One Pot, Mildly Acidic Digestion Protocols for Peptide Mapping Using Lys-C and RapiZyme™ Trypsin

Significance of the Topic

Peptide mapping of therapeutic monoclonal antibodies is critical for confirming their primary structure and monitoring post-translational modifications that define safety and efficacy. Conventional digestion workflows often introduce artifacts such as deamidation and oxidation under alkaline conditions, complicating data interpretation and quality assessment.

Objectives and Study Overview

This study evaluates a streamlined one-pot digestion protocol combining RapiZyme Trypsin with MS-grade Lys-C at mildly acidic pH to reduce method-induced modifications, eliminate desalting steps, and achieve high digestion efficiency for the NISTmAb reference material.

Methodology and Instrumentation

The sample preparation involved denaturation and reduction of NISTmAb with guanidine hydrochloride and TCEP, alkylation with iodoacetamide, followed by ten-fold dilution into histidine buffer at pH 5.5, 6.0, or 6.5. RapiZyme Trypsin and Lys-C were added at enzyme:protein ratios of 1:5 and 1:50, respectively. Digestions were carried out at 37 °C for 0.5, 1, or 3 hours and quenched with 1% formic acid.

• LC–MS platform: ACQUITY Premier UPLC with CSH C18 column (2.1×100 mm, 1.7 µm) at 60 °C and Xevo G2-XS QTof in ESI+ mode.
• Detection: UV (214 nm) and high-resolution MS (100–4000 m/z).

Main Results and Discussion

Expected peptide cleavages increased with both higher pH and longer digestion times, with a 3 h digest at pH 6.5 matching the benchmark at pH 7.5, 0.5 h. Missed cleavages declined under the same conditions. Non-specific cleavages remained below 2.5% across all conditions. Unidentified species, likely peptides with mixed cleavages, diminished with increased pH and time. Method-induced deamidation (PENNYK peptide) and oxidation (MISR peptide) were consistently lower at acidic pH than the conventional workflow. Addition of Lys-C enhanced digestion completeness but modestly increased low-level autolysis species, as confirmed by blank digests under optimal conditions.

Benefits and Practical Applications

This one-pot, mildly acidic protocol simplifies sample preparation by removing desalting, reduces artifactual modifications, and delivers high-quality peptide maps suitable for routine QA/QC and biopharmaceutical characterization.

Future Trends and Applications

• Integration into automated, high-throughput platforms for biologics analysis.
• Extension to targeted peptide mapping workflows and other protease combinations.
• Further optimization of pH and incubation parameters to balance completeness and minimal autolysis.

Conclusion

Combining RapiZyme Trypsin with Lys-C at mildly acidic pH in a one-pot workflow offers an efficient, artifact-minimizing approach for mAb peptide mapping. The method streamlines preparation, maintains enzyme integrity, and supports robust assessment of critical quality attributes.

References

1. Chelius D, Rehder DS, Bondarenko PV. Anal Chem. 2005;77:6004.
2. Cao M et al. J Pharm Sci. 2019;108:3540–3549.
3. Ippoliti S, Zampa N, Yu YQ, Lauber MA. Waters Application Note 720007840EN. 2023.
4. Hanna CM et al. Waters Application Note 720008019EN. 2023.