By Juliana Coronel, Aishwarya Patil, Ahmad Al-Dali, Thomas Braß, Nicole Faust, and Silke Wissing, CEVEC Pharmaceuticals GmbH, Cologne, Germany

 

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Recombinant adeno-associated virus (rAAV) is currently used as vector in about 250 clinical trials for gene therapy (Bulcha et al., 2021). Nonetheless, there are still several challenges concerning the production process. Due to limitations of the production platforms, such as manufacturing scale and viable cell densities, the yield of rAAV is often insufficient to meet later market demands.

This challenge was successfully addressed with an intensified process in perfusion mode using CEVEC’s proprietary stable producer cells from the ELEVECTA® platform. The platform consists of human suspension cells with stable integration of all components necessary to produce AAV: adenovirus helper functions, AAV replicase and capsid genes, as well as the gene of interest (GOI). Production in this system is induced via doxycycline. The power of this approach was demonstrated with the production of an AAV8 vector with GFP (GOI) by a proof-of-concept clone.

Perfusion bioprocessing has become more popular in recent years for several applications. Perfusion mode means continuous processing with cell retention within a bioreactor, enabling higher cell densities and the benefit of exchanging the spent medium with fresh medium thereby reducing the concentration of byproducts inside the bioreactor. The employed perfusion set-up consisted of a lab-scale stirred tank bioreactor connected to an ATF-2 system. After an initial cell growth phase in batch mode, perfusion was initiated to enable growth to high cell density. AAV production was induced upon reaching the targeted viable cell density, and perfusion mode was maintained during the production phase (Figure 1). The cell suspension was harvested from the retentate at 5 days post-induction.

CEVEC Fig1
Figure 1: Cultivation of ELEVECTA® producer cell line in stirred-tank bioreactor for stable rAAV production. Comparison between ATF-based perfusion and conventional batch processes. Viable cell density (VCD) and viability. Two independent runs in perfusion mode and two runs in batch mode were performed. In the ATF perfusion process, perfusion was initiated 3 days after inoculation and production of rAAV was induced after 7 days (dashed vertical line). In the batch process, production of rAAV was induced 3 days after inoculation (dashed vertical line).

The perfusion-based production process led to high viral genome (1015 vg/L) and capsid titers (Figure 2-A). Compared to the reference process in batch mode (n=2), the volumetric production of AAV determined by qPCR was 40-fold higher in perfusion mode (n=2), whereas the VCD at time of induction was 5-fold higher. The cell-specific yield (vg/cell) in perfusion mode was thus 8-folder higher. It is noteworthy that the perfusion process with a fully stable AAV producer cell line also resulted in high percentages of full particles (30–40 %) (Figure 2-B).

CEVEC Fig2
Figure 2: Production of rAAV (cell suspension samples) in perfusion mode shown at different time points after induction. A) AAV8 genome titers determined by qPCR. Capsid titers determined by ELISA. B) Percentage of full capsids (ratio of genome titer to capsid titer).

Taking the scalability of the ATF system and bioreactors into account, the described approach using a stable helper-virus-free system in perfusion offers unique possibilities for large-scale production of  high-titer and high-quality rAAV.

 

Reference
Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct. Target. Ther. 2021 Feb 8; 6(1): 53. doi: 10.1038/s41392-021-00487-6.