Stable transfection of cells and microorganisms is the cornerstone of therapeutic protein manufacturing. In culture, stably transfected mammalian cells expand and pass their protein-producing traits on to their progeny. In transient transfection, new genes enter cells and assume some protein-making capability without becoming part of the organism’s genome. Daughter cells therefore lack the ability to manufacture the desired protein.
Numerous physical and chemical methods exist for achieving transient transfection, including gene guns, electroporation, and cell membrane-disrupting surfactants. All these techniques impose physical limits on per-cell and volumetric productivity of transient production.
Yet transient transfection has one advantage over stable integration of new genes: speed. Where stable transfection takes weeks or months, transient transfection is accomplished in days, thereby providing rapid, reliable access to small quantities of protein for characterization and experimentation.
Traditional, surfactant-based approach
For antibodies used as biochemical reagents, transient transfection may be all that is needed to produce enough protein to conduct months’ or years’ worth of experiments, or for a vendor, several years’ supply of a commercial product.
GenScript specializes in transient protein expression services, which have been referenced in numerous publications over the last several years. The company also maintains an online selection guide to types of transient transfection/expression.
“Transient expression has traditionally been used to obtain anywhere from a few milligrams to a hundred milligrams of protein,” says Bo Wu, Ph.D., director of protein production at GenScript. But now, technical advancements have pushed the production boundaries from milligrams to gram quantities.”
Think of transient transfection and expression as a short-cut from the tried-and-true hybridoma production route, which in addition to stable incorporation of genes requires lengthy clone selection. “With hybridomas, the clone you select sometimes becomes unstable,” Wu adds, “and that is the basis in many instances for moving from hybridomas to transient production. There are also lots of ways to improve titers in transient systems.”
GenScript is capable of transfecting many different cell types but is expert with CHO and HEK cells that are widely used in large-scale protein expression through stable transfection. GenScript’s transfection method uses liposomes of polyethyleneimine (PEI), a cationic polymeric surfactant, to encapsulate the transfection plasmid into positively charged species. The PEI-plasmid complex attaches to negatively charged cell surfaces, from where the cell internalizes them into the cytoplasm.
Companies expressly seeking proteins with uniform, drug-like properties should think twice before undertaking a transient transfection, Wu warns. “Transient transfection is only relevant when you’re interested in a protein’s sequence and basic binding behavior, when you don’t care about things like post-translational modifications.”
Many expression systems, many methods
A web search for “transient transfection” turns up numerous vendors. Some, like MaxCyte, specialize in one transfection technology (but may offer alternatives). Many firms, like Agilent Technologies, offer a line of vectors and transfection reagents that addresses the diversity of cell line work generally. Agilent’s product line includes GeneJammer Transfection Reagent, AdEasy Adenoviral Vector, SureVector, and pSG5 & pMC1Neo vectors. Each product is somewhat specialized.
SureVector is used to create custom mammalian expression vector —the construct that delivers the gene to the cell. A transfection kit would also be used in the downstream workflow after building the vector and propagating in E. coli. SureVector also operates in mammalian expression systems.
AdEasy Adenoviral Vector System is optimized to work with AD-293 cells, the MBS Mammalian Transfection Kit operates in CHO and HEK293 cell lines, and GeneJammer works with many cell lines, including both adherent and suspension cells.
“These systems can be used in a single cell that goes through the workflow, if the researcher desires,” says Laura Whitman, global product manager at Agilent Technologies. In other words developers can use the same cell line from discovery through production.
Both GeneJammer and the MBS Mammalian Transfection kit work with CHO cells, but productivity varies significantly depending upon the protein being expressed and the various tags and promoters used.
The one-cell-line idea
The biomanufacture of proteins for research, development, and therapy occurs through the culture of a wide variety of cells and microorganisms. Most projects begin with a “discovery” phase for which transient protein expression provides limited quantities of protein for characterization and experimentation. Since working with the same cell line from discovery through production is highly desirable, so is a broadly applicable transfection method covering both transient and stable protein expression, for example MaxCyte’s flow electroporation-based transfection system.
MaxCyte’s underlying technology, Flow Electroporation®, loads genes or proteins into cells at scales of between 500,000 and 200 billion cells per transfection.
During electroporation, cells suspended in buffer experience brief electrical pulses, which generate reversible permeability in cell membranes to allow entry of DNA, RNA, and protein. MaxCyte has developed electroporation protocols for more than 80 cell types, including primary cells, production cells (including CHO, HEK, Vero, BHK21, and Perc.6), and stem cells.
Image: The MaxCyte STX® Scalable Transfection System uses Flow Electroporation™ Technology to load DNA, mRNA, or other molecules into a wide variety of cell types for a range of biomanufacturing applications, including recombinant protein production and viral vector manufacturing.
“CHO cells offer multiple advantages,” says James Brady, Ph.D., vice president, technical applications at MaxCyte. “They may grow to densities exceeding ten million cells per milliliter through fed-batch cultures that extend for more than two weeks. Media and feed are adjustable to control for protein quality, including post-translational modifications.”
HEK cells are used for manufacturing some viral vectors and vaccines, but their short culture times and low cell densities limit their suitability for recombinant protein production. Historically, transient protein expression was conducted in HEK cells because transfecting CHO via conventional transfection methods, such as lipids, CaPO4, or PEI, is difficult. Vero and BHK21 cells also present challenges for standard transfection methods.
“The advantage of flow electroporation is that electroporation protocols can be optimized for individual cell types, allowing users to achieve high transfection efficiencies with cells relevant to their specific production needs, including multiple strains of CHO cells.”
Working with one cell line from discovery through development and beyond brings a level of familiarity that extends both to protein expression and purification. For monoclonal antibodies this means CHO cells, of which drug sponsors, vendors, and contract manufacturers have developed numerous individual lines like CHO-S, CHO-K1, CHO-DG44, CHOZN®, and ExpiCHO.
“Host cells exert significant influences on product quality,” Brady says. “Therefore, during early discovery and development phases, working with a cell line and production media that closely match those used for commercial production is highly desirable. Flow Electroporation® allows researchers to perform transient expression in a range of CHO cells lines and to culture the transfected cells in multiple media/feed formulations, thus allowing continuity of product quality throughout discovery, development, and commercialization.”
Transient transfection is a shortcut, which means it presents tradeoffs compared with stable transfection methods. A good deal of variability exists within and among methods as well, particularly when they are paired with specific cells and culture conditions.
“The success of transfection depends on transfection efficiency, low cytotoxicity, and reproducibility,” says Whitman. “To ensure high transfection efficiency researchers need reliable transfection technology and reagents to optimize cell culture conditions.”
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