Advantages of optimized AAV harvest in fixed-bed bioreactors

Adeno-associated viruses (AAV) are popular viral vectors for gene therapy which can be explained by their

  • natural occurrence of several serotypes of AAVs with various surface tropisms due to which they can more selectively target specific tissues and cells;
  • ability to transduce dividing as well as nondividing cells with the potential for long-term transgene expression;
  • site-specific integration that allows to insert the therapeutic gene into a specific location in the genome;
  • replication-incompetence, resulting in the absence of disease associated with the wild-type virus;
  • feature of evoking relatively low immune response compared to retroviruses (e.g. LV) and

Certain therapeutic applications with AAVs require a high amount of viral vectors which in turns requires a highly effective, large-scale manufacturing process. Given the current progression of clinical studies with AAV, it will be difficult to meet the future market demand due to the known challenges of process scale-up with established technologies (STR, flatware). Next-generation fixed-bed bioreactors such as the scale-X™ bioreactors offer an alternative approach which not only facilitates process development and scale-up with suspension and adherent cells, but also enables an advanced harvest strategy with high product recovery, small volumes, reduced host cell impurities and increased full/empty capsid ratio to address time and capacity gaps.

Influencing parameters for AAV harvest

AAV are small, non-enveloped viruses that show relatively high stability compared to other viruses under standard bioprocessing conditions. However, AAVs are susceptible to sticking to hydrophobic surfaces and tend to aggregate in liquid phase, which both can cause product loss. Additionally, in a standard production process the AAVs can be found intra- and extracellularly, creating the need for cell lysis during the harvest step. To retrieve as much product as possible despite the hurdles mentioned, Univercells Technologies developed an optimized AAV harvest strategy in the scale-X bioreactor considering the
virus characteristics.

Figure 1. Parameters and their influences and limitations for
a successful AAV harvest.

Figure 1. Parameters and their influences and limitations for a successful AAV harvest.

The most well-known parameters that are often considered in standard harvest protocols are a combination of:

  • lysis detergent,
  • DNAse,
  • temperature,
  • addition of a stabilizer.

Yet, those protocols often neglect other essential factors such as:

  • buffer composition,
  • pH,
  • ionic strength of buffer.

A suitable buffer composition is key for DNAse activity as well as AAV stability. The right pH is required for optimal DNAse activity but also supports AAV stability and avoids their aggregation. Additionally, high ionic strength in the buffer is advantageous for AAV recovery but its inhibiting effect on the DNAse must be considered.

Unlike non-optimized protocols, the AAV harvest protocol of Univercells Technologies takes into account all parameters mentioned above resulting in significantly higher viral particle recovery. The combination of the reagents with optimal pH, ionic strength and temperature at the right time as well as the cell immobilization inside the fixed-bed play an important role in the successful harvest. Additionally, most of the cell debris, together with other impurities such as HCP and HC-DNA, are removed from the harvest due to the natural filter effect of the fixed-bed.

Advantages of optimized harvest protocol in the scale-X bioreactors

The harvest protocol developed by Univercells Technologies was applied in an AAV-2 production run
in scale-X hydro bioreactor using suspension HEK293F cells in serum free medium BalanCD (Learn more about growing serum-free, suspension HEK293 cells in a fixedbed bioreactor? Read more here. As a result, the harvest was highly concentrated as cells grew trapped at high concentration in a small volume and its HCP and HCDNA content was 5- and 10-fold reduced, while 98% of the total AAVs were recovered from the bioreactor.

If you are interested in using a scale-X bioreactor for AAV production or you would like to optimize your AAV harvest strategy for simplified scale-up and facilitated downstream, reach out to us.

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