September 1, 2010 (Vol. 30, No. 15)

Pfenex System Repositioned to Support Early-Stage Research

Change is under way at Pfenex as the company works to expand its bacterial-based protein-expression system. Originally used to manufacture biotherapeutics, vaccines, and protein-based reagents, Pfenex had been working with companies to express a protein of interest for late-stage clinical development. Now the company is exploring how the system can be used for discovery.

“In our initial business model, we developed a production strain and licensed it back to the client for production of clinical materials and commercial products,” explains Patrick Lucy, vp of business development at Pfenex.

But when Bertrand Liang, M.D., Ph.D., joined Pfenex in 2009 as CEO, he scrutinized the company’s business model. A veteran of big biotech, Dr. Liang recognized that protein-expression problems are not limited to clinical development, but start back in the early drug discovery phase.

Accordingly, Pfenex has repositioned its protein-expression technology to support early-stage challenges related to the discovery of large and small molecules. Discovery scientists want to move programs forward rapidly to increase the value of a company’s pipeline while minimizing costs. “To do this, scientists need soluble, active protein on demand,” says Lucy.

Pfenex expression technology can be applied across the product-development continuum.

Agricultural Origin

The Pfenex platform is based on a strain of Pseudomonas fluorescens, first isolated 25 years ago from a lettuce leaf by scientists at Mycogen, part of Dow AgroSciences. Mycogen used the microbe for large-scale production of insecticidal proteins for agricultural use. P. fluorescens adeptly produces large amounts of protein at low costs, according to the company.

Dow Chemical acquired Mycogen in 1998, and its researchers enhanced the flexibility of P. fluorescens for large-scale production of industrial enzymes. During the past decade, the Pfenex team turned the strain into the Pfenex Expression Technology™ platform for protein production.

The Pfenex Expression Technology platform assesses the performance of hundreds of host-strain plasmid combinations in a high-throughput, parallel manner to rapidly identify the best strain for high-yield expression of an active target protein. The platform includes an extensive library of expression tools and P. fluorescens host strains with phenotypes designed to enhance quality and stability of proteins expressed recombinantly.

Prior to launching the platform, Lucy and colleagues at Dow spent several years perfecting it to manufacture large amounts of recombinant proteins efficiently and in a soluble, properly folded form. Dow launched the Pfenex Expression Technology platform in 2004 to perform strain-engineering work for pharmaceutical companies. Then in 2009, Pfenex became independent of Dow.

Discovery Projects

When seeking bacterial strains to manufacture vaccines or biologics, or for discovery and research projects, Pfenex scientists evaluate several hundred to one thousand production strains over five weeks. Screening includes robotics and running assays in 96-well plates.

The researchers are evaluating alternative approaches to screen thousands of strains in the same amount of time. Depending on the desired product, broader screens will provide gram quantities for lead-protein work or milligrams for earlier-stage work.

“Instead of looking for a single gene coding for a lead protein, we screen a thousand strains and look for five to ten genes that are potential lead proteins,” Lucy says. In the near future, Pfenex will increase throughput four- to fivefold by running assays in 384-well plates.

For example, in vaccine development, thousands of antigens are potential candidates against a pathogen, but only a few are formulated. When researchers try to express a likely candidate, the antigen is rejected if its expression is poor. “The antigen is thrown out strictly on the basis of whether it can be expressed and manufactured,” notes Lucy.

The Pfenex Expression Technology system improves the search by providing milligram amounts of several antigens for animal testing to narrow down the choice based on biological results. Starting with putative antigens, thousands of strains are screened to find ones that can make milligram batches of each antigen in small-scale fermentors.

“We leverage the Pfenex platform to express small amounts of a variety of antigens to enable partners to conduct animal testing faster. This allows discovery scientists to open up their pipeline and find more vaccines,” Lucy says. The same approach to expression-strain development is applied to protein-engineering projects as well.

Small molecule drugs, which are chemically designed to counterattack a protein in the body, are also identified by the Pfenex platform. Protein targets are often difficult to express, and researchers need milligrams of a target protein for further x-ray and crystallographic analysis to understand its biological function.

An additional advantage of the Pfenex Expression Technology is scalability. Traditional protein-expression systems, such as yeast or E. coli, sometimes produce small amounts of a protein for discovery yet fail during scale-up to make larger batches for clinical trials. Once Pfenex identifies a production strain, it can be used for discovery, preclinical and clinical testing, and large-scale fermentation.

“We are confident that our clients will not have to switch hosts in order to meet increasing demand as the product progresses through development and into commercialization,” says Lucy.

Pfenex has an internal pipeline of biosimilars, including human growth hormone and interferon beta-1b. These and many other biosimilars are manufactured inefficiently in traditional systems like E. coli, Lucy says. The expressed proteins are insoluble and do not fold like native proteins. In contrast, the high-quality biosimilars made in Pfenex host strains are soluble, active, and expressed at significantly higher levels than in E. coli, he adds.

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