IMPROVED BIOPHARMACEUTICAL PEPTIDE MAPPING WORKFLOWS USING A NOVEL AUTOLYSIS-RESISTANT TRYPSIN ENZYME

Significance of Peptide Mapping

Peptide mapping is a cornerstone assay in biopharmaceutical analysis, enabling detailed confirmation of protein identity through sequence coverage and monitoring of post-translational modifications. As multi-attribute methods gain traction for quality control and product release, robust and reproducible digestion workflows are essential to minimize artifacts such as missed cleavages and enzyme autolysis.

Study Objectives and Overview

This study evaluates a novel, autolysis-resistant trypsin variant (RapiZyme™ Trypsin) against an established MS-grade trypsin across four digestion protocols. Protocols include an accelerated 30-minute high enzyme-to-protein (E:P) ratio digestion, a traditional 3-hour digestion, an overnight low-ratio digestion, and a one-pot no-desalting protocol.

Methodology and Instrumentation

Denaturation and reduction were performed with guanidine hydrochloride and dithiothreitol, followed by alkylation with iodoacetamide. Three desalting approaches or a single-pot workflow were explored before incubation under varying E:P ratios, temperatures, and durations. Digests were quenched and analyzed by LC-MS using an ACQUITY™ Premier Peptide CSH C18 column (60 °C, 1–35 % B gradient) coupled to a Xevo™ G3 QTof MS in MSE mode. Data processing utilized the UNIFI™ peptide mapping workflow.

Main Results and Discussion

• Sequence coverage exceeded 95 % in all conditions, matching the standard protocol.
• Accelerated digestion with RapiZyme Trypsin at 1:5 E:P for 30 min yielded ~94 % of total ion current for expected peptides versus 88 % for the competitor.
• RapiZyme Trypsin showed 2–3 % fewer missed cleavages across protocols and undetectable autolysis species, even at high enzyme loadings.
• Chromatographic baselines remained clean with minimal unmatched peaks, simplifying data interpretation.

Benefits and Practical Applications

Autolysis resistance permits rapid, high-ratio digestions without elevated temperature or extended incubation, enhancing throughput for routine peptide mapping and multi-attribute monitoring. The versatility across protocols supports diverse laboratory workflows, from accelerated tests to one-pot methods, while maintaining high-quality, reproducible data.

Future Trends and Opportunities

Advancements in protease engineering and automation will further streamline peptide mapping. Integration into comprehensive MAM platforms and workflows promises real-time quality monitoring. Emerging trends include multiplexed digestion strategies and tailored enzyme formulations for challenging targets.

Conclusion

RapiZyme™ Trypsin delivers robust performance in peptide mapping, combining high enzymatic activity with exceptional autolysis resistance. Its application across flexible workflows offers improved data quality, reduced analysis times, and streamlined sample preparation for biopharmaceutical characterization.

References

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