Versatile and Rapid Digestion Protocols for Biopharmaceutical Characterization Using RapiZyme™ Trypsin

Importance of the Topic

Peptide mapping is a cornerstone assay for biopharmaceutical characterization, enabling confirmation of protein identity and the assessment of site-specific modifications. Traditional trypsin digestions can be lengthy and prone to artifacts such as missed cleavages, non-specific cleavages, autolysis peaks, and artificial modifications. Rapid and reliable digestion methods reduce turnaround times and simplify data analysis, supporting robust quality control in biotherapeutic development.

Objectives and Study Overview

This application note evaluates RapiZyme trypsin, a homogeneously methylated recombinant porcine enzyme with enhanced thermal stability and autolysis resistance. The study compares RapiZyme to a leading MS-grade trypsin under four digestion protocols: a traditional 1:20 enzyme:protein (E:P) ratio, a rapid 30-minute high-ratio (1:5) digestion, an optimized overnight 1:100 E:P protocol, and a one-pot workflow that omits desalting.

Methodology

Protein samples (Remicade mAb) were denatured in guanidine hydrochloride, reduced with DTT, alkylated with iodoacetamide, and buffer-exchanged into Tris or histidine buffers at pH 7.5 or 6.5. Digestions were performed at various E:P ratios, incubation times (30 min to overnight), temperatures (ambient to 37 °C), and in some cases in the presence of guanidine without prior desalting. Acid quenching and dilution prepared samples for LC-MS analysis.

Instrumentation

• LC System: ACQUITY UPLC I-Class PLUS
• Detector: ACQUITY TUV and ACQUITY RDa
• Column: ACQUITY Premier Peptide CSH C18, 130 Å, 1.7 µm, 2.1 × 100 mm
• Vials: QuanRecovery with MaxPeak HPS
• Data Software: UNIFI v3.0 with waters_connect v2.1

Key Results and Discussion

• Traditional Digestion (1:20 E:P, 3 h at 37 °C): RapiZyme matched competitor performance, achieving >93 % sequence coverage and minimal artifacts.
• Accelerated Digestion (1:20 E:P, 1 h): Equivalent peptide maps obtained in one hour, reducing sample prep time by two-thirds.
• Rapid High-Ratio Digestion (1:5 E:P, 30 min): RapiZyme produced clean peptide maps with <1 % missed cleavages and <0.1 % autolysis, whereas the competitor showed significant autolysis peaks.
• Overnight Low-Ratio Protocol (1:100 E:P, pH 6.5, ambient): Complete digestions with <1 % missed or non-specific cleavages and minimal deamidation, outperforming higher pH/temperature conditions.
• One-Pot Workflow (1:5 E:P, 2 h, 0.6 M guanidine): RapiZyme achieved 96 % of total signal from expected peptides, demonstrating resilience to denaturant and enabling desalting-free workflows.

Benefits and Practical Applications

• High autolysis resistance enables the use of elevated enzyme concentrations and rapid digestions.
• Clean baselines reduce unmatched peaks, accelerating data interpretation.
• Versatility across traditional, rapid, overnight, and one-pot protocols supports diverse lab workflows.
• Reproducible batch-to-batch performance ensures method robustness.

Future Trends and Opportunities

Integration of RapiZyme trypsin into multi-attribute monitoring (MAM) workflows can further streamline quality control in biomanufacturing. Future developments may include automated high-throughput platforms, continuous-flow digestion systems, and expanded one-pot chemistries that enhance sample throughput while maintaining data quality.

Conclusion

RapiZyme trypsin delivers robust, efficient, and artifact-minimized peptide mapping across a range of digestion protocols. Its enhanced autolysis resistance and high activity support rapid workflows and simplified sample preparation, making it a versatile tool for biopharmaceutical characterization and quality control.

References

1. Ranbaduge R, Yu YQ. A Streamlined Compliant Ready Workflow for Peptide-Based Multi-Attribute Method (MAM). Waters Application Note, 720007094. 2020 Dec.
2. Mouchahoir T, Schiel JE. Development of an LC-MS/MS peptide mapping protocol for the NIST mAb. Anal Bioanal Chem. 2018;410:2111–2126.
3. Ren D et al. An Improved Trypsin Digestion Method Minimizes Digestion-Induced Modifications on Proteins. Anal Biochem. 2009;392:12–21.
4. Millian-Martin S et al. Inter-laboratory Study of an Optimized Peptide Mapping Workflow Using Automated Trypsin Digestion for Monitoring Monoclonal Antibody Product Quality Attributes. Anal Bioanal Chem. 2020;412:6833–6848.
5. Evans AR et al. ID-MAM: A Validated Identity and Multi-Attribute Monitoring Method for Commercial Release and Stability Testing of a Bispecific Antibody. Anal Chem. 2021;93(26):9166–9173.
6. Song YE et al. Automated Mass Spectrometry Multi-Attribute Method Analyses for Process Development and Characterization of mAbs. J Chrom B. 2021;1166:122540.
7. Hao Z et al. Multi-Attribute Method Performance Profile for Quality Control of Monoclonal Antibody Therapeutics. J Pharm Biomed Anal. 2021;205:114330.
8. Cytiva. Instruction Manual for PD-10 Desalting Columns. 2022.