Protein similarity is a quantitative approach for detecting small changes in protein secondary structure by analyzing and comparing the amide I band spectra between proteins. As the amide I band is very sensitive to changes in protein secondary structure as discussed previously, the ability to measure small differences in the spectra can be a powerful tool in monitoring the biosimilarity of a protein. A number of algorithms have been proposed for this comparison, including the correlation coefficient and the area of overlap. For the purposes of this paper, the area of overlap method was used to calculate protein similarity. These results can be compared to published results using other methods to assess the sensitivity of the MMS method relative to more traditional methods such as FTIR or UV-CD.
Figure 1 shows the overlaid spectra of BSA at four concentration levels (0.1, 1.0, 10 and 200 mg/mL) as acquired by the MMS analyzer. In the middle concentration range, similarity exceeds 97% with a drop off at the high and low ends of the range.
For comparison, FTIR values found in the literature show a mean similarity of 86.37% +/- 7.98% at a single concentration of 10 mg/mL for HEWL. Using FTIR, protein similarity values at the 97% level could only be obtained at a concentration of 50 mg/mL. Thus, RedShiftBio MMS analyzer achieves better similarity with less deviation over a concentration range that far exceeds the measurement capability of FTIR, while also addressing the limitations of UV-CD at higher concentrations that may require additional workflow and dilutions.
|Sample Concentration [%]||Similarity [%]|
1 Kendrick BS, Dong AC, Allison SD, Manning MC, Carpenter JF. 1996. Quantitation of the area of overlap between second-derivative amide 1 infrared spectra to determine the structural similarity of a protein in different states. J Pharm Sci 85(2):155–158.
2 Jennifer D’Antonio, Brian M. Murphy, Mark Cornell Manning, Wasfi A. Al-Azzam. 2012. Comparability of Protein Therapeutics: Quantitative Comparison of Second-Derivative Amide I Infrared Spectra. J Pharm Sci 101(6):2025–2033.
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Application Note on Microfluidic Modulation Spectroscopy
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