Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product through Mass Photometry

November 02, 2023 2 min read

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product through Mass Photometry

About AAV (adeno-associated virus) Analysis

AAV sample characterization is a critical step in research, development and manufacturing processes of gene therapies involving viral vectors. There are several analytical approaches available to assess critical quality attributes (CQAs) for AAV samples, such as capsid content (empty/full ratios) and titer.

 

Use of MP (Mass Photometry) Analysis for AAV Samples

The new paper titled “ Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product” published by Boehringer Ingelheim explore the benefits of MP analysis for assessing aggregate content in AAV samples. 

Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids.

This revelation is important as it confirms that MP is an accurate and robust method to determine the molecular mass of AAV capsids including genomic content on a single particle level, resolving capsid species with differing genome size. 

 

Compatibility of mass photometry (MP) measurements with commonly used buffer conditions and excipients.

 

Benefits of MP Analysis 

Right off the bat, MP was found to be compatible with various excipients and a wide range of buffer conditions. This compatibility is a major benefit of MP, eliminating the needs for complex sample preparation or even excipient exchanges. In the long run, it saves researchers both time and resources. 

Good for us, MP can be operated with capsid titers as low as 8 × 1010 cp mL−1 with a CV < 5% using just 10 µL total sample volume.

Another advantage of MP is the single particle-level analysis enabling a differentiation between empty, filled, and double-filled species. In comparison to the data from SV-AUC, the MP data was better which can be considered as the gold standard for this purpose. 

It was found that MP outperforms other methods of assessing empty/filled capsid content. Due to its low sample consumption and user-friendly operation, MP is an indispensable instrument in the research and development environment. 

 

Further Studies on the use of MP for AAV Analysis 

Another paper, taken from Takeda, a 3rd party study and benchmarking of MP for AAV Analysis titled “Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry” further confirms the benefits of MP over other commonly adopted methods to quantify AAV byproducts that are co-produced during the manufacturing process. 

The paper elaborates on the use of analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) for quantifying AAV subspecies. However, these were associated with long turnaround times, low sample throughput and complex data analysis.

Mass Photometry is indeed a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaptation of method parameters. 

Here’s a video from our leading partner, Refeyn on the application of mass photometry for AAV sample characterization in GMP-regulated environments. 

 

 



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