In recent years, adeno-associated virus (AAV) vectors have emerged as the leading delivery platform in clinical gene therapy. This is due to their broad tissue tropism, low immunogenicity, and stable long-term expression, which make AAV gene therapy applicable to a broad range of indications. Since the approval of the first AAV gene therapy (Glybera®) in 2012, several further AAV gene therapies have received market authorization, including products for the treatment of inherited blindness, hemophilia, and spinal muscular atrophy. Yet, creating a safe and effective AAV gene therapy remains difficult, not least due to the complexity of the purification process. This article discusses key challenges for AAV purification, before delving into the importance of monitoring AAV titers and host cell nucleic acid contamination. It also highlights some novel products for performing these types of measurements.
A typical AAV production workflow involves multiple steps. First, the viral producer cells (usually a mammalian cell line such as HEK293) are expanded in culture, prior to their transfection with one or more AAV-encoding plasmids. Besides the cell culture conditions, factors requiring optimization include the plasmid concentration, choice of transfection reagent, and incubation time, all of which must be carefully assessed for their impact on transfection efficiency and AAV yields. Following transfection, the cells are lysed and the supernatant is harvested for AAV purification. Batches must then be concentrated to produce an effective dose, often by as much as 100 to 10,000 times. During this process, a variety of critical quality attributes (CQAs) is measured to support product release, including viral titers and the concentration of residual host cell nucleic acid.
Purification is a critical step in the AAV production workflow, serving to ensure that the resultant vectors have a good clinical safety and efficacy profile. However, AAV purification is notoriously difficult, often necessitating that methods be developed on a serotype-dependent basis to accommodate divergent AAV characteristics without compromising yields. Other challenges for AAV purification center on improving efficiency (many established methods involve multiple labor-intensive steps that risk product loss), minimizing the impurity burden associated with cell lysis and clarification, and scaling up operations. In addition, the high cost of many purification methods can limit the development and commercialization of AAV.
Put simply, the viral titer is the physical concentration of viral particles, which is commonly reported as the number of capsids per mL. It is used during optimization of the AAV production workflow, and for calculating AAV yields once the process is up and running, as well as represents a prerequisite for carrying out preclinical and clinical research. Ultimately, the viral titer functions as a CQA for AAV gene therapy to ensure that patients receive an effective dose. Other types of titers that are routinely measured during AAV production include the infectious titer, which is the concentration of viral particles capable of transducing cells, and the ratio of full to empty viral capsids, which is influenced by the experimental conditions.
The presence of host cell nucleic acids in AAV gene therapies can cause tumorigenesis or immunotoxicity, as well as may bring about infectivity, making it essential that such product-related impurities are removed. An established way of achieving this is to use one of the genetically engineered broad-spectrum nucleases derived from the Gram-negative bacterium Serratia marcescens, which can degrade all forms of DNA and RNA, including single-stranded, double-stranded, linear, circular, native, and denatured nucleic acids. Researchers should, however, exercise caution when working with these types of products, as many of them lose activity in the high salt concentrations (400–500 mM) that are often used to increase purification yields.
With AAV gene therapy still in its infancy, novel products are vital for it to reach its full potential. With that in mind, ACROBiosystems has recently launched its AAV3 Titration ELISA Kit and AAV5 Titration ELISA Kit, both of which benefit from highly specific detection (no cross-reaction with other AAV serotypes), high sensitivity (1.70E+08 capsids/mL), and a broad detection range (1.70E+08 to 1.09E+10 capsids/mL), with results available in just 2 hours 20 minutes. These products are complemented with a new GMP-grade high-salt tolerant all-purpose nuclease, GMP Salt Active GENIUS™ Nuclease, which maintains high enzyme activity under 0–500 mM NaCl conditions.
To learn more about how ACROBiosystems can support your AAV gene therapy project, visit acrobiosystems.com