ONTARIO - Enabling Biotech Innovation

Ontario’s bioprocessing sector is fostering vital R&D

By Deborah Komlos

In different ways and to varying degrees, bioprocessing spans the gamut of the diversity that is biotechnology. Not only does it underlie efforts to keep us healthy from within by working toward life-changing drugs and technologies, but it also looks to the outside, aiming to minimize environmental hazards such as greenhouse gases and contaminated water.

An underpinning for other biotech sectors and itself an independent realm, bioprocessing has much to boast about. In Ontario, research efforts are helping to keep the sector strong, and to further boost Canada’s current capacity.

“It’s enabling the biological requirements or criteria to be satisfied for various things — whether it’s making an artificial kidney . . . or making an antibiotic in a fermentor or cleaning up waste water in an activated sludge plant,” explains Murray Moo-Young, PhD, professor of chemical engineering at the University of Waterloo (UW) (Waterloo, ON), about bioprocessing. “They’re based on the same bioprocessing principles — material transfer, fluid mechanics, reaction kinetics — to allow the biological system to function properly.”

Regarding the bioprocessing sector per se, “(it) has its own challenges to develop generic procedures and techniques, so that in itself is an end product,” Moo-Young says. In terms of fermentation technology, he says computerized assistance and increasing productivity by high cell density systems, and using mathematical tools for metabolic engineering are some new developments.

Ontario’s current activities in bioprocessing include projects affiliated with Cellnet — the Cell-Factory Bioprocessing Research Network, funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) (Ottawa, ON) — for which Moo-Young is director.

Headquartered at UW, Cellnet involves 16 faculty members at six Canadian universities, among which 10 are in Ontario, including C. Perry Chou, PhD, an associate professor in UW’s department of chemical engineering.

Protein Machines

Chou, who was awarded a Canada Research Chair in Novel Strategies for High-Level Recombinant Protein Production earlier this year, is using recombinant DNA technology, working with the bacterium E. coli, to mass produce valuable proteins.

“We’re trying to optimize the host-vector system,” Chou says, “such that the production efficiency will be enhanced.”

Of focus for Chou is penicillin acylase, an important industrial enzyme that is required for the production of several γ-lactam antibiotics.

“One of the very big bottlenecks that we encounter is the protein folding problem,” Chou says. In order for proteins to function properly, he explains, their 3-D structures must be intact, and some key degenerative diseases — including Alzheimer’s, Huntington’s and mad cow disease — are related to protein misfolding.

“When we try to abuse this living cell by asking it to produce such high amounts of a protein, the cell won’t be happy,” Chou says, “and that’s why it causes a lot of physiological burden on the cell.” Consequently, he adds, cell culture growth is inhibited and many cells lyse, which limit fermentation capacity.

Chou’s team recently had a breakthrough in relation to the protein misfolding issue, finding a new inducer for the lac or lac-derived promoter system, “that not only can induce this promoter system, but also can significantly improve the protein misfolding problem,” he says.

The finding is significant, Chou says, because many labs use the lac or lac-derived promoter system to induce gene overexpression. “They always encounter the problem of protein misfolding when they overexpress the gene,” he adds.

Customized Enzymes

For Wing Sung, PhD, the application value of research has been notably realized through a collaborative agreement with Iogen Corp. (Ottawa, ON).

A principal research officer at the Institute for Biological Sciences, National Research Council of Canada (NRC-IBS) (Ottawa, ON), Sung was the inventor of four generations of an engineered xylanase called BioBrite® — launched by Iogen since 1997— for the production of bleached pulp.

Working with xylanase, an enzyme that occurs naturally in the fungus Trichoderma, Wing modified three amino acid positions, permitting the enzyme to function at 65 C, up from its original 55.

The collaboration with Iogen began in 1993, Sung recalls, when the firm approached the NRC-IBS to improve its xylanase, by allowing it to work at a higher temperature.
He explains that in the early ’90s, pulp mills changed their operations, requiring production at 65 C. The initial thrust for this change, he says, was a discovery by Canadian researchers in 1985 that PCB was being leached from paper cartons into milk. Subsequently, chlorine dioxide was identified in the pulping process as superior to the traditional bleaching agent, chlorine, in mitigating PCB production.

“With the application of chlorine dioxide to replace chlorine, it reduced almost all of the PCB on the surface of paper,” Sung says. Moreover, with less PCB being produced, less of it leaks into the waste water that results during the production process.

In order to implement use of chlorine dioxide, pulp mills needed to raise their operating temperature, Sung says, because the chemical is a slower reacting agent that requires a higher temperature.

Equally important, he says, was the 1985 discovery by scientists in Finland that pulp pretreated with xylanase would require less bleaching agent. Xylanase works to ease the release of lignin, which is trapped within the pulp by xylan and gives pulp its brown colour.

The outcome of the NRC-IBS work — invention of BioBrite® — produced the first industrial enzyme to be engineered in Canada, Sung says.

The first three generations of BioBrite are currently processing four million tons of pulp in Canada and the U.S. annually, Sung says, creating a production cost net saving of $500,000 to $1 million per mill per annum. He adds that the usage also avoided an accumulative total of 40,000 tons of undesirable organohalide byproducts in 1997-2002.

The latest BioBrite will be launched this year, Sung says, and operates at a temperature above 75 C, which should cover all of the operating requirements of the pulp mills in North America.

Meanwhile, Sung has already changed gears, having begun a collaboration last year with Vancouver, B.C.-based Hemptown Clothing Inc. He is working to help improve the efficiency of the enzymatic processing of hemp fibres. A fast-growing crop, hemp has long, strong fibres that are of interest for the production of high-quality textiles, as well as for the reinforcement of composite material, Sung says.

Per acre, hemp absorbs four-and-a-half times more carbon dioxide than one acre of forest, providing fast biomass buildup, he says. “The idea is, let’s grow (hemp) and save the trees as the carbon sink,” he adds. “Hemp requires less or no pesticide, fertilizer, herbicide, et cetera, because it grows so fast. Dandelion doesn’t have a chance.”

Sung is also continuing his research interest in plant fibre extraction in an initiative involving flax that has received funding through the Canadian Biomass Innovation Network (CBIN) (Ottawa, ON), which supports applied R&D in bioenergy, biofuels, industrial bioproducts, and bioprocesses.

Switching Carbohydrate Sources

To align with the Canadian government’s desire to reduce greenhouse gases, and to make the best use of plant biomass, Hung Lee, PhD points to the importance of sugars found in structural carbohydrates.

An environmental biology professor at the University of Guelph (Guelph, ON), Lee is researching the bioconversion ability of the pentose-fermenting yeasts Pachysolen tannophilus and Pichia stipitis, studying some of the key enzymes involved. Better understanding of the biochemistry of pentose metabolism will support a long-term goal of improving the functionality of the enzymes, Lee says.

Although Saccharomcyes cerevisiae — the traditional yeast used to make products such as beer and wine — is very well-studied and has been genetically modified to ferment pentose sugars, Lee says this function is still quite weak relative to its glucose fermentation capability.

“The discovery of pentose-fermenting yeasts occurred shortly after the second oil embargo, and it generated worldwide interest in biomass conversion,” Lee explains.

“However, the relatively low cost of oil through most of the 1980s led to diminished government interest in alternative fuels or bioenergy.”

The conclusion of the first Gulf War in the early 1990s kept the oil price low for another decade, he continues. Due to reduced funding for bioenergy in Canada, many researchers left the field, Lee says, as there was little economic incentive to keep along those research lines.

“When there’s no economic incentive, then people just won’t bother looking at (bioenergy),” Lee notes. “That’s why I think in this current climate of high gasoline prices, maybe that’s going to drive some of these technologies eventually.”

Scaling Up

Saving costs while augmenting bioprocessing potential is also a strong driver for novel technologies being developed by the industry, including those of Nysa Membrane Technologies Inc. (Burlington, ON)

Based on technology that originated at McMaster University (Hamilton, ON), the structured gel membranes produced by Nysa are “a truly disruptive technology,” says president and CEO Lisa Crossley, PhD.

Although resin chromatography is the workhorse of the biotech industry, this technique has significant limitations at the large scale, Crossley explains, including cost of hardware, as well as cleaning, storage and regulatory compliance requirements.

“So the unmet need there is a low-cost, inherently disposable high-capacity membrane that can perform like resin-based chromatography in terms of capacity, but like membranes in terms of high throughput and ease of operation,” she says.

Unlike conventional membranes, which usually have a polypropylene or polyethylene support, and are coated to make them hydrophilic and bear a binding surface, Nysa’s porous membrane support is fully impregnated with a polymer-based gel.

“You end up with much higher binding capacities, so you can bind your molecules of interest at much higher concentrations,” Crossley says. Both the raw materials and the manufacturing process are very inexpensive, she adds, and the materials are very hydrophilic, which facilitates purification.

The target market for the technology is large-scale bioprocessing for purification of biological drugs, Crossley says. The firm expects its pilot line for continuous product manufacturing online by mid-August.

Boosting Biopharma Capacity

For the Centre for Biopharmaceutical Manufacturing (CBM) of the Ottawa Life Sciences Council (OLSC) (Ottawa, ON), maximizing Canada’s biopharma output is key, and is central to the Canadian Bioprocessing Initiative (CBI), a proposed national strategy being overseen by the CBM.

The CBI comprises a series of recommendations that were released to government and industry last December for a $450 million Canada-wide investment over the next seven years. The focus: to build capacity in research, biomanufacturing and training.

“Part of the issue around trying to build capacity in biopharmaceutical manufacturing is the lack of early Phase I through Phase III clinical trial manufacturing capacity for those products in those phases,” says Ken Lawless, president and CEO of OLSC and chair of the CBI Founders Committee.

“We’ve invested somewhere in the neighbourhood of $15 billion over the last seven or eight years in public research into drug discovery and in our companies,” Lawless continues. “We have a pipeline of somewhere in the neighbourhood of about 350 to 400 products in this biotechnology space. At issue is that the companies are finding it difficult to find manufacturing capacity in Canada, and therefore are going offshore, and as soon as they do, that manufacturing never comes back to Canada.”

Also critical, Lawless highlights, is that with the global industry tripling in size, there is a huge need for highly qualified personnel, and it is the already established and quickly growing centres — particularly in the U.S. — that are attracting Canadian talent.

Among the recommendations is the creation of 30 new Canada Research Chairs to increase research capacity, as well as a focus on building international collaborations and increasing access to industry-relevant training.

Financially, the importance of bolstering Canada’s biopharma capacity is crucial, Lawless says.

The estimated current pharmaceutical trade deficit in Canada is $5.5 billion — of which Ontario’s share is $4.2 billion — and is anticipated to grow close to $11 billion by 2010, he says.

“Ontario represents a significant proportion of the biotechnology industry and our opportunity exists not only to capture the research and clinical development, but we need to (also) capture the manufacturing,” Lawless says. “We have a window of opportunity for Ontario and for Canada to shine in this space.”

To learn more about Ontario’s bioprocessing sector, please visit the following Web sites:

Biomass Microbial Processing Network

http://www.biocap.ca”>www.biocap.ca

The Canadian Biomass Innovation Network

http://www.cbin.gc.ca”>www.cbin.gc.ca

Cell-Factory Bioprocessing Research Network

http://www.cellnet.ca”>www.cellnet.ca

Ottawa Bioproducts Business Network

www.bioplan.ca”>www.bioplan.ca