Pseudomonas-based protein expression to rival E. coli?

By Kirsty Barnes

- Last updated on GMT

Related tags: Protein, Biotechnology

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

The US firm said it has seen over a 100 per cent increase in the adoption of its Pf​enex 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 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 Pf​enex that can enhance microbial fermentation. Dowpharma's commitment to its Pf​enex 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, Pf​enex 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 Pf​enex 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 Pf​enex'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 Pf​enex 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 Pf​enex 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 Pf​enex is now in Phase II trials and Hoeprich estimates that the first Pf​enex-produced product may be on the market within three to five years.

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