"For cancer therapy, or treating an autoimmune disease," observed Stefan Wildt, director of strain development at GlycoFi Inc. in Lebanon, N.H., "you need metric tons for any given antibody per year. About 500 candidates are currently in clinical trials. And with a drug manufacturing capacity already stressed out," he added, "our approach might provide biotech companies with a way to generate antibodies in very large amounts. That's why those molecules are the largest class of protein product of interest to us."
Wildt is co-senior author of a paper in the Proceedings of the National Academy of Sciences (PNAS), dated April 17, 2003. It's titled: "Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris." Its other co-senior author is Tillman Gerngross, GlycoFi's chief scientific officer.
"Our overall finding in the PNAS paper," Wildt told BioWorld Today, "is that one can really and truly re-engineer fungal systems so they are capable of producing therapeutic glycoproteins - and the emphasis is on glyco.' We really managed to eliminate fungal-specific glycosylation, which clearly showed the first step toward a complete human glycan sugar. If you take a therapeutic protein, say an antibody for instance," he explained, "and you produce it in a fungal system, nonmodified and in a mammalian cell, you will get back two molecules that look fairly identical, with the exception of the N-glycan, with sugar chains attached to very specific locations.
"The one produced in the fungal system will be recognized by our immune system as something that's foreign," he continued, "because it has a fungal-specific N-glycan on it. That N'," Wildt noted, "stands for asparagines,' a shorthand initial for that amino acid. You could, in theory, make it in a bacterium or a yeast, but only if it has an appropriate N-glycan so it functions as desired.
"That is true for about 70 percent of all therapeutic glycan proteins that are currently on the market," Wildt pointed out. "They completely depend on proper glycosylation to fold correctly and thus create an active therapeutic compound. There are hundreds in the pipelines. That's why we feel that this is such a breakthrough. It is awfully expensive to produce therapeutic proteins. If a large company decides today that they would like to bring a new drug on line, that usually means they also have to bring on line new manufacturing capacity. And what that means in turn is that they have to commit up to $400 million to build a new plant, because the current manufacturing facility is at capacity. Whenever a large company, such as Pfizer or Bristol-Myers, decides to move a drug candidate from the pipeline toward clinical studies," Wildt went on, "they have to make sure that they can also produce them. And that is a major bottleneck for these companies."
Recruiting Pichia pastoric Plus Fungal Proxies
"Now what we did," Wildt recounted, "is decide to look at fungal systems. They are per se better production vehicles, because they go faster, cheaper and produce more protein per cell. And the only drawback we could see using fungal systems was actually that they were not capable of producing these therapeutic glycoproteins - such as antibodies.
"When we started to test this approach," he recalled, "people in the industrial know said, This is really intriguing, because if you can come on line with a fungal system, we would need to build smaller protein production plants, or we could even get away with inserting that technology into what we already have.' And that saves them potentially hundreds of millions of dollars.
"Scientists have tried since the early 90s to re-engineer fungal systems, because the advantages were so obvious. What has been done in the past was that most research groups we know of pursued a sequential approach - knocking out a gene here and knocking in another gene there, then seeing what happens. We pretty much decided the sequential strategy does not really work that well. So we proceeded to devise a library approach - a tour de force, so to speak.
"We said, We will not attach one enzyme after the other in a fungus. We will create large libraries of enzymes and get a high-throughput read-out of the concepts we engineered, which actually worked, rather than spend years on testing five or six different permutations.' So we went in and tested literally thousands of DNA constructs. Nobody else found them before us."
From ER To Golgi To Human Cell
"In the PNAS article, yeasts are the fungal subspecies. We do this in a yeast called Pichia pastoris, which is a well-established protein production host. It is known as a very robust yeast that is typically used for production of nontherapeutic proteins. For example, collagen, cellulases, food enzymes.
"The steps from RNA to protein take place at the endoplasmic reticulum (ER). We genetically re-engineered the secretory pathway of P. pastoris to perform sequential reactions that mimic early processing of N-glycans in humans and other higher mammals. This represents the first report of a yeast able to synthesize hybrid glycans in high yield, and opens the door for engineering yeast to perform complex human-like glycosylation.
"What also happens at the ER is the RNA translation. While the protein is being synthesized, it is being inserted into the ER, where it penetrates the membrane. It is within the ER that a complex of enzymes can exit, and will attack an N-glycan. That glycosylation is co-translational. One piece of the protein is still being synthesized, while its first segment is already inserted into the ER, and being modified by attachment of those sugar chains. The ER is in any mammalian cell - as well as eukaryotic cells, with the exception of what typically happens on the outside of the ER. Then the protein moves from the ER into the Golgi apparatus - a process complexly identical between humans and yeast.
"Right now we're moving from a proof-of-concept to real product development, the really expensive things in a biotech company's life - animal studies and clinical trials. One of the major challenges for us is now to inform the FDA about what is coming."