The baculovirus expression vector system (BEVS) is widely used to produce recombinant proteins, virus-like particles (VLPs), and viral vectors such as recombinant adeno-associated virus (rAAV) (1,2). As processes scale, consistent cell growth, predictable infection, and reliable productivity are critical.
This study evaluates HyClone™ CD BEVS complete medium, a chemically defined (CD), protein-free, animal-derived component-free formulation, across Sf9 workflows from shake flasks to stirred-tank bioreactors.
The medium supports reproducible and scalable BEVS processes through:
- Rapid cell growth and high viable cell densities (> 2 × 107 cells/mL).
- High pre-infection viability (> 95%).
- Consistent protein expression across scales.
Introduction
The insect cell (IC)–BEVS is widely used in vaccine and therapeutic manufacturing. Compared with mammalian systems such as Chinese hamster ovary (CHO) cells, BEVS enables faster recombinant protein production without the need for stable cell line development (3). Insect cell cultures also operate under simpler conditions, including lower temperature, minimal CO₂ requirements, and tolerance to a broader range of culture conditions.
As processes scale, the focus shifts from speed to reproducibility and product consistency. Culture medium plays a central role in achieving these outcomes by influencing cell growth, infection efficiency, metabolic stability, and productivity. While traditional insect media often rely on hydrolysates and other undefined components, CD and animal-derived component-free formulations provide improved consistency, process control, and regulatory alignment.
HyClone CD BEVS complete medium is designed to support consistent insect cell culture performance from early development through manufacturing scale.
Materials and methods
We cultured Sf9 cells in HyClone CD BEVS complete medium under standard conditions in shake flasks and stirred-tank bioreactors. Growth kinetics, viability, and infection performance were evaluated across multiple passages and scales.
Cultures were infected with a baculovirus expression vector encoding recombinant IgG at a defined multiplicity of infection (MOI). After the infection, we monitored productivity, cell diameter, and viability.
All the comparisons we performed were done against commonly used insect cell culture media under equivalent process conditions.
Results
Growth kinetics and culture consistency
HyClone CD BEVS complete medium supported consistent growth across passages, with an average population doubling time (PDT) of 24 ± 1.9 h, showing low variability (Fig 1).
Relative to alternative CD media with hydrolysates, these cultures showed faster early growth, higher peak viable cell densities, significantly lower PDT (< 0.0001), and stable late-stage viability.
These results indicate stable metabolic behavior and support predictable culture expansion and process timing.
Fig 1. PDT profiles of Sf9 cells in two media, representing multiple lots with QC assessments across manufacturing sites. Average PDT was 33.6 ± 7.5 h with HyClone SFM4Insect (n = 22) versus 24.0 ± 1.9 h with HyClone CD BEVS Complete (n = 6), indicating a shorter PDT and lower lot-to-lot variability with CD BEVS Complete.
Cell growth performance across scales
Using HyClone CD BEVS complete medium, we observed consistent growth performance in both shake flasks and stirred-tank bioreactors. Peak viable cell density exceeded 20 × 106 cells/mL in both formats, with viability above 95% throughout the growth phase (Fig 2). In addition, the growth profiles were comparable across the formats, with the bioreactors showing slightly higher peak densities, likely due to improved environmental control.
Fig 2. Sf9 cells in HyClone CD BEVS complete medium reached > 20 × 106 viable cells/mL and consistent high cell viability (> 95%) through Day 9 at both culture scales. Shake flask cultures were performed in triplicate (n = 3) against a single bioreactor run (n = 1) with technical replicates.
Baculovirus infection performance
Following infection with BEVS, the cultures showed synchronized growth arrest and consistent post-infection behavior. We observed cell viability declining and cell diameter increasing, indicating infection progression (Fig 3).
Overall the infection behavior and productivity remained consistent. However, bioreactor cultures showed a modest delay in production kinetics relative to shake flasks, reflecting differences in process dynamics.
Fig 3. IC-BEVS infection kinetics (MOI 0.1) and culture response. Viability, cell diameter, and growth arrest profiles confirm consistent infection behavior across culture formats.
Productivity and protein expression performance
HyClone CD BEVS complete medium supported efficient production of recombinant IgG protein, with IgG titers increasing steadily through 168 h post-infection in both culture formats, with shake flask cultures reaching a slightly higher final titer than the 2 L stirred-tank bioreactor (Fig 4). This modest offset is consistent with the slightly slower production kinetics observed in the bioreactor.
Sustained cell viability through the post-infection phase (Fig 3) supported this extended production window. Direct benchmarking of IgG productivity against commonly used insect cell culture media is presented in the following section (Fig 6).
Fig 4. Recombinant IgG protein production following infection (MOI 0.1) with an in-house baculovirus-expressing recombinant human IgG. IgG titers increased steadily through 168 h post-infection in both shake flask and 2 L stirred-tank bioreactor cultures in HyClone CD BEVS complete medium, with comparable expression kinetics across the two formats.
Benchmark comparison across media
We performed direct comparisons between HyClone CD BEVS complete medium and commonly used insect cell culture media. Comparator media showed slower growth rates, earlier viability decline, and reduced overall performance (Fig 5). HyClone CD BEVS complete medium showed clear performance advantages with faster growth rates (22.2 ± 2.3 h PDT), higher peak VCD (2.0 × 107 ± 0.66 cells/mL), and improved late-stage viability.
Fig 5. Batch growth promotion study (n = 3 for each condition) seeded at 0.75 × 106 viable cells/mL comparing HyClone CD BEVS complete medium with commonly used insect culture media. Dunnett’s test demonstrated significantly improved growth relative to all other conditions (p < 0.0001; viability p < 0.01).
IgG expression performance
We performed protein expression studies to analyze expression kinetics and productivity. HyClone CD BEVS complete medium showed higher cumulative IgG production and faster production kinetics compared to benchmark media, with consistent results across replicates analyzed by one-way ANOVA with Dunnett’s multiple comparisons test against HyClone CD BEVS complete medium (n = 3, p < 0.0001) (Fig 6).
Fig 6. IC-BEVS IgG expression across different media. All conditions were infected at 3.0 × 10⁶ viable cells/mL with an MOI of 0.1. Data represent the mean of three replicate cultures (n = 3); differences between media were assessed by one-way ANOVA with Dunnett’s multiple comparisons test against HyClone CD BEVS complete medium (p < 0.0001).
Scalability in single-use systems
Insect media have traditionally been supplied in small containers or dry formats, as large liquid formats have been limited by lipophilic components, which can absorb to single-use bag films and introduce variability in effective concentrations and culture performance.
The chemically defined formulation of HyClone CD BEVS complete medium minimizes component adsorption, enabling stable and scalable liquid formats in single-use systems. This allows delivery in dry powder media (DPM), ready-to-use 1000 mL PETG bottles, and 1, 10, 20, and 200 L bioprocess bags, showing consistent performance and scalability across formats (Fig 7).
Fig 7. Consistent IgG expression performance across single-use bag formats post-infection. IgG titers generated in HyClone CD BEVS complete medium across 10, 20, and 200 L single-use bioprocess bags exhibit minimal variability, with no statistically significant differences observed between scales (n = 3), (ANOVA p < 0.001).
Discussion
In this study, HyClone CD BEVS complete medium consistently supported rapid Sf9 cell growth, high viability, efficient baculovirus infection, and robust recombinant protein production across multiple culture formats. The medium achieved faster growth rates and higher cell densities than benchmark insect media, providing a strong foundation for productive BEVS workflows.
These growth advantages complemented higher recombinant IgG titers and faster production kinetics following infection. Consistent infection behaviour and protein expression across shake flasks and stirred-tank bioreactors demonstrate that performance is maintained during scale-up, reducing development risk when transferring processes to larger manufacturing environments.
The medium also showed consistent performance across single-use bioprocess bag formats, supporting its use from process development through commercial manufacturing. Together, these findings indicate that HyClone CD BEVS complete medium can help simplify scale-up while improving productivity and process consistency.
Conclusions
Our study demonstrates three key benefits of HyClone CD BEVS complete medium for BEVS-based protein production:
- Accelerate cell expansion
Rapid growth kinetics, high viable cell densities, and sustained viability support efficient seed train expansion and predictable process performance. - Increase recombinant protein productivity
Higher IgG titers and faster production kinetics compared with benchmark media support improved process output. - Enable scalable manufacturing
Consistent performance from shake flasks to bioreactors and across single-use bag formats supports reliable scale-up and technology transfer.
References
- Hong M, Li T, Xue W, et al. Genetic engineering of baculovirus-insect cell system to improve protein production. Front Bioeng Biotechnol. 2022;10:994743. doi:10.3389/fbioe.2022.994743
- Liu S, Li J, Peraramelli S, et al. Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells. Mol Ther. 2024;32(1):74-83. doi:10.1016/j.ymthe.2023.11.022
- Jarvis DL. Developing baculovirus-insect cell expression systems for recombinant glycoprotein production. Virology. 2003;310(1):1-7. doi:10.1016/S0042-6822(03)00120-X
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