Empty adenoassociated virus (AAV) capsids pose a significant problem for viral-vector gene therapies. High levels of empty particles can increase patient immune responses while adding no contribution to the clinical efficacy of the treatment. Therefore, efficient characterization tools are needed to quantify empty AAVs. Racha Majed (technical sales specialist at Refeyn) and Quentin Bazot (innovation and process development team leader at ABL) presented a mass photometer for AAV analysis in a January 2024 webinar. The SamuxMP mass photometer enables users to analyze low amounts of samples and calculate empty/full capsid ratios accurately within minutes.

The Presentation

During AAV manufacturing, upstream processes generate some empty capsids. Empty/full AAV capsid ratios are important metrics that should be characterized as soon as possible during process development. Mass photometry enables quick, accurate characterization of such metrics.

Technology Assessment: ABL scientists compared the SamuxMP mass photometer with other analytical methods, analyzing the percentage of full capsids for five commercially available standards of different serotypes. The SamuxMP mass photometer results were similar to those from static light scattering ultraviolet-absorbance (SLS-UV) and charge-detection mass spectrometry (CDMS) methods. The ABL team also evaluated viral titer estimation with the SamuxMP equipment, comparing data from that process with results from enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR) methods. On average, the SamuxMP mass photometer calculated 1.5× lower titers than the ELISA method and about the same titers as the qPCR method.

Early Characterization: ABL scientists tested early characterization possibilities with the SamuxMP mass photometer using two AAV9 samples produced with the same plasmids, transfection reagents, and cell line. The percentage of full AAV capsids measured by the SamuxMP mass photometer matched results from qPCR/ELISA ratios. The SamuxMP system also assessed sample purity, identifying differences from the use of multiple elution buffers for the samples during affinity capture. It also quantified partially filled capsids for samples using different plasmid systems, which the qPCR/ELISA method could not.

Next, the team performed two studies to confirm the mass of encapsulated genomic cargo with the SamuxMP mass photometer. In the first study, they tested an AAV with an expected mass of 1,417 kDa. From the difference between the average masses of the empty and full peaks, the team calculated the average mass of the cargo with a standard error of <5% using the SamuxMP mass photometer. For a different sample, which presented three mass peaks, the same approach determined that the middle peak corresponded to AAVs containing one copy of the genome while the peak with the greatest mass corresponded to AAVs containing two copies.

Optimization of Polishing Steps: Polishing steps often require lengthy downstream processes and analyses to ensure enrichment of full AAVs. Mass photometry helps to optimize such processes to save time during polishing. In an experiment, the SamuxMP mass photometer analyzed all fractions obtained from anion-exchange (AEX)chromatography. This analysis took only about two hours and helped the ABL team improve polishing processes.

The SamuxMP mass photometer optimizes AAV manufacturing with its quick turnaround, taking <2 minutes per sample. It requires only a small sample volume and works with a variety of buffers. The use of mass photometry greatly decreased AAV development time in ABL’s case studies and lowered analytics costs.

Refeyn offers three software applications for use with the SamuxMP mass photometer in facilities compliant with good manufacturing practices (GMPs): The Refeyn ManageMP platform provides a solution for user authentication, access controls, and system configuration. It also enables review and export of audit trails. The Refeyn AcquireMP platform operates the mass photometer and processes raw data. And the Refeyn EvaluateMPS platform analyzes AAV data and exports reports electronically. With this Refeyn software package, the SamuxMP mass photometer can be implemented in GMP-compliant quality control (QC) and release assays.

Questions and Answers

What is the minimum molecular mass that can be detected, and what sample volume is required for analysis? The SamuxMP mass photometer can detect a minimum molecular mass of 500 kDa. The system uses 10–20 μL of a sample for analysis.

What buffers are suitable for SamuxMP measurements? There are no special requirements. The system can work with an array of different buffers.

Find the full webinar online at www.bioprocessintl.com/category/webinars.