GlycoFi, Inc. Reports the First Successful Production of a Human Protein with Complex Glycosylation in a Genetically Modified Yeast
LEBANON, N.H.--(BUSINESS WIRE)--Aug. 28, 2003--Yeasts and other fungi are reliable, cost-effective workhorses for the production of many industrial enzymes. However, biopharmaceutical companies currently manufacture few human therapeutic proteins using these organisms. The inability to correctly decorate human proteins with complex sugar molecules - a process known as glycosylation - has been the main obstacle that has prevented the use of fungal hosts for the expression of human therapeutics.
Today a team of scientists at GlycoFi, Inc. and Dartmouth College report the successful reengineering of the glycosylation pathway in the yeast Pichia pastoris. Published in the latest issue of Science, these results constitute the first fungal organism able to secrete a human glycoprotein with complex human glycosylation.
"The vast majority of therapeutic proteins are glycosylated and most require complex, human-like glycosylation to ensure therapeutic efficacy," said GlycoFi's Stephen Hamilton, Ph.D., lead author of the publication. "Mammalian cell lines can replicate human-like glycoprotein processing to a large extent, and so have traditionally been used to produce most protein therapeutics. However, mammalian cell culture systems have significant drawbacks including low protein yields, long fermentation times, production of a mixture of protein glycoforms, and ongoing viral contamination issues."
"Being able to produce human glycoproteins with homogenous N-glycan structures in a fungal host allows us to take advantage of the inherent commercial advantages of yeast and other fungal production systems," says Tillman Gerngross, Ph.D., GlycoFi chief scientific officer and associate professor of biochemical engineering at Dartmouth College. "Moreover, the ability to produce a homogeneous glycoprotein in yeast offers the biopharmaceutical industry a new tool for further understanding the structure-function relationships of glycoproteins and for potentially creating safer, more effective therapeutics."
Humanizing the Yeast Glycosylation Pathway
The GlycoFi team began their work with the yeast P. pastoris, a robust organism commonly used in fermentation processes, which can be grown to high cell density in a chemically defined growth medium. This yeast normally produces non-human N-glycans of the high mannose type, which have no therapeutic value for humans. The scientists modified the yeast by first eliminating endogenous yeast glycosylation pathways, while sequentially engineering into the organism five active eukaryotic proteins, including mannosidases I & II, N-acetylglucosaminyl transferases I & II and UDP-N-acetylglucosamine transporter. The targeted localization of these enzymes enabled the generation of a synthetic in vivo glycosylation pathway that enabled the yeast to produce a complex human N-glycan, GlcNAc2Man3GlcNAc2, in vivo. However, unlike the glycosylation pathway in mammalian cell lines, which typically produces an array of glycoforms, the genetically modified yeast yielded essentially homogeneous glycoforms.
"We have essentially been able to humanize the yeast, where it is able to make a single glycoform of exceptional uniformity," Dr. Gerngross said. "Unfortunately, using mammalian cell culture, it is difficult to isolate individual protein glycoforms and even more difficult to produce specific structures at a commercial scale. The ability to express a single protein in a library of genetically engineered yeasts - each producing a defined and uniform glycoform - will enable the generation of glycoprotein libraries that can be used both to elucidate specific structure-function relationships and to identify the most efficacious molecule for a particular therapeutic use. Moreover, once identified, a particular protein glycoform can be readily produced at industrial scale using the relevant yeast, due to the well-established rapidity with which yeast fermentations can be scaled up."
About GlycoFi
GlycoFi, Inc., privately held, was founded in 2000 with the mission of dramatically improving the capacity and cost of producing human therapeutic proteins, while simultaneously enhancing their efficacy and safety as therapeutics. The company harnesses the inherent advantages of yeast and other fungal-based protein expression systems by engineering these systems to produce correctly glycosylated human therapeutic proteins, thus greatly increasing the efficiency, fidelity and scalability at which those proteins can be made. For more information on GlycoFi, please visit the company's website at www.glycofi.com. |
