Automating Rapid High-Throughput LC-MS mAb Subunit Screening of Microbioreactor Cell Culture Samples

Importance of the Topic

Monoclonal antibodies (mAbs) are critical biopharmaceuticals requiring rapid, high-throughput analytical methods to monitor key product quality attributes early in clone selection and process development. Automated workflows that combine efficient sample preparation with robust LC-MS analysis help overcome bottlenecks, reduce analyst workload and accelerate development timelines.

Objectives and Study Overview

This application note describes a fully automated protocol for the purification, digestion and subunit-level LC-MS screening of mAbs directly from microbioreactor cell culture supernatants. Using low sample volumes (20–100 µL), the workflow integrates magnetic Protein A purification, IdeS (FabRICATOR®) digestion, DTT reduction and rapid LC-MS analysis on a Waters™ BioAccord™ system with waters_connect™ informatics.

Methodology and Instrumentation

Sample Preparation and Automation

• Sampling of spent Chinese hamster ovary (CHO) cell culture media spiked with trastuzumab at 0.5 µg/µL.
• Automated Protein A capture on Magne® beads, low-pH elution and neutralization in a 96-well format using the Andrew+™ Pipetting Robot with Shaker+ and Magnet+ devices.
• On-deck IdeS digestion (FabRICATOR®, 2 units/µL) and DTT reduction (40 mM) at 37 °C for 60 min to generate Fc/2, Fd′ and light-chain subunits.

Instrumentation

• Waters™ ACQUITY™ UPLC I-Class PLUS with BioResolve™ RP mAb Polyphenyl column (2.1 × 100 mm, 450 Å, 2.7 µm) at 80 °C.
• BioAccord™ LC-MS System: ESI+ on ACQUITY RDa™ detector, 50–2000 m/z, 2 Hz scan rate, capillary voltage 1.5 kV.
• Mobile phases: 0.1% formic acid in water (A) and in acetonitrile (B); 4.5 min gradient at 0.4 mL/min.
• Data handled in waters_connect™ with INTACT Mass™ App and UNIFI v2.1.2.14.

Key Results and Discussion

The automated workflow processed up to 48 samples in under three hours, delivering subunit spectra with <20 ppm mass accuracy. Protein A purification improved chromatographic quality versus direct analysis of unpurified media. Automated and manual protocols yielded comparable relative quantification of Fc glycoforms, Fd′ and light chains. The method proved robust at low mAb loads (0.5–10 µg) and demonstrated consistent recovery and digestion efficiency across replicates.

Benefits and Practical Applications

• High throughput: up to 96 samples per run in a single 96-well plate format.
• Minimal sample requirement: suitable for microbioreactor volumes.
• Reduced hands-on time: fully automated on Andrew+ robot versus 1–5 manual interventions on other platforms.
• Broad applicability: supports clone screening, process optimization and quality control in biopharmaceutical development.

Future Trends and Potential Applications

Integration of advanced informatics (e.g., AI-driven data interpretation) and higher-throughput LC-MS platforms will further accelerate mAb screening. Expanded automation for multi-attribute monitoring, including glycan profiling and oxidation mapping, could enable real-time process control in continuous biomanufacturing. Miniaturized sample handling and higher multiplexing may support single-cell clone evaluation.

Conclusion

This study demonstrates a robust, fully automated workflow for mAb subunit analysis directly from cell culture media using minimal sample volumes. Combining magnetic Protein A purification, IdeS digestion and rapid LC-MS on the BioAccord system streamlines high-throughput screening of critical quality attributes, facilitating faster biotherapeutic development.

References

1. Lu RM, Hwang YC, Liu IJ et al. Development of Therapeutic Antibodies for the Treatment of Diseases. J Biomed Sci. 2020;27:1.
2. Dong J, et al. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring. Anal Chem. 2016;88:8673–8679.
3. Shion H, et al. INTACT Mass™ – waters_connect™ Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics. Waters Application Note 720007547. 2022.
4. Morrison L, et al. Direct LC-MS Characterization of Glycoform Distribution and Low Molecular Weight Impurities in mAb Process from Ambr® 15 Bioreactors. Sartorius Application Note. 2022.
5. Wagner-Rousset E, et al. Antibody-Drug Conjugate Model Fast Characterization by LC-MS Following Ides Proteolytic Digestion. mAbs. 2014;6(1):173–184.