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Volume 84 Issue 33 | pp. 15-25
Issue Date: August 14, 2006

Cover Stories

Enzymes At Work

Rapid screening and optimization of enzymatic activity, along with available, easy-to-use enzymes, are making biocatalysis a handy tool for chiral synthesis
Department: Science & Technology

Chemists have been practicing organic chemistry for hundreds of years; microbes have been at it even longer. Microbial and other enzymes are superbly enantio-, chemo-, and regioselective across a diverse range of reactions under mild conditions of pH, temperature, and pressure. Why, then, has it taken chemists so long to put aside a dislike of "bugs" and use their enzyme catalysts?

[+]Enlarge
Optimized
Degussa has invested in developing enzymatic reactions as a technology platform and has now made its Service Center Biocatalysis part of its Exclusive Synthesis & Catalysts business unit.
Credit: Degussa
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Optimized
Degussa has invested in developing enzymatic reactions as a technology platform and has now made its Service Center Biocatalysis part of its Exclusive Synthesis & Catalysts business unit.
Credit: Degussa

The question is especially pertinent when it comes to making pharmaceuticals. When scientists at GlaxoSmithKline, AstraZeneca, and Pfizer examined 128 syntheses from their own companies, they found that as many as half of the drug compounds made by their process R&D groups are not only chiral but also contain an average of two chiral centers each (Org. Biomol. Chem. 2006, 4, 2337). And to meet regulatory requirements, enantiomeric purities of 99.5% were found to be necessary.

"When it comes to wanting selectivities of 98% or higher, you are probably bound to a bioprocess, because getting beyond 95% otherwise is really tough," says Kurt Faber, professor of chemistry at the University of Graz and a member of the Research Center for Applied Biocatalysis (RCAB) in Austria. Biocatalysis can also open up new or even greener reaction routes (J. Mol. Catal. A 2006, 251, 66).

The pharmaceutical researchers also pointed out that more than half the time process chemists purchased chiral starting materials rather than generating the chiral centers themselves. Putting two and two together, fine chemicals producers are taking advantage of biocatalysis to produce the needed high-purity chiral intermediates. They do so on both a custom and catalog basis; BASF, for example, has its ChiPros chiral building blocks, and DSM offers intermediates under the Chiralitree label.

BASF and DSM, Degussa, Lonza, NPIL Pharma (through its acquisition of Avecia's business), and many Japanese companies actually have long histories of using large-scale fermentations and biotransformations. Enzymes are end products and processing aids in many industries and are used to make bulk food ingredients and specialty chemicals (C&EN, April 3, page 69). Other fine chemicals companies, such as Dowpharma, Cambrex, and Archimica (the former Clariant pharmaceutical fine chemicals unit), have added biocatalysis capabilities largely through acquisitions.

Within the past 10 or so years, fine chemicals firms have begun integrating biocatalysis into their custom synthesis offerings. "We see it as one part of the toolbox to address our customers' problems as broadly as possible," says Wolfgang Wienand, head of Degussa's Service Center Biocatalysis. SCB was created in 2004 after the company invested for three years in one of its Project House technology development initiatives. In February, SCB became part of the company's Exclusive Synthesis & Catalysts business unit.

Most have built technology platforms around several enzyme types to offer different chemistries. For example, resolution using hydrolase enzymes, such as lipases, is a well-developed approach to separating racemic mixtures, although its inherent 50% maximum yield can be a drawback (see page 29). Enzymatic reactions that generate chiral centers are valuable, because they convert prochiral substrates into single-enantiomer products and offer 100% theoretical yield.

For more than a decade, BASF has used lipases to resolve racemic alcohols and amines by selective enzymatic acylation of one enantiomer to yield easily separable products. More recently, the company has been using new dehydrogenase enzymes to make optically active styrene oxides and aliphatic alcohols. And it is expanding its access to other new enzymes through a relationship with Diversa.

BASF, Enzis (recently acquired by Codexis), the start-up company Oxyrane, and others have also been exploring epoxide hydrolases for making chiral intermediates. These enzymes have generated interest because they can compete, for example, with metal-catalyzed Jacobsen hydrolytic kinetic resolution in resolving epoxides with high enantioselectivity (C&EN, Oct. 24, 2005, page 27).

In other work on epoxides, BASF has collaborated with Dick B. Janssen's group at the University of Groningen, in the Netherlands, whose work also supported Enzis, on halohydrin dehalogenases. These enzymes typically catalyze the reversible ring closure of vicinal haloalcohols to yield epoxides and halide ions. But the enzyme can accept other small negatively charged nucleophiles, such as azide, cyanide, and nitrite ions, to yield β-substituted alcohols and nitriles (Adv. Synth. Catal. 2006, 348, 579).

C−C bond-forming reactions are fundamental synthetic tools, and DSM has a strong position using lyases and aldolases, says Marcel Wubbolts, DSM's program director for white (industrial) biotechnology. For example, DSM researchers have modified a deoxyribose aldolase enzyme to increase its activity and substrate tolerance (Biotechnol. J. 2006, 1, 537) and thereby make it more synthetically useful for producing an intermediate for the blockbuster drug Lipitor (see page 26).

DSM also makes enantiopure cyanohydrins on a large scale from hydrogen cyanide and aldehydes or ketones using optimized (R)- and (S)-hydroxynitrile lyases (HNLs). These cyanohydrins then go into intermediates such as (R)-2-chloromandelic acid for a cardiovascular drug and (R)-2-hydroxy-4-phenylbutyric acid for angiotensin-converting enzyme inhibitors, Wubbolts explains.

DSM and RCAB researchers also have applied HNLs to the kilogram-scale production of 1,2-amino alcohol intermediates. They have developed a chemoenzymatic process for making (R)-2-amino-1-(2-furyl)ethanol to demonstrate a convenient general route (Org. Process Res. Dev. 2006, 10, 618). An HNL enzyme stereoselectively generates the chiral center in one step from a cheap starting material and avoids the need for protection/deprotection steps.

On the other hand, to produce (R)- or (S)-alcohols on an industrial scale, Degussa has engineered a two-enzyme, whole-cell system containing either an (R)- or (S)-alcohol dehydrogenase and either formate dehydrogenase (FDH) or glucose dehydrogenase (GDH), Wienand says. The company collaborated with BRAIN AG (Biotechnology Research & Information Network) and German universities and government organizations to develop the technology under a government grant for sustainable bioproduction methods.

Ketoreductases or alcohol dehydrogenases are attractive means to synthesize a chiral center directly, but they require nicotinamide adenine dinucleotide (NADH) or NAD phosphate (NADPH) as a hydrogen source. Two-enzyme systems, either in cells or using isolated enzymes, have become the prevailing solution to recycle catalytic amounts of the expensive cofactors. As the second enzyme, FDH oxidizes formate to CO2 or GDH converts glucose to gluconolactone to regenerate NADH or NADPH, respectively.

Daicel Chemical Industries has created a recombinant whole-cell system containing GDH and an optimized tropinone reductase, originally isolated from a plant rather than from more typical microbial sources, explains John R. Peterson, president of Thesis Chemistry, Daicel's U.S. representative. Daicel uses it to reduce 3-quinuclidone to (R)-3-quinuclidinol, an intermediate for urinary incontinence drugs. Improving on the yield and selectivity of competing chemocatalytic and enzymatic approaches, the process is now industrially useful for making this and other intermediates, he adds.

Although converting a ketone to a single chiral hydroxyl group is very useful, a greater challenge is achieving regioselective reactions in multifunctional molecules while avoiding the need for protecting groups. Faber's group and collaborators from Ciba Specialty Chemicals have investigated the reduction of diketones and oxidation of diols using an alcohol dehydrogenase they discovered. They found that the regio- and stereoselectivity obtained depended on the relative position of functional groups (Eur. J. Org. Chem. 2006, 1904).

Earlier this year, DSM started collaborating with enzyme developer IEP, which has commercialized more than 10 bioreduction processes. Whereas oxidoreductases are now appearing in industrial processes to more efficiently make chiral alcohols, Wubbolts believes that "there is still a lot of work to do on very selective hydroxylations or oxidations within complex molecules and on addition and elimination reactions, as with lyases."

Other desirable intermediates include amino acids, especially nonnatural variants that can't be made by fermentation. Many fine chemicals companies produce these on several-hundred-kilogram or ton scales via hydrolytic resolution. Other enzymatic approaches are reductive amination or amino-group transfer. Excelsyn's biocatalytic capabilities, which came with its acquisition of the former Great Lakes fine chemicals business, include making nonnatural amino acids using transaminases, amino oxidases, and ammonia lyases.

Expressive
Dowpharma used its Pfenex expression technology to produce large quantities of enzymes.
Credit: Dow Chemical
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Expressive
Dowpharma used its Pfenex expression technology to produce large quantities of enzymes.
Credit: Dow Chemical

Another approach is the reductive amination of α-ketoacids to l-amino acids. Degussa and collaborators at the University of Stuttgart, in Germany, have created a cellular system with leucine dehydrogenase and FDH that uses ammonium formate as the cosubstrate to catalyze a reductive amination leading to the bulky amino acid l-neopentylglycine in more than 99% enantiomeric excess (ee) and 95% conversion (Org. Process Res. Dev. 2006, 10, 666). The technology is an extension of one Degussa developed to produce l-tert-leucine on the ton scale.

Dowpharma combines chemocatalysis and enzymes to make unusual amino acids, says Karen Holt-Tiffin, head of biocatalysis. One approach uses a transition-metal catalyst to asymmetrically hydrogenate an N-acyl enamide, followed by removal of the N-acyl group with an aminoacylase under mild conditions. Analogously, enamides can be hydrogenated to make enantiopure N-acyl amines, then deprotected with secondary amidases to yield chiral amines. The company is also working to combine its hydroformylation technology for making a range of achiral aldehyde precursors with enantioselective enzymatic reactions to produce α-amino acids.

"Because customers usually ask us to supply a specific intermediate and are not worried about how we make it, we have the ability to objectively identify the best single technology or combination of technologies for a specific molecule," Holt-Tiffin says. Along these lines, she says many of the "myths" surrounding biocatalysis, such as limited productivity, high costs, and the instability and unavailability of enzymes, are not true for the majority of cases. "The biggest hurdle is still trying to change the mind-set of the organic chemists so they are not worried about using enzymes and actually try them as a method of choice."

Many enzymes can be isolated and even immobilized, making them more stable and easier to handle, even separable and recoverable, and usable in standard reactors. Enzyme engineering can make them more robust and tolerant of organic solvents, unusual substrates, or other reaction conditions, which in turn allows for high substrate concentrations and greater productivity. Scale-up considerations are comparable with those of chemocatalytic processes, many suppliers say. "Many biocatalytic reactions are very cost competitive," DSM's Wubbolts adds.

New technologies are being developed to avoid problems such as substrate or product inhibition, as well as aid product recovery. For example, one process uses an in situ absorbent resin that acts as both a preloaded substrate feed and eventual product sink to control substrate and product concentrations. The technique has been used in scaling up Baeyer-Villiger oxidations catalyzed by a cyclopentanone monooxygenase as a greener approach to making cyclic lactones (Org. Process Res. Dev. 2006, 10, 599).

Fine chemicals producers say having established biocatalysis platforms allows them to respond quickly to customer requests. High-throughput screening and parallel experimentation tools have made it easier to find enzymes that can carry out a given reaction. Most practitioners say once a hit is found, development takes just one to three months when the enzyme is available. All this, they hope, will go far in dispelling the idea that biocatalysis development requires long lead times and is amenable only to second-generation manufacturing processes.

If a new enzyme must be discovered, cloned, and expressed, or an existing one optimized for a substrate or set of reaction conditions, the timeline can stretch to several months. Nevertheless, customers are often willing to start their testing with material from not-yet-optimized processes or even from 50%-efficient resolutions if they can get the product in hand quickly, suppliers say. When a compound moves into later stages of testing or beyond, customers may then invest in enzyme and process optimization for more efficient and cost-competitive production.

A key starting point for biocatalysis suppliers is a diverse range of enzymes to address different chemical reactions. Most of these companies have extensive strain and enzyme collections for in-house screening and development. Some also have gene expression technology for rapidly scaling up and producing quantities of enzymes. For example, Dowpharma has a Pseudomonas fluorescens-based system called Pfenex, whereas DSM has a production system called PluGbug, employing several different, well-characterized microorganisms.

To expand their capabilities, companies collaborate with small technology providers and partner with leading biocatalysis centers. In addition to several university groups, hot spots include RCAB, which involves 14 institutes in Austria; Germany's Research Center J??lich; and the Innovations Center for Biocatalysis in Hamburg. BASF, Degussa, DSM, and Lonza participate in the Bioconversion-Chemistry-Engineering Interface (BiCE) program at University College London. And DSM is part of the Netherlands' Integration of Biosynthesis & Organic Synthesis (iBOS) program.

Ciba, Fluka, Lonza, Novartis, Roche, and others have created the Swiss Industrial Biocatalysis Consortium. The group believes biocatalysis is being impeded by a lack of broad availability of microbial strains and enzymes, report Lonza researchers Hans-Peter Meyer and James E. Leresche (Org. Process Res. Dev. 2006, 10, 572). The member companies intend to pool their strain collections to advance the development of industrial biotechnology.

At the start of this year, the U.K. Centre of Excellence for Biocatalysis, Biotransformation & Biocatalytic Manufacturing (CoEBio3) opened its doors at the University of Manchester, with satellite labs in York and Scotland. The center has already attracted bigger companies such as AstraZeneca, BASF, Dowpharma, Excelsyn, GlaxoSmithKline, and Lonza, along with smaller enzyme technology players including Ingenza, Novacta Biosystems, and Codexis subsidiary J??lich Chiral Solutions.

"The center is very well connected to the needs of industry," says Nicholas J. Turner, professor of chemistry at Manchester and the center's research program director. "We can go from 'genes to kilos,' or all the way from very basic blue-sky research to production capability." The latter is possible through an alliance with the National Industrial Biotechnology Facility being built at the Centre for Process Innovation in Wilton. The facility will be available in early 2007 and will allow large-scale production of biocatalysts and scale-up of bioconversion processes.

Not only does this capability make the center's offering unique, Turner says, but he also believes it will be important to further the adoption of biocatalysis. "You add enormous value if you can demonstrate that the technology works on a production scale," he explains. "Because then, in the mind of the chemist and people who want to use it, you can translate the idea from something interesting in the lab to a product they can see."

What chemists, particularly those in pharmaceutical process R&D, think about biocatalysis varies. Although pharmaceutical companies-especially those that have sold natural-product drugs produced through fermentation-are very familiar with biotransformations, several years ago many stepped away from such products and scaled down their related efforts. Bristol-Myers Squibb (BMS), GlaxoSmithKline, Merck, Pfizer, and Schering-Plough are the major drug companies mentioned most often for having dedicated bioprocess efforts.

Recently, on the basis of the impact of biocatalysis on the synthesis of chiral small-molecule drug candidates, several pharmaceutical process R&D groups have placed a greater emphasis on biocatalysis for organic synthesis and have expanded or reinitiated activities in this area, says Ramesh N. Patel, who heads the BMS group. He can offer dozens of examples where biocatalysis has been successful (Current Org. Chem. 2006, 10, 1289).

BMS's multidisciplinary effort was created about 20 years ago to find means to access chiral intermediates, Patel explains. Today, the BMS group has extensive enzyme and culture collections and can purify, sequence, clone, and express enzymes in its own production facilities. The group also works with coworkers in chemistry and chemical engineering in process R&D.

"When we consider any route, safety and quality are very important, and then cost," Patel says. "We want an efficient and economical process, and whatever looks best goes on to further development." High enantio- and regioselectivities of bioprocesses are key considerations, as are the mild operating conditions, he says, which can help avoid racemization, epimerization, and rearrangements that might otherwise plague synthetic processes.

At the same time, high-throughput enzyme screening and mutagenesis capabilities, generally dubbed "directed evolution," allow the BMS group to improve the activity, productivity, and stability of enzymes, as well as solvent and substrate tolerance, Patel explains. "Most pharmaceutical intermediates are not an enzyme's natural substrate, but with directed evolution we can tailor-make the enzyme to work with the desired substrate," he says. "Initially we may start with a wild-type enzyme, but in parallel we will optimize the enzyme and scale up the process."

A good illustration can be found in two routes Patel and coworkers developed for making the R and S enantiomers of 6-hydroxybuspirone, a metabolite of the company's anxiety drug Buspar and a potential drug candidate itself. First, they used an l-amino acid acylase to hydrolyze racemic 6-acetoxybuspirone to (S)-6-hydroxybuspirone with 95% ee after 45% conversion; after separation, the remaining (R)-6-acetoxybuspirone was converted to (R)-6-hydroxybuspirone by acid hydrolysis. Then they reported the direct synthesis of either enantiomer using an (R)- or (S)-ketoreductase to reduce 6-ketobuspirone, achieving greater than 95% yield and 99.9% ee. For this, they cloned and expressed both reductases and cofactor-regenerating enzymes (Enzyme Microb. Technol., DOI: 10.1016/j.en zmictec.2006.03.033).

Durable
Kaneka has found a robust formate dehydrogenase enzyme for cofactor recycling in oxidation-reduction processes.
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Durable
Kaneka has found a robust formate dehydrogenase enzyme for cofactor recycling in oxidation-reduction processes.

In contrast, Merck's bioprocess R&D department has reported successes with commercially available enzymes. One was the pilot-scale synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol using a ketoreductase (Tetrahedron: Asymmetry 2006, 17, 554). Both enantiomers are intermediates for P/neurokinin 1 receptor antagonists; the R isomer already is used in Merck's new antinausea drug Emend, and the S isomer is under study. In another case, the group used a ketoreductase from enzyme supplier Biocatalytics to resolve one enantiomer of a bridged bicyclic ketone intermediate that had been accessed previously by chromatography (J. Mol. Catal. B 2006, 38, 158).

Several suppliers, including Amano Enzyme, Biocatalytics, J??lich Chiral Solutions, and Novozymes, produce enzymes used in the chemical synthesis market. Some have begun selling off-the-shelf, ready-to-use products, including sets of biocatalysts for screening. Biocatalytics, for example, offers a ketoreductase kit with about 100 enzymes having either R or S selectivity that can work with a range of different substrates (Tetrahedron 2006, 62, 901).

"We've focused on formulating the enzymes so that someone who has never used an enzyme before would be able to use it easily," says J. David Rozzell, president of Biocatalytics. "For example, our ketoreductases come preformulated with the cofactor system so all you have to do is add water and ketone and look for product in an assay." The company produces a wide variety of enzyme types and has launched a set of 16 ene reductases (EREDs), which selectively reduce C=C bonds in the presence of other reducible functionalities, such as carbonyls.

Biocatalytics works closely with RCAB, having set up its European subsidiary in Graz. It also collaborates with Austrian biotech company Eucodis to create genetically improved or novel enzymes. If a customer finds a hit, Biocatalytics can supply enzymes up to the commercial scale. "In 2005, we supplied enzymes for six different drugs that were in some stage of clinical trials," Rozzell says. "Enzymes can be very cost effective; if they weren't, they wouldn't be used" in so many industrial-scale processes.

Access to enzymes and to tools to screen and manipulate them has helped biocatalysis play an important role in the synthesis of several Pfizer drugs, including the cholesterol-lowering drug Lipitor and the nerve pain treatment Lyrica. The company's initial commercial route for Lyrica used stoichiometric amounts of (S)-mandelic acid to resolve the desired enantiomer. Nearly 70% of the process material, including intermediates, reagents, and solvents, became waste.

A new enzymatic route being implemented uses an inexpensive enzyme, operates at high substrate concentrations, and eliminates organic solvents from all three reaction steps. The undesired enantiomer of the starting material is continuously recycled to increase overall yield. Pfizer's group cloned and optimized a nitrilase, which converts a nitrile into a carboxylic acid, for making a chiral intermediate for the Lyrica active ingredient (J. Mol. Catal. B 2006, 41, 75).

The approach Pfizer researchers have taken is evident in their chemoenzymatic synthesis of the antiangiogenesis agent pelitrexol (Org. Lett. 2006, 8, 1653) and a chiral intermediate for an HIV protease inhibitor (Org. Process Res. Dev. 2006, 10, 650). In both cases, chemical changes to the substrate, such as adding an ester moiety or changing a protecting group, had significant effect on the selectivity or productivity, respectively, of enzymatic hydrolysis. Reaction or medium engineering has played a role in these and other routes they have developed (Org. Process Res. Dev. 2006, 10, 655).

"It's about thinking more like a process chemist when optimizing an enzymatic step," says Junhua Tao, who until recently headed Pfizer's biotransformations group in La Jolla, Calif. Last year, Pfizer restructured its R&D organization and moved its biocatalysis unit to Connecticut, where it had traditional fermentation capabilities. Tao; Kim Albizati, former Pfizer executive director of chemical R&D; and others stayed behind to create a new company called BioVerdant.

"Fast screening tools really allow you to take advantage of enzymes," Tao agrees. But once a biocatalyst is found-and he says there are many already available to choose from-he believes substrate modulation and reaction engineering can often help surmount obstacles faster than directed evolution can. Although directed evolution approaches are powerful, Tao says, people often overemphasize biological approaches to making enzymatic reactions work.

"What BioVerdant is trying to do is very different, and that is to integrate modern chemical R&D and biocatalysis for drug synthesis," he says. "We will use retrosynthetic analysis to deconstruct molecules and think about both organic synthesis and biocatalysis from the very beginning." Initially trained as a chemist, Tao says organic chemists are "pretty stubborn because they are comfortable with the tools they have, but we need to break that barrier."

Within most pharmaceutical companies, organic chemists vastly outnumber scientists working with biocatalysis. As a result, Tao and many others say, biocatalysis hasn't made as large an impact as it could. Biocatalysis is often turned to as a last resort when traditional chemical methods fail, putting even more pressure and time constraints on achieving results, or just to "fix one step," which has little impact on overall manufacturing routes or costs.

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Automated enzyme discovery and optimization are tools that are accelerating the use of biocatalysts.
Credit: Degussa
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Searching
Automated enzyme discovery and optimization are tools that are accelerating the use of biocatalysts.
Credit: Degussa

"This is problematic because people working in biocatalysis don't get as much opportunity to work on molecules," Tao says, "and so the view is that biocatalysis never offers much." As product pipelines thinned, many companies have made cutbacks, especially in process chemistry, and thus the resources aren't there for discovering, engineering, and handling enzymes. Some pharmaceutical companies see biocatalysis as an area for outsourcing.

In response, Tao and others see excellent opportunity in offering multidisciplinary services to the pharmaceutical industry. Competition is clearly growing: Along with BioVerdant, other small companies offering similar services include Chiralix, based on technology from Raboud University Nijmegen, in the Netherlands, and CLEA Technologies, a spin-off of Delft University founded by chemistry professor and former DSM R&D director Roger A. Sheldon. Many others, such as Biomethodes in France, c-LEcta in Germany, and Novacta, focus on enzymatic process development, and others lean toward enyzme discovery or engineering.

Proteus, a protein technology company, and fine chemicals firm PCAS created PCAS Biosolution earlier this year to develop chemobiocatalytic routes. "There are always many options possible, and by combining expertise, we can rapidly select the routes where biocatalysis would provide a competitive edge," says Jean-Marie Sonet, director for business development and marketing at Proteus. "We want to integrate the biocatalytic approach from the earliest stage possible, when the inquiry comes to the table."

Of course, the larger fine chemicals and custom manufacturing firms with biocatalysis capabilities are stepping up as well. BASF, for example, decided to expand its business with a service offering for outside customers. It will offer screening of its enzyme collections, optimization of enzymes and related process development, and biocatalyst production and scale-up.

"Biocatalysis is gaining momentum within pharmaceutical companies," Degussa's Wienand adds. "Many are getting very good at applying it, but they do not usually do the molecular biology behind it and that is where we can really offer something as a service center."

Most fine chemicals companies don't sell their enzymes, although they may provide them for screening or license them for production. They see value instead in offering intermediates, custom manufacturing, and process development. Some large manufacturers may not even use purified enzymes but instead work with cellular systems or crude enzyme preparations, such as cell lysates, which can keep production costs down.

For example, PCAS Biosolution has access to production capabilities up to the industrial scale and Proteus' microbial collection and technologies for screening, engineering, producing, and formulating enzymes. Whereas directed evolution creates variants through cycles of screening and mutagenesis, Proteus uses gene shuffling to recombine several genes. "You do not introduce additional mutations but create different combinations, and that enables you to explore a much broader sequence space in a shorter time," Sonet explains. "It also lets you avoid reaching certain limits by introducing negative mutations or ending up with an inactive enzyme.

"Testing existing enzymes on a new substrate is generally very disappointing because of the specificity of the enzymes," Sonet explains. "That they are highly specific is a strong benefit in having selective reactions, but it is a disadvantage because they are not universal." Instead of endless searching to find one that works, he says, Proteus' technology enables the rapid design of new enzymes. "Rather than changing a process to use a known enzyme, we optimize the enzyme to develop a new process, and that was impossible to do 10 years ago because the tools were not there."

Several changes over the past five to 10 years have been making biocatalysis more accessible to process chemists, and without these, Tao, Patel, and many others point out, biocatalysis wouldn't be where it is today. These changes include advances in high-throughput enzyme screening, parallel experimentation, genomics and proteomics for inexpensive sequencing, and directed evolution techniques for optimization (Org. Process Res. Dev. 2006, 10, 562). Another big factor is stable, preformulated enzymes that can be used more simply like familiar reagents.

Still, even though they needn't get involved in the biology of preparing enzymes, many process chemists might find enzymes frustrating, suggests Andrew Wells, senior scientist at AstraZeneca. "Chemists may buy an enzyme powder and find it works in a reaction, but when they scale the reaction up, they buy the enzyme from another source and the results go out the window," he explains.

To highlight these inconsistencies, he compared 14 commercial lipases and found that not only did many differ, in some instances containing multiple or impure enzymes, but some were exactly the same and just repackaged by different distributors (Org. Process Res. Dev. 2006, 10, 678). Understanding the nature of the catalyst and the optimum process conditions is crucial for successfully identifying and scaling up a biocatalytic process, just as it is for any catalyst, he adds.

AstraZeneca has a small group of mostly synthetic organic chemists within its process R&D department who have been exploring biocatalytic processes, Wells says. Although they are expanding work in this area, they don't have a culture collection or capabilities for modifying and producing enzymes. "If we need to access these techniques, we go outside," he says. The company is one of the founding member companies of CoEBio3 and participates in BiCE. "We stay focused on our core capabilities in organic chemistry-route design, optimization, and scale-up. We see enzymes as yet another catalyst in our toolbox, and that's key."

For now it appears that wider acceptance of biocatalysis in pharmaceutical manufacturing will have to be pushed by the technology suppliers, such as Codexis. About 18 months ago, Codexis acquired Jülich Fine Chemicals. Now called Jülich Chiral Solutions, it sells a variety of enzymes and supplies intermediates, such as chiral alcohols (Eng. Life Sci. 2006, 6, 125). J??lich Chiral Solutions has a manufacturing collaboration with Wacker-Chemie and works with CLEA on enzyme formulation and with Iceland's Prokaria to obtain diverse enzymes, as well as with many academic collaborators. It also is an outlet for products from Codexis.

Codexis, meanwhile, focuses largely on strategic alliances in which it optimizes enzymes through its molecular breeding technology and creates biocatalytic routes for pharmaceutical partners. "One idea behind the acquisition was to see if we can accelerate adoption by seeding the marketplace with off-the-shelf or wild-type enzymes that are easier for our customers to access," explains Tassos Gianakakos, Codexis senior vice president for business development. He believes they will then turn to companies like Codexis for more sophisticated optimization and process development.

"Our target market, for better or worse, has been relatively slow to adopt biotechnological approaches to traditional chemical process R&D," he continues. "It's probably not surprising since there's been a lot of very good chemistry done over the past 30-40 years within pharmaceutical companies, and layered on top of that is the fact that, historically, biocatalysis hasn't delivered the promise." He anticipates there will be less dissatisfaction and more results now that the tools exist to make enzymes work with most substrates and in most reactions.

"People have to have faith that this is actually going to be fruitful," Gianakakos explains. The company's technology platform includes millions of mutated enzymes and a dozen or more different reaction platforms. "We can screen compounds against actual enzymes and our databases," he says. Bioinformatics capabilities allow Codexis scientists to accelerate its directed evolution process by predicting the amount of gene shuffling and specific mutations needed, along with the likelihood and length of time for reaching a target.

"Awareness is increasing as chemists take the first step by bringing in off-the-shelf enzymes to use," he says. "It just takes time for disruptive technologies to get adopted." To shorten the adoption curve, Codexis tries to make its biocatalytic processes user-friendly, familiar, and efficient. "We make our processes look just like chemical processes: The substrate concentrations are extremely high, the enzyme catalyst loading is very low, and separations are very fast," Gianakakos says.

"There's a confluence now where some of the drug developers will look at biocatalysis sooner, but by and large it still is a niche technology," he continues. Success in the marketplace will be another measure to demonstrate value: After about five years of work, Codexis' technology is now used in seven commercial products. These include two Pfizer products, the animal health product doramectin and the side-chain intermediate for Lipitor, and with DSM the 7-amino-3-deacetoxycephalosporanic acid intermediate for β-lactam antibiotics.

Contributing to the growth of biocatalysis are cost pressures, lower R&D productivity, the need to address selectivity and complex chemistry for high-purity chiral compounds, and a desire to access greener technologies. Codexis believes biocatalysis can often help shorten syntheses from as many as 12 steps to just three or four, cut manufacturing costs 40-60%, and reduce capital expenditures by more than 25%, while significantly reducing the environmental footprint. There is also the potential to create new intellectual property by tailor-making enzymes and routes for given substrates. And new enzymes catalyzing more and different reactions are expected to emerge.

Ultimately, whether a manufacturing process uses traditional chemistry, biocatalysis, or a combination will depend on the efficient and economic performance of a technology to produce a given target. Routes may be largely biocatalytic or simply contain one such step, and thus it's difficult to predict the size of the market. It consists of enzyme sales, process R&D development services, and income or royalties on product sales. Codexis, for example, generates royalties on five products, and through a toll manufacturing relationship, provides material for another two.

"The market is approaching a period where it will certainly grow at substantial double-digit rates, as much as 25% per year in the next two to three years," Gianakakos says. The drivers will be adoption of the technology as well as commercialization of more biocatalytic processes. "We may still have one or two more years of awareness-building before you start seeing people move from being early adopters to more traditional users, but once that happens, you will see the rapid introduction of biocatalytic processes."

 
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