Characterization of Viral Vector Particles Using the Agilent 6545XT AdvanceBio LC/Q-TOF

Importance of the Topic

Adeno-associated virus (AAV) particles serve as leading delivery vehicles in gene and cell therapies. Accurate characterization of their capsid proteins and associated post-translational modifications (PTMs) is critical for ensuring safety, efficacy, and regulatory compliance of these biotherapeutics.

Objectives and Study Overview

This study presents a comprehensive LC/Q-TOF mass spectrometry workflow to determine critical quality attributes (CQAs) of intact AAV capsids and to identify PTMs on individual capsid proteins (VP1, VP2, VP3). The approach integrates intact mass analysis with peptide mapping to achieve high sequence coverage and site-specific PTM localization.

Used Instrumentation

• Agilent 1290 Infinity II LC system (high-speed pump, multisampler, thermostatted column compartment)
• Agilent AdvanceBio columns for intact and peptide mapping analyses
• Agilent 6545XT AdvanceBio LC/Q-TOF with dual Jet Stream source
• Agilent MassHunter software: Acquisition (B.09.00) and BioConfirm 10.0

Methodology

Sample Preparation:

• Intact analysis: buffer exchange with 10 kDa cutoff filters into 5 mM TCEP, 20 % ACN, 0.1 % FA
• Peptide mapping: denaturation, reduction, alkylation, digestion with trypsin and rAsp-N

Chromatography and MS Conditions:

• Mobile phases: water/0.1 % FA (A), ACN/0.1 % FA (B)
• Gradients optimized for intact and peptide mapping analyses
• Q-TOF in standard mass mode; large molecule SWARM autotune for intact runs; iterative MS/MS for peptide mapping
• Data processing with BioConfirm 10.0, 1 % FDR, <10 ppm mass error threshold

Main Results and Discussion

Intact Mass Analysis:

• Baseline resolution of VP1, VP2, VP3 capsid proteins with minimal adduct interference after buffer exchange
• Accurate mass determination of native and phosphorylated VP1 and VP2 (<10 ppm error), and unmodified vs. acetylated VP3 proteoforms (<6 ppm error)

Peptide Mapping:

• Sequence coverage: VP1 97.7 %, VP2 98.5 %, VP3 100.0 %
• Site-specific identification of three phosphorylation sites on VP1 and at least two on VP2
• Detection of N-terminal acetylation on VP3 and low-level methionine oxidation and asparagine deamidation

Benefits and Practical Applications

The described workflow offers high sensitivity and specificity for AAV capsid CQA assessment. It supports robust quality control, accelerates process development, and meets emerging regulatory requirements for detailed structural characterization of gene therapy vectors.

Future Trends and Potential Applications

Advances may include integration of ion mobility separation, native MS for intact virus analysis, automated high-throughput workflows, and expansion to other viral vector platforms. Enhanced software capabilities for PTM profiling and real-time QC monitoring are also anticipated.

Conclusion

This integrated LC/Q-TOF workflow with Agilent 1290 Infinity II and 6545XT AdvanceBio systems, combined with MassHunter BioConfirm, enables accurate mass measurements and comprehensive PTM mapping of AAV capsid proteins, providing a reliable solution for gene therapy vector characterization.

Reference

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