Automated High-Throughput Clone Screening of Therapeutic Antibodies: From Automated IdeS Digestion to Intact Fc/2-Glycosylation Analysis by MALDI-TOF

Significance of the Topic

Glycosylation of the Fc/2 fragment in monoclonal antibodies is a pivotal quality attribute in biopharmaceutical development.
Early detection and selection of clones with optimal glycan patterns enhance therapeutic efficacy, safety and manufacturability.

Objectives and Study Overview

This work describes a fully automated, high-throughput workflow for screening therapeutic antibody clones by combining IdeS digestion and intact Fc/2 glycosylation analysis via MALDI-TOF.
The key goals were to eliminate manual bottlenecks, achieve reproducible sample preparation and deliver rapid data processing for large sample sets.

Methodology and Instrumentation

The protocol integrates all steps on a robotic platform:

• IdeS enzymatic digestion to cleave IgG1 into Fc/2 and F(ab')2 subunits.
• Quenching and dilution with TFA to suppress non-specific signals and salts.
• Automated MALDI matrix preparation using 2,5-DHAP/DAC and glass beads to induce co-crystallization by high-frequency shaking.
• Direct spotting of the matrix–analyte suspension onto a 384-spot AnchorChip plate for MALDI-TOF analysis.

Instrumentation Used

• Agilent Bravo Automated Liquid Handling System with heating and shaking modules.
• Bruker autoflex maX MALDI-TOF mass spectrometer operating in positive linear mode (5–20 kDa).
• Bruker BioPharma Compass 2021 for automated data processing and glycoform profiling.

Key Results and Discussion

The workflow processed approximately 2 × 384 samples per day without the need for LC purification, leveraging the direct MALDI-TOF readout of the Fc/2 fragment (~m/z 13,000).
Six major glycoforms (G0F, G0F-N, G1F, G2F, G2F+1aGal, G2F+2aGal) were consistently identified and quantified.
Performance metrics:

• Cosine similarity scoring against a reference glycan profile ensured rapid pass/fail assessment (green > 0.9, yellow 0.6–0.9, red < 0.6).
• Mass accuracy was typically better than ±5 Da across 33 replicates.
• Relative abundance coefficients of variation for high- and low-abundance glycoforms ranged between 1 % and 6 %, indicating excellent precision.

Benefits and Practical Applications

This automated MALDI-TOF approach delivers:

• High throughput and reduced hands-on time compared to LC-ESI methods.
• Robust glycoform screening without chromatographic cleanup.
• Standardized data reporting and traffic-light quality assessment for rapid clone ranking.

Future Trends and Potential Applications

The methodology can be further scaled by:

• Implementing 384-well vial plates and multi-channel pipetting heads for parallelized processing.
• Extending the platform to other antibody isotypes or engineered Fc variants.
• Integrating real-time data analytics and machine-learning algorithms for predictive glycosylation profiling.

Conclusion

The presented automated protocol streamlines IdeS digestion and MALDI-TOF sample preparation, achieving rapid, reproducible Fc/2 glycosylation analysis at high throughput.
By eliminating manual crystallization steps and chromatographic cleanup, the workflow accelerates early clone selection and supports robust quality control in antibody development.

Reference

1. Haslam C. et al. The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond. J Biomol Screen. 2016;21(2):176-186.
2. Ritorto MS. et al. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat Commun. 2014;5:4763.
3. Wenzel T. et al. 2,5-Dihydroxyacetophenone: a matrix for highly sensitive MALDI-TOF analysis of proteins using manual and automated techniques. Rapid Commun Mass Spectrom. 2006;20(5):785-789.
4. Cosine similarity. Wikipedia. Last visited Feb 18, 2020.
5. LabTie™ Bead Loader. BIOSPEC Product Information.