Adeno-associated virus (AAV) analysis from titer to payload: Chromatography-based solutions
What are AAVs
Adeno-associated viruses are non-pathogenic viruses that are used as gene-delivery tools for gene-therapies. For that, it is taken advantage of AAVs transferring their genetic material into dividing and quiescent cells. The transferred gene is translated into proteins by the cell's translation machinery, but does not integrate into the genome. Thus, the genetic material is not passed on to daughter cells and is mainly used for cell types that do not divide. This way, non-functional or missing proteins resulting from genetic defects can be replaced.How is an AAV structured and which characteristics are important?
An AAV particle is made up of a protein shell and the payload. The protein shell (capsid) is formed from three viral proteins that come together to form the icosahedral particle. This envelops the genetic material: single-stranded DNA, which in wild-type viruses encodes for capsid proteins and in therapeutically used AAVs for the protein of interest.- Important characteristics to analyze in AAVs:
- Analyzable by chromatographic methods:
- Titer (SEC-MALS)
- Full-empty-ratio (Anion-exchange, SEC-MALS and UV)
- Purity (SEC-MALS)
- Analyzed by non-chromatographic methods
- Identity of the genetic material (e.g. qPCR)
- Analyzable by chromatographic methods:
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Analysis of AAVs by HPLC
AAV titer and purity by SEC-MALS
The titer (concentration) of AAVs is important at different steps of AAV production because it reports on the yield. It is also important in the final quality control to confirm that the desired number of particles is contained in the medication and thus to ensure safe and efficient use. The AAV titer can be analyzed by combining size exclusion chromatography (SEC) and Multi-Angle Light-Scattering (MALS). In SEC, the AAVs are separated from impurities. The subsequent detection via MALS is more sensitive to the virus particles compared to other HPLC detectors and allows the quantification of the AAV titer.
Calibration curve of different concentrations of AAV5.
SEC-Chromatogram of dilution series of AAV5 separated on TSKgel GMPWXL SEC column and detected by LenS3-MALS detector.
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Full-empty analysis of AAVs
Only capsids containing the complete genomic content are therapeutically effective. Therefore, analyzing the amount of full and empty capsids in an AAV sample is essential. Anion-exchange chromatography can separate full from empty capsids since full capsids elute later compared to empty capsids. A study published in 2023 (Kurth et al. 2023) used the TSKgel Q-STAT anion exchange column and succeeded in replacing the toxic compound TMAC with choline chloride for full-empty AAV separations. A new application note summarizes the method presented in this study and demonstrates how further method development can enhance serotype-dependent analytical quantitation and process development.
Separating AAV6 full and empty capsids using a Q-STAT AEX column with choline-Cl. Mixtures of 100 % empty capsids, 50:50 mixtures, and 100 % full capsid samples were prepared. A choline-Cl gradient (100–350 mM) was applied for elution of empty and full capsids, and a fluorescence signal was used for detection.
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AAV payload content
An alternative way of measuring the average fill state of an AAV sample is by combining SEC-MALS with different detectors such as UV at 260 nm and 280 nm. The AAV analysis module in the SECview 3.0 Software of the LenS3 MALS detector automatically calculates the average fill and titer of an AAV sample.