Investigating process-related post-translational modifications in NISTmAb RM 8671 using high-throughput peptide mapping analysis

Significance of the topic

Peptide mapping is a fundamental analytical workflow for characterizing monoclonal antibodies (mAbs) and biotherapeutics. It provides detailed information on primary amino acid sequence, post-translational modifications (PTMs), and structural integrity, which are critical for ensuring drug safety, efficacy, and regulatory compliance. High-throughput, reproducible methods reduce assay variability and enable efficient batch-to-batch comparability in biopharmaceutical development.

Study objectives and overview

This investigation evaluated the performance of the Thermo Scientific™ SMART Digest™ kit for rapid enzymatic digestion of the NISTmAb RM 8671 reference material. A time-course study (15–75 minutes) assessed digestion efficiency, sequence coverage, and identification and relative quantification of key PTMs such as deamidation, oxidation, glycation, and glycosylation. The overall goal was to demonstrate a streamlined workflow suitable for high-throughput peptide mapping with minimal sample preparation–induced artifacts.

Methodology and instrumentation

The study employed a bottom-up peptide mapping approach combining the SMART Digest heat-stable immobilized trypsin workflow with high-resolution LC-MS analysis. Sample preparation involved dilution of the NISTmAb to 2 mg/mL, enzymatic digestion at 70 °C with agitation for 15, 30, 45, 60, or 75 minutes, resin removal, disulfide reduction with DTT, and acidification prior to UHPLC-MS injection.

Instrumentation used:

• Vanquish™ Flex Binary UHPLC system with Acclaim VANQUISH C18 column (2.1 × 250 mm, 2.2 µm)
• Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer
• BioPharma Finder™ and Xcalibur™ software for data acquisition and peptide identification

Main results and discussion

• Sequence coverage for both heavy and light chains reached 100 % across all digestion times (15–75 min) with high confidence (≥ 95 %) and mass accuracy (≤ 5 ppm).
• Chromatographic reproducibility was excellent, with retention time precision (RSD) ≤ 0.2 % for monitored peptides.
• Complete digestion was achieved in as little as 30 minutes, yielding over 1 300 identified components and robust MS signal intensity.
• Sample preparation–induced PTMs remained low: N-terminal pyroglutamate formation exceeded 99 %, glycosylation profiles (A2G0F, A2G1F, A2G2F, Man5) matched expected NISTmAb patterns, oxidation levels of methionine and tryptophan remained < 3 %, and glycation on lysine residues was < 1.7 %.
• Deamidation exhibited a predictable increase with extended digestion time, rising from ~ 6.95 % (N318) at 15 min to ~ 10.62 % at 75 min, reflecting temperature- and pH-dependent kinetics.

Benefits and practical applications

• Rapid, automated digestion reduces total assay time and labor compared to traditional in-solution protocols.
• High sequence coverage and low artifact levels enhance confidence in PTM profiling for QA/QC and comparability studies.
• The workflow supports high-throughput screening of mAb candidates and manufacturing batches with minimal manual intervention.

Future trends and opportunities

Advancements may include integration of multi-enzyme digestion strategies, further automation of sample preparation, real-time online monitoring of enzymatic reactions, and application of AI-driven data analysis to accelerate identification of low-abundance PTMs. Emerging high-resolution instruments and improved software algorithms will enable deeper structural characterization and faster turnaround times for biopharmaceutical development.

Conclusion

The SMART Digest kit combined with Vanquish UHPLC-Orbitrap MS provides a robust, high-throughput peptide mapping platform for mAb characterization. Rapid digestion, complete sequence coverage, excellent reproducibility, and minimal preparation-induced modifications make this workflow an attractive option for biopharmaceutical research, development, and quality control.

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