Fluorescence-based thermal stability screening is concentration-dependent and varies with protein size

Abstract

Proteins are used in many areas including diagnostic and therapeutical applications. Screening protein stability is an essential step for production, pharmacokinetic/pharmacodynamic studies, and determination of storage conditions. Instability of proteins can cause serious problems such as activity loss and unexpected adverse effects, so determination of sensitive and reliable methods for protein stability measurement is crucial. There are several “gold-standard” protein stability tests such as differential scanning calorimetry (DSC), but they are usually not suitable for high-throughput settings and consume large amounts of proteins. Instead, more high-throughput methods such as fluorescent-based assays can be used and validated to make stability screening process more straight-forward, easier, and lower-cost. Here, two methods were systemically compared to see whether their measurements depended on protein sizes. DSC and Sypro Orange dye-based fluorescent assay were compared for various proteins with different sizes and quaternary structures. This is the first systemic comparison of these two methods for thermal stability testing for different ranges of proteins in the literature. It was shown that protein melting temperature (Tm) measured by fluorescent assay highly depends on protein concentration and protein size. Larger proteins with multi-domain structures such as monoclonal antibodies gave more deviated and lower than expected Tms compared to small proteins. It has been concluded that fluorescent-based thermal stability assays are more suitable for smaller proteins, but protein concentrations used are still needed to be optimized in their settings for more reliable results.

Keywords

• Differential scanning calorimetry
• fluorescent dye
• thermal melt
• thermal stability
• protein stability

References

1. Acharya, P., & Rao, N. M. (2003). Stability studies on a lipase from Bacillus subtilis in guanidinium chloride. Journal of Protein Chemistry, 22(1), 51-60.
2. Akbarian, M., & Chen, S. H. (2022). Instability challenges and stabilization strategies of pharmaceutical proteins. Pharmaceutics, 14(11).
3. Al-Ghobashy, M. A., Mostafa, M. M., Abed, H. S., Fathalla, F. A., & Salem, M. Y. (2017). Correlation between dynamic light scattering and size exclusion high-performance liquid chromatography for monitoring the effect of ph on stability of biopharmaceuticals. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1060, 1-9.
4. Anraku, M., Yamasaki, K., Maruyama, T., Kragh-Hansen, U., & Otagiri, M. (2001). Effect of oxidative stress on the structure and function of human serum albumin. Pharmaceutical Research, 18(5), 632-639.
5. Araya, C. L., Fowler, D. M., Chen, W., Muniez, I., Kelly, J. W., & Fields, S. (2012). A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proceedings of the National Academy of Sciences of the United States of America, 109(42), 16858-16863.
6. Boll, B., Bessa, J., Folzer, E., Rios Quiroz, A., Schmidt, R., Bulau, P., Finkler, C., Mahler, H. C., Huwyler, J., Iglesias, A., & Koulov, A. V. (2017). Extensive chemical modifications in the primary protein structure of IgG1 subvisible particles are necessary for breaking immune tolerance. Molecular Pharmaceutics, 14(4), 1292-1299.
7. Cardamone, M., & Puri, N. K. (1992). Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochemical Journal, 282(Pt 2), 589-593.
8. Elgendy, M. (2017). Assessment of modulation of protein stability using pulse-chase method. Bio-Protocol, 7(16), e2443.

9. Engrola, F. S. S., Paquete-Ferreira, J., Santos-Silva, T., Correia, M. A. S., Leisico, F., & Santos, M. F. A. (2023). Screening of buffers and additives for protein stabilization by thermal shift assay: a practical approach. In: Sousa Â., Passarinha L. (eds) Methods in Molecular Biology (Vol. 2652, pp. 199-213). Humana.
10. Fiedler, S., Cole, L., & Keller, S. (2013). Automated circular dichroism spectroscopy for medium-throughput analysis of protein conformation. Analytical Chemistry, 85(3), 1868-1872.
11. Gill, P., Moghadam, T. T., & Ranjbar, B. (2010). Differential scanning calorimetry techniques: applications in biology and nanoscience. Journal of Biomolecular Techniques, 21(4), 167-193.
12. Gromiha, M. M. (2010). Protein folding, stability and interactions. Current Protein & Peptide Science, 11(7), 497.
13. Groves, M. R., Muller, I. B., Kreplin, X., & Muller-Dieckmann, J. (2007). A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye. Acta Crystallographica Section D: Biological Crystallography, 63(Pt 4), 526-535.
14. Hawe, A., Sutter, M., & Jiskoot, W. (2008). Extrinsic fluorescent dyes as tools for protein characterization. Pharmaceutical Research, 25(7), 1487-1499.
15. Huynh, K., & Partch, C. L. (2015). Analysis of protein stability and ligand interactions by thermal shift assay. Current Protocols in Protein Science, 79(1), 28-29.
16. Jana, K., Mehra, R., Dehury, B., Blundell, T. L., & Kepp, K. P. (2020). Common mechanism of thermostability in small alpha- and beta-proteins studied by molecular dynamics. Proteins: Structure, Function, and Bioinformatics, 88(9), 1233-1250.
17. Jung, F., Frey, K., Zimmer, D., & Muhlhaus, T. (2023). DeepSTABp: A deep learning approach for the prediction of thermal protein stability. International Journal of Molecular Sciences, 24(8), 7444.
18. Kalyoncu, S., Yilmaz, S., Kuyucu, A. Z., Sayili, D., Mert, O., Soyturk, H., ... & Inan, M. (2023). Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris. Scientific Reports, 13(1), 5224.
19. Kazlauskas, E., Petrauskas, V., Paketuryte, V., & Matulis, D. (2021). Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein-ligand binding and protein stability. European Biophysics Journal, 50(3-4), 373-379.
20. Kirley, T. L., & Norman, A. B. (2022). Critical evaluation of fluorescent dyes to evaluate the stability and ligand binding properties of an anti-cocaine mAb, h2E2. Journal of Immunological Methods, 508, 113323.
21. Kopra, K., Valtonen, S., Mahran, R., Kapp, J. N., Hassan, N., Gillette, W., ... & Härmä, H. (2022). Thermal shift assay for small GTPase stability screening: evaluation and suitability. International Journal of Molecular Sciences, 23(13), 7095.
22. Lavinder, J. J., Hari, S. B., Sullivan, B. J., & Magliery, T. J. (2009). High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. Journal of the American Chemical Society, 131(11), 3794-3795.
23. Layton, C. J., & Hellinga, H. W. (2011). Quantitation of protein-protein interactions by thermal stability shift analysis. Protein Science, 20(8), 1439-1450.
24. Manning, M. C., Patel, K., & Borchardt, R. T. (1989). Stability of protein pharmaceuticals. Pharmaceutical Research, 6(11), 903-918.
25. Miotto, M., Olimpieri, P. P., Di Rienzo, L., Ambrosetti, F., Corsi, P., Lepore, R., Tartaglia, G. G., & Milanetti, E. (2019). Insights on protein thermal stability: a graph representation of molecular interactions. Bioinformatics, 35(15), 2569-2577.
26. Nemergut, M., Zoldak, G., Schaefer, J. V., Kast, F., Miskovsky, P., Pluckthun, A., & Sedlak, E. (2017). Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering. Protein Science, 26(11), 2229-2239.
27. Oh, J., Durai, P., Kannan, P., Park, J., Yeon, Y. J., Lee, W. K., Park, K., & Seo, M. H. (2023). Domain-wise dissection of thermal stability enhancement in multidomain proteins. International Journal of Biological Macromolecules, 237, 124141.
28. Puglisi, R., Brylski, O., Alfano, C., Martin, S. R., Pastore, A., & Temussi, P. A. (2020). Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy. Communications Chemistry, 3(1), 100.
29. Redhead, M., Satchell, R., McCarthy, C., Pollack, S., & Unitt, J. (2017). Thermal shift as an entropy-driven effect. Biochemistry, 56(47), 6187-6199.
30. Rufer, A. C., & Hennig, M. (2020). Biophysical assessment of target protein quality in structure-based drug discovery. In: Renaud J. P. (ed) Structural Biology in Drug Discovery: Methods, Techniques, and Practices (pp. 143-164). Humana.
31. Schuster, J., Koulov, A., Mahler, H. C., Detampel, P., Huwyler, J., Singh, S., & Mathaes, R. (2020). In vivo stability of therapeutic proteins. Pharmaceutical Research, 37(2), 23.
32. Stourac, J., Dubrava, J., Musil, M., Horackova, J., Damborsky, J., Mazurenko, S., & Bednar, D. (2021). FireProtDB: database of manually curated protein stability data. Nucleic Acids Research, 49(D1), D319-D324.
33. Tresnak, D. T., & Hackel, B. J. (2023). Deep antimicrobial activity and stability analysis inform lysin sequence-function mapping. ACS Synthetic Biology, 12(1), 249-264.
34. Vetri, V., Canale, C., Relini, A., Librizzi, F., Militello, V., Gliozzi, A., & Leone, M. (2007). Amyloid fibrils formation and amorphous aggregation in concanavalin A. Biophysical Chemistry, 125(1), 184-190.
35. Vuorinen, E., Valtonen, S., Eskonen, V., Kariniemi, T., Jakovleva, J., Kopra, K., & Harma, H. (2020). Sensitive label-free thermal stability assay for protein denaturation and protein-ligand interaction studies. Analytical Chemistry, 92(5), 3512-3516.
36. Warrender, A. K., Pan, J., Pudney, C. R., Arcus, V. L., & Kelton, W. (2023). Constant domain polymorphisms influence monoclonal antibody stability and dynamics. Protein Science, 32(3), e4589.