Carrying Proteins into A New Sphere

---

By Deborah Komlos

Separating from water much like cream from milk, safflower oil and its inherent properties have given Maurice Moloney, PhD and his Calgary-based company reason to aim for the top.

Founded by Moloney in 1994 as a University of Calgary spinoff, SemBioSys Genetics Inc. offers a unique approach to producing, purifying, formulating and delivering protein-based pharmaceuticals through genetically transforming safflower (Carthamus tinctorius L.) seed. The company’s platform technology — Stratosome™ Biologics System — involves attaching commercially relevant proteins to the seed’s oil-storing organelles (oil bodies) as an oleosin fusion or via affinity capture. The company has also developed a means of recovering the non-transgenic oil bodies for dermal- and personal-care use, referred to as the DermaSphere™ Ingredient System.

Success with the safflower oil bodies stemmed from initial research on canola in 1990. Funded federally and provincially, Moloney and his group of three or four researchers at the University of Calgary examined how oil seeds, like canola, can package vegetable oil within individual seed cells. The purpose of this, explains Moloney, who is also a professor at the university’s Department of Biological Sciences in addition to his role as chief scientific officer of SemBioSys, was to find novel means of improving oil quality or quantity.

During that research, the team discovered the fundamentally interesting property that oil bodies function as an important protein repository in the seed, in addition to storing oil. The group then set to work on examining how proteins are organized on the oil bodies. “In the course of doing that, I guess I came up with a cute idea — that you could potentially attach other proteins to the oil body,” Moloney says.

While on sabbatical at a U.K. research institute in 1993, Moloney developed the concept of forming SemBioSys. His plan materialized the following year through the University of Calgary’s corporation, University Technologies International Inc. Outside funding followed from connecting with several companies, including initially with Ciba-Geigy Canada Ltd. (later becoming Novartis Pharmaceuticals Canada Inc.), and eventually with Dow AgroSciences Canada Inc. The latter became — and still is — the company’s only equity stakeholder, aside from the university and Moloney.

Prior to pursuing research on seed-specific gene expression, herbicide resistance and the plant cell cycle at the University of Calgary, Moloney had worked on agricultural chemicals at Imperial Chemical Industries Ltd. in England, which became Zeneca Group PLC and ultimately part of AstraZeneca PLC. Moloney then joined California-based Calgene Inc. (later acquired by Monsanto Co.), where he used canola as a model to develop the first transgenic oilseed plants. This work resulted in a landmark plant biotechnology patent and eventually became the basis of the canola varieties Roundup Ready® and Liberty Link®.

SemBioSys now comprises 45 members, including 35 in R&D, and moved to its own 25,000-square-foot, off-campus facility in June 2001.

With several products in the pipeline and already bearing a strong intellectual property portfolio comprising nine issued U.S. patents and another nine applications pending, “The longer-term focus is definitely towards therapeutic proteins,” Moloney says. “The unfortunate thing about this technology is it can be applied to a very wide range of problems and we have had to make some strategic decisions about what are we going to focus on in the next little while.”

Selecting Product Candidates

Perhaps not as lucrative but posing less of a regulatory challenge relative to pharmaceuticals, a personal-care product is the first the company hopes to launch, Moloney says, towards the end of this year or early 2004.

“We’ve taken the view that we would like to get this company into a break-even mode in a relatively short term; we’re much more investible if we’re in that situation,” he says.

Another project involves a food-processing protein, which the company intends to launch sometime next year. Significant work is also underway using the affinity reagent Protein A as a tool for antibody purification. The first product based on that work is hoped to be launched by the end of 2004.

“Therapeutic antibodies have enormously increased in their presence in the marketplace and the single biggest problem with the production of therapeutic antibodies is purification,” Moloney says. In fact, he adds, this step consumes as much as 70 per cent of funds spent on production.

In this regard, the idea to use oil bodies as a vector for protein production in seed turned out to be quite valuable, Moloney says. As the oil fraction is lighter than water, natural separation occurs allowing ease of purification and not requiring costly chromatography steps.

Oil bodies comprise a triacylglyceride (vegetable oil) core surrounded by a half-unit phospholipid membrane and an outer shell of proteins called oleosins (Fig. 1). The group uses the conventional technology of Agrobacterium-based transformation on leaf and stem pieces to introduce a copy of the native oleosin protein with an attached target protein (an “oleosin fusion”) into the oilseed crop (Fig. 2). A cleavage site may be included to permit enzymatic or chemical cleavage of the target protein during purification. As the seed develops, the oleosin fusion is targeted to oil bodies. Using SemBioSys’ proprietary method, the seed is processed through a series of steps including grinding, washing and centrifugation to extract the protein-rich fraction.

“The oil bodies, when they’re produced purified, look like a beautiful white ointment,” Moloney says. A single oil body measures from 0.5 to 2 µm in diameter. Hundreds of these organelles are found in a typical cell of 50 µm. A safflower seed itself is slightly oval-shaped and about five to six millimetres long.

A large number of companies are very interested in trying to incorporate the oil bodies into their products, Moloney says. Various therapeutic agents, such as anti-oxidants like vitamin E or steroids that are used to treat hyper-inflammatory conditions, are possible applications. Through clinical trial work on human subjects, his group has demonstrated significantly less resulting irritation when oil bodies are used with each of hydroquinone, retinoic acid and salicylic acid, instead of the conventional personal-care bases.

In cases where the final product is an ointment, there is an advantage to having the protein of interest as an intrinsic part of the oil body, fused to its membrane protein, Moloney explains. However, when the desired end result is protein purification, having the oil body function as a magnet to pick up proteins floating freely in the cell by affinity capture (covalent bonding) is preferred.

Developing from seed to seed in about four months and requiring a relatively small acreage, safflower presents several advantages as a protein production system. For instance, Moloney says, the crop of their food processing protein will at maturity require roughly 1,000 hectares from which about two tons of protein can be made per year. This will deliver about a quarter of the world’s supply of the protein. “When you look at the volumes and numbers you have to deal with, from an agricultural point of view they’re not outrageous,” Moloney says.

Critical Issues Faced

Despite the various reasons for optimism, Moloney admits that SemBioSys still faces a rather large roadblock. Unlike in the United States, he explains, there is currently no Canadian system in place to regulate commercial production. Fortunately, Canadian specifics exist for field trials, such as allowed acreage. The company has thus been able to conduct such trials, with main sites in southern Alberta and southern Saskatchewan. To permit counter-seasonal growth, the company also has sites in California, Mexico and Chile.

The key now, Moloney says, is having the Canadian Food Inspection Agency recognize transgenic plants as a new category of crops. Trepidation naturally surrounds cultivating a crop that contains biologically active materials, which then raises the level of scrutiny and custody management of the material by several orders of magnitude. “It isn’t normal agriculture,” he says. This was why they decided to work on safflower as it is considerably less extensively grown and is not a food crop — aside from some grown for bird seed — compared to canola, which is a major oilseed in Canada. Thus, sufficient spatial separation from any food crops could be ensured.

If within the next few months the appropriate regulations are not established and implemented, Moloney says the company’s “biggest concern” is having to resort to “plan B”: moving production to the U.S.

“We’ve been fastidiously Canadian nationalists about this company from day one as anybody who knows us can tell you,” Moloney says.

Reflecting on the success of SemBioSys to date, Moloney says a key question he initially faced was how to obtain funding beyond the regular use of university grants. Crucial as well, “Is you figure out pretty early on that this is not a one-person show and you’ve got to surround yourself with some pretty good people who can take on responsibilities of all different kinds,” he says.

Canada is very fortunate in having extremely competitive educational institutions producing excellent researchers across the range of biological sciences, Moloney says. But the challenge is to find more business professionals who have the necessary skills to build a company. “It is helpful to have an MBA, but I still think an MBA on its own can be fairly theoretical until you’ve actually gone in and managed a small company and built it up,” he says.

For this reason, “We take people straight out of post-docs and give them a lot more responsibility than they expected to have and they become involved in the patenting side, the intellectual property side,” Moloney says.

“Although we were doing a lot of bench-level science at Calgene, I learned a lot about the patent system very quickly; a lot about negotiating deals with third-party companies,” he says. “So I was kind of thrown in the deep end, and so that’s what we do with our people.”

It’s important, Moloney says, to have individuals such as SemBioSys’ president and CEO Andrew Baum, with whom he worked at Calgene to develop the Roundup Ready® concept. Baum has worked in the field of plant biotechnology for about 17 years.

“Now look around Canada and find somebody who’s got that level of experience – it’s difficult,” Moloney says. “Ultimately what we need is a core of people who have worked in Canada, trained in Canada, are familiar with what we do in Canada,” he says. Having more expertise in an organization also increases the opportunity for everybody to learn from that expertise and minimize the chance of making “big” mistakes.

Coupled with training and attracting suitable personnel, Moloney says the issue of establishing concrete regulatory rules is necessary and will give the industry a chance to flourish in Canada. “At the same time it gives the general public the sense that the regulators are absolutely on top of this,” he says.

Also critical, though, is increasing the comfort level of people and the average investor with the notion of unconventional crops. “I think we require a certain amount of demystification before people will be more comfortable,” Moloney says.

There’s generally less apprehension, he says, about investing in the drug industry or something such as oil and gas, where people don’t have an issue with that business and how it works. “The main thing you have to keep your eye on is what are oil prices doing, what factors at work drive it up or down,” he says.

Studies using plant material lack the same ethical concerns that are often associated with animal-based systems. However, it does sound very different to people when genes are inserted into “this and that” in order to have various substances expressed, Moloney says.

“Now in fact, eventually people will remember that half of our pharmaceuticals come from plants,” he says, giving the examples of aspirin, codeine and morphine.

Until then and along the way, Moloney says that creating products by plant transgenics which have clearly beneficial effects will encourage a greater acceptance of this novel mode of manufacturing, and offer a sense of demystification.

“People can (then) see an example of what this whole biotechnology thing is about.”