A high-resolution accurate mass multi-attribute method for critical quality attribute monitoring and new peak detection

Significance of Topic

Monoclonal antibody (mAb) therapeutics demand rigorous monitoring of critical quality attributes (CQAs) to ensure safety and efficacy.
High-resolution accurate mass (HRAM) multi-attribute methods (MAM) integrate identification, quantitation, and new peak detection (NPD) into a single workflow.
This approach aligns with Quality by Design principles and regulatory expectations for biopharmaceutical process development and lot release.

Study Objectives and Overview

The primary goal was to develop and optimize an HRAM MAM workflow to monitor multiple CQAs of the NISTmAb standard.
Key targets included glycosylation, deamidation, isomerization, succinimide formation, oxidation, C-terminal lysine truncation, N-terminal pyroglutamate, and glycation under normal and stressed conditions.
The method’s capability for NPD was assessed using spiked peptide retention time calibration (PRTC) standards and stress-induced impurities.

Methodology and Instrumentation

Sample Preparation and Digestion

• Denature in 7 M guanidine HCl, pH 8.3; reduce with DTT; alkylate with iodoacetamide; buffer exchange into 50 mM Tris pH 7.9.
• Trypsin digestion (1:10 enzyme:substrate) at 37 °C for 30 min; quench with 10% formic acid.
• Thermal stress: 60 °C for 120 h; oxidative stress: 0.03% H₂O₂ for 24 h followed by identical digestion.

Liquid Chromatography–Mass Spectrometry

• UHPLC: Thermo Scientific Vanquish Horizon with Accucore Vanquish C18+ column (1.5 µm, 2.1 × 150 mm) at 50 °C; flow 0.25 mL/min; 6–70% acetonitrile in 64 min gradient.
• MS: Q Exactive Plus Hybrid Quadrupole-Orbitrap; full MS at 140 000 resolution (m/z 200), AGC 3e6, 300–1800 m/z; Top5 DDA MS/MS at 17 500 resolution for peptide mapping.
• Data processing: Thermo Scientific BioPharma Finder 3.1 for discovery and peptide mapping; Chromeleon 7.2.10 for targeted quantitation and NPD.

Key Software Parameters

• Mass tolerance 8 ppm for quantitation; XIC integration using ICIS algorithm with retention time matching.
• NPD frame width 10 ppm and 0.5 min, peak intensity threshold 1e6, ratio filter ≥1000 for PRTC spike, ≥20 for stress comparisons.

Main Results and Discussion

Sequence coverage of NISTmAb light and heavy chains exceeded 96%.
HRAM at 140 000 resolution resolved overlapping isotopes (e.g. deamidated vs native peptides), enabling accurate CQA quantitation.
Mass tolerance optimization (5–10 ppm) prevented misintegration of closely spaced peptides.
Reproducibility studies across ten injections, multiple instruments, and different users showed <5% variation for most CQAs.
Low-level host cell protein peptide TFTTQETITNAETAK (~10–15 ppm) was reliably quantified with intensities down to 2e4 counts.
Thermal stress increased deamidation at N289 (0.06→0.78%, 13-fold) and isomerization at D283 (>60-fold) in FNWYVDGVEVHNAK.
Oxidative stress elevated methionine oxidation (e.g. M255 in DTLMISR from ~2% to ~14%), while glycosylation profiles remained stable.
NPD detected all 15 spiked PRTC peptides with fold changes >1000 relative to control; stress-induced variants (oxidation, deamidation, isomerization) were also identified as new peaks.

Benefits and Practical Applications

The HRAM MAM workflow consolidates multiple conventional assays (HILIC, SEC, CEX, CE, RP-HPLC, ELISA) into a single LC-MS run.
Enables simultaneous monitoring of diverse CQAs with high specificity and sensitivity.
Integrated NPD detects impurities and modifications not targeted a priori, supporting quality control and comparability studies.
Software integration (BioPharma Finder → Chromeleon) streamlines method transfer from research to GMP-compliant environments.

Future Trends and Opportunities

Automation of sample preparation and data processing to increase throughput.
Machine-learning algorithms for advanced peak detection and characterization of unknown modifications.
Extension of MAM workflows to other modalities (e.g. bispecifics, ADCs) and real-time process monitoring.
Standardization of MAM protocols and regulatory acceptance for release testing.
Integration with high-throughput platforms and cloud-based reporting.

Conclusion

The Thermo Scientific HRAM MAM workflow based on Vanquish UHPLC and Q Exactive Plus Orbitrap offers a robust, flexible platform for CQA quantitation and NPD.
High resolution and mass accuracy ensure reliable integration of coeluting species and low-abundance modifications.
Software solutions support end-to-end GMP compliance from instrument control to reporting.
This MAM approach reduces assay complexity, accelerates decision making, and enhances biotherapeutic quality assessment.

References

1. Rogers R, et al. Development of a quantitative mass spectrometry multi-attribute method for characterization, quality control testing and disposition of biologics. MAbs. 2015;7(5):881–90.
2. Rogers R, et al. A view on the importance of “multi-attribute method” for measuring purity of biopharmaceuticals and improving overall control strategy. AAPS J. 2018;20:7.
3. Li WZ, et al. Structural elucidation of post-translational modifications in monoclonal antibodies. In: State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization. 2015;2:119–45.
4. Ren D, et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Anal Biochem. 2009;392(1):12–21.