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Pseudomonas-based protein expression to rival E. coli?

By Kirsty Barnes , 08-Nov-2006

Drug developers are increasingly turning to Pseudomonas-based expression technology for their hard-to-express proteins, according to contract manufacturer Dowpharma.

The US firm said it has seen over a 100 per cent increase in the adoption of its Pfenex expression technology in the first half of 2006 over the same period last year and expects this growth to continue as more and more protein and antibody-derived vaccines come through the pharma pipelines. "We have nearly tripled the number of new customers using this technology since it was first made available at the beginning of 2005," Kurt Hoeprich, global commercial director of Biopharmaceuticals at Dowpharma told In-PharmaTechnologist.com. Although it predicts a continual increase in business for the future, the company has no expansion plans as yet as it has been able to triple its in-house efficiencies over the past year through the use of automated technology. According to Research and Markets, the biopharmaceutical market is predicted to reach $2.6bn (€2bn) this year, an increase from $1.4bn in 2003, with the primary driver of this market being mammalian cell culture. Mammalian cell culture is a platform that can be utilised by biotechnology companies, pharmaceutical companies moving into the biopharm space, as well as cell reagent manufacturers and contract manufacturers trying to maintain a competitive edge and meet future trends. However, Dowpharma elected in 2004 not to go ahead with plans to build a mammalian cell production facility at its Smithfield site in the US, which has since been closed, in favour of concentrating on other technologies, such as Pseudomonas-based Pfenex that can enhance microbial fermentation. Dowpharma's commitment to its Pfenex technology is indicative of the changes currently occurring within the biotech industry, specifically around mammalian cell culture technology, with analysts forecasting a shift from mammalian cell culture technologies to microbial fermentation techniques. Currently the gold standard for non- mammalian cell protein expression is E. coli, however, Dowpharma claims that the success of its new technology to date means it has the potential to knock E. coli from the top spot. "We have been able to genetically engineer a unique form of Pseudomonas that enables the system to outperform E. coli," said Hoeprich. "A number of companies are offering E. coli- and yeast-based services and are continually trying to optimise them but we are the only company offering this unique Pseudomonas-based service." According to the firm, Pfenex can achieve soluble, correctly folded proteins with a yield improvement that can be over ten times greater than E. coli. "One of the biggest problems with E. coli is that the protein comes out in an insoluble, inactive form and a series of steps then need to be undertaken to refold it correctly - this can be extremely cumbersome and does not always produce very high yields of correctly folded therapeutic protein," explained Hoeprich. "On the other hand with Pfenex, the vast majority (75-80 per cent) of proteins that we have expressed so far have folded completely the first time - there are very few proteins that have not and even then they have still come out partially folded." Hoeprich believes that because of this there is a good chance that Pfenex will increasingly replace E. coli as a first-line expression technology as the complexity of new drug candidates also continues to increase. "We are already seeing a trend of more and more candidates that require complex folding - this now makes up more than 50 per cent of our Pfenex business," he said. In addition, it is claimed that Pfenex's effectiveness enables a faster route through process development and an easier transition into manufacturing. "We've identified what we call a high-throughput strain identification system with Pfenex," said Hoeprich.


"In most cases we can identify 10-20 different forms of Pfenex for a given protein and deliver a result within eight to twelve weeks - with an insoluble protein using E. coli this process can take up to a year." Furthermore, Dowpharma said these time and quality efficiencies also bring cost savings too. "The majority of money is saved with Pfenex primarily because of the much greater yields it is able to produce per fermentation batch compared to E. coli," said Hoeprich. "In addition, because the protein comes out in a soluble form it is easier to handle and allows a clearer path through the purification process." Importantly, the Pfenex technology also fits directly into customers' existing E. coli equipment, allowing for a "direct swap out," requiring no extra investment and it is operationally very similar to the E. coli process, Hoeprich pointed out. The downside of Pfenex is that like all prokaryotes, including E. coli, Pseudomonas can't glycosylate and therefore there are a huge number of glycosylated proteins and antigens that can still only be made using mammalian cell systems. Dowpharma has its bioprocessing plant in San Diego, with systems ranging from 5-1500L, where it runs and documents a customer's protein, creating them a complete recipe that they can then use when they transfer the technology to their own in-house facilities to begin making clinical trial quantities of the drug.


The most advanced drug product being manufactured by a customer using Pfenex is now in Phase II trials and Hoeprich estimates that the first Pfenex-produced product may be on the market within three to five years.

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