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.