stable and discreet structural misfold. The ability of circular dichroism (CD) and two infrared spectroscopic techniques, transmission Fourier transform infrared (FTIR) spectroscopy and microfluidic modulation spectroscopy (MMS), along with various spectral comparison algorithms, were assessed for their ability to detect the presence and quantify the amount of the misfolded structure.