Proteins are comprised of chains of amino acids which, in specific order, dictate structure and function as the primary structure. Aggregation is most notably associated with β-sheet formation, a structure that is present in protein biomolecules in varying amounts. Other structures that contribute to the overall secondary structure, folding, function and activity of proteins include alpha-helices, turns, and unordered regions.
As demonstrated above, the formation of intermolecular β-sheet is key to irreversible protein aggregation. However, the attention that researchers in the biopharmaceutical space pay to protein secondary structure and intermolecular β-sheet does not match their importance. This is primarily due to the limitations of traditional tools, especially their low sensitivities. That is to say, no meaningful conclusion can be drawn unless there is a very large change in protein secondary structure that goes beyond the system error.
KBI has published a paper to compare the Limit of Quantification (LOQ) of traditional Fourier-transform infrared (FTIR) spectroscopy, Circular dichroism (CD) and Microfluidic Modulation Spectroscopy (MMS) by Brent S. Kendrick et al. LOQ is defined as the smallest amount or the lowest concentration of a substance that is possible to be determined by means of a given analytical procedure with the established accuracy, precision, and uncertainty. MMS is 30 times more sensitive than traditional FTIR and 5 times more sensitive than CD.
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Determining Spectroscopic Quantitation Limits for Misfolded Structures
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Abstract
“Protein secondary structures are frequently assessed using infrared and circular dichroism spectroscopies during drug development (e.g., during product comparability and biosimilarity studies, reference standard characterization, etc.) However, there is little information on the lower limits of quantitation of structural misfolds and impurities for these methods. A model system using 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, transmission Fourier transform infrared spectroscopy and microfluidic modulation spectroscopy, along with various spectral comparison algorithms, were assessed for their ability to detect the presence and quantify the amount of the misfolded structure.”