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Adeno-associated Virus (AAV) Titration Service

Adeno-associated-Virus-AAV-Titration-Service

Determination of viral titer is vital for the AAV related drugs development. There are two measurement categories: physical titer determination and biological titer determination. Physical titer includes viral particle titer (or capsid titer) and genomic titer; and biological titer used to present as infection titers and transduction titers.

Physical Titer of AAV

  • Viral particle titer

Viral particle concentration titer, also known as capsid titer, takes the number of AAV particles as the unit of measurement. The detection techniques can measure solid AAV particle titer, hollow AAV particle titer, and total AAV particle titer. It is challenge of assuring a low empty shell rate of the AAV manufacturing.  In general, the empty shell rate of wild-type AAV is over 50%. For the modified AAVs which lack of packaging-related cis-elements, the empty shell rate would be higher. There are many methods for AAV titration and quality monitoring virus particle titer or particle titer ratio, such as ELISA, Transmission Electron Microscopes (TEM), High-performance liquid chromatography (High-performance liquid chromatography, HPLC), analytical ultracentrifugation (Analytical Ultracentrifugation, AUC), etc. People use TEM and AUC to detect the ratio of hollow shell AAV particles to solid AAV particles in AAV products.

  • ELISA

ELISA measures AAV particle titers by developing color by reacting with an enzyme-catalyzed substrate attached to the specific antibody that specifically binds to the AAV capsid. The most common is the antibody sandwich ELISA method. The AAV capsid is composed of a mixture of three proteins, VP1, VP2 and VP3. On average, each virion contains 60 VP molecules in a 5:5:50 ratio of VP1/ VP2 / VP. The 96-well plate provided in the kit is pre-coated with capture antibodies that can bind to the three VPs. When AAV samples or standards are added to the 96-well plate, their AAV capsid proteins will bind to the capture antibodies. After incubation and plate washing, AAV VP capsid antibody and HRP-labeled secondary antibody are added respectively, and the unbound antibody will be eluted. After color development of the substrate, the absorbance at 450 nm was measured, and finally the AAV capsid protein concentration in the unknown sample was calculated by the AAV standard curve, and then the AAV titer in the sample was calculated.

  • TEM

The diameter of AAV particles is about 20~26nm. The AAV particles can be observed intuitively through transmission electron microscopy (TEM), and AAV particles can be counted directly. TEM is inappropriate for viral absolute titer determination, due to the artifacts. TEM is a classic method of evaluating the ratio of the content of empty virus shells to the content of solid virus particles. The AAV particles without genomic DNA are low density and show holes (black dots) in the middle of the particle image under the electron microscope. While the virus particles containing genome are solid. In addition, the genomes packaged in some AAVs are incomplete, which is between empty shell particles and solid particles. To exclude the impurities background of original product, the purified AAV samples are benefit to the accurate identification result.

  • HPLC

HPLC is a fast and convenient analytical technique. It is very common in the quantitative analysis of AAV, which is able to distinguish the content of hollow capsids and complete capsids in AAV products. HPLC can be applied to high throughput and real-time quantitate of various serotypes of AAVs. Size exclusion chromatography coupled with 18-angle laser light scattering detector (SEC-MALS) has been widely used in the process of biological products, and it is a powerful analytical tool.

  • AUC

AUC technology is a method of studying the sedimentation characteristics and structure of biological macromolecules. It applies special rotors and detection to continuously track the sedimentation process of substances in a centrifugal field. Utilizing ultraviolet absorption or Raleigh interference, AUC can real-time detect the change process of the sedimentation profile distribution of each component and calculate the sedimentation coefficient of each component. Based on the difference in sedimentation coefficients of empty AAV and solid AAV, the ratio of empty AAV titer to that of solid AAV containing the complete genome can be accurately quantified. Only purified AAV preparations can be detected by AUC, requiring large amount of testing samples. In addition, the AUC detection cycle is long and low throughput.

  • Genome titer

The genome titer is based on the amount of genome contained in the AAV capsid as a quantitative basis, and the concentration of AAV containing the target genome is determined. Genomic titer determination routes are PCR, Dot-blot and spectrophotometry. The pre-processing of digesting the AAV protein is essential for the accuracy quantitation.

  • q-PCR

Creative Diagnostics provides the AAV titration service based on various viral types. For the viruses carrying fluorescence protein, we can titrate the virus load by fluorescence intensity. For the precise titration, we determined AAV titers by directly extracting viral genomes from lysed viral particles, using qPCR to quantify the copy number of the viral genome. AAV particles are very stable, even stay at room temperature for several days. We also offer the purifying and concentrating service for the AAV samples before or after titration.

Biological Titer

  • Infectious titer

TCID50 is one of the widely used infection titer assays on virology field. For AAV titration, adenovirus is needed as a helper virus. In the presence of the helper virus, AAV is diluted 10-fold and then infected with AAV-rep and AAV-rep in a 96-well plate. The cap expressing cell line was then detected by PCR to determine the number of negative and positive wells, and the infection titer was calculated by TCID50.

  • Transduction titer

The transduction titer is determined by infecting the experimental cells with serial diluted AAV samples and calculating infection positive/negative cell wells by transgene detection. Its measurement unit is the transduction unit (TU). To improve the detection sensitivity, helper virus co-infection is often used to increase the expression rate of AAV transgene, and its measurement unit is enhanced transduction unit (ETU). The transduction titer is generally carried out by in vitro experiments on cell lines.

Quotations and Ordering

Quotations-and-Ordering

References:

  1. Subramanian, S., Maurer, A. C., Bator, C. M., Makhov, A. M., Conway, J. F., Turner, K. B., ... & Hafenstein, S. L. (2019). Filling adeno-associated virus capsids: estimating success by cryo-electron microscopy. Human gene therapy, 30(12), 1449-1460.
  2. Gimpel, A. L., Katsikis, G., Sha, S., Maloney, A. J., Hong, M. S., Nguyen, T. N., ... & Braatz, R. D. (2021). Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Molecular Therapy-Methods & Clinical Development, 20, 740-754.

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