Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF

Significance of the Topic

Analysis of intact monoclonal antibodies (mAbs) provides vital information on molecular weight, glycoform distribution, and post-translational modifications to ensure product quality and consistency.
This approach accelerates decision-making in biopharmaceutical development and manufacturing.

Objectives and Study Overview

This study presents a high-throughput workflow integrating Agilent 1290 Infinity II UHPLC, 6545XT AdvanceBio LC/Q-TOF, and MassHunter BioConfirm software.
The goal is to achieve accurate, reproducible characterization of intact mAb standards, therapeutic antibodies, and antibody-drug conjugates (ADCs).

Methodology and Instrumentation

Intact mAb samples were diluted to 1.0 µg/µL and analyzed without desalting.
A 4-minute gradient on a PLRP-S 2.1 × 50 mm column at 0.5 mL/min separated the proteins.
MS data were acquired in positive mode over 100–10 000 m/z at 1.0 spectra/s and deconvoluted using maximum entropy to obtain zero-charge mass spectra.

Instrumentation

• Agilent 1290 Infinity II UHPLC system
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual JetStream source
• PLRP-S 1000 Å, 2.1 × 50 mm, 5 µm column
• MassHunter BioConfirm B.08 software

Main Results and Discussion

NIST mAb analysis revealed six primary glycoforms with average mass errors below 0.5 ppm.
Herceptin and other therapeutic mAbs showed consistent low-ppm mass accuracy across major glycoforms.
ADC (T-DM1) deconvolution identified nine conjugation states (D0–D8) with an average DAR of 3.5.
Mirror plots of innovator versus biosimilar rituximab highlighted nearly identical glycoform profiles, enabling rapid comparability assessment.

Benefits and Practical Applications

• Mass accuracy at single-ppm level for intact proteins
• Resolution of major and minor glycoforms and PTM variants
• High throughput: 4-minute LC gradient allows 96 samples in 8 hours
• Quantitative ADC analysis with automated DAR calculation
• Direct comparability studies for biosimilar development

Future Trends and Potential Applications

Advances in Q-TOF sensitivity and software automation will enable deeper PTM profiling and subunit analysis.
Integration with machine learning may further accelerate data interpretation.
Applications will expand to personalized therapy monitoring and high-throughput bioprocess analytics.

Conclusion

The combined UHPLC/Q-TOF workflow with automated deconvolution provides rapid, precise characterization of intact mAbs and ADCs.
Its high mass accuracy and throughput support critical quality control and biosimilar comparability in the biopharmaceutical industry.

References

• Intact Protein Analysis Using an Agilent 6550 Q-TOF Mass Spectrometer, Agilent Technologies (2015)
• Monoclonal Antibody Analysis with Agilent 1290 LC and 6530 Q-TOF, Agilent Technologies (2012)
• Automated ADC DAR Calculation Workflow, Agilent Technologies (2016)