October 1, 2007 (Vol. 27, No. 17)
Sue Pearson Ph.D. Freelance Writer GEN
Gleaning Greater Yields with Novel Approaches and Fine-tuned Tools Is One Solution
Protein-based drugs are luxury goods, according to François Arcand, CEO, ERA Biotech (www.erabiotech.com). “Since most have the same market size as Cartier watches, I believe no health system in the world has the financial means to supply its general population with drugs made in mammalian cells. They are just too expensive to be widely prescribed,” he says.
With the average cost of being treated with Herceptin estimated at $60,000– 70,000, for example, it’s not hard to see what Arcand is driving at.
An area where many biotech and pharma companies are looking to reduce expenses is in their manufacturing. Scientists from ERA Biotech and a number of other firms addressed this issue at Cambridge Healthtech’s “Protein Expression Europe” conference in Prague last month. A whole raft of technologies is arising that promise greater yields and as a consequence reduced cost of goods.
One such system from ERA Biotech is a storage organelle technology triggered by assembler peptides of the Zera® family (originally derived from corn) that can accumulate proteins in any eukaryotic cell type.
“Express a genetic fusion of Zera and a protein of interest, and the cell will form membrane-bound structures, or StorPro organelles, containing high concentrations of proteins with the same solubility and structure as the native version,” Arcand explains.
The fact that the recombinant protein is not denatured as it would be in an inclusion body makes Zera technology appealing to manufacturers, Arcand adds.
The first advantage of using a protein accumulation system is that target proteins are encapsulated in vivo by the user’s choice of cell, Arcand explains. “This gives both yield and quality, as the protein is protected from metabolic and proteolytic activities, and the host cell is not exposed to any toxic effects of the recombinant protein. This encapsulation also enables production of difficult-to-express proteins.”
The second advantage is much simpler purification strategies. Since the organelles are so dense, they can be concentrated by homogenization and centrifugation, according to Arcand. This means less downstream processing steps. Further, each remaining step uses much smaller chromatography columns and other equipment, he says.
“When you think of it, the current secretion paradigm implies spending a fortune fishing out proteins from a vast proteolytic soup. Instead, the StorPro organelles allow for accumulation and concentration of an intact protein, and can therefore deliver significantly reduced manufacturing costs.”
Zera technology is currently being explored by 30 academic and commercial partners, Arcand reports. “They confirm that StorPro organelles can be induced to form in most eukaryotic cells including CHO cells, insect cells transfected using baculovirus, filamentous fungi, plant cells, whole insects, and whole plants.”
“The next step for ERA is to allow production scientists to quantify, protein by protein, the dollar-per-gram advantage of Zera/StorPro accumulation over secretion.”
Easier to Use Baculovirus
For scientists interested in trying baculovirus systems, Oxford Expression Technologies (www.expressiontechnologies.com) asserts that its flashBAC technology promises greater yields.
“What puts most scientists off using the baculovirus system is selecting recombinants by plaque assay,” notes Linda King, Ph.D., director. “We have overcome this limitation by deleting the replication elements and only adding them in with the gene of interest so nonrecombinants will not replicate.
“We have also deleted nonessential virus genes such as chitinase, cathepsin, and p10 to produce a plasmid that has all the relevant baculovirus elements, but can easily be grown in a bacterial host before being introduced into insect cells,” Dr. King adds. “This also means we can automate the work on a robotic platform, a process which has been validated in 24-well plates at NextGen Sciences.”
Removing nonessential genes not only reduces the size of the final vector but improves expression, according to Dr. King. “Removing the chitinase gene means chitinase protein doesn’t accumulate in the endoplasmic reticulum, enabling secretion of other proteins. Also p10 produces a protein that can degrade the nucleus, so removing this allows cells to live longer and produce more protein.”
Dr. King reports that the flashBAC system expresses proteins in the mg/L region in such a way that “scientists with little experience can use it with confidence, making this technology accessible and affordable for most labs to try.”
E.coli and Yeast—Winners for Some Proteins
For those companies that want to manufacture large amounts of smaller proteins such as antibody fragments, cytokines, and recombinant vaccines as quickly and cost-effectively as possible, E.coli and yeast expression systems really score highly.
Avecia Biologics’ (www.avecia.com) E. coli-based protein production platform called pAVEway offers a combination of molecular biology, strains, and fermentation protocols, according to Ian Hodgson, Ph.D., head of molecular biology. “The vectors we have developed are designed specifically for large-scale processes rather than research only and are based on tightly regulated E. coli promoters instead of T7.
“This has several advantages,” Dr. Hodgson continues. “Firstly it means we can fully suppress expression, so there isn’t any protein produced before induction and we can modulate expression levels by varying inducer concentration. In this way we can tightly regulate protein expression for production of toxic proteins, for example.
“Additionally we are not tied into using lDE3-based E. coli strains, and users don’t have to pay licensing costs of using a T7 promoter.”
The main benefit of using pAVEway is the time it takes to get process scale expression up and running. “With pAVEway we can have intracellular single-chain proteins being expressed at 5–10 g/L and Fab fragments at 500 mg/L in process-scale fermenters in a three-day fermentation cycle, within four weeks of starting the gene cloning,” Dr. Hodgson points out. “When you compare this with CHO, which can take up to a month to get this kind of yield, it shows pAVEway could potentially reduce the cost of running a plant by around 75 percent, so its not hard to see why using E. coli could have a major impact on cost of goods.”
To rival the E. coli expression systems in terms of production costs are a number of good yeast-based platforms including Hansenula polymorpha, offered by Artes Biotechnology (www.artesbiotechnology.com). Yeast has a number of advantages over E. coli. It can correctly assemble 3-D and 4-D structures containing disulfide bounds and glycan structures for better protein solubility.
“Hansenula is often the next choice after E. coli when looking at producing therapeutic proteins or technical enzymes inexpensively and it is already being used successfully to manufacture some marketed drugs,” notes Jens Klabunde, Ph.D., project manager. Dr. Klabunde is referring to examples of interferon-a-2a, a treatment of chronic hepatitis C being produced by Minapharm Pharmaceuticals and a hepatitis B vaccine being made by Dynavax Europe.
Squeezing More From Mammalian Cells
Using an integration strategy like Artes, Selexis (www.selexis.com) offers a way of improving expression in mammalian cells with its SURE Cell Line Development process. “We have developed novel human genetic elements to remodel the organization of chromatin,” explains Igor Fisch, Ph.D., president and CEO. “Selexis Genetic Elements allow integration of 50 to 100 copies of a transgene into a single chromosomal locus, and by boosting transcription, higher recombinant protein expression levels can be measured.”
The suspension-adapted cell lines are stable enough that they can be grown without selective pressure or serum in a chemically defined medium, according to Dr. Fisch. Within six weeks of transfection, Dr. Fisch reports that the company obtained titers in shake flasks exceeding 1 g/L without any media optimization.
Selexis SURE Cell Line Development process has been used to develop CHO, HEK293, and BHK cell lines as well as stem cells. It can improve expression of mAbs, cytokines, EPO, and membrane receptors. “In fact, there will be two therapeutic mAb products in Phase I trials by the end of this year that have been produced using our SURE Cell Line Development process and should help improve perception of its benefits,” remarks Dr. Fisch.
“We are seeing 2- to 10-fold higher expression and yields of greater than 2 g/L in 300 L bioreactors using our cell lines,” Dr. Fisch states. Additionally, since the technology works with existing processes, he continues, there is no need to re-engineer the R&D laboratory or the pilot plant or even make significant changes to culture media or purification methods.
Though baculovirus, yeast, and E. coli expression systems are attractive, there are also technologies to remodel eukaryotic cell lines to improve expression. Many think the conservatism of process development and manufacturing scientists is holding back these innovations from being more widely adopted.
Discussing Hansenula’s acceptance, Dr. Klabunde remarks, “For example, we did a feasibility study for one company where we were achieving promising productivity that was higher than their established production method. They still did not switch because they were worried about investment costs to adapt production plants and regulatory hoops they would have to go through.
“However, these issues are often in people’s heads rather than being a reality, as the production only requires a switch of media to culture the yeast, and the purification would mainly be fine tuning of the existing columns to achieve the correct pH. We estimated to do this and the validation would be much less than the savings in production costs, but it required too large a shift in mindset for the manufacturers to accept.”
Arcand adds, “Biopharma operates today with two validated manufacturing tools: microbial systems for small and simple proteins and CHO cells for expensive antibody-like proteins. Both are too expensive for 90 percent of the world’s population, and there are scores of product classes out there they cannot make—at any cost. I believe open-minded companies that are risking using novel production tools now will pretty soon be saving lives, as well as making a lot of money.”