Towards a scalable bioprocess for rAAV production using a HeLa stable cell line

Abstract

The majority of recombinant adeno‐associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale‐up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa‐based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3‐based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3‐4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity‐based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time‐efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System

“…In general, the 100 kDa MWCO PES Sartocon ® Slice 200 flat sheet cassette outperformed the hollow fiber in most parameters evaluated (i.e., total particle recovery, total protein removal, permeate fluxes) (Table 2). The DNA removal was 1.8-fold higher in the hollow fiber when compared with the flat sheet cassette; however, this impurity clearance can be further increased with the later additional downstream purification steps such as affinity capture chromatography [19,20].…”

Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV

“…rAAV2 particles were generated in HeLaS3-rAAV2 producer (clone H32D2) cell line, purified and stored as described before (Escandell et al, 2023). Samples were boiled at 56 °C for 10 min prior to transduction, to eliminate any possible Adv contaminants.…”

High‐yield recombinant adeno‐associated viral vector production by multivariate optimization of bioprocess and transfection conditions

“…Several techniques exist for producing rAAVs, including triple transfection of human embryonic kidney (HEK) 293 cells, herpes simplex virus‐mediated infection of HEK293 cells, baculovirus‐based infection of Spodoptera frugiperda (Sf) 9 cells, and the use of stable producer cell lines 14–17 . The most commonly used technique for rAAV production is transient triple transfection of HEK293 cells 18–20 .…”