Adeno-associated virus capsid protein characterization and host cell protein profiling using micro-flow UHPLC-Orbitrap MS

Significance of the Topic

The detailed analysis of adeno-associated virus (AAV) capsid proteins and related host cell protein (HCP) impurities is essential to guarantee the safety and efficacy of gene therapy products. Comprehensive sequence confirmation, PTM profiling, and sensitive HCP detection support quality control and regulatory compliance in biopharmaceutical manufacturing.

Objectives and Study Overview

This work aimed to establish a micro-flow UHPLC-Orbitrap MS/MS workflow for:

• Full sequence coverage and site-specific PTM identification of AAV6 capsid proteins
• Relative quantification of capsid PTMs
• Identification and quantitation of residual HCPs in crude harvest and purified AAV6 samples

Methodology and Instrumentation

Micro-flow reversed-phase UHPLC separations were performed on a PepMap C18 column (1.0 × 150 mm, 3 µm) at 80 µL/min. AAV capsid proteins were digested with pepsin under acidic and heated conditions, while HCP samples were reduced, alkylated, and trypsin-digested. Data-dependent MS/MS acquisition used the Orbitrap Exploris 480 with high-resolution settings for both full MS (60,000) and MS/MS (15,000) scans. Peptide mapping and HCP workflows were processed in Thermo Scientific™ BioPharma Finder™ 4.1 software.

Instrumentation

• Thermo Scientific™ Vanquish™ Horizon UHPLC system
• Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer
• Thermo Scientific™ PepMap™ 100 C18 column (1.0 × 150 mm, 3 µm)

Main Results and Discussion

• Pepsin digestion achieved 100% sequence coverage of AAV6 VP1 in a single run and confident identification of VP1, VP2, and VP3 N-termini.
• Low-abundance PTMs were detected and quantified: phosphorylation (~0.4%), oxidation (~0.6%), and deamidation (~0.33%) of targeted peptides.
• Crude AAV6 harvest analysis identified 817 HCPs (≥3 unique peptides); purified AAV6 showed only 30 HCPs, demonstrating effective host protein removal by POROS AAVX affinity resin.
• Spiked-in protein standard allowed absolute estimation of low-level HCPs (e.g., 3.6 pmol/mL) with <12% CV across triplicate analyses.

Benefits and Practical Applications

• The micro-flow UHPLC-Orbitrap method combines high sensitivity, throughput, and robustness for viral vector characterization.
• Ready-to-use LC-MS/MS templates and a unified informatics workflow simplify peptide mapping and HCP screening in biomanufacturing environments.
• Quantitative PTM and impurity profiles support critical quality attribute monitoring and process optimization.

Future Trends and Opportunities

Emerging approaches may integrate targeted PRM assays for ultra-sensitive PTM quantitation, machine learning-driven spectral interpretation, and expanded host proteome libraries for deeper HCP coverage. Automation of sample preparation and real-time data feedback could further streamline viral vector analytics.

Conclusion

The developed micro-flow UHPLC-Orbitrap MS/MS workflow delivers comprehensive AAV capsid protein mapping and sensitive HCP profiling with high reproducibility. This platform addresses key analytical challenges in gene therapy vector characterization and aligns with industry requirements for safety and quality control.

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