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Mass photometry recognized for effective AAV analysis

Updated: Nov 8


Illustration showing empty, partially filled and full AAV capsids

The importance of effective adeno-associated virus (AAV) analysis – and how mass photometry can support this – has been recognized in recent publications.  

 

The need is clear for rapid, efficient, user-friendly and compliant analytical tools for AAV characterization to accelerate AAV research, development and production. These tools also support the assessment and monitoring of critical quality attributes (CQAs) of AAV gene therapy products to ensure their quality, safety and efficacy. AAV CQAs include relative capsid content (e.g. empty/full capsid ratios) of all serotypes, the detection of impurities and aggregation, and capsid titer quantification.   





Two of the following new publications discuss how mass photometry is addressing these needs and the value that it brings to analytical testing strategies for AAV-based gene therapy development and production. One paper, meanwhile, explores how mass photometry can be used to probe the effects of thermal stress on AAVs. 



Quantifying AAV capsid titer


A recent article, written by a principal scientist in gene therapy programs for Regeneron, Dr. Marilia Barros, provides a useful assessment of current and emerging technologies for quantification of AAV capsid titer.  

 

Accurate capsid titer quantification is crucial for determining correct dosing, as well as ensuring therapy efficacy and safety. Analytical speed and sample size are also key, given the tight clinical timelines and short supply of material within AAV gene therapy programs. Current quantification methods, such as ELISA, can therefore prove challenging and so the assessment and optimization of additional orthogonal AAV capsid titer methods are needed. 

 

As part of the article’s evaluation of the most commonly used methods for this AAV analysis, the effectiveness of mass photometry was summarized: “Mass photometry is a rapid and sensitive analytical technique that requires minimal sample volumes and concentrations. This makes it particularly advantageous for evaluating AAV samples, especially when working with small batch sizes.  

 

A notable feature of mass photometry is its titer estimation tool, which provides comprehensive data on all AAV capsid populations within a sample, including empty, full, and partially filled capsids. This capability allows for a detailed assessment of the sample’s composition and aids in more accurate characterization of AAV products.” 

 

Strategies for quantifying AAV capsid titer are also the focus of a recent webinar by Dr. Barros. She was joined in the webinar by Refeyn senior application scientist Dr. Matt Ranaghan, who gave an overview of how mass photometry can be used in AAV analysis more broadly.  




Viral vector analysis in regulated environments


A manager from MilliporeSigma’s viral vector CDMO analytical development team, Dr. Michael Born, recently discussed the value of mass photometry in another article advising on viral vector testing. This useful article aims to provide a playbook for reference when designing an analytical testing strategy for viral vectors, either internally or with a CDMO partner, so ensuring these therapeutics meet regulatory requirements and industry standards. 

 

The application of mass photometry is advocated in a number of steps of this seven-step strategy. For example, during clinical manufacturing process development, which is key to ensuring product consistency, mass photometry is recommended for analysis and optimization of purification processes. In another step, the importance of quality control data is highlighted along with the need for testing strength, purity and quality attributes supported by NGS, mass photometry and mass spectrometry for consistency and compliance. 

 

The stability of the final product, even if exposed to environmental or physical stressors, is also considered key, and formulation is crucial in ensuring this. Discussing formulation testing, the author observes: “This step can include stability, solubility and compatibility tests with buffer excipients; using mass photometry for rapid and detailed analysis of viral vector subpopulations; and optimizing manufacturing conditions.”



Probing thermal stress effects on AAVs


A recent research paper has found mass photometry to be a useful tool for studying the effects of thermal stress on AAVs, such as the disintegration or aggregation of these capsids at elevated temperatures. Publishing in Molecular Therapy: Methods & Clinical Development researchers from the Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, used our SamuxMP system, which is optimized for AAV characterization. The studies aimed to support the better understanding of AAV-transgene release. 

 

Data generated using nuclease treatment in combination with mass photometry demonstrated that the thermal uncoating process is highly AAV-specific and can be influenced by serotype, genome and host system. The SamuxMP proved useful for these studies as it enables accurate identification of AAV subpopulations that could otherwise be misidentified. This is due to its single-molecule measurement of capsids in solution with fast data acquisition. 


Schematic showing that outcomes of exposing AAV capsids to heat were analyzed with mass photometry

Ebberink et al. (2024) used mass photometry to assess the stability of empty and filled AAV preparations exposed to heat stress. (The image is the paper’s graphical abstract, used under CC-BY 4.0).










Interested in Refeyn’s AAV analytics solutions?  





 

Author: Catie Lichten, PhD, Refeyn Scientific Communications Manager

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