Scott Pattison, Ph.D., director of molecular biology at Kemp Biotechnologies (www.kempbiotech.com), says that the need to develop novel reagents and processes for their contract research service projects has led to the ongoing development of a complete toolbox of reagents, which are used for production of recombinant proteins in mammalian cells.
Acquired by Pelican Life Sciences in 2006, Kemp Biotechnologies is a contract cell culture, molecular biology, protein expression, and purification services provider. Many of the tools that Kemp Biotechnologies uses for transient mammalian cell protein production are being converted into products to be sold by Gene Choice (www.genechoice.com), another Pelican Life Sciences company.
“We had spent a great deal of time finding ways to use transient instead of stable transfection to produce recombinant proteins and thought that others could benefit from our efforts,” Dr. Pattison says. When protein production clients came to us requiring protein expression in mammalian cells we were at one time confined to using stable cell lines. These lines generally take at least four months to produce and characterize. Three years ago we asked ourselves—how viable would a transient expression platform be for these clients? By successfully developing a reliable transient process, we could realize significant time and cost savings in delivering a protein.”
Kemp scientists came up with a lipid-free reagent for transfection and developed a HEK293-based cell line that could grow well in serum-free suspension culture. “We have repeatedly delivered in excess of a gram of purified protein to our clients in eight weeks or less (from microgram quantities of plasmid to purified protein), using our transient expression process, compared to the four or five months generally required for stable cell line development,” he says.
Gene Choice now markets Transfectol™-LS Transfection Kits, which the company says work with a wide variety of expression vectors in HEK293 cells as hosts. Proteins produced using this transient system have included secreted proteins such as Fc fusions, intracellular proteins including kinases, and cell surface proteins such as ion-channel proteins and GPI-anchored proteins. The company is working on adapting CHO and NSO cell lines as acceptable hosts for large-scale transient production.
Dr. Pattison describes a protein production challenge illustrating the time and cost benefits of Kemp’s transient mammalian expression process. “A client came to us requesting that we produce one gram of recombinant immunoglobulin. The original process that they had developed at their facility used adherent serum-containing cultures of a HEK293-derived cell line. Their procedure involved transfecting multiple batches of 50 T180 flasks of these cells, utilizing a lipid-based transfection reagent.
“While reasonable product yields were achieved, the process was an extremely labor-intensive and costly approach. We were able to apply our bioreactor-scale transfection process in which we could transfect the same number of cells (~2e9 cells) in a single 20-L serum-free culture, with our Transfectol-LS transfection reagent. The final mean volumetric yield for the project was 15 mg of purified product per liter of cell culture. It took us six weeks to deliver 1.1 grams of their purified IgG, and the product met the client’s endotoxin and bioactivity specs. Implementing our bioreactor-scale process cut the number of hands-on research scientist hours needed to complete the project at least in half, compared to the original protocol.
“Utilizing a serum-free process minimizes concerns over adventitious agents, is a major cost-saver, and makes life much more pleasant for downstream protein recovery and purification.”
Dendritic Nanotechnologies (www.dnanotech.com), a subsidiary of Starpharma Holdings, entered a license and supply agreement with EMD Biosciences in February for its PrioFect™ Transfection reagents based on the company’s Priostar dendrimers. Dendritic Nanotechnologies says that its dendrimers are well-defined, highly branched synthetic macromolecules synthesized by surrounding a core particle with layered branching elements produced through a proprietary synthetic process.
According to Lori Reyna, Ph.D., senior scientist and manager of life sciences, “What distinguishes our dendrimers is that we have unique control over the size of the nanodendrimers we produce.”
Citing issues including the lack of ability of existing reagents to infect a variety of cell types, poor targeting, and toxicity, the company says its dendrimer production technology affords unique size control in nanometer increments, thereby allowing optimization of siRNA delivery into individual cell lines. “While we don’t at the moment clearly understand why we are able to transfect a variety of cell lines using our dendrimers between two and tennanometers, depending on the cell type, we do know we can greatly increase transfection efficiencies through our ability to control dendrimer size.”
Dendrimers are synthesized as a single molecular entity with high structural and chemical homogeneity and offer a precisely controlled macromolecular surface, according to Dr. Reyna. The company says that such dendrimers can be used in an “uncapped” state or functionalized by conjugation of active groups to its surface for use as potential pharmaceuticals as well as targeted nucleic acid transfection reagents.
“A lot of the development work we have done is for commercialization for siRNA and DNA transfection platforms. But because of the dendrimer structure, we can add properties such as nuclease protection, a very important characteristic. We can also target the dendrimers by adding small molecules or antibodies,” explains Dr. Reyna. The size of the particles, she says, also allows passive targeting, for example, in in vivo nucleic acid delivery applications, because nanodendrimer biodistribution varies based on size.
In a pharmaceutical application, Starpharma’s VivaGel, a vaginal microbicide, is based on the antiviral properties of a lysine-based dendrimer with naphthalene disulphonic acid surface groups and has been successfully tested in Phase I clinical trials for the prevention of HSV-2 infection. The product is currently being tested in an expanded Phase II trial in Kenya, Puerto Rico, and San Franciso.