HyClone™ boost expression supplement is a two-part, chemically defined, animal component–free formulation designed to improve transient adeno-associated virus (AAV) vector production in HEK 293 cells such as the ELEVECTA™ cell line from Cytiva. Optimized for use with HyClone prime expression and HyClone peak expression media, it is also compatible with other basal media supporting HEK 293 suspension cultures (Fig 1).
HyClone boost expression supplement represents a novel approach to enhance AAV vector productivity in the cell and gene therapy field.
Introduction
HEK293 cells are widely used to produce viral vectors used in cell and gene therapy. Viral vector customers are looking for solutions to improve productivity in their process which can be rapidly deployed to clinical manufacturing. We have developed a cell boost product for the HEK293 platform, HyClone boost expression supplement, which demonstrates improved AAV titer with prime expression medium using different HEK293 cell lines and across multiple AAV serotypes.
We used HyClone prime expression medium for a transient AAV process in two HEK293 cell lines, namely ELEVECTA transient, and Thermo VPC 2.0. Minimum virus titer improvement criteria were set to two-fold increase over no boost control. Evaluation included four AAV serotypes (2, 5, 8, 9) using both cell lines. We monitored percentage full capsids for any significant changes in full capsid with the optimal-performing supplement boost condition(s).
The workflow we used in this study is summarized in Figure 2.
Fig 1. HyClone boost expression supplement is available in liquid or powder form in pack sizes suitable for small-volume cell culture as well as large-scale bioprocessing applications.
MATERIALS AND METHODS
Cell culture and maintenance
HEK293 suspension cells (ELEVECTA transient cells or VPC 2.0 cells) were used for recombinant adeno‑associated virus (rAAV) production. We maintained cells in HyClone prime expression medium under standard suspension culture conditions with routine passaging. For optimal AAV production, we used cells with passage numbers between P4 and P15 .
Frozen cell stocks were thawed directly into prime expression medium and expanded for at least three passages prior to transfection. We monitored cell densities and viabilities throughout the process to ensure healthy growth.
Seeding and culture conditions
Three days prior to transfection, cells were seeded into shake flasks at a density of approximately 4–5 × 10⁵ cells/mL in prime expression medium. Cultures were maintained under shaking conditions appropriate for HEK293 suspension cells.
On the day of transfection (Day 3), cultures were adjusted to a target cell density of approximately 3.3 × 10⁶ cells/mL using fresh prime expression medium considering the volume of the 10% transfection complex for a final cell density of 3 × 10⁶ cells/mL after transfection.
Triple plasmid transfection
Recombinant AAV production was performed using a standard triple plasmid transient transfection approach. Cells were transfected using PEI‑MAX as the transfection reagent at a final cell density of 3 × 10⁶ cells/mL.
Plasmid DNA was added at a total concentration of 2 µg/mL (0.66 µg/10⁶ cells) for a 30 mL culture volume, consisting of a pAAV-Rep-Cap plasmid, helper plasmid, and gene of interest/green fluorescent protein (GOI/GFP)-plasmid at a 1:1:1 molar ratio. DNA–PEI-MAX complexes were prepared using a DNA‑to‑transfection reagent ratio of 1:2. Complexes were formed in prime expression medium at a complexation volume of 3 mL per 30 mL final culture volume and incubated for 15 min at room temperature prior to addition to the cell culture.
Supplement feeding
Performance evaluation experiments were done with hydration of boost expression a and b powder. pH and osmolality were adjusted according to the IFU. Approximately 18 to 24 h post‑transfection (Day 4), HyClone boost expression supplements were added to the cultures. Boost expression supplement a and supplement b were added at final concentrations of 10% (v/v) and 1% (v/v), respectively.
Note: It is important to note that boost expression supplements a and b should be added to the culture vessel as individual solutions and should not be mixed in advance as this will cause precipitation.
Harvest and cell lysis
We harvested cells three days post‑transfection (Day 6). Cultures were collected and subjected to cell lysis followed by nuclease (denarase/benzonase) enzyme treatment to digest residual host cell and plasmid DNA. Clarified lysates were aliquoted and stored at –80 °C until further analysis.
Analytical methods
We quantitated viral genome titers using droplet digital PCR (ddPCR). Viral particle and total capsid concentrations were determined using ELISA‑based assays. Results were used to assess improvements in AAV productivity and full capsid percentage following supplement addition.
Figure 2 describes the methods.
Fig 2. Transient triple transfection process for AAV production. The boost expression supplement is added only once 18 to 24 h after transfection.
Results
Incorporation of the HyClone boost expression supplement into the AAV production workflow resulted in a two- to nine-fold increase in viral genome titers compared to baseline values. This benefit was consistently attained across multiple serotypes (AAV2, AAV5, AAV8 and AAV9) and HEK 293-derived cell lines (ELEVECTA transient and VPC 2.0). Additionally, minimal impact on packaging percentages was observed.
The following graphs demonstrate the performance of the supplement (Fig 3–6) in ELEVECTA and VPC cell lines (Fig 7–10).
Results using ELEVECTA transient cells
Fig 3. ELEVECTA cell line AAV2 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated little lower VCD, but comparable viability compared to control on the day of harvest, and near six-fold increase in AAV2 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid post-transfection between control and supplement addition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Fig 4. ELEVECTA cell line AAV5 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated comparable VCD and viability to control on the day of harvest, and two-fold increase in AAV5 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
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Fig 5. ELEVECTA cell line AAV8 VCD, viability, titer, and cell specific productivity rate (n = 4): The supplement demonstrated comparable VCD and viability to control on the day of harvest, and near two-fold increase in AAV8 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Fig 6. ELEVECTA cell line AAV9 VCD, viability, titer, and cell specific productivity rate (n = 4): The supplement demonstrated comparable VCD and viability to control on the day of harvest, and near three-fold increase in AAV9 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Results using VPC 2.0 cells
Fig 7. VPC 2.0 cell line AAV2 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated significantly lower VCD, but comparable viability compared to control on the day of harvest, and nine-fold increase in AAV2 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates a modest lower full capsid percentage with supplementaddition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Fig 8. VPC 2.0 cell line AAV5 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated significantly lower VCD, but comparable viability compared to control on the day of harvest, and two-fold increase in AAV5 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Fig 9.VPC 2.0 cell line AAV8 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated lower VCD, but comparable viability compared to control on the day of harvest, and two-fold increase in AAV8 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell-specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Fig 10. VPC 2.0 cell line AAV9 VCD, viability, titer, and cell-specific productivity rate (n = 4): The supplement demonstrated significantly lower VCD, but comparable viability compared to control on the day of harvest, and over 2.5-fold increase in AAV9 titer compared to the control. Full capsid percentage shown in the secondary Y-axis of the middle graph demonstrates comparable full capsid between control and supplement addition. Cell-specific productivity is shown in the right graph. The error bars represent SD, and p ≤ 0.05 between control and boost expression supplement.
Conclusion
HyClone boost expression supplement represents a novel approach to enhance AAV productivity within the cell and gene therapy field. Demonstrated performance across multiple cell lines, and serotypes underscores the cell boost’s utility as a robust tool for advancing viral vector development and large-scale manufacturing.
- Ready-to-use HyClone boost expression supplement removes the need for additional culture materials and makes the virus production process more robust. The supplement is compatible with the current process of rAAV production using Cytiva base media.
- The supplement consistently delivers a two-fold or more improvement in transient AAV viral genome titers across multiple serotypes in both the ELEVECTA and VPC 2.0 cell lines.
- The titer enhancement was achieved without compromising product quality, as full capsid percentages remained comparable compared to those of no control across all tested serotypes and cell lines.
ACKNOWLEDGMENTS
We would like to thank the entire Cytiva team across Genomic Medicine (GenMed) and Cell Culture Media (CCM) business units for giving helpful and constructive feedback while writing this application note.