Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

Improved biopharmaceutical peptide mapping workflows using a novel autolysis-resistant trypsin enzyme (#163)

Samantha Ippoliti 1 , Nick Zampa 1 , Caryn Hepburn 2 , Heather Patsiouras 2 , Sviat Eliseenko 2 , Ying Qing Yu 1 , Matthew A Lauber 1
  1. Waters Corporation, Milford, Massachusetts, USA
  2. Waters Australia, Rydalmere, NSW, Australia

Peptide mapping provides a wealth of information about a protein or antibody therapeutic. Through digestion of protein chains down to 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 trypsin enzyme in various desired protein digestion protocols.

Samples were digested with both RapiZymeTM 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. Data independent acquisition MS detection (MSE) was performed via TOF and QTOF mass spectrometers. 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 Enzyme:Protein (E:P) ratio of 1:20 for 3 hours at 37 oC. Comparable performance was observed. 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 RapiZyme 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 RapiZyme 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 RapiZyme trypsin gave <4% of undesired peaks in the TIC, while the competitor enzyme 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.

 

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