SBIR-STTR Award

The pharmacological efficacy of a protein often depends on the protein's glycosylation structure, and successful manufacturing relies on host systems that produce glycoforms similar to those in humans. The overall objectives of this project are to develop tools for the metabolic engineering of fungal glycosylation pathways and to use these tools toward the creation of fungal strains capable of producing and secreting large quantities of recombinant therapeutic proteins with human-like glycosylation structures. The tool development strategy centers on a library-based approach that will allow rapid identification of properly targeted and expressed glycosylation enzymes in a high throughput manner. PROPOSED COMMERICIAL APPLICATIONS: The current method of glycoprotein production based on mammalian cell culture systems, specifically Chinese hamster ovary cell lines and mouse fibroblast/myeloma cell lines, presents manufacturers with the problems of low production levels, high cost, and safety. In its present state, protein production technology cannot keep up with research and development efforts, making manufacturing the growth-limiting factor for the biotechnology industry. With 84 biological drugs on the market and well over 500 in clinical development, the proposed project will generate technology toward the manufacturing of glycosylated proteins with high production levels in a safe and cost-effective manner, thereby alleviating the production bottleneck and allowing more products to reach the patients who need them

Through metabolic engineering of the yeast glycosylation pathway, GlycoFi is developing yeast capable of secreting recombinant proteins with human-like glycosylation. In Phase I, a toolkit of cloning vectors, strains, libraries and protocols were developed for targeting glycosylation enzymes to the yeast secretory pathway. The feasibility of this approach was demonstrated by the functional expression of sigma-1,2,-mannosidase in vivo, resulting in the production of a recombinant protein with 65 percent human-like Man5glycosylation structure. This Phase II proposal aims to develop an automated, high-throughput approach capable of screening 3000 samples per week for highly efficient enzyme catalytic domains. These enzymes will be incorporated into a production strain generating commercially important proteins with a human-like Man5glycosylation structure. Proposed Commercial Application: A shortage of manufacturing capacity has emerged as the single largest barrier preventing therapeutic proteins in the development pipeline from reaching the clinic. GlycoFi's novel yeast-based protein production platform will alleviate this bottleneck and allow the bio-pharmaceutical industry to bring their products to market in a faster and more cost effective manner.

Thesaurus Terms:
biotherapeutic agent, drug design /synthesis /production, glycosylation, high throughput technology, mannosidase, protein engineering, technology /technique development Golgi apparatus, alpha 1 antitrypsin, chimeric protein, endodeoxyribonuclease, endoplasmic reticulum, fermentation, glycoprotein, interferon beta, protein purification, recombinant protein Drosophilidae, Moniliales, bioengineering /biomedical engineering, biotechnology, high performance liquid chromatography, matrix assisted laser desorption ionization, peptide library