Analysis of Adenoviral Vector Proteins by RPLC, Native Fluorescence, and Online MS

Importance of the Topic

Adenoviral vectors play a critical role in modern biopharmaceutical applications, including vaccines against infectious diseases and gene therapies for monogenic disorders. Detailed profiling of viral capsid proteins supports product quality, informs copy number assessment, and ensures batch consistency in advanced therapy medicinal products.

Objectives and Study Overview

This application note demonstrates a rapid reversed-phase liquid chromatography method with native fluorescence detection and online mass spectrometry to characterize the intact protein components of a human adenovirus 5 vector expressing GFP. The approach aims to resolve proteins across a broad abundance range and confirm molecular weights directly.

Methodology and Instrumentation

This study combined difluoroacetic acid ion pairing with a superficially porous phenyl-bonded stationary phase to enhance chromatographic resolution while maintaining MS sensitivity. Sample injection is direct, without prior cleanup. A 20-minute gradient from 10 to 50% acetonitrile (0.1% DFA) was applied on a 2.1×150 mm, 2.7 µm BioResolve RP mAb Polyphenyl column at 80 °C and 0.4 mL/min.

Instrumentation

• Liquid chromatography ACQUITY UPLC H-Class PLUS Bio System; fluorescence detection at Ex 280 nm, Em 360 nm
• Mass spectrometry Vion IMS QTof with ESI+; m/z 1000–4000, desolvation at 650 °C

Main Results and Discussion

The method achieved peak capacities of 200–480 within a 20-minute run. Native fluorescence detected protein peaks spanning three orders of magnitude in copy number. Online MS delivered total ion counts up to 2×10^7, enabling deconvolution of protein masses from small peptides (1.3 kDa, 3.6 kDa) to large capsid proteins (10–110 kDa). Identifications included protein VI (22.1 kDa, endosome lysis protein), protein IX (~14.37 kDa likely representing sequence variation or acetylation), and the hexon protein (~107.9 kDa variant).

Benefits and Practical Applications

• Direct analysis of formulated viral samples without extensive sample preparation
• DFA ion pairing optimizes chromatographic separation with minimal MS ion suppression compared to TFA
• Superficially porous phenyl column technology provides high recovery and compatibility with UPLC/UHPLC systems
• Simultaneous fluorescence and MS detection supports copy number assessment and mass confirmation in a single run

Future Trends and Applications

Advances may include deeper proteomic profiling of viral vectors through integrated peptide mapping, monitoring sequence variants and post-translational modifications, and extending this platform to other viral delivery systems. High-throughput adaptation and regulatory implementation will support accelerated development of next-generation vaccines and gene therapies.

Conclusion

The presented RPLC–native fluorescence–MS workflow enables rapid, sensitive, and direct analysis of adenoviral vector proteins. This method offers a solid foundation for characterization and quality control of AdV-based biotherapeutics, facilitating regulatory compliance and product consistency.

References

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