SMART Digest compared to classic in-solution digestion of rituximab for in-depth peptide mapping characterization

Significance of the topic

Peptide mapping by liquid chromatography–mass spectrometry is a critical analytical tool in the biopharmaceutical industry for confirming the primary structure of monoclonal antibodies (mAbs) and monitoring post-translational or chemical modifications that impact safety and efficacy. Streamlining this workflow accelerates quality control and comparability assessments while reducing artefacts introduced during lengthy sample preparation.

Study objectives and overview

This study compared the performance of the Thermo Scientific SMART Digest kit with two classic in-solution digestion protocols (urea-denaturation and heat-denaturation) for peptide mapping of the therapeutic mAb rituximab. Key metrics included sequence coverage of heavy and light chains, reproducibility, and the identification and relative quantification of critical modifications such as deamidation, oxidation, glycosylation, and carbamylation.

Methodology and instrumentation

• Sample preparation: Rituximab at 2–10 mg/mL was reduced, alkylated, and digested. In-solution methods used 7 M urea or 70 °C heat for denaturation followed by trypsin overnight at 37 °C. SMART Digest used immobilized trypsin at 70 °C for 15–75 min.
• LC conditions: Thermo Scientific Vanquish Flex UHPLC with Acclaim VANQUISH C18 column (2.1 × 250 mm, 2.2 µm) at 0.3 mL/min, 50 °C, gradient from 4% to 100% organic.
• MS detection: Thermo Scientific Q Exactive HF Hybrid Quadrupole-Orbitrap with HESI-II source; full MS at 60,000 FWHM and top-5 data-dependent MS2 at 15,000 FWHM.
• Data processing: Chromeleon CDS 7.2 and BioPharma Finder 1.0 algorithms with 5 ppm mass tolerance and standard PTM search parameters.

Main results and discussion

• Sequence coverage: All six digestion conditions achieved 100% coverage of heavy and light chains. The SMART Digest protocol reached complete coverage in as little as 15 min.
• Reproducibility: SMART Digest replicates by different operators showed <5% RSD in retention times and peak areas, demonstrating robust performance.
• PTM profiling: Major N-glycoforms at N301 (A2G0F, A2G1F, A2G2F) and N-terminal pyro-glutamate formation were quantified consistently across methods. SMART Digest minimized deamidation and carbamylation artefacts compared to urea-based protocols.
• Modification factors: Heat-denatured in-solution digestion gave the highest deamidation (factor ~8.8) and oxidation (factor ~2.9). SMART Digest at 15 min showed the lowest factors (deamidation factor = 1, oxidation factor = 1) and only gradual increases at longer times.
• Peptide-level insight: Sensitive sites in peptides V306–K321 (N319) and G375–K396 (N388) were monitored by extracted ion chromatograms and isotopic shifts, confirming accurate detection of low-level deamidation.

Benefits and practical applications

• Time efficiency: Digestion time reduced from overnight to minutes without compromising data quality.
• Reduced artefacts: Elimination of urea and optimized pH minimized artificial modifications.
• High throughput: Compatibility with automated UHPLC-MS platforms supports routine QC and comparability studies.
• Standardization: The kit design ensures consistent enzyme activity and sample-to-sample reproducibility.

Future trends and possibilities

• Integration of on-line or automated digestion modules to further streamline workflows.
• Expansion to other therapeutic proteins, biosimilars, and complex biologics for comparability and stability studies.
• Advancements in high-resolution MS and data-analysis software to enable deeper characterization of low-abundance PTMs.

Conclusion

The SMART Digest kit combined with Vanquish Flex UHPLC and Q Exactive HF MS delivers a rapid, reproducible, and artefact-minimized peptide mapping workflow. It matches or exceeds classic in-solution methods in sequence coverage and PTM profiling while drastically reducing digestion time and chemical modifications.

Reference

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