Rapid Antibody Digestion Enabled by Automated Reversed-Phase Desalting on the Agilent AssayMAP Bravo Platform

Significance of Topic

This application note addresses a critical bottleneck in biotherapeutic antibody analysis by mass spectrometry: lengthy trypsin digestion under denaturing conditions. Automated, high‐throughput workflows that integrate protein desalting, cleanup, and digestion can accelerate decision making in analytical and QC laboratories, enabling same‐day peptide mapping for monoclonal antibodies.

Study Objectives and Overview

The main goals of the study were to:

• Develop an automated reversed-phase desalting protocol on the Agilent AssayMAP Bravo platform.
• Optimize cartridge priming, sample loading, washing, and elution parameters to minimize denaturant concentration.
• Compare digestion times and peptide recovery for diluted samples, RP-W cartridge desalting, and size exclusion chromatography (SEC).
• Demonstrate a complete automated workflow—from antibody capture to peptide analysis—requiring less than eight hours.

Methodology and Instrumentation

The workflow combined three Agilent AssayMAP Bravo applications:

• Affinity Purification (protein G cartridges) to isolate antibodies from CHO cell supernatants.
• Protein Cleanup with RP-W reversed-phase cartridges to desalt denatured, reduced, and alkylated antibodies.
• In-Solution Digestion to add trypsin and incubate for fixed periods on deck.

Key instrumentation and consumables included:

• Agilent AssayMAP Bravo liquid handler and cartridges (PG-W and RP-W resins).
• Agilent 1290 Infinity LC system and Agilent 6550 iFunnel Q-TOF MS with Jet Stream ESI.
• Poroshell 300SB-C18 and AdvanceBio Peptide Mapping columns for peptide separation.

Main Results and Discussion

An optimized protocol was established with these highlights:

• Cartridge priming with ≥ 20% acetonitrile ensured quantitative capture and minimal carryover.
• RP-W cartridges bound up to 75 µg of denatured antibody per 5 µL bed with <10% breakthrough.
• Optimal elution consisted of 60% acetonitrile in 0.1% TFA, requiring ~12 µL total volume to recover >90% of bound protein.
• Sample loading flow rates up to 20 µL/min and volumes from 200–1,000 µL showed consistent recovery when eluent was added just before elution.
• Comparison of denaturant removal methods demonstrated that RP-W desalting or SEC enabled complete trypsin digestion within 1–2 hours, versus >18 hours when samples were only diluted.
• Reproducibility studies across 48 replicates yielded coefficient of variation (CV) values below 5% for protein cleanup and below 10% for peptide peak areas in LC/MS analyses.

Benefits and Practical Applications

The workflow delivers:

• Same‐day turnaround for antibody peptide mapping and post‐translational modification analysis.
• Reduced hands-on time through full automation from purification to digestion.
• Scalable parallel processing of up to 96 samples with highly reproducible recovery.
• Compatibility with large volumes and diverse antibody sources without extensive revalidation.

Future Trends and Opportunities

Emerging directions include:

• Integration with targeted quantitation methods such as MRM/SRM for absolute antibody titer measurements.
• Extension of the platform to other protein classes and post‐translational modification workflows (glycosylation, phosphorylation).
• Enhanced resin chemistries to further reduce sample loss and extend binding capacity.
• Deeper coupling with artificial intelligence for automated method optimization and data interpretation.

Conclusions

This study demonstrates that automated reversed‐phase desalting using AssayMAP Bravo significantly reduces the time required for antibody digestion while maintaining high recovery and reproducibility. The end‐to‐end automation integrates affinity capture, cleanup, and digestion into a workflow with total processing time under eight hours. This platform empowers analytical and QC laboratories to accelerate biotherapeutic antibody characterization and supports higher throughput and more consistent results.

References

1. Ren, et al. An improved trypsin digestion method minimizes digestion-induced modifications on proteins. Analytical Biochemistry 2009, 392, 12–21.
2. Bovee, M.; Russell, J.; Murphy, S. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform. Application Note, Agilent Technologies, 2014.
3. Jiang, X.; et al. The effect of various S-alkylating agents on the chromatographic behavior of cysteine-containing peptides in reversed-phase chromatography. Journal of Chromatography B 2013, 915-916, 57–63.