Biocatalysis: Transformations in the Making

24 June 2002 00:00  [Source: ICB Americas]

Once viewed as a questionable new approach to chemical synthesis, biocatalysis has become a key component of the chemist's toolbox for developing economical and efficient routes to both fine chemicals and pharmaceuticals. Specialty enzyme manufacturers and leading chemical companies are focused on developing novel enzymes capable of transformations not easily achieved through traditional methods. Effective integration of biocatalysis within complex synthetic routes will be the key to fully leveraging this emerging technology.

The merchant enzyme industry reached roughly $1.8 billion in sales in 2000 and is growing at about 14 percent per year, according to Clyde Payn, president of The Catalyst Group, a Spring House, Pa.-based consultancy. The captive market comprises an additional 15 to 20 percent over and above the merchant market.

The industrial enzyme market is valued at $1.5 billion, with the global merchant market for biocatalysts used in fine chemical/pharmaceutical applications estimated between $120 million and $150 million in 2001, according to Jane Zhou, senior research associate, Technology Catalysts Inc. (TCI), a Little Falls, Va.-based consultancy. Annual growth over the last three to five years has been between 8 and 9 percent. Similar growth rates are expected for the next three to five years as growth is being driven by the push to single enantiomer drugs.

There are two aspects to the biocatalyst market, the industrial enzyme segment and the specialty enzyme market. Industrial enzyme manufacturers include large players such as Novo-zymes, Genencor, DSM, Degussa and smaller but still important producers, such as Danisco and Chr. Hansen. These companies manufacture enzymes for food, animal feed, pulp and paper, detergents and other industrial applications.

The specialty segment includes enzyme developers such as Diversa, Maxygen, and MediChem. These companies' enzyme products find applications in diagnostics, asymmetric synthesis and other specialty applications. In addition, there is a large group of companies involved in fine chemicals and pharmaceuticals that develop and use their own enzymes in house.

Over the last five years, the main change affecting the speed of development in new biocatalysis technology has been directed evolution or mutagenesis capabilities, says The Catalyst Group's Mr. Payn. This technology involves the shuffling of amino acids encoding the RNA and DNA of enzymes to make the products more active and specific for desired transformations and reaction conditions. "Ten years ago enzyme evolution was Darwinian," he explains. "Today it is synthetic, and the technology is not only widely accepted but also available to most players, which has dramatically changed the economics for using enzymes in synthesis."

In the future, Mr. Payn believes, biocatalyst developers will be looking to techniques such as proteomics to enable them to predict how to shuffle genes in order to obtain novel biocatalysts with particular attributes. He also points to two key trends for the use of biocatalysis in chemical synthesis-the greater availability of selective enzymes that are much more highly active and tailored to specific transformations and the development of much more sophisticated and higher productivity technologies.

Others also point to the technology gains in biocatalysis. Karlheinz Drauz, vice president, technology andR&D management, Degussa Fine Chemicals, notes that in the last decade biotechnology and especially biocatalysis have developed faster and more broadly than classical chemistry. "The design of metabolic pathways and the combination of different genetic information in one production strain (for discrete molecules as well as for functional proteins) will provide us with superior processes and will open an area of real economically and environmentally favorable processes," he says.

In the long-term, focusing on areas where chemistry struggles is key to commercialization, says Patrick McCroskey, senior director, business development, Diversa Corp. "To be competitive with chemical synthesis, novel and enabling enzyme platforms will be needed that can be utilized off the shelf. We expect to see major advancements for the microbial production of biopharmaceuticals and large-scale production of bulk chemicals," he adds.

Marcel Wubbolts, competence manager biocatalysis and biotransformations, DSM Fine Chemicals, sees the growing use of increasingly complex building blocks, such as two chiral centers instead of one, driving biocatalysis to becoming a more important tool to chemists than it is now. Other trends are the integration of disciplines, such as biocatalysis and homogeneous catalysis in one reactor, and process intensification, such as multiple steps in one reactor.

Michael Petersen, manager, commercial development, Lonza AG/ Biotec, expects further integration of more and more enzymatic steps into one biocatalysis step. "Hence, several enzymes will work simultaneously and form a kind of artificial metabolic pathway," he explains. Lonza sees this technology as having large potential in both fine chemicals and industrial chemicals.

Jim Lalonde, director of biocatalysis and chemical development, Altus Biologics, points to the trend for pharmaceutical companies to outsource biocatalysis R&D rather than to maintain internal groups with this expertise. He also sees changes within specialty enzyme developers, which often start out with an enzyme-based business model but later migrate to a drug discovery or chemical production model.

In order to be successful, biocatalytic capabilities must be integrated into the overall synthesis chain, notes Bob Holt, biocatalysis research manager with Avecia. "The ultimate objective is to create a molecule. To be successful is to bring all available technologies to bear on the problem, including genetic engineering, physical separations, microbiology, biocatalysis, synthetic organic chemistry, and others," he says.

Significant recent commercial processes using biocatalysts include DuPont's bio-based polyester product (1,3-propanediol feedstock) and Dow/Cargill's polylactic acid product. In addition to a host of pharmaceuticals and fine chemicals using biocatalytic techniques, biopharmaceuticals are also becoming very important and significant. Out of 37 new pharmaceutical products introduced in 2001, 17 (more than one-third) were bio-based, according to IMS Health, a Plymouth Meeting, Pa.-based consultancy. The Catalyst Group's Mr. Payn says these new technologies will eventually spill over and be available in the industrial chemicals/polymer areas in the not too distant future.

Other current applications of biocatalysts include the production of high fructose corn syrup, aspartame, semi-synthetic penicillins and certain cancer drugs, according to TCI. "Novel biocatalytic technologies combine the advantages of biocatalysts, i.e., stereoselectivity, regioselectivity, mild reaction conditions, and variable solubility, with the advantages of combinatorial chemistry, i.e., rapid generation of novel drug compounds," Ms. Zhou explains. Other potential advantages are a reduction in the number of required steps for a transformation as compared to traditional chemical synthesis, reduced solvent use, fewer by-products, and ease of recovery and reuse.

Drug discovery and production and oligonucleotide synthesis are two key areas where biocatalytic methods are in development for fine chemical applications, says TCI. Combinatorial chemistry, a commonly used methodology to generate a large number of compounds with potential therapeutic activity, is being applied to biocatalysis to produce novel compounds with high regio- and stereoselectivity. Contract research organizations are also using biocatalysis to optimize production processes, offering expertise many chemical companies have not yet acquired.

The synthesis of long-chain oligonucleotides is critical for applying knowledge gained from bioinformatics research. Novel biocatalytic methods are in early stages of development that enable production of longer oligonucleotide chains (greater than 50 nucleotides) as compared to synthetic procedures or biological methods.

With so much potential at hand, both large and specialized players are increasing their investment and proprietary positions in biocatalysis.



Strategic Positioning:

Altus Biologics

Atlus Biologics is involved in biocatalysis for chemical synthesis in three ways: enzyme sales, process development services and chemical product sales. The company specializes in developing processes for the fine chemical and pharmaceutical industries that can combine biocatalysis with traditional organic chemistry. These processes may be based on isolated enzymes, cross-linked enzyme crystals (proprietary CLEC immobilization/stabilization technology) or whole-cells. In these processes, the biocatalyst is used to establish chirality or to perform a selective transformation under mild conditions.

"Our expertise is in developing processes which are scalable and cost-effective based on readily available enzyme or microbes," says Jim Lalonde, director of biocatalysis and chemical development with Altus. "We have found that by providing not only enzymes, but also biocatalytic process development expertise to the industry, we can have a very high rate of success." The availability of relatively low-cost liquid handling systems and analytical instruments allows screening of several hundred enzyme or microbial reactions/day using microtiter plates .

Recently developed processes at Altus have included kinetic resolution using ester hydrolases, mild oxidations of C-H and C-N bonds, and couplings of peptides and beta-lactam antibiotics in organic solvents. The company has recently developed a CLEC form of Candida antarctica B lipase, the single most commonly used lipase in biocatalysis, and has also recently expanded its biocatalyst collection to include microorganisms with the ability to mildly hydrolyze nitriles, oxidize primary amines to ketones, asymmetrically reduce ketones to chiral alcohols, and to hydroxylate unactivated C-H bonds. "These oxidative transformations are extremely useful in the production of interesting scaffolds for drug discovery and for the production of preparative amounts of drug metabolites," notes Mr. Lalonde.

Altus has expanded its capability to produce chemicals in kilogram quantities internally and in production scale through strategic partners. The company has recently invested in fermentation and microbial screening and is building a large collection of microbes that express useful catalytic activities.

Avecia

For Avecia, biocatalysis has become an integral part of the company's toolbox for developing multi-step synthetic route, giving researchers extra scope to be innovative. "Most of the biocatalysts we use are developed in house with specific transformations in mind," says Avecia's Mr. Holt. The company has an extensive program to screen microorganisms for specific reactions and possesses a collection of about 6,000 organisms.

Alternatively, Avecia can, using selective culture techniques, isolate microorganisms from environmental samples to obtain novel biocatalysts that can perform the reaction of interest. "At this point we are considering whether we need to add additional diversity or whether or not 6,000 organisms are enough," says Mr. Holt. Each one of the organisms contains several enzymes and therefore is individually a potential source of hundreds of different biocatalysts. "The bigger question we are now facing is how to harness and take advantage of the resources we currently have."

In addition to its active program for developing technologies in-house, Avecia is interested in licensing technology and in acquisitions or collaborations. "We want to help people outside Avecia to maximize the possible opportunities for new biocatalytic technologies so that we can take advantage of novel developments as well," says Mr. Holt. "We have extensive experience and expertise within Avecia that gives us the ability to evaluate the potential of activities on the outside," he adds.

Avecia has developed and operates at ton-scale biocatalytic reactions for the resolution of esters as starting materials for chiral alcohols and acids. The company has also discovered and developed processes using microorganisms to selectively reduce ketones to enantiomerically pure secondary alcohols. All of these transformation have proven difficult to achieve with any type of selectivity using traditional chemical methodologies.

Avecia now has two bioreduction processes currently manufacturing at multi-ton scale for asymmetric reduction of ketones. "The ability to make these processes commercially successful is a significant development for us," says David Moody, director of new technology ventures with Avecia Pharmaceuticals. "Most people would not have considered this technology achievable, and pharmaceutical companies have expressed great interest in these processes."

The company is currently investigating additional ketone reduction processes and a new deracemization technology that will be an enantioselective route to three specific functional groups that are important in pharmaceuticals. Avecia is also developing new technologies for enzyme stabilization which the company says have significant commercialization potential.

As a specific example, Avecia has developed a five step route to BHA (6-hydroxymethyl-2,2-dimethyl-[1,3]-dioxan-4-yl)-acetic acid tert-butyl ester, a key intermediate in the synthesis of HMG CoA reductase inhibitors, which act to lower cholesterol (scheme above).

The first step is asymmetric reduction of a ketone, followed by regioselective protection of a primary alcohol, and both are enzyme-catalyzed reactions. The diol is converted to an acetonide, which is crystalline and can be purified prior to the final deprotection step. Both the bioreduction and trans-esterification steps could not be achieved with the required selectivities using traditional synthetic methods.

Degussa

Degussa has applied biocatalysis and biotransformations using enzymes and whole-cell biocatalysts for fine chemicals development since the early 1980s. The company uses commercially available biocatalysts as well as proprietary ones for captive use, which are developed via screening and evolution.

One top development in biocatalysis at Degussa includes kinetic dynamic enzymatic resolution systems having a racemase which can, when combined with a hydantoinase and a carbamoylase or an acylase, transform a racemate in one pot and in situ completely into the desired single enantiomer without any external racemization. A second technology involves use of cofactor dependent enzymatic reactions such as oxidations, reductions and some lyase reactions. Degussa is also investigating highly effective, stable and cheap hydrolases that provide cost effective processes. This class of enzymes is the most important one also for industrial chemicals.

Following this work, in 1990s, Degussa developed a cofactor-dependent reductive amination to make L-amino acids with bulky side chains. L-tert.-leucine, a highly useful non- natural amino acid, is the most prominent example. Both of the enzymes used within this process are genetically modified by classical or evolutionary methods (an amino acid dehydrogenase and formate dehydrogenase). In 2001 Degussa introduced its new L-hydantoinase process for the conversion of monosubstituted racemic hydantoins to L-amino acids.

Degussa has also developed other processes that use esterases, lipases and dehydrogenases for synthesizing chiral alcohols, amines, acids, and amino and hydroxy acids and plans to extend its dehydrogenase platform and add new racemases to enzyme systems that are limited by nature to kinetic resolution.

"Having achieved access to a broad range of biological diversity and improved methods to characterize function of detected and isolated genes, we expect to constantly find new enzymatic activities, which might be the basis for future industrial applications," says Degussa's Mr. Drauz.

In 2001 Degussa Fine Chemicals acquired the Chinese company Nanning Only Time, which has biotechnology platforms in fermentation and biocatalysis. Also, in an effort to position itself as a leading company in the field of biocatalysis, Degussa at the beginning of 2001 concentrated its biocatalysis activities in the Project House Biotechnology.

In the Project House Biotechnology, about 30 employees of eight Degussa business units work together on the development of biocatalytical processes. "They share their experience and hardware and together they cover all the necessary competencies from screening and evolution to fermentation and biotransformation," says Stefan Buchholz, Degussa AG, general manager, Project House Biotechnology.

To be able to scale up newly developed processes quickly, the Project House recently invested in a pilot scale fermenter (300 liters) and state-of-the-art down-stream processing equipment. The internal resources of the Project House are complemented by a strong network of university groups and biotech companies such as Proteus, Thermogen, Diversa and Brain. The Project House team works on new methods for the production of pharmaceutical intermediates and on the development of biocatalytical routes for producing nutraceuticals, care specialties, feed additives, and texturant systems.

Diversa

As one of the leading specialty enzyme manufacturers, Diversa develops enabling biocatalysis platforms and processes targeted at high value fine chemical processes with an emphasis in pharmaceutical intermediates. Diversa develops biocatalysts for sale or license and uses them to produce chemical products with partners or toll manufacturers. "Our programs leverage our discovery, high throughput screening and evolution capabilities, analytical sciences and chemical process development skills," says Patrick McCroskey, senior director, business development for Diversa.

Diversa has been a pioneer in the discovery of extremophiles and the evolution of enzymes to perform under commercial process conditions, and the company targets its activities toward discovery of biocatalysts that enable routes that traditional chemical methodologies are unable to achieve. "Enantio- and regoselective catalysts that can operate under mild or extreme conditions-those that are high temperature or high or low pH for example-are key to expanding the scope of opportunities for biocatalysis," adds Mr. McCroskey.

The company's DiscoveryPoint Nitrilase platform, introduced atthe Synthetic Organic Chemical Manufacturers Association's Informex trade show in February of this year, is a new advancement for using biocatalysis for alpha-hydroxy acids and alpha-amino acids. The DiscoveryPoint Nitrilase platform includes over 200 nitrilases (only 10 to 15 were known previously) that are being investigated for use in the synthesis of intermediates for angiotensin-converting enzymes (ACE) inhibitors, Pfizer's Lipitor (atorvastatin) and other key pharmaceutical products.

Diversa recently completed construction of a 60,000 square foot building primarily to house expansion of its biocatalysis and chemical process development activities. It already has a 76,000 square foot building.

DSM

DSM makes use of both commercially available enzymes and, increasingly, of enzymes that have been discovered, developed and produced in house. The genomics and bioinformatics programs at DSM (e.g. the Aspergillus niger genome was recently completed by DSM) provide the tools for rapid identification and development of new biocatalysts in high throughput. "As a result, development times for large-scale production and application of new biocatalysts are reduced, taking away one of the most important barriers for biocatalysis process development in the past," says DSM's Mr. Wubbolts.

Top new developments at DSM include the integration of techniques to resolve racemic mixtures by kinetic resolution that at the same time racemize remaining substrate (dynamic kinetic resolution, or DKR), for instance by enzymatic racemization or racemization by homogeneous catalysis in one reactor. Like the rest of the industry, DSM strives for the chiral synthesis of a desired enantiomer in 100 percent theoretical yield starting from nonchiral staring materials.

DSM recently introduced a set of enantio-complementary biocatalysts that catalyze the addition of HCN to aldehydes and/or ketones (hydroxynitrile lyase, or HNLs), yielding either (S)- or (R)-cyanohydrins at high enantiomeric excess. The (S)-HNL and (R)-HNL enzymes can thus be used to produce the corresponding cyanohydrins, which represent extremely useful building blocks for chiral synthesis. A particular example is the synthesis of synthetic pyrethroids for application in agriculture.

Another technology platform that DSM has recently explored is that of acylases. Several acylases, some of which have been developed at DSM Life Science Products (DSM Anti-Infectives) for the synthesis of semi-synthetic penicillins and cephalo-sporins, have now also been applied to the synthesis of chiral amines.

The amidase technology platform that DSM has developed for the production of non-natural L-amino acids, such as L-tert-Leucine, has large synthetic utility as illustrated by the synthesis of a range of unsaturated glycine derivatives such as propargylglycines and allylglycines. These novel, functionalized amino acid building blocks have been developed in collaboration with the University of Amsterdam.

Lonza

Lonza offers process development to its customers by means of classical chemistry and biotechnology. For biocatalysis, Lonza uses isolated enzymes and whole cells. The company mainly produces its biocatalysts internally and also offers biocatalyst identification and strain development services through a research group dedicated to construction of high-performance industrial biocatalysts using advanced methods in molecular biology and classical microbiology.

Most of the projects using biocatalysis within Lonza are custom manufacturing arrangements. The company does, however, use biocatalysis for producing vitamins and vitamin-like compounds such as niacinamide and L-carnitine on the thousands-of-tons scale for food and feed applications.

In developing and introducing new biotechnology processes on an industrial scale, Lonza is focused on serving the needs of the pharmaceutical, biotech, agrochemical, cosmetics and food/feed industries. Currently the company is working on projects integrating different enzymatic activities into one biocatalyst, according to Mr. Petersen. He expects the introduction on an industrial scale some time in 2003.

Lonza recently announced plans to expand its Kourim biotech plant in the Czech Republic. New bioreactors with a volume of 150 cubic meters will be installed, which will be in addition to existing capacity of 300 cubic meters at the site. The investment will total SFr 85 million ($56 million) and includes additional downstream-processing capabilities and supporting utilities. Rene Imwinkelried, head of Lonza Biotec, says that the new plant forms part of a global expansion program in Lonza's biotechnology custom manufacturing services.

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