Top-Down CDR Sequencing of Native Intact mAbs Through Deconvolution of HC+LC Mixture Spectra

Significance of the Topic

The accurate sequencing of antibody variable regions is crucial for therapeutic development, quality control, and structural biology applications. Top-down mass spectrometry offers rapid confirmation of complementarity-determining regions without enzymatic digestion artifacts, enabling native intact monoclonal antibody analysis.

Objectives and Overview

This study introduces a workflow for deconvoluting heavy chain (HC) and light chain (LC) mixture spectra from electron capture dissociation (ECD) of native mAbs. It aims to confirm CDR sequences within a single top-down spectrum and to validate an automated “Ion Matchmaking” algorithm in ExDViewer software.

Methodology and Instrumentation

Intact NIST monoclonal antibody was buffer-exchanged into 100 mM ammonium acetate and infused by static nanospray into an Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer modified with an ExD cell for ECD. Spectra were acquired in MS1 mode with low-mass cutoff and supplemental collision energy. Data analysis was performed using ExDViewer freeware, featuring automated peak picking, deconvolution, charge-state averaging, and the Ion Matchmaking algorithm for ambiguous ion assignments.

Main Results and Discussion

ECD fragmentation produced extensive series of c- and b-ions across CDRs. Variable region coverage reached 75–85% for LC and 45–50% for HC. Implementation of Ion Matchmaking reduced false-positive assignments in overlapping m/z regions, notably within CDR3 domains, and accelerated manual validation. Multiple charge states (3+ to 7+) were consistently observed for key fragment ions.

Benefits and Practical Applications

• Fast, direct confirmation of mAb variable regions without digestion or reduction.
• Native analysis preserves disulfide bonds, reducing sample handling artifacts.
• Simultaneous interrogation of HC and LC simplifies sequencing workflows.
• Suitable for QC in biopharmaceutical production and research on antibody structure.

Future Trends and Applications

Advances may include integration with ultraviolet photodissociation, enhanced machine-learning algorithms for automated assignments, extension to middle-down workflows, and high-throughput screening for PTMs. Real-time monitoring and cloud-based data analysis platforms are expected to streamline antibody characterization further.

Conclusion

The combination of native top-down ECD and automated Ion Matchmaking in ExDViewer enables rapid, reliable sequencing of mAb CDRs directly from intact antibody spectra. This approach minimizes artifacts, enhances throughput, and supports stringent quality control in therapeutic antibody development.

References

1. Shaw JB et al. Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation. Anal Chem. 2020;92(1):766–773.
2. Vasilev YV et al. Top-down characterization of native monoclonal antibodies obtained with electron capture dissociation on Q-TOF instruments. ASMS 2023.
3. ExDViewer. Agilent proprietary software, freeware download at exdviewer.agilent.com.