Using recombinant adeno-associated viruses (rAAV) to deliver therapeutic genes has emerged as a viable treatment option for a range of diseases, as demonstrated by the approval of multiple rAAV-based in vivo gene therapies. However, developing scalable, high-titer GMP-compatible production processes remains a significant challenge across the industry.
Previous work has shown that the ELEVECTA™ transient cell line grows in a single-cell suspension, reaches high cell density, and supports high-titer rAAV production for multiple serotypes. It also significantly reduces levels of host cell DNA (hcDNA), including encapsidated, compared to another commercially available HEK293 cell line (1).
Building on this work, we scaled up the previously established process to 50 and 200 L in the Xcellerex™ X-platform single-use bioreactor. Scalability was demonstrated up to 200 L with comparable cell culture performance and titer.
Together, the next-generation Xcellerex™ X-platform single-use bioreactor, the ELEVECTA™ transient cell line and optimized, GMP-grade, offer a robust solution for scalable, high-titer manufacturing of high-quality rAAV products—from R&D through to commercial manufacturing.
Introduction
Our ELEVECTA™ transient cell line is a HEK293-derived suspension cell line that harnesses the power of transient transfection to deliver high titers across multiple rAAV serotypes. In this application note, we demonstrate that the cell line is robust and scalable, performing consistently from 15 mL microbioreactors up to 50 L and 200 L Xcellerex™ X-platform bioreactors.
Additionally, the production process also delivered comparable results across two bioreactor systems: Xcellerex™ X-platform and Xcellerex™ XDR bioreactors, highlighting its transferability across bioreactor platforms. To support this process, we developed an optimized, GMP-grade HyClone™ prime expression medium offering that enables high cell density and efficient transfection processes.
The ELEVECTA™ transient cell line showcases a novel feature: a significant reduction of host cell DNA (hcDNA), including encapsidated hcDNA, in the final virion product. This reduction is achieved through inactivation of DFFB (DNA fragmentation factor subunit beta), which reduces DNA fragmentation during cell death. Residual DNA is a process-related impurity that includes both free and encapsidated DNA impurities (such as plasmid DNA or hcDNA) and may have adverse effects on product quality and safety, according to FDA guidance (2).
In two 50 L runs, the ELEVECTA™ transient cell line demonstrated an average of 17.7 nanograms (ng) hcDNA per 1014 vg (viral genomes). By using the ELEVECTA™ transient cell line, rAAV processes could achieve significant reduction in packaged hcDNA–improving product quality and safety.
Materials and methods
Table 1. Plasmids and reagents used in this study
| Description | Type | Supplier | Size (kb) |
| pALD-AAV9 | Plasmid | Aldevron | 6.083 |
| pALD-HELP | Plasmid | Aldevron | 11.584 |
| pAAV-LUC-GFP | Plasmid | Aldevron | 8.502 |
| ELEVECTA™ transient cell line | Cell line | Cytiva | N/A |
| HyClone™ prime expression medium | Reagent | Cytiva | N/A |
| RevIT AAV enhancer | Reagent | MirusBio | N/A |
| PEI MAX | Reagent | Polysciences | N/A |
| FreeStyle 293-F Cells | Cell line | Thermo Fisher | N/A |
| Expi293 expression medium | Reagent | Thermo Fisher | N/A |
Microbioreactor testing
For microbioreactor testing, we used the Ambr 15 microbioreactor system (Sartorius). We performed three independent experimental runs with luciferase: green fluorescent protein (LUC-GFP) as transgene with duplicates (total n = 6).
We seeded the ELEVECTA™ transient cells at 4.0 × 105 viable cells per mL in HyClone™ prime expression medium without centrifugation, in 13.7 mL working volume. Three days after seeding, we diluted the cultures to obtain 3.3 × 106 viable cells in 12.9 mL. We then added the transfection mixture to achieve a final cell density of 3 × 106 viable cells in 14 mL production volume.
RevIT AAV enhancer (Mirus Bio) was added directly to specific cultures immediately following complex addition, at a 1:1000 ratio. We harvested the bioreactors 72 h post-transfection. Microbioreactor parameters and transfection conditions are listed in Table 2.
Table 2. Microbioreactor setpoints and parameters
| Parameter | Value |
| Production volume (mL) | 14 |
| pH range | 7.3 ± 0.10 (post-transfection), PID control |
| Dissolved oxygen (%) | 40 (PID control, O2 on demand) |
| Temperature (°C) | 37 |
| Sparger airflow (VVM)1 | 0.013 |
| RevIT AAV enhancer | Reagent |
| Agitation (W/m3) | 120 |
| Growth and production medium | HyClone™ prime expression medium |
| Base | 1 M sodium bicarbonate |
| Transfection | |
| Plasmids | pALD-HELP, pAAV-LUC-GFP, pALD-AAV9 |
| Plasmid ratio | 1:1:1 molar ratio |
| Transfection reagent | PEI MAX |
| Complexation volume (%) | 10 |
| Target transfection cell density (cells/mL) | 3.0 × 106 |
| Plasmid DNA/cell | 0.67 μg DNA/106 cells |
| Plasmid DNA to transfection reagent ratio | 1:4 |
| Complexation medium | HyClone™ prime expression medium |
| Complexation time (min) | 15 to 20 |
| Day of transfection | 3 |
| Production phase/harvest | 3 d post-transfection |
| Lysis buffer | 10×: D-PBS with 10% detergent |
| Endonuclease | Denarase (C-Lecta), 60 U/mL |
| Lysis + endonuclease time (min) | 30 |
| Lysis + endonuclease temperature (°C) | 37 |
1 VVM: Volumetric gas flow per minute
Bioreactor scale-up
We performed scalability studies using Xcellerex™ X-platform X-50 and X-200 bioreactors and the Xcellerex™ XDR-50 bioreactor. ELEVECTA™ transient cell line cells were first expanded in shake flasks and a ReadyToProcess WAVE™ 25 rocking bioreactor, then inoculated at a cell density of approximately 0.25 × 106 cells/mL. Starting volumes were 40 L for the 50 L bioreactors and 160 L for the 200 L bioreactor.
We transfected the cultures three days post-inoculation, when they reached the target density of 3.3 × 106 cells/mL. RevIT enhancer was added to the diluted DNA prior to complexation, at a 1:1000 ratio. We then added transfection complex solution to the bioreactors, resulting in a final working volume of approximately 45 L for the 50 L bioreactors, and 180 L for the 200 L bioreactor, reaching target transfection final cell density of 3 × 106 cells/mL.
Harvest was performed 72 h post-transfection, following the addition of endonuclease and 10× lysis buffer. Table 3 outlines the bioreactor setpoints and parameters.
Table 3. Bioreactor setpoints and parameters
| Parameter | Value | |
| pH range | 7.3, +/- 0.1 (PID control) | |
| Dissolved oxygen (%) | 40 (PID control, O2 on demand) | |
| Temperature (°C) | 37 | |
| Sparger airflow (VVM)1 | 0.02 | |
| CO2 maximum gas flow rates (L/min) | 50 L | 0.5 |
| 200 L | 2.0 | |
| O2 maximum gas flow rates (L/min) (W/m3) | 50 L | 0.5 |
| 200 L | 0.9 | |
| Agitation (W/m3) | 50 | |
| Growth and production medium | HyClone™ prime expression medium | |
| Base | 1 M sodium bicarbonate | |
| Transfection | ||
| Plasmids | pALD-HELP, pAAV-LUC-GFP, pALD-AAV9 | |
| Plasmid ratio | 1:1:1 molar ratio | |
| Transfection reagent | PEI MAX | |
| Complexation volume (%) | 10% of total volume post-transfection | |
| Target transfection cell density (cells/mL) | 3.0 × 106 | |
| Plasmid DNA/cell | 0.67 μg DNA/106 cells | |
| Plasmid DNA to transfection reagent ratio | 1:2 | |
| Complexation medium | HyClone™ prime expression medium | |
| Complexation time (min) | 15 to 20 | |
| Day of transfection | 3 | |
| Production phase/harvest | 3 d post-transfection | |
| Lysis buffer | 110× buffer formulation: 20 mM MgCl2, 10% Tween 20, 10 mM Tris | |
| Endonuclease | Denarase (C-Lecta), 25 U/mL | |
| Lysis + endonuclease time (min) | 60 | |
| Lysis + endonuclease temperature (°C) | 37 | |
1 VVM: Volumetric gas flow per minute
Purification
We clarified lysate pools using a three-stage filtration process, consisting of PDP8 and Bio10 depth filters, followed by Supor™ EKV membrane in Kleenpak™ Nova capsules run in series. Clarified pools were concentrated to a target 10-fold volumetric concentration factor, using a Cadence™ single-use 100Kda Omega TFF module.
Affinity chromatography
We performed affinity chromatography using an ÄKTA™ avant 150 chromatography system. Clarified lysate was loaded onto a commercially available prepacked 5 mL column. The membrane was equilibrated over 10 column volumes (CV) with 50 mM Tris, 0.5 M NaCl, pH 8.0.
Prior to loading, we spiked the feed with EDTA to a final concentration of 10 mM to help prevent precipitation. After sample loading, we washed the column with 10 CV of 50 mM Tris, 0.5 M NaCl, pH 8.0, followed by 10 CV of 50 mM Tris, pH 8.0. Elution was performed using 100 mM glycine, pH 2.5, over 5 CV. The eluate was neutralized with bis-Tris propane (BTP), pH 9.0 at a ratio of 1:5 (neutralizing buffer: eluate).
Analytical methods
Viral genome titer measurements
- We measured viral genome titers using quantitative PCR (qPCR). Samples were pretreated with DNase and proteinase K digestion, followed by dilution. We quantitated using a primer and probe set from Integrated DNA Technologies, targeting the SV40 amplicon of rAAV, due to the presence of the SV40 polyadenylation signal.
Capsid titer measurement
- Intact capsid titers were determined using the PROGEN PRAAV9 quantitative ELISA kit, following the manufacturer’s instructions. This sandwich ELISA method enables accurate quantitation of rAAV particles.
hcDNA analysis
- We quantitated the hcDNA using the resDNASEQ quantitative HEK293 DNA kit (Applied Biosystems), a qPCR-based method. Sample preparation was performed using either the HighPure nucleic acid kit (Roche) or the PrepSEQ™ residual DNA sample preparation kit (Applied Biosystems), both of which include proteinase K treatment to release encapsidated hcDNA. We used all kits according to the manufacturers’ protocols.
Experimental work in Ambr15 was performed from Nov 2023 to March 2024 and data is held at Cytiva Cologne. Experimental work in X-platform, XDR, and STR was performed from Dec 2024 to March 2025 and data is held at Cytiva Westborough and Cytiva Uppsala.
Results
Significant reduction in encapsidated hcDNA with the ELEVECTA™ transient cell line
To help us understand the ELEVECTA™ transient cell line performance, we quantitated hcDNA levels (including encapsidated hcDNA) and compared to a reference FreeStyle 293-F control cell line.
Levels of hcDNA found in affinity-enriched rAAVs derived from the ELEVECTA™ transient cell line at the 50 L scale averaged 17.7 ng per 1014 vg and were similar to results achieved at 3 L and 10 L scales (Fig 1).
3 L and 10 L conditions were run in triplicate and the 50 L condition was run in duplicate. Statistical comparison of the 3 L conditions was performed using ANOVA, with p-value of 0.009. Full protocols and data are available upon request.
Fig 1. Average hcDNA levels (including encapsidated) after purification of viral vectors for (A) ELEVECTA™ transient cell line (n = 3) and 293-F cell line (n = 3) at 3 L scale, and (B) for ELEVECTA™ transient cell line at 3 L and 10 L (both n = 3) and 50 L (n = 2). Data shows mean values ± S.D. Two asterisks (**) represent a p-value of < 0.01.
Process scalability assessment
To evaluate process scalability, we carried out rAAV9 production with RevIT AAV Enhancer addition in 50 and 200 L stirred-tank bioreactors and compared to 15 mL microbioreactor performance. Across all production scales, we observed robust and consistent performance in terms of growth, viability, and titer (Fig 2).
Cultures in the Ambr 15 mL microbioreactor were seeded at 0.4 × 10⁶ cells/mL and diluted to 3.3 × 10⁶ cells/mL on day 3 prior to transfection. In contrast, we seeded the Xcellerex™ 50 and 200 L cultures at 0.25 × 10⁶ cells/mL to reach the same transfection density without dilution. Slightly faster and more consistent growth was observed in Xcellerex™ X-platform and XDR bioreactors, potentially due to differences in scaling parameters or seeding density.
Fig 2. (A) Viable cell density (VCD) and viability data of ELEVECTA™ transient cells in four bioreactor formats: Ambr 15 microbioreactor (n = 6), and the Xcellerex™ X-50 (n = 3), X-200 (n = 1), and XDR-50 (n = 2) bioreactors with RevIT enhancer. (B) Doubling time in hours from day 0 to day 3 for the bioreactor cultures. Data shows mean values ± S.D.
We harvested the bioreactors after a three-day production phase, where cells were lysed and analyzed for genomic and capsid titers via qPCR and ELISA, respectively. As shown in Figure 3, we found that genomic titers were consistent across scales:
- 2.9 × 10¹¹ vg/mL at 37% full capsids (15 mL)
- 2.9 × 10¹¹ vg/mL at 27% full capsids (X-50)
- 2.0 × 10¹¹ vg/mL at 26% full capsids (X-200)
- 2.3 × 10¹¹ vg/mL at 23% full capsids (XDR-50)
These results were highly comparable, confirming successful scale-up and process robustness.
Fig 3. Comparison of rAAV9 titers and capsid fullness across 15 mL Ambr (n = 6), X-50 (n = 3), X-200 (n = 1), and XDR-50 (n = 2) bioreactors with RevIT AAV enhancer. Titers were analyzed by qPCR and ELISA. Data shows mean values ± S.D.
Discussion
The ELEVECTA™ transient cell line is a robust, fast-growing, easily handled cell line delivering relevant titers and percent full capsids, while exhibiting low encapsidated hcDNA. We demonstrated scalability up to 50 and 200 L working volumes, achieving titers of 2.9 × 1011 rAAV9 vg/mL and 27 % full capsids on average in the 50 L X-platform bioreactor.
With increasing regulatory pressure to reduce encapsidated hcDNA in rAAV virions to below 10 ng per dose (3), our ELEVECTA™ transient cell line offers a promising path to reducing packaged impurities, supporting improve product safety and quality.
Conclusion and summary
In this application note, we demonstrate that the ELEVECTA™ transient cell line, in combination with HyClone™ Prime expression medium, provides a robust and scalable rAAV production process. Some key outcomes include:
- Low residual hcDNA, including encapsidated, (17.7 ng/1014 vg) for rAAV9 at 50 L scale.
- Reproducible and consistent titer across multiple scales and bioreactor systems.
- rAAV9 titer > 2.0 × 1011 vg/mL and > 23% full capsids across 15 mL, 50 L, and 200 L scales.
- A streamlined process, yielding high quality vectors, from development to manufacturing.
References
- Application note: novel transient cell line to enable high yield and scalable rAAV production with low encapsidated host cell DNA; https://cdn.cytivalifesciences.com/api/public/content/7b_M5iXXRHWOGlM
- U.S. Department of Health and Human Services, Food and Drug Administration. Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs): Guidance for Industry. 2020;29-30.
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