“…Although the AAV titers obtained in batch cultures are within reported ranges for this production system (extending from 0.1–3.58 × 10 4 VG/cell) ( Blessing et al, 2019 ; Guan et al, 2022 ), these were considerably lower (up to 3−fold) than the ones obtained with perfusion-mocking. This set of experiments shows the potential advantages of implementing perfusion methodologies and corroborates other reported studies on the subject ( Vázquez-Ramírez et al, 2018 ; Fernandes et al, 2021 ; Escandell et al, 2022 ). Nevertheless, some optimization is still possible through 1) optimizing medium exchange rates to sustain VCC TOT of 10 × 10 6 cells/mL and balance the nutrient consumption associated with doubling the cell concentration after transfection ( Fernandes et al, 2021 ), 2) exploring different plasmid ratios, proportions of total plasmid DNA and transfection reagent per cell ( Chahal et al, 2014 ; Meade et al, 2021 ; Wosnitzka et al, 2021 .…”
Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector’s overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.
“…Although the AAV titers obtained in batch cultures are within reported ranges for this production system (extending from 0.1–3.58 × 10 4 VG/cell) ( Blessing et al, 2019 ; Guan et al, 2022 ), these were considerably lower (up to 3−fold) than the ones obtained with perfusion-mocking. This set of experiments shows the potential advantages of implementing perfusion methodologies and corroborates other reported studies on the subject ( Vázquez-Ramírez et al, 2018 ; Fernandes et al, 2021 ; Escandell et al, 2022 ). Nevertheless, some optimization is still possible through 1) optimizing medium exchange rates to sustain VCC TOT of 10 × 10 6 cells/mL and balance the nutrient consumption associated with doubling the cell concentration after transfection ( Fernandes et al, 2021 ), 2) exploring different plasmid ratios, proportions of total plasmid DNA and transfection reagent per cell ( Chahal et al, 2014 ; Meade et al, 2021 ; Wosnitzka et al, 2021 .…”
Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector’s overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.
Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.
“…As an example, rAAV vectors were employed as a delivery system in 39% of all gene therapy clinical studies in the United Kingdom in 2021, with this number increasing to 90% in in vivo gene therapy clinical trials (Catapult report, 2021). However, the doses required for systemic treatments in clinical trials, such as those for Duchenne muscular dystrophy, are beyond the current capabilities of Chemistry, Manufacture, and Control (CMC) if the commercial production (reviewed in Escandell et al, 2022). The primary manufacturing bottleneck is the use of transient production platforms, mainly using the HEK293 cell line (reviewed in (Escandell et al, 2022).…”
Section: Introductionmentioning
confidence: 99%
“…However, the doses required for systemic treatments in clinical trials, such as those for Duchenne muscular dystrophy, are beyond the current capabilities of Chemistry, Manufacture, and Control (CMC) if the commercial production (reviewed in Escandell et al, 2022). The primary manufacturing bottleneck is the use of transient production platforms, mainly using the HEK293 cell line (reviewed in (Escandell et al, 2022). While this technology seems cost-effective in delivering the rAAV product quantity needed for phase I/II clinical trials and/or nonsystemic dosages, it isn't viable at large scale since it lacks robustness and due to high cost of raw materials, such as Good Manufacturing Practices grade plasmid DNA.…”
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.
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