BIOCATALYSIS

Enzymes can be useful catalysts for a wide variety of chemical reactions. But when it comes to small-molecule pharmaceutical chemicals, they do something especially useful: They make single-isomer compounds.

	ON THE HUNT Biocatalysis plays a key role in DSM's research efforts. BASF PHOTO

The major custom manufacturers serving the pharmaceutical industry are doing everything they can to amass synthesis techniques that yield single-enantiomer compounds, be they drug intermediates or active pharmaceutical ingredients (APIs). While enantiomerically selective organic synthesis is the traditional approach, using enzymes and enzyme-containing microrganisms to biocatalyze a reaction is becoming increasingly important.

Recognizing this importance, fine chemicals companies that don't have biocatalysis expertise are taking steps to gain it, and those that already possess it are trying to cement their technological leads.

Degussa recently launched an $18 million program called Project House Biotechnology that seeks to apply biocatalysis across the company's 21 business units. Stefan Buchholz, head of the biotechnology house, says a key target of the effort is Degussa's fine chemicals business.

Dow Chemical's bid to acquire Ascot, the British fine and specialty chemicals firm, was largely motivated by a desire to obtain Ascot's ChiroTech chiral chemistry subsidiary. ChiroTech practices both biocatalytic and organic chiral synthesis methods, including the enzyme-based production of (-)-lactam, an intermediate for carboxylic nucleosides such as the AIDS drug abacavir sulfate.

George J. Biltz, global business leader for Dow's contract manufacturing services and fine chemicals business, notes that ChiroTech's biocatalysis expertise complements his company's emerging strength in biomanufacturing. "Dow is clearly positioning itself as a leader in this industry," he says.

Clariant, which joined the fine chemicals big leagues last year with the acquisition of BTP, has embarked on a home-grown program to develop its own biocatalysis expertise. Meanwhile, established players such as Lonza, DSM, Avecia, and Great Lakes Fine Chemicals are taking steps to expand their capabilities.

Most involved in pharmaceutical fine chemicals say biocatalysis is still a market niche, although one that is growing at a double-digit rate--faster than the business as a whole. Hard sales and growth figures are difficult to come by, but one indication of the technology's potential came in an investor presentation recently given by Sergio Marchionne, chief executive officer of Lonza, one of the longest standing practitioners of biocatalysis.

Marchionne said Lonza's Biotec microbial fermentation unit has 17 products in its pipeline of compounds developed for pharmaceutical and "nutraceutical" customers. Seven of these are in various phases of the drug development process, while the balance are already launched but generally haven't reached their peak sales potential. Marchionne said these products are potentially worth close to $100 million in sales for Lonza.

ONE REASON companies don't quote specific sales figures is that a biocatalytic transformation can be just one of many reactions in a multistep API synthesis.

Bob Holt, research manager for biocatalysis at Avecia, says he can't quantify his company's biocatalysis-based sales for this reason. However, the technique is clearly critical to Avecia: According to Holt, synthesis of five of the firm's top 10 commercial pharmaceutical intermediates involves at least one biotransformation step.

Likewise, Ellen de Brabander, global R&D director for DSM Fine Chemicals, says that 30% of the more than 80 pharmaceuticals in development for third parties at DSM involve biocatalytic steps. De Brabander says this number doesn't tell the whole story, either, since DSM often scales up products based on a customer-developed technology package and doesn't have the opportunity to select the most competitive route.

Growing drug industry demand for enantiomerically pure compounds is the driver most companies cite for pursuing biocatalytic technology. Richard Wisdom, who heads the biocatalysis effort at Clariant, says chirality is behind his firm's effort. "It's a key area when you examine the types of molecules pharmaceutical companies are looking to make," he says.

Steve Taylor, who leads discovery at ChiroTech, notes that biocatalysis can also be used to improve the purity of single-isomer products made with other chiral technologies.

For example, he says ChiroTech makes a number of D- and L-amino acids using asymmetric hydrogenation. But at the end of the reaction, rather than removing the required N-acetyl protecting group with the traditional acidic approach, it uses a d- or L-acylase enzyme; this yields a neutral amino acid with enantiomeric purity greater than 99%. "We put the chirality in with hydrogenation, then sharpen up the quality with enzymes," Taylor says.

Chirality isn't always the driver, however. René Imwinkelried, head of Lonza Biotec, adds that biocatalysis can be used to carry out conversions that would otherwise require difficult or multiple synthetic chemistry steps. In such cases, biocatalysis can be the preferred route even if chirality is not desired. As an example, Imwinkelried points to the enzymatic hydrolysis of cyanopyridine to the vitamin niacinamide that Lonza carries out at large scale in a two-year-old plant in Guangzhou, China.

Lonza's niacinamide process is typical of the role biocatalysis plays in the synthesis of fine chemicals: It's generally used in multistep processes in concert with more traditional production techniques.

Indeed, most of the sector's players emphasize that biocatalysis is only a tool, and one that may or may not be used depending on the production challenge at hand. "We don't claim that biocatalysis is superior to chemical synthesis or vice versa," Imwinkelried says. "When we get an inquiry we can offer both tools."

Typically, he says, Lonza scientists will work with the customer to determine the best route to a desired molecule. If no clear approach emerges from a desk evaluation, Lonza may then conduct lab-scale biocatalytic and synthetic reactions in parallel to tease out the best solution. "Which way we go is very much case by case," Imwinkelried says.

Even its proponents acknowledge that biocatalysis isn't always the best approach. Holt gives the example of a 100-kg lot of a chiral intermediate that Avecia made with an enzymatic resolution because it already possessed "off the shelf" technology that allowed for quick response to a customer request.

However, anticipating subsequent, larger orders, Avecia also assessed both asymmetric chemistry and diastereomer crystallization methods of obtaining the desired isomer. All three approaches worked, but Holt says the company ultimately chose the crystallization as the best option for long-term manufacture.

More and more, though, biocatalysis seems to be winning out. DSM's de Brabander says she expects its role in the fine chemicals business to increase in part because of new technology coming to play in the biological sciences.

The explosion in bioinformatics and high-speed screening techniques is allowing companies to survey greater numbers of microorganisms for desired biocatalytic activity, she says. Advances in "directed evolution" methods for tailoring these organisms also play a role.

Such techniques, de Brabander says, allow companies to survey more potential biocatalysts faster than ever before. For example, DSM says it recently responded to a customer inquiry by producing lab quantities of a biocatalyzed product in three months--a process that included the identification and preparation of a new enzyme.

Broadly speaking, biocatalysis is used to carry out two distinct types of transformations: Ones in which pairs of enantiomers are resolved from each other, and ones in which a racemic compound is stereoselectively transformed into a single isomer derivative.

Resolutions generally employ isolated enzymes. They are pretty basic technologically, but can be very effective. ChiroTech's Taylor points to his firm's process for the specialty analgesic (S)-(+)-naproxen, where bioresolution is followed by recycle of the unwanted enantiomer. "It's a nice example because it's generic, where cost pressure is everything," he says. "It shows resolution with recycle can compete with chemistry."

STEREOSELECTIVE REACTIONS can also be based on isolated enzymes, but are often conducted by a microorganism that was grown by fermentation. The enzyme is, in effect, left inside the organism, along with cofactors that are required in complicated transformations such as redox reactions.

Among stereoselective reactions, the biocatalytic production of "unnatural" amino acids is the focus of several firms, partly because amino acids are versatile building blocks for a number of drug families, including protease inhibitors. In particular, L-tert-leucine is being pursued through a surprisingly wide variety of production means.

Great Lakes Fine Chemicals, for example, is currently scaling up production of L-tert-leucine using a process developed by NSC Technologies, the biocatalysis company it acquired in 1999.

According to Simon Sellers, vice president for technology and commercial development, Great Lakes starts with a whole-cell bacteria strain produced by fermentation. The transaminase-containing cells are then fed a substrate that they biotransform to tert-leucine. Great Lakes already makes amino acids such as D-phenylalanine and D-tryosine using similar technology.

DSM produces L-tert-leucine through a combination of chemical and biocatalytic means. According to de Brabander, the company makes D,L-tert-leucamide with Strecker chemistry and converts this into the L-amino acid with the enzyme L-amidase.

Degussa employs an isolated leucine dehydrogenase enzyme that works in concert with cofactor regeneration--an industry first, according to Buchholz. Consuming a cofactor would be prohibitively expensive but, in the Degussa scheme, it is returned to its original state with the help of a second enzyme, formate dehydrogenase.

Buchholz says Degussa has a second amino acid platform based on L-acylase that it uses to make L-methionine. In development is a process in which L-hydantoinase and two other enzymes are co-expressed in a genetically engineered cell. Degussa plans to use it as a source of d- and L-amino acids of interest to pharmaceutical customers.

Buchholz believes there are four fundamental skills in biocatalysis: The screening techniques for finding microorganisms that contain desired enzymes; the ability to optimize the organism with directed evolution; the capacity to grow the optimized organism through fermentation; and the technology to apply the biocatalyst--either as isolated enzyme or whole cell--in a chemical reaction.

"Our goal is to have expertise in all four fields," he says. Some of these skills exist already in Degussa; some have been acquired, such as the chiral technology group of Aventis Research; and some will be developed during the three-year run of the Project House. Buchholz adds that for a specialty chemical maker like Degussa, the key skill is the application of biocatalysis in chemical synthesis. "For us, enzymes are just a means to an end," he says.

Buchholz makes a distinction between companies that buy enzymes and companies that grow their own. Although enzymes can be purchased from a number of sources, he contends a company that can produce its own is at a competitive advantage.

Avecia's Holt agrees. "You can buy enzymes for use in resolutions or regioselective chemistry," he says. "But there are just a limited number available. So a key strength is to be able to develop novel enzymes yourself that have novel selectivity and higher reaction rates. But that requires microbiological skills and fermentation capability, which not everyone has."

Three of Avecia's major products, he points out, are based on the asymmetric reduction of ketones to secondary alcohols in whole cells. "You cannot buy enzymes off the shelf that do this," Holt says.

He notes that Avecia's experience in microbiology dates back to the 1970s, when its predecessor company ICI developed a single-cell animal feed protein called Pruteen that grew on methanol. "It was a technical success and a commercial disappointment," Holt says, but it left the company with a lot of knowledge about fermentation.

Others agree that biocatalytic expertise is critical, but contend that possessing fermentation capacity isn't.

Clariant, although new to biocatalysis, has expertise. Wisdom, for example, ran the biocatalysis program at ChiroScience, the U.K. biotechnology company that once owned ChiroTech. And Clariant's biocatalysis program falls under its life science molecules synthesis center, which is headed by Graham Howe, ChiroTech's former director of operations.

HOWE POINTS OUT that there's ample merchant market fermentation capability that can be hired to produce biocatalyst-yielding microorganisms. Libraries of novel enzymes are available as well, from specialist firms and universities.

"Our initial strategy will be to access biocatalysts from outside parties. We won't make them ourselves," he says. "Our strength will be the development of biotransformations and their integration into our own chemistry and synthesis stream."

Likewise, NSC Technologies historically went to third parties for its fermentation steps. Great Lakes continues to be comfortable with this approach, according to Sellers, although it has brought many of the previously tolled biotransformation and purification steps into existing facilities.

Biocatalyst experts are also of two minds about the importance of specialized companies such as Diversa, Maxygen, Protéus, and Thermogen that have developed libraries of novel or "extremophile" organisms, often culled from exotic sources such as geothermal vents and volcanoes.

Through its recently formed alliance with the drug development company MediChem, Degussa has access to the expertise of MediChem's Thermogen industrial enzyme subsidiary, Buchholz notes. Degussa's care and silicones units are working with the French enzyme-discovery firm Proteus as well.

Howe acknowledges that such firms are a potential source of novel biocatalysts for Clariant as its program progresses. "We are evaluating a number of organizations and what they can provide," he says.

Imwinkelried says Lonza has had discussions with such companies and notes that it has a similar alliance on organic chemistry catalysts with the combinatorial chemistry firm Symyx. "We know the companies and if we feel they can contribute something then we will go with them," he says.

TAYLOR'S EXPERIENCE so far is that simple in-house screening and enzyme growth techniques yield good, very cheap biocatalysts. He notes that ChiroTech has a fruitful cooperation with the University of Exeter in England for developing extremophile enzymes. However, he's wary of the costs of an alliance with an outside company. "Those alliances can sometimes require a lot of investment and you're not guaranteed a successful outcome," he says.

Holt, at Avecia, is also cautious regarding the current fad for extremophiles, although the company is actively supporting research at the Centre for Extremophile Research at the University of Bath in England. "There are a lot of people doing a lot of trips around the world, but in most cases it's simply not necessary," he maintains. "From an industrial point of view, it's often the microbe in the garden that will do the job."

What all parties do agree on, however, is that the role of biocatalysis in the synthesis of fine chemicals is on the rise. Imwinkelried notes that the understanding and appreciation of biocatalysis is growing among customers in the pharmaceutical industry. "Some now recognize that involving biotechnology tools very early on is an advantage," he says. "But there is further potential to be exploited."

BREWING Great Lakes Fine Chemicals' lab in Mount Prospect, Ill., is the source of several transaminase breakthroughs.
GREAT LAKES FINE CHEMICALS PHOTO

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