Deciphering the Biophysical Effects of Oxidizing Sulfur-Containing Amino Acids in Interferon-beta-1a using MS and HDX-MS
Damian J. Houde1°, George M. Bou-Assaf1*, Steven A. Berkowitz1#
1 Technical Development, Biogen, 225 Binney St, Cambridge, MA 02142
° Current affiliation and address: Codiak Biosciences, 500 Technology Square, Cambridge, MA 02139
# Currently independent consultant
* Corresponding author
Running title: Characterization of biopharmaceuticals by MS
Address reprint requests to: George M. Bou-Assaf, 225 Binney St, Cambridge, MA 02142 – Ph: (617) 914-5399 – Email:
SUPPLEMENTAL INFORMATION
Supplemental Figure 1
HDX-MS butterfly (A & C) and difference (B & D) plots for oxidized (A & B) [1] and NEM-alkylated (C & D) [2] INFβ-1a relative to the control at pH 7.2. Based on the conclusions from our present work and earlier work [3], the major conformational changes indicated by both data sets can be attributed to the modification of the free sulfhydryl group of Cys17 in both situations. At first glance, the changes in deuterium uptake are fairly comparable in terms of the peptides that exhibit a difference. The extent of those differences are somewhat more pronounced in the oxidized sample compared to the NEM-alkylated one. Nevertheless, given this similarity, our previous work showed that the biological function of the oxidized sample is fairly similar to that of the control whereas that of the NEM-alkylated sample is significantly reduced compared to the control [3]. On closer evaluation of the HDX-MS data, the alkylated INFβ-1a does show a subtle, but significant different in comparison to the control in the area of peptides #6 & 7 that is absent in the oxidized INFβ-1a, while the latter shows a significant difference in HDX-MS data relative to the control in the area of peptide #26, which does not exist when the alkylated and control INFβ-1a samples are compared. With this detailed analysis, we conclude that the differences observed in peptides #6 & 7 of alkylated INFβ-1a could be the key structural change responsible for the difference in biological activity relative to either the control or oxidized INFβ-1a which we observed in our previous work [3]. On the other hand, the observed difference in peptide #26 between the oxidized and the control INFβ-1a has no impact on this particular biological function.
References:
1. Houde, D., Berkowitz, S. A., Engen, J. R.: The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies. J. Pharm. Sci. 100, 2071-2086 (2011)
2. Berkowitz, S. A.: Analytical characterization: Structural assessment of biosimilarity. In: Endrenyi, L., Declerck, P. J., Chow, S.-C. (eds). Biosimilar Drug Product Development, CRC Press, Boca Raton, FL (2017)
3. Bobst, C. E., Abzalimov, R. R., Houde, D., Kloczewiak, M., Mhatre, R., Berkowitz, S. A., Kaltashov, I. A.: Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches. Anal. Chem. 80, 7473-7481 (2008)
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