Choosing between Stable and Transient Transfection

Stable transfection of mammalian and microbial cells, and their subsequent expansion for large-scale production of therapeutic proteins, is a hallmark bioprocessing operation. Thanks to advances in culture media and feed strategies, process analytics/understanding, and unit operation optimization, specific titers of up to 10 grams of monoclonal antibody (mAb) per liter are now routinely achieved.

The main drawback of stable transfection is that it takes time. A typical fed-batch culture extends over several weeks and demands attention and resources during that time. Transient expression protocols deliver protein in as little as 24 hours post-transfection, a timeframe that allows for studying short-term gene expression, RNA interference, and gene silencing. Most significantly, transient expression gets protein into the hands of analytical groups quickly for early characterization studies.

Other factors to consider when deciding between transient and stable transfection:

Stable transfection

• Uses DNA transfection only
• Gene incorporated into genome
• Selective screening required
• Low copy number integration
• Can use inducible promoters

Transient transfection

• Uses DNA or RNA transfection agents
• No incorporation into genome; effect is halved with each generation
• Selective screening not required
• Relatively high incorporation
• No inducible promoters

Versatile platforms

In addition to a wide range of therapeutic proteins and mAbs, transient expression is suited to rapid production of advanced antibody-derived products, according to Dr. Shuting Xu, Head Field Application Scientist at GenScript Europe.

“Examples include nanobodies, which are composed of a single-domain antibody fragment that binds to antigens, and bispecific antibodies, which are artificial proteins that simultaneously bind to two different antigens or two epitopes on the same antigen. Both may be expressed through either transient or stable expression.”

Post-translational modifications (PTMs), a current area of intense focus due to their potential effects on antigenicity, circulating half-life, and activity, do not differ substantially whether the protein is expressed through stably or transiently transfected cells. “PTMs strictly depend on the expression system, that is the cell lines utilized,” Xu adds. PTMs are almost entirely a function of the expression system, not the transfection method.

Expression yields or titers are typically higher when stably transfected cells are used, Xu explains, because stably-expressing cell lines are selected based on their expression capacity, which they pass on to offspring as the cells are passaged and the culture expanded. “However, with advanced technologies and expression platforms, transient expression can reach very high expression yield as well.”

According to GenScript, its NextGen CHO expression product achieves up to 3 g/L via transient expression. Those titers were the upper limit of stable transfection systems not too long ago, and are still considered high for some proteins manufactured in fed-batch systems.

Consistency—bridging discovery and production

Every development project focuses intently on quality, which is often mentioned in the same conversation as patient safety. Xu tells Biocompare that, at least theoretically, there should be no great differences in purity/impurity profiles between proteins expressed through transiently vs. stably transfected cells, as product purity depends on purification methods.

“However, stable expression offers the benefit of higher batch-to-batch consistency. Even with the same plasmid and the same cell line, transiently produced batches will vary a bit. That is why stable expression is normally used when large quantities of protein are desired, while transient expression is preferred during early research phases because of its faster timeline and flexibility with respect to modifications in the design of target proteins.”

Whenever possible, protein drug developers try to use the same expression cell for transient and stable expression, in order to maintain similarity and reduce uncertainty in moving from discovery through development and beyond. This helps to maintain consistency of target protein PTMs and, it is hoped, protein function as well. GenScript’s NextGen CHO platform, for example, allows this from high-throughput expression for screening through stable cell-line generation for gram- or even kilogram production levels.

Despite the benefits and drawbacks of both stable and transient expression, there is no “formula” or algorithm for selecting which one to use. Both methods involve getting foreign (target) genes into cells. Since this protein-manufacturing capability is diluted with each cell division, transient transfection may be the way to go when protein requirements are low, or when your colleagues running assays need material “yesterday.” Transient gene expression for mAbs takes six to ten days post-transfection, whereas the equivalent from stably transfected cells requires several weeks. “HEK293 cells normally achieve higher transfection rates and higher yields when polyethylenimine, an inexpensive, high-charge cationic polymer, is used as the transfection reagent,” Xu says.

Note that even stable transfection is a transient process at initiation whereby, through the process of selection and amplification, stable clones emerge in which the gene becomes part of the host genome, and replicates along with cells as they expand.

“Because the stable transfection of cells is a longer and more arduous process, it is the most practical approach for recombinant antibody production at larger scales,” Xu explains. “During initial selection, when research quantities on the order of ten to 1000 mg will suffice and speed is of the essence, large-scale transient expression is an option. But for large-scale production of therapeutic antibodies, stable gene expression is preferred as it allows for greater process consistency and control of the final product quality.”

Conclusion

Transient protein expression for therapeutic protein discovery and characterization is well-established, with global demand for reagents and related services estimated at upwards of $540 million. Leading players include ProteoGenix, Absolute Antibody, Thermo Fisher Scientific, Merck KgaA, Lonza Group, Agilent Technologies, Takara Bio, Mirus Bio, Bio-Rad Laboratories, Qiagen, HiMedia Laboratories, Promega, and Sino Biological.

While most of the news (and new products) in transient expression involve workhorse mammalian cell lines like CHO or HEK, the transfection of plant-based systems is also of interest. Producing valuable proteins in plants or plant culture typically involves gene expression followed by standard downstream purification.

Several products of such “molecular farming” are now commercial, and the approach seems compelling enough, but success has been limited by “inertia driven by perceptions of low productivity, the high cost of downstream processing, and regulatory hurdles that create barriers to translation.”