Transient Protein Expression Gaining Ground

Transient protein expression exploits non-genomic gene expression to produce proteins for early characterization studies. Because the expressed gene is not heritable, protein production falls off rapidly as transfected cells age and die. That is why, at least for now, transient expression is at best limited to yields in the tens of milligrams range.

Transient expression platforms rely on chemical transfection, which is quicker, requires far less development, and is much less costly than genome-inserting viral vectors. The technology is well established for common animal cell expression systems, like CHO (Chinese hamster ovary) and HEK (human embryonic kidney), and also works in plant cells but, due to differences between cell membranes and a plant’s cell wall, the methods differ significantly.

An interesting twist to transient methods is cell-free protein expression, which uses a cell’s protein-producing apparatus or ingredients—enzymes, feedstock, cofactors, and genes—minus the cell and organelles—to generate small quantities of investigational proteins.

According to market research firm OpenPR, demand for transient protein expression systems will rise at about 5.6% per year, from about $410 million today to about $503 million in 2027.

Selecting a transient platform

Once developers decide that transient expression suits their situation, they face a decision tree similar to those encountered for stable transfection.

The first consideration is whether to use a prokaryotic or eukaryotic expression system. “If the desired protein is relatively small and simple then prokaryotic platforms, such as E. coli or yeast, are excellent choices as they are cheap, fast, and productive,” says Ian Wilkinson, Ph.D., CEO of Gamma Proteins. “But prokaryotes have difficulty expressing complex proteins with multiple domains, disulphide bonds, or post-translational modifications—most notably glycosylation.

Antibodies are obvious examples of proteins unsuited for expression in prokaryotic platforms. “In these situations, you will typically get higher-quality protein, with the correct folding and post-translational modifications, from a eukaryotic system,” Wilkinson says. The higher cost and more complex, time-consuming development and production for eukaryotic expression may be the only option, however, given the high premium on high-quality protein.

Assuming the eukaryotic route is highly preferred if not forced, the next choice is between mammalian or insect platforms. Many mammalian lines are available, each conferring its unique set of attributes to expressed proteins. HEK293 and CHO cells are most widely used, Wilkinson notes, “but CHO is preferred for clinical-grade material.”

Gamma Proteins bases its business not on any novel expression technology but on the premise that research proteins should not be prohibitively expensive. “During the past 20 years the cost of DNA sequencing and gene synthesis have fallen by more than a hundredfold and the productivity of mammalian transient expression systems has similarly increased,” Wilkinson tells Biocompare. “But despite these advances, the cost of recombinant proteins has barely changed. We focus on recombinant Fc receptors because they are a critical reagent in the research and development of therapeutic antibodies.”

The main advantage to this approach is the ability to create a developable product from a sequence in weeks.

For example, during the COVID-19 pandemic, Wilkinson proposed using transient expression to progress rapidly from the antibody discovery phase to having material sufficient for a phase 1 study.

“You could then run stable cell lines in parallel so that they are ready for later clinical phases,” he says. But scaleup is the real challenge with mammalian transient expression, as it has rarely been carried out at the 10-liter scale, much less at several hundred liters, which would be required to make 50–200 gram batches sufficient for a small phase 1 study.

“That is why in practice transient expression is ideal for early-stage research where small amounts of material suffice. But once larger quantities are needed then stable cell lines are the way to go,” Wilkinson says.

Cell-free systems

Heterologous, or stable transfection based on E. coli, HEK293, CHO, and sf9 cells, is tried and true yet we are seeing accelerated interest in cell-free expression systems, according to Michael Chen, Ph.D., CEO and Founder of Nuclera, which specializes in tools for protein discovery.

Operating without living cells, cell-free expression systems utilize extracted cellular machinery to make proteins.

One of the primary benefits of this approach is speed, Chen explains. “Cell-free systems can produce proteins within hours, bypassing the complexity of DNA transformation/transfection as well as the lengthy growth cycles of cells. Additionally, cell-free systems make protein purification easier, removing the need for cell lysis and clarification.”

Cell-free systems, especially those derived from the familiar and well-characterized E. coli expression platform, are well suited for today’s protein expression and purification screening challenges, Chen adds. “In an environment where the user requires construct screening of many proteins, adoption of cell-free protein synthesis technologies dramatically simplifies the cost and operational requirements of protein screening.”

Cell-free systems also provide greater control over protein folding and reaction conditions, allowing for the addition of disulfide bond-forming additives, facile incorporation of non-natural amino acids, and synthesis of complex or toxic proteins that kill or disable intact-cell expression systems. Plus the open environment of cell-free systems facilitates easy monitoring of the expression process.

“Cell-free systems have drawbacks compared with cell-based systems, however. Where cell-free systems excel for screening end applications, they are typically not a feasible option when scaling protein production due to the expense of purified components,” Chen says. “Cell-free systems are also not known to produce human-like glycosylation for proteins limiting the use of cell-free systems for certain users.”

By contrast, expression in living cells offers a more cost-effective and scalable solution since cells naturally handle complex post-translational modifications crucial for the functionality of many proteins. But they are slower, requiring significant time for cell growth and protein expression. They are also less flexible, with limited ability to incorporate non-standard amino acids.

“Ultimately, the choice between cell-free and cell-based expression is not binary,” Chen tells Biocompare. “Cell-free systems excel at screening, and cell-based systems excel at scaling. The key is to understand your protein requirements and use the appropriate tools.”