Tuesday, June 7, 2005

By Angelo DePalma

Interest in characterizing novel proteins has led to an explosion in gene expression techniques. Whether used for therapy, experimentation, or biomanufacturing, gene expression is about producing proteins.

Andrew Breile and Jay Wong prepare an in vitro coupled transcription-translation reaction for protein expression

Stable expression of protein-coding genes is the gold standard for bioproduction systems. However, stable transfection takes time. The alternative, transient gene expression, offers a rapid, convenient route to any protein whose precursor gene can be synthesized. Transient gene/protein expression delivers protein anywhere from three to ten days after the gene becomes available, compared with six months for stable transfection. The down-side is that since transient transfection vectors operate independently of genomic chromosomes, the induced trait extinguishes as cells reproduce. Protein production from transient transfection therefore only lasts for a few days to perhaps two weeks.

Med-chem on proteins?
Biotech firms may prepare hundreds of variants of a target protein during drug development, in much the same way as small-molecule drug developers create analog libraries. Since each of these discovery-stage proteins must be produced and tested quickly, developers use transient gene/protein expression techniques.

"Transient expression systems are the best way to get through the large number of proteins we need for a typical development project," says Tina Etcheverry, Ph.D., Senior Scientist at Genentech (South San Francisco, CA).

Culturing mammalian cells in a 65 liters bioreactor. Photo copyright 2005, Biotechnology Research Council National Research Council Canada (NRC-BRC)

Protein expression doesn't end after researchers settle on a clinical candidate. Investigators may wish to create variants for different animal models, or to produce what Dr. Etcheverry calls protein "reagents," primarily receptors, used to assay the protein product. After settling on the target, researchers will then affinity-mature it for higher efficacy, which means more proteins need to be made.

Moreover, targeted therapies require a separate diagnostic molecule, usually a monoclonal antibody, which must be developed in much the same way as therapeutic molecules.

More than perhaps any other biotech company, Genentech has demonstrated that transient expression systems are scalable, from Petri dish-sized cultures up to 100-liter bioreactors that churn out grams of material for primate and other studies.

Large-scale transient expression was rare until recently, not because of a lack of technology but due to the cost of reagents and for generating genes. The cost hurdle still exists for stable gene transfection. For example, adenoviruses used in gene therapy and for transfecting mammalian cells require separate manufacturing and validation efforts.

"Viral vectors need to be produced, amplified, and purified," says Yves Durocher, Ph.D., Group Leader at the National Research Council Canada (NRCC; Montreal, Quebec). "Plus afterward you have to assure those viruses were removed from whatever you make."

By using simple vehicles like calcium phosphate and cationic lipids, transient expression eliminates the cost hurdle and add-on manufacturing associated with adenoviruses.

The path to scale-up
As one of the world's leading cell culture experts, Florian Wurm, Ph.D. of the Swiss Federal Institute of Technology (Lausanne) has a keen interest in transient gene expression, particularly for mammalian cell systems. Wurm was part of a group at Genentech (which included Tina Etcheverry) charged with creating an improved version of tissue plasminogen activator (TPA). The molecule eventually approved, TNKase-TPA, was the result of hundreds of codon substitutions on the gene, each performed separately and expressed as single amino acid changes in the protein.

Harvesting a 20 liters bioreactor culture using a continous-flow centrifuge. Photo copyright 2005, Biotechnology Research Council National Research Council Canada (NRC-BRC)

During this project Dr. Wurm's group needed more variants of TPA than they could hope to create through stable transfection. So they became experts at transient gene/protein expression — not just for producing experimental quantities of protein, but for preclinical production as well. "Transient expression gives you protein almost as quickly as you can make DNA," says Dr. Wurm. "So I thought, why not scale this up?"

During Wurm's tenure at the company, his group used calcium phosphate as the gene delivery vehicle. By the time he left for academe the company was routinely using transient gene expression at the 2-3 liter scale. At Lausanne, Wurm's group has run transient transfections at the 100-liter scale in bioreactors. Genentech went on to perfect several methods based on cationic lipid gene delivery, and is now also capable of running hundred-liter cultures on transiently-transfected cells.

Although yields are low compared with stable transfection, transient methods could one day be employed for manufacturing. The key will be discovery of proteins whose efficacy is significantly higher than that of the native material. For example, TNKase-TPA is more than three times as effective per milligram as TPA, with approximately the same safety profile. Dr. Wurm believes that with additional improvements through mutagenesis, a protein with 100 times the efficacy of TPA might have been discovered. "But the realities of corporate drug discovery dictated that the research project had to end at some point," he notes.

Culturing mammalian cells in a 180 liters bioreactor. Photo copyright 2005, Biotechnology Research Council National Research Council Canada (NRC-BRC)

A biologic that is 100 times more powerful than the native protein need only be administered at 1% the dose of the original. Protein expression can be carried out in small bioreactors rather than in mega-facilities, and downstream purification is significantly compressed.

"The key to biotech economics is potency," notes Wurm. "MAb manufacturers struggle to produce hundreds of kilograms of product, but the overwhelming majority of the dose never makes it to the tumor. If we can improve monoclonal antibody potency, to the point where a patient only needs a few milligrams per day instead of grams per day, we will need to manufacture considerably less of these drugs to meet market demand."

Yves Durocher at NRCC agrees. "The biggest issue in moving beyond the 100-liter scale is producing enough plasmid DNA," he says, noting that the one milligram per liter of DNA required should not be too difficult to manufacture since the technology is established and the material is already made, under GMP conditions, for gene therapy.

The mechanisms of transient gene expression are not well-characterized. What is known is that if genes can get into cells there is a good chance they will be utilized by ribosomes. How the gene enters cells is not that critical. When mixed with DNA, calcium phosphate, by far the least expensive vehicle, forms particles which the cell ingests. Cationic lipids, by contrast, break through the cell membrane and allow genes to enter that way.

Transient expression will grow in importance as new protein drugs transform medicine. "The most serious bottleneck in biotechnology today is protein production," says Dr. Wurm. "Not the kilograms or hundreds of kilograms for any individual protein, but the number of different proteins we need to express to develop these drugs. Transient gene expression helps overcome that hurdle."

Look Ma, no cells!
Cell-free methods, which have been used since the early days of molecular biology, provide the most straightforward route to proteins. Cell-free extracts containing ribosomes and other crucial protein-generating ingredients from wheat germ, rabbit reticulocyte, or E. coli produce modest quantities of protein when spiked with DNA or messenger RNA.

Density, sensitivity and resolution of microarrays are just some of the factors that affect queality of results. How current the content is can speed up or slow down the journey to discover the novel targets or biological process of desease. (Photo courtesy of Agilent Technologies.)

Benefits of cell-free techniques include speed and latitude for incorporating labels at desired locations in the target protein. Cell-free expression is also the way to go for proteins that inhibit cell growth or are toxic to stably-transfected cultures. Such products may often be produced in cell-free systems. The bad news is that extracts only operate for a few minutes or hours, compared with live cells which can be kept alive and productive for days or weeks.

Cell-free methods allow expression of proteins that may be toxic to living cells. Plus extracts may contain protease inhibitors to limit protein degradation, another strategy that would not work with living cells.

Several companies provide kits for cell-free protein expression. Roche Applied Science (Indianapolis, IN) introduced its RTS (Rapid Translation System) cell-free expression technology about five years ago. Roche claims that RTS generates a hundred milligrams or more of protein per run.

Cell-free protein expression has a reputation for being a research tool, but commercial possibilities are intriguing. For example Ambergen (Boston, MA) has turned its cell-free method into a medical diagnostic tool.

AmerGen's diagnostic technology, which it calls the ELISA Protein Truncation Test (PTT), is also based on cell-free protein synthesis. Ninety-five percent of diseases caused by genetic mutations involve chain truncation. Examples include polycystic kidney disease, neurofibromytosis, an inherited form of muscular dystrophy, and bcr1/bcr2-active breast cancers. Thirty such diseases have been identified.

Truncation assays compare normal proteins, which contain an intact C- and N-terminus, with abnormal proteins that lack the C-terminus. Traditionally these assays employ radioactive tags and gel electrophoresis, which are difficult to use in a clinical setting. AmberGen's variation on this theme is a routine ELISA test. Researchers supply separate tags for each terminus, but mutant proteins lacking the C-terminus are also missing that specific signal.

Ambergen has also developed proprietary niche technologies for generating proteins from genes in vitro. The first method is non-radioactive labeling through transfer RNA (tRNA), which delivers amino acids to proteins as they grow. TRAMPE (tRNA-mediated protein engineering), as the technique is called, uses fluorescently-labeled amino acids affixed to tRNA and added to the cell-free expression medium. The other innovation is a photocleavable form of biotin, PC Biotin, which assists in isolating proteins with biotin affinity.

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