Therapeutic monoclonal antibodies are the fastest-growing class of biological agents generating yearly sales of approx. USD 220 billion in 2023 which is expected to triple over the next decade [1]. With the growing market significance of therapeutic antibodies, analytical methods allowing reproducible and metrologically traceable quantification need to be developed. The serin protease trypsin has become the gold standard enzyme for digesting intact protein into peptides for LC-MS analysis. However, many protocols exist that often lead to different results depending on the digestion procedure [2]. In particular, the type and amount of detergents added during sample preparation for protein unfolding before digestion is known to create undesirable structure modifications of proteins (e.g. carbamylation) and significant bias in measurement results by inhibiting enzyme activity [3]. The enzymes themselves can also have an influence, whereby immobilized variants are increasingly being used to improve their stability, minimize self-digestion and enable a higher substrate-enzyme ratio [4].
Here, we studied the implementation of a thermostable[5] and surface-functionalized trypsin-variant for improved antibody digestion. Applying this enzyme should allow digestion at elevated temperatures where the protein is naturally unfolding, thereby increasing enzyme accessibility and reducing the impact of detergents on the overall degree of unfolding. For this purpose, the inactive proenzym was recombinantly produced in inclusion bodies of E. coli and successfully folded back into functional conformation via multi-stage dialysis using a cysteine-cystine redox system. Only the enzyme showed activity after cleavage of the propeptide, which strongly reduces the possibility of self-digestion during production and subsequent immobilization steps. To further enhance enzyme stability, the enzyme was coupled covalently onto magnetic particles and investigated with regard to changes in activity. Compared to wild-type and a non-immobilized enzyme, the surface-functionalized thermostable variant showed increased activity at temperatures above 80°C. Thus, this novel type of trypsin particles promises to not only improve antibody digestion at high temperatures, but also to prevent self-digestion and enable the separation of trypsin from target peptides in LC–MS analysis.
Ref.:
[1] Monoclonal Antibodies Market Outlook (2023 to 2033). https://is.gd/d9oVOT.
[2] Burkhart, J. M., et al. J Proteomics 75.4 (2012): 1454-1462. DOI: 10.1016/j.jprot.2011.11.016.
[3] Proc, J. L., et al. J Proteome Res 9.10 (2010): 5422-5437. DOI: 10.1021/pr100656u.
[4] Naldi, M. et al. J Pharm Biomed Anal, (2018): 160, 222-237. DOI: 10.1016/j.jpba.2018.07.051.
[5] Xiao, T., et al. Process Biochemistry 130 (2023): 227-235. DOI: 10.1016/j.procbio.2023.04.024.