Developing the analytics and analytical workflows supporting the analysis of the next generation of biotherapeutic and gene therapies

Importance of the Topic

Gene therapies represent a frontier in biopharmaceutical development, demanding precise analytical tools to guarantee product safety, potency, and purity. Adeno-associated virus (AAV) vectors, with their complex capsid architecture and critical empty/full ratios, necessitate robust workflows for characterization, process monitoring, and quality control throughout development and clinical manufacturing.

Objectives and Study Overview

This work establishes harmonized analytical workflows for AAV capsid analysis. The primary objectives are to:

• Optimize size-exclusion chromatography (SEC) for aggregate and monomer profiling across multiple AAV serotypes.
• Develop anion-exchange chromatography (AEX) assays for accurate quantification of empty versus full capsids.
• Implement reverse-phase liquid chromatography–mass spectrometry (RPLC-MS) methods for in-depth capsid protein identity, ratio, and post-translational modification analysis.

Methodology and Instrumentation

Chromatographic separations were conducted on Waters ACQUITY UPLC H-Class Plus and Arc systems using:

• BEH450 SEC columns (4.6×300 mm, 2.5 µm) with phosphate buffer and KCl to minimize secondary interactions.
• ProteinPak Hi Res Q columns (4.6×100 mm) for AEX separation with quaternary ammonium gradients.
• BEH C4 columns (2.1×100 mm) and DFA-modified mobile phases on a BioAccord LC-MS system for capsid protein profiling.
• μDAWN multi-angle light scattering (MALS) and QDa/RDa detectors for molecular weight confirmation and intact mass measurement.
• Fluorescence detection to extend sensitivity in low-level protein assays.

Main Results and Discussion

• SEC methods achieved clear separation of AAV monomers and aggregates, with reproducible performance across serotypes using optimized buffer conditions.
• AEX assays demonstrated linear quantification of empty/full ratios (0–100%). Serotype-specific gradient refinements were required to resolve capsid charge variants accurately.
• RPLC-MS with DFA modifiers produced high-resolution separation of VP1, VP2, and VP3 proteins, enabling intact mass deconvolution and detection of phosphorylation (+80 Da) on capsid subunits.
• Fluorescence detection provided over 20× signal-to-noise improvement compared to UV absorbance, allowing trace-level protein monitoring with minimal sample consumption.

Benefits and Practical Applications

• Stage-appropriate analytics accelerate gene therapy development, reducing time and sample requirements for key quality attribute assessment.
• Harmonized workflows support method life-cycle management in compliance with USP <1220> and ICH Q12/Q14 guidelines, ensuring ongoing method suitability.
• Integrated LC-MS strategies deliver orthogonal data on capsid integrity, empty/full distribution, and protein composition, enhancing decision-making in process development and QC release.

Future Trends and Possibilities

Emerging chromatographic materials (e.g., larger-pore and monolithic columns) will accommodate larger viral vectors beyond AAV. Native MS and charge detection MS (CDMS) offer direct measurement of viral particles with minimal preparation. Automation of sample handling and advancement of informatics platforms will further enable high-throughput, data-integrity-compliant analytics aligned with digital transformation in biopharma.

Conclusion

The established SEC, AEX, and RPLC-MS workflows provide robust, scalable analytical solutions for critical quality attribute profiling of AAV vectors. These methods support streamlined gene therapy development and regulatory compliance by delivering accurate, high-resolution data on capsid composition and integrity.

References

1. Fu X et al. Accurate quantification of AAV empty/full capsids by ion-exchange chromatography. Hum Gene Ther. 2019;30(4).
2. Ujma J et al. High-resolution cyclic ion mobility separations in analytical chemistry. Anal Chem. 2019;91(6):3574–3582.
3. Roots Analysis. Gene therapy market report and analysis. 2020.