Biocatalysis Advances in Pharmaceutical Development

Biocatalysis in synthesizing pharmaceutical intermediates and active pharmaceutical ingredients (APIs) is becoming an increasingly important part of the toolbox of fine chemical and pharmaceutical companies. These companies are forming alliances with firms specializing in biocatalysis and research groups to optimize reactions for improved stereoselectivity, yield, and reaction conditions.

Biocatalysts may be used in a variety of reactions. For example, dehydrogenases, transaminases, and reductive aminases may catalyze different amino acids and amines (see Figure 1). Ketoreductases may be used in stereoselective ketone reduction to produce chiral alcohols. Amidases and nitrilases are used for stereoselective hydrolytic reactions of amides and nitriles (1).

Figure 1

Specialists in biocatalysis

Reflecting growing interest in biocatalysis, several pharmaceutical majors are joining with specialty firms. Schering AG (Berlin, Germany), to be integrated into Bayer Schering Pharma, signed a pact in May with the Biotechnology Research and Information Network AG (BRAIN AG, Zwingenberg, Germany) to develop and optimize microorganisms for producing intermediates for steroid drugs.

BRAIN AG also recently concluded a collaboration with Degussa AG (Düsseldorf, Germany) for identifying and supplying alcohol dehydrogenases used to produce enantiopure alcohols.

Sandoz (Holzkirchen, Germany) is part of the Research Center Applied Biocatalysis (RCAB) at the University of Graz (Graz, Switzerland), whose research includes the synthesis of chiral compounds and bioactive molecules. Sandoz is one of 16 industrial partners in RCAB. BASF AG (Ludwigshafen, Germany), Degussa, DSM (Heerlen, Netherlands), and the specialty companies BioCatalytics, Inc. (Pasadena, CA) and Eucodis GmbH also are members (Vienna, Austria).

RCAB recently cloned a new and highly active Penicillin V acylase from Pectobacterium carotovorum. Penicillin acylases are enzymes used in producing 6-aminopenicillanic acid, an intermediate in semisynthetic penicillins. The new acylase can be further improved for specific activity, pH optimum, and stability to meet process conditions (2).

Company Web sites

AstraZeneca (London) and Merck & Co., Inc. (Whitehouse Station, NJ) are among the industrial partners for the Center of Excellence for Biocatalysis, Biotransformations, and Biocatalytic Manufacture (CoEBio3) at the University of Manchester (Manchester, UK). The CoEBio3 opened earlier this year and is focused on industrial biotechnology processes in chemical manufacturing for fine chemicals, pharmaceuticals, and biopolymers. The CMOs and fine chemical companies Dowpharma (Midland, MI), Lonza (Basel, Switzerland), SAFC (St. Louis, MO), BASF, and DSM also are part of the center.

DSM partnered earlier this year with IEP GmbH (Wiesbaden, Germany) for screening and developing biocatalysts for making pharmaceutical intermediates and APIs. DSM plans to combine IEP's enzyme technology with its process-scale expertise to expand the chiral alcohols that DSM can manufacture at a commercial scale. IEP's bioreduction technology is used for synthesizing chiral alcohols and derivatives such as chiral epoxides.

IEP also is licensing bioprocessing technology to SK Energy & Chemicals, Inc. (Fair Lawn, NJ) for synthesizing chiral products for advanced intermediates and APIs. SK Energy & Chemicals's chiral portfolio includes enzymatic processes for (S)-2-tetrahydrofuroic acid and (R)-2-tetrahydrofuroic acid and bioprocesses to produce chiral alcohols.

Merck and AstraZeneca, along with DSM, Degussa, and Lonza, also participate in the Bioconversion-Chemistry Engineering Interface Program at the University of London. The group consists of researchers interested in integrating biocatalysis in pharmaceutical chemical development. Target research involves evaluating catalytic and enzymatic methods using a three-step conversion involving carbon–carbon bond formation to create a ketodiol, amination to create an aminodiol, and selective oxidation.

Novartis (Basel, Switzerland), Roche (Basel, Switzerland), and Lonza are among the companies in the Swiss Industrial Biocatalysis Consortium. The group is addressing the need to increase the availability of new strains and enzymes that may be applied in industrial manufacturing, including routes for synthesizing intermediates and APIs. The group's first priority is to focus on oxidoreductases, NADH-dependent dehydrogenases for the asymmetric reduction of ketones, ketoacids, and olefins, mono-oxygenases, and asymmetric carbon–carbon formation by aldolases and hydroxynitrile lyases (3).

Schering-Plough Corporation (Kenilworth, NJ) joined with Codexis, Inc. (Redwood City, CA) earlier this year. Codexis developed a biocatalytic process for a key intermediate for an undisclosed pharmaceutical compound. Codexis also formed a pact with Bristol-Myers Squibb Company (New York, NY) last for year for developing a biocatalytic route for an undisclosed compound.

Other start-up firms include Ingenza, Ltd. (Edinburgh, Scotland). The company was spun off in 2002 from the School of Chemistry at the University of Edinburgh. It focuses on using biocatalysis in manufacturing chiral amines and amino acids at high enantiomeric purity.

BioVerdant (San Diego, CA) was founded by two former researchers at Pfizer, Inc. (New York, NY): current CEO Kim Albizati and Alex Tao, vice- president of R&D and chief scientific officer. While at Pfizer, Albizati built and led a 70-person chemical research and development group. Beginning in the late 1990s, he created, with Tao, one of the first dedicated groups in the pharmaceutical industry to use enzymatic biotransformations in chemical process development. Tao is former associate research fellow and creator and head of the biotransformations group at Pfizer.

And Venkiteswaran Subramanian, former global R&D director, biotechnology and bioprocessing at Dow Chemical Company (Midland, MI) was named director of the University of Iowa's Center for Biocatalysis and Bioprocessing (Coralville, IA) in 2005. His previous work included integrating biocatalysis into existing chemical technology for the contract manufacturing of pharmaceuticals and intermediates.

Biocatalysis at work

In a recent example of biocatalysis at work, Pfizer researchers developed an enzymatic process for producing (2S)-4,4-difluoro-3,3-dimethyl-N-Boc-proline, a key intermediate for the synthesis of HIV protease inhibitors. The group needed to scale up the process to produce multi-kilogram quantities for further clinical trials, and to do so, developed an alternative strategy for producing an enantiopure acid in the synthesis. The approach was developed by switching the protective group of the proline ester from a Boc to a benzyl moiety and a pig-liver esterase was found to be the most active and enantioselective enzyme (4).

References

1. P. Van Arnum, "Achieving Enantioselectivity in Synthesizing APIs and Intermediates," Pharm. Technol. 30 Technology Outlook: APIs, Intermediates, and Formulation supplement, s20–s25 (2006).

2. Research Center of Applied Biocatalysis, University of Graz, Third Scientific Report July 1, 2004–June 30, 2005, (Graz, Switzerland, 2006).

3. H.P. Meyer and T. Münch, "Swiss Industrial Biocatalysis Consortium," BioWorld Europe 1, 15 (2005).

4. S.Hu et al., "Efficient Enzymatic Process for the Production of (2, S)-4,4-Difluoro-3,3-dimethyl-N-Boc-proline, a Key Intermediate for the Synthesis of HIV Protease Inhibitors," Org. Process Res. Dev. 10 (3), 650–654 (2006).