June 1, 2014 (Vol. 34, No. 11)
The Microbe’s Methylotrophic Ways Still an Inspiration for Protein Expression Platforms
James M. Cregg, Ph.D., of the Keck Graduate Institute came to engineering Pichia pastoris for biopharmaceuticals through a circuitous route. After more than 30 years, he remains a pioneer in using Pichia to produce commercial antibodies.
Dr. Cregg’s interest in Pichia began while he was working at SIBIA (Salk Institute Biotechnology Industry Associates). About a decade before, Philips Petroleum had invested in Pichia for manufacturing inexpensive animal feeds. Philips had a nearly endless supply of methanol—the yeast’s preferred carbon source—through the conversion of refinery off-gases, mainly methane.
The oil shock of the early 1980s changed the economics unfavorably, so Philips tasked its multimillion dollar Pichia plant toward manufacturing growth hormone for pigs. Lacking direct expertise, the company approached SIBIA, and before long Dr. Cregg had a process and enough hormone to test, but because of the oral delivery method the product didn’t work.
“The pigs didn’t grow any faster,” Dr. Cregg says. “But at least we had an expression system, so we started looking around for applications.”
Philips eventually terminated its interest in pharmaceuticals and dropped Pichia entirely. Dr. Cregg began collaborating with Merck, and eventually moved on to the Oregon Graduate Institute, where his Pichia work continued.
In collaboration with Seattle-based Alder Biopharmaceuticals, Dr. Cregg helped to commercialize that company’s Mab Xpress® Antibody Production System, the manufacturing platform for Alder’s two Phase II monoclonal antibodies.
Pichia has several advantages compared with animal cell culture: shorter time of cell- line selection (one month versus up to nine months), shorter doubling time (90 minutes versus 1 day), more compact fermentation cycle (one week versus one month), larger potential production scale (160,000 L versus 25,000 L), adaptation to relatively inexpensive culture media, and no requirement for viral clearance and related validation. Cell culture virus studies can take as long as nine months.
Worldwide regulators have approved at least 16 products expressed in Pichia. These include large peptides and recombinant proteins (such as Kabitor® from Dyax, albumin, heparin-binding EGF-like growth factor, insulin, interferon-alpha), enzymes (trypsin, phytase, nitrate reductase), a vaccine (Shanvac™ hepatitis vaccine from Shantha/Sanofi), and two antibody fragments (Nanobodies ALX00171 and ALX0061 from Belgium-based Ablynx). But thus far, no fully functional antibodies.
Purifying Pichia-produced proteins is simple compared with CHO. The organisms excrete recombinant proteins into the medium but release very few native proteins. “The product is virtually the only protein in the medium,” Dr. Cregg explains. Purification consists primarily of spinning down intact yeast cells, and perhaps a flow-through step to remove impurities.
Pichia conducts many post-translational modifications common in higher eukaryotes, such as protein folding, disulfide formation, and glycosylation. Pichia would have no serious faults except that its glycosylation consists entirely of mannose glycans, compared with the variety of sugars employed by more advanced organisms. Native Pichia mannose-based glycosylation is associated with adverse immune responses in humans, whereas the sialic acid glycosylation of CHO cells improves circulating half-life.
The significance of the differences between human-like and nonhuman-like glycosylation remains controversial. It is not an issue for research reagents or at least some recombinant protein therapeutics, but glycosylation affects stability, solubility, bioavailability, activity, pharmacokinetics, and immunogenicity. The story can be even more complicated, as some marketed products contain nonhuman glycoforms. Also, human-like glycosylation is highly heterogeneous, differing noticeably from batch to batch.
Alder overcame the glycosylation peculiarities of Pichia by engineering the yeast—changing a serine to an alanine at the C-terminus—in a way that renders the organisms incapable of accepting N-linked glycans at that location. (Due to an impending stock offering, Alder was unable to confirm the nature of glycosylation at other locations on their clinical-stage antibodies.)
Research groups have achieved varying degrees of success in humanizing Pichia glycosylation by modifying glycans enzymatically post-expression, changing culture conditions, and through reengineering Pichia’s glycosylation pathways. One such approach, Pichia Glycoswitch®, developed by researchers at the University of Ghent, is available through license from Research Corporation Technologies.
The most concerted commercial effort at glycan humanization is occurring at Glycofi. Co-founded by Tillman Gerngross, Ph.D., an engineering professor at Dartmouth, Glycofi was acquired by Merck in 2006 for $400 million. Glycofi achieved complete humanization by deleting 4 yeast genes and introducing 14 nonnative genes that control glycosylation.
Merck continues to publish in journals on this work, but since the acquisition industry-level news from its Glycofi subsidiary has nearly come to a standstill.
If Pichia is such a great expression system why hasn’t every biomanufacturer jumped on it?
Simply: The yeast’s glycosylation issues have not been solved to the industry’s satisfaction. “It’s still considered new,” says Dr. Cregg, “and drug companies are very conservative.” For example, Alder took 10 years to bring its monoclonal antibodies to the clinic. And several Pichia-produced drugs have failed, but whether the molecule or the expression system was at fault is unclear.
Other issues involve intellectual property and royalty fees, which Francisco Valero Barranco, Ph.D., professor of chemical engineering at the University of Barcelona, says are extremely high. “Avoiding patent infringement in this field is very difficult,” he laments. Dr. Barranco believes that plain vanilla Pichia is fine for producing nondrug proteins such as enzymes, but for now barriers are too high for human therapeutics.
So impervious is the barrier to accessing humanizing technology that Dr. Barranco, himself a renowned Pichia expert, has been thwarted in attempts to collaborate with industry.
Another potential drawback is Pichia’s disposition toward an ineffective unfolded protein response (UPR). Many expression systems compensate for UPR by temporarily shutting down protein production and bringing in chaperone proteins to address misfolded products. The inability to deal with UPR leads to unusable product and premature cell death.
Yet Dr. Barranco is betting long-term on Pichia. If glycosylation problems are overcome, he predicts that Pichia “could be an excellent substitute for CHO.”
In Pichia’s favor are streamlined process development that is even simpler than for platform CHO processes. The CHO-based processes await the perfect wave of media/feed protocols; Pichia processes need just water and methanol.