(T1230-09-60) Improved Biopharmaceutical Peptide Mapping Workflows Using a Novel Autolysis-Resistant Trypsin Enzyme

Purpose: Peptide mapping analysis provides a wealth of information about a protein or antibody therapeutic. Through digestion of protein chains down to the peptide level, it is possible to establish identity via sequence coverage and assess post-translational modifications (PTMs) at specific amino acid sites. This assay is increasingly implemented for multi-attribute monitoring (MAM)1-2 and product release testing, which means that it must be reliable and robust. The data generated from peptide mapping is already relatively complex when one considers only the expected digestion components. It is unnecessarily complicated by missed cleavages (under-digestion), non-specific cleavages (over-digestion), and autolysis peaks (self-digestion of the enzyme). This study demonstrates the utility of a new completely and homogenously methylated porcine autolysis-resistant MS-grade trypsin enzyme in various desired protein digestion protocols.

Methods: Samples of infliximab were denatured, reduced, and alkylated, then either buffer exchanged or diluted in digestion buffer. Samples were digested with both the novel trypsin and another prominent MS-grade trypsin in various enzyme:protein ratios, incubation times, temperatures, and pH conditions. Samples were acidified and analyzed via RPLC-MS using a C18 column (1.7μm, 2.1 x 100 mm) maintained at 60 oC for a 50-minute linear gradient of 1-35% B (80-minute total time). Mobile phase A (MPA) was 0.1% formic acid in water and mobile phase B (MPB) was 0.1% formic acid in acetonitrile. DIA MS detection (MSE) was performed via TOF and QTOF mass spectrometers. Data analysis was carried out via an app-based informatics platform.

Results: Many traditional trypsin digestion protocols1-8 utilize some version of denaturation, reduction, alkylation, and desalting prior to trypsin digestion. The digestion is commonly performed at an enzyme:protein (E:P) ratio of 1:20-1:25 (w/w) for up to 4 hours at elevated temperature. The aim of this study was to evaluate the performance of the novel trypsin compared to another industry-leading MS grade trypsin. Parameters indicating a successful digestion include > 90% sequence coverage and low levels of the following: missed cleavage, non-specific cleavage, trypsin autolysis, unmatched peaks, and artificial PTMs. The initial comparison was performed on a denatured, reduced, alkylated, and desalted sample of infliximab at an E:P ratio of 1:20 for 3 hours at 37 oC. Comparable performance was observed—each gave >93% sequence coverage and >95% of the integrated TIC area is comprised of infliximab peptides with expected tryptic cleavage. The same conditions were also used for a shortened digestion time of 1 hour, and it was observed that equivalent (and repeatable) peptide profiles were achieved for infliximab using novel trypsin. Traditionally, the longest step in peptide mapping protocols is enzyme digestion. To save time and minimize artificial PTMs, many analysts aim to shorten digestion time. Since the novel trypsin is resistant to autolysis, it was tested at a higher E:P ratio of 1:5 with 30-minute incubation time. A significant difference in the quality of resulting data is observed. The near-complete digestion of infliximab using the novel trypsin gave < 4% of undesired peaks in the TIC, while the competitor showed ~ 12%. Finally, many scientists prefer a longer overnight digestion. However, at optimal conditions for trypsin activity (pH 7-8 and elevated temperature), a prolonged digestion yields artificial PTMs such as deamidation. We also investigated a 1:100 E:P ratio digestion performed overnight at ambient temperature and mildly acidic pH, with favorable results.

Conclusion: RapiZyme Trypsin, a novel homogenously methylated porcine trypsin, has proven utility to generated reproducible high quality peptide mapping data, due to its high activity and autolysis resistance.
• RapiZyme Trypsin can be used flexibly in a variety of protocols, from accelerated digestions, overnight and simple 1-pot digestion

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