An Integrated LC-MS Platform for Multi-Attribute Monitoring of CQA’s of mAbs at the Subunit Level

Importance of the Topic

An accurate and efficient approach for monitoring critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is essential for ensuring product safety, efficacy, and consistent performance. Subunit-level analysis simplifies complex glycoforms and oxidation products while delivering actionable data to support Quality-by-Design (QbD) strategies, process optimization, and regulatory compliance in biopharmaceutical development.

Objectives and Study Overview

This application note describes the development and demonstration of an integrated liquid chromatography–mass spectrometry (LC-MS) platform combined with automated data processing for multi-attribute monitoring (MAM) of mAb subunits. The study focuses on Fc-core fucosylation, aglycosylation, and high-mannose glycoforms of the therapeutic antibody trastuzumab, assessing method repeatability and instrumentation robustness.

Methodology and Instrumentation

Sample Preparation:

• Trastuzumab was digested with IdeS (FabRICATOR) to yield scFc and F(ab) fragments.
• Endoglycosidase (Endo S or Endo S2) treatments generated simplified glycoforms: afucosylated scFc (+1 GlcNAc), fucosylated scFc (+1 GlcNAc+1 Fucose), and aglycosylated forms.
• Final peptide concentration adjusted to 0.01 mg/mL in 3% acetonitrile, 0.1% formic acid for injection.

Chromatography and Mass Spectrometry:

• Column: ACQUITY UPLC BEH C4 at 80 °C with a 3 min gradient (0.1% FA in water to 0.1% FA in ACN).
• MS system: Vion IMS QTof operated in positive ESI mode, m/z 500–4000, capillary voltage 2.75 kV, desolvation at 600 °C.
• Lock-mass correction using Glu-Fibrinopeptide B.

Data Processing:

• UNIFI Scientific Information System with intact protein workflow and MaxEnt1 deconvolution.
• Custom field formulas calculated fucosylation%, aglycosylation%, and high-mannose content by comparing Endo S and Endo S2 digests.
• Automated integration of total ion chromatogram peaks and relative abundance reporting.

Main Results and Discussion

• UPLC separation yielded a single scFc peak at 2.6 min, enabling clean MS analysis.
• Deconvoluted mass spectra showed <5 ppm mass accuracy for aglycosylated, afucosylated, and fucosylated forms.
• Relative abundances for two trastuzumab batches were consistent, with fucosylation levels around 85–90% and aglycosylation below 1%.
• Instrument-to-instrument and day-to-day repeatability exhibited RSD values under 10% for all measured subunit modifications.
• Differential afucosylation between Endo S and Endo S2 digests provided a quantitative measure of high-mannose and hybrid glycan structures (~3.9%).
• UNIFI’s customizable reporting enabled generation of compliant-ready summaries, tables, and plots with minimal manual intervention.

Benefits and Practical Applications of the Method

• Streamlined sample prep and rapid 3 min LC-MS analysis facilitate near-real-time CQA monitoring.
• Automated data workflows and customizable calculations minimize user input and errors.
• High mass accuracy and robust repeatability support late-stage development and quality control release testing.
• Platform flexibility allows expansion to other modifications (e.g., oxidation, deamidation) and glycan profiling.

Future Trends and Possibilities

• Integration of high-throughput automation and robotic sample handling to further accelerate analysis.
• Application of ion mobility separation to resolve isomeric glycoforms and oxidation products.
• Coupling with machine-learning algorithms for predictive quality assessment and real-time process control.
• Expansion of MAM platforms to biosimilar comparability studies and multi-product pipelines.

Conclusion

The described integrated LC-MS platform combining ACQUITY UPLC, Vion IMS QTof, and UNIFI software provides a robust, high-accuracy method for multi-attribute monitoring of mAb subunits. With simplified workflows, rapid analysis, and automated reporting, this approach meets QbD and regulatory requirements, offering a powerful tool for process development, QC testing, and therapeutic antibody characterization.

Reference

1. Xu W, Jimenez RB, Mowery R, et al. A Quadrupole Dalton-Based Multi-Attribute Method for Product Characterization, Process Development, and Quality Control of Therapeutic Proteins. mAbs. 2017;9(7):1186–1196.
2. Bomans K, Haberger M, Bonnington L, et al. Multi-Attribute Monitoring of Antibody Modifications by Semi-Automated LC-MS Peptide Mapping. Am Pharm Rev. 2016.
3. Upton R, Bell L, Guy C, et al. Orthogonal Assessment of Biotherapeutic Glycosylation: Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action. Anal Chem. 2016;88(20):10259–10265.
4. Haberger M, Bonnington L, Bomans K, et al. Application of ESI-MS in a Quality Control Laboratory. Am Pharm Rev. 2016.
5. Sokolowska I, Mo J, Dong J, Lewis MJ, Hu P. Subunit Mass Analysis for Monitoring Antibody Oxidation. mAbs. 2017;9(3):498–505.
6. Dong J, Migliore N, Mehrman SJ, et al. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC–MS Analysis. Anal Chem. 2016;88(17):8673–8679.
7. Chung S, Quarmby V, Gao X, et al. Quantitative Evaluation of Fucose Reducing Effects in a Humanized Antibody on FcγR Binding and ADCC Activities. mAbs. 2012;4(3):326–340.
8. Shields RL, Lai J, Keck R, et al. Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves FcγRIII Binding and ADCC. J Biol Chem. 2002;277(30):26733–26740.
9. Waters Corporation. Biopharmaceutical Platform Solution with UNIFI – Key Applications. App Note 720004887EN. 2017.