Dow extracts the stress from bioprocessing

The researchers from The Dow Chemical Company and Pfizer used glycol ether solvents to selectively extract a proprietary therapeutic protein from genetically engineered Pseudomonas fluorescens ( P. fluorescens ) bacteria, dramatically increasing the purity of the product and reducing the amount of downstream purification needed.

The research was published by as an early view article in the journal Biotechnology Progress and builds on recent work by Dow in using glycol ethers to recover proteins from whole fermentation broth or hydrophilic compounds such as carboxylic acids from aqueous solutions.

Dow has developed the Pf enex expression technology based on the P. fluorescens bacterium as they can be readily modified to produce proteins within a compartment known as periplasmic space, which is located between the inner cytoplasmic membrane and the outer cell wall.

This system can produce yields ten times greater than Escherichia coli ( E. coli ) and also correctly folds the proteins which are often misfolded by E. coli.

"The Pfenex system consistently gives good yields of high quality protein and this new recovery technology enhances that, and really adds to the lower cost of goods that is so often a benefit of this system," said Dr Chuck Squires, senior director of biopharmaceutical services division at Dow.

Cell lysis is commonly used to release therapeutic proteins from host cells, but because this ruptures the cell walls various impurities such host cell proteins, nucleotides and endotoxins are also released.

Various purification steps are then needed to remove the impurities.

Bioprocess designers have tried various ways to improve this step and reduce the impurities released, including bacterial capture and lysis before specific elution of the products .

This new method developed by Dow uses glycol ether solvents to increase the permeability of the cell wall and allow isolation of the proteins within the periplasmic without rupturing the inner cell wall and releasing the impurities held within.

"This technique leaves behind about 80 per cent of the cell leading to a five-fold purification of the protein," said Dr Squires.

According to the lead author of the report, Dr Timothy Frank, scientist and senior technical leader at The Dow Chemical Company's Engineering and Process Sciences Laboratory, the paper discusses the extraction of a proprietary Pfizer protein and is just one example of the systems that the company has studied using the technique.

He continued by explaining that Dow has several World Patent applications pending, including WO, 2005/087791, which describes the use of the extraction technique for use with any Gram-negative bacteria used as a host cell for the production of therapeutic proteins.

In addition, the researchers also found that heating the glycol ether extract to 55°C caused the solution to split into an aqueous layer and an organic layer with the aqueous layer containing essentially all of the recovered protein in a volume of less than 15 per cent of the original extract volume.