Advances in Insect Cell-Expression Systems

Scientist pipetting biological samples of cells into a 96-well microplate. Researcher experimenting with a diagnostic plate in wells with culture medium | Image credit: ©angellodeco/Stock.Adobe.com

Successful production of recombinant proteins and viruses requires use of the optimum expression system for the target product. Mammalian cell culture predominates due to its ability to produce biomolecules with post-translational modifications that closely match those of human proteins. Microbial fermentation is quicker, but mostly suited for the production of smaller molecules such as peptides and antibody fragments. Insect cell culture generates high levels of recombinant proteins and can be easily adapted to high-density suspension culture. It also offers advantages for expressing intracellular proteins and multi-protein complexes that are difficult to obtain using mammalian and bacterial systems. In addition, with the ability to insert multiple genes into the baculovirus vector, it is possible to express multiple proteins/protein subunits simultaneously. Numerous advances in technology are improving the performance of insect cell-culture systems and increasing their attractiveness for the large-scale production of therapeutics and vaccines.

Improving efficiency, yield, and quality

Advances in technology have improved the efficiency of baculovirus vectors, enhancing protein yield and product quality, according to Yuxiang Hu, chief scientific officer at SignalChem Biotech (part of Sino Biological). The traditional insect cell-baculovirus expression vector system (IC-BEVS) comprises a recombinant baculovirus engineered to deliver a transgene encoding a desired protein that is used to infect an insect cell line, typically lepidopteran, which then expresses the product.

These advances, continues Hu, include optimized viral promoters and modified viral genomes that enable more efficient and higher-level expression of recombinant proteins, optimized cell-culture media, and new insect cell lines adapted to grow in the newly developed media with higher density.

Greater understanding of insect cell lines has also led to advances, observes Thera Mulvania, president of Expression Systems, an Advancion company. “There has been a progression away from thinking of insect cells as a tool analogous to E. coli [Escherichia coli] and understanding that the cell lines require characterization and defined passage protocols,” she explains. “These protocols,” Mulvania says, “differ from site to site, but the key is reproducibility in handling of the cultures. Additionally, the passage stability of recombinant baculoviruses is being addressed by defining the limits of passage as well as interrogating for genetic stability and expression.”

The safety of insect cell systems has also been improved, notes Mulvania, through the development of cell lines that do not contain endogenous viruses of concern, specifically Sf-rhabdovirus in Spodoptera cell lines and alphanodavirus in Trichoplusia cell lines. “Commercially available options for cell lines absent these viruses, combined with the FDA’s preference for using these cells, are eliminating the related viral clearance requirements in Phase I, saving time and money,” she comments.

Cell-line engineering

The capabilities of a given host cell line have a direct impact on the overall performance of a cell-culture system with respect to productivity and product quality. Insect cell-culture systems are no exception. Advances in clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and other genome-editing tools have, therefore, been beneficial for improving the production of protein therapeutics and vaccines using insect-based platforms. “Access to new techniques for gene editing has resulted in improved cell lines that can be optimized for higher productivity, better protein folding, and other attributes,” Hu says.

Mulvania highlights the work of Don Jarvis of the Department of Molecular Biology at the University of Wyoming. “His group has focused on the modification of glycosylation pathways in insect cell lines to yield patterns closer to those achieved with mammalian cell culture,” she comments. Efforts across academia and industry have also been geared, according to Mulvania, to identifying and deleting nonessential genes in the baculovirus genome to enable better protein production.

Development of stable insect cell lines is another area of research, according to Hu, with the goal of eliminating the need for transient transfection/infection with baculovirus, which is currently required with traditional insect cell lines (Sf9, Sf21, and High Five). Mulvania highlights the development of producer cell lines such as OneBac that minimize the number of baculoviruses needed to generate adeno-associated viral (AAV) vectors.

“Stable cell lines reduce the variability seen with transient expression and improve scalability and consistency,” Hu observes. However, she cautions that developing stable expression cell lines is a time-consuming process and thus typically pursued only for proteins with high market demand.

Cell-culture media

The composition of the cell-culture medium is another important factor impacting protein expression for both mammalian and insect cell systems. From a development standpoint, however, insect cell media has historically lagged mammalian cell-culture media, observes Mulvania. “Although there is a trend toward chemically defined media for insect cell culture, available options have previously not compared to hydrolysate-containing media formulations,” she says.

Some new media products designed for insect cell culture have addressed that deficiency, but they come with certain recognized performance challenges, such as the need for enhancers and issues with cell-line compatibility, according to Mulvania. Expression Systems introduced a media formulation specifically developed to support superior cell growth and expression without the need for supplementation. “The new chemically defined cell-culture media allows for easy adaptation of insect cell lines, providing the industry with a true plug-and-play platform that reduces the time and effort required for development of protein manufacturing processes,” she contends.

Such advances in insect cell culture media have been a major factor in boosting cell growth and protein production, agrees Hu. “Optimized media now provide better nutrient balance, minimize waste products, and increase cell longevity in culture, all of which are critical for large-scale production,” she states.

Process optimization

One of the biggest advantages of insect cell systems, according to Mulvania, is the reduced need for monitoring and control systems. “As a lytic manufacturing process, the unique process conditions for insect cell systems are cell-culture density and defined baculovirus titering, allowing for precision in the production kinetics,” she explains. Optimizing mix speeds and aeration requirements are similar to other systems, but pH management is often unnecessary. Efforts to increase cell density and thus per-liter yield continue to benefit the industry, Mulvania says.

Increased yield is being achieved through access to new cell lines, new cell-culture media, and more advanced process monitoring and control, according to Hu. In addition, she notes that the employment of single-use (disposable) bioreactor technologies has facilitated the rapid scale-up of insect cell cultures while maintaining control over environmental factors such as pH, oxygen, and nutrient levels. The result is not only increased flexibility at large-scale, but also improved batch-to-batch product quality and stability.

More engineering needed

One of the major limitations of insect cell expression systems is their inability to achieve post-translational modifications (PTMs) like those of mammalian expression systems. PTMs have a direct impact on the safety and efficacy of recombinant protein-based therapeutics and vaccines, and generally need to be similar to those that occur naturally on human proteins. Both Hu and Mulvania believe that further cell-line engineering/genome editing of baculovirus systems is needed to produce proteins with desirable quality attributes, such as mammalian glycosylation profiles.

Will advances drive greater use?

Although a number of clinical products have been manufactured using IC-BEVS technology, such as the COVID-19 vaccine from Novavax, the influenza vaccine FluBlok from Sanofi, and a number of commercial gene therapies, Mulvania remains surprised by the lack of penetration of the system in the market. “Insect cell culture is an accepted and successful manufacturing platform that deserves more widespread adoption,” she stresses.

One issue that must be overcome, according to Mulvania, is the use of academic protocols rather than processes designed for large-scale production. “Expectations regarding scalability can be incorrect, and as a result the true potential of the platform is not being realized,” she says. “Expression Systems strives to bring greater awareness of the potential of BEVS by developing a user-friendly, effective manufacturing platform that provides consistent, scalable results,” she adds.

There is, notes Hu, an expectation that additional advances in cell-line engineering, cell-culture media formulations, and process optimization strategies will further improve the performance and widen the applicability of insect cell culture for the production of protein therapeutics and vaccines.

About the author

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.

Article details

BioPharm International®
Vol. 38, No. 1
January/February
Pages: 18–20

Citation

When referring to this article, please cite it as Challener, C.A. Advances in Insect Cell-Expression Systems. BioPharm International 2025 38 (1).