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Scientists from DSM and Kaneka discuss various techniques in this roundtable moderated by Patricia Van Arnum.
This article is part of PharmTech's supplement "API Synthesis and Formulation 2009."
Achieving high yields with desired stereoselectivity is an ongoing goal in synthesizing active pharmaceutical ingredients (APIs), and biocatalysis is an important tool in this regard. To gain a perspective, Pharmaceutical Technology's Senior Editor Patricia Van Arnum asked leading experts to share recent applications of biocatalysis. Hans Kierkels, senior scientist, and Oliver May, corporate scientist of biocatalysis, both with DSM (Geleen, The Netherlands) discuss the development of a nonanimal pig-liver esterase used in the synthesis of the API aliskiren and the application of ammonia lyases for producing amino acids and derivatives. Masahiko Yamada, senior researcher with Frontier Biochemical & Medical Research Laboratories, a research division of Kaneka (Osaka, Japan), provides various biocatalytic approaches to making chiral amines.
PharmePLE (DSM) is a nonanimal-derived, pure enzyme used in the synthesis of optically pure compounds. (IMAGE COURTESY OF DSM)
Applications of Biocatalysis
By Hans Kierkels, senior scientist, and Oliver May, corporate scientist of biocatalysis, DSM Pharmaceutical Products
A special challenge in manufacturing pharmaceuticals is the increasing complexity that requires many steps in the synthesis of a given molecule. On average, eight steps are required for the synthesis of an API, according to a recent analysis (1). The dynamics of drug development are challenging as well. The high attrition rate of drug candidates usually does not justify extensive route scouting and process development in early-development phases. In these phases, the focus is on speed of delivery rather than on manufacturing cost-efficiencies. This limited focus often leads to suboptimal routes and poorly developed processes for manufacturing clinical trial material.
A changing toolbox
Given the limited number of rapidly accessible biocatalysts and the prohibitive timelines and costs for developing new or improved enzymes, biocatalysis had been a niche technology. But much has changed. During the last decade, breakthroughs in molecular biology, analytics, bioinformatics, and gene synthesis brought about an increasing spectrum of readily available biocatalysts faster and cheaper than ever before (1–4). It now takes only a few mouse clicks to identify new enzymes in exponentially growing databases using bioinformatic tools, and another mouse click to order genes or even mutant libraries in custom-made expression systems. Within a few weeks, those enzymes are shipped to laboratories where they are rapidly produced. The PluGbug expression system (DSM) is an example of a system that uses these tools. Such activities previously took several years, but now they take only several weeks. As a result, the enzyme toolbox is growing rapidly. DSM, for example, increased its enzyme collection from several hundred enzymes to more than 2000 enzymes during the last decade. Access to a rapidly growing spectrum of readily available biocatalysts, increasing cost pressures, and changing mindsets, will increase opportunities for biocatalysis. This change will happen throughout all phases of drug development, including preclinical and early clinical phases, where first-time-right processes using enzymes (see Figure 1) will be used in these shorter timelines.
Figure 1: Opportunities for enzymatic processes due to advances in biocatalysis. (FIGURE COURTESY OF DSM)
The mindset of scientists involved in route scouting and process research and development is changing slowly, and only a few pharmaceutical companies have captured those developments. Flexible and open partnering approaches with enzyme-service providers and contract manufacturers that provide access to a broad enzyme toolbox and offer interdisciplinary route-scouting expertise are just emerging. The main impact of biocatalysis is still in developing second-generation processes for late clinical phase or launched APIs. Two recently introduced processes highlight the benefit of biocatalysis.
A biocatalytic route to aliskiren
An excellent example of a successful process substitution was recently reported for producing an intermediate used in the synthesis of aliskiren, a renin inhibitor used to treat hypertension. A key step in the synthesis of aliskiren is an enzymatic resolution catalyzed by pig-liver esterase (PLE). PLE is a versatile biocatalyst for organic chemists because it has a broad substrate spectrum and excellent enantio- and regio-selectivity.
The commercially available PLE is animal derived. Its quality can vary significantly from batch to batch, and it is therefore not safe or suitable for pharmaceutical applications. To address this problem, DSM and its collaboration partner, the Graz University of Technology in Austria, identified different isoforms of PLE. Using capabilities in enzyme development and production, a highly efficient and patented microbial expression system and fermentation process was developed for different isoforms of PLE that runs at a 25,000-L scale at DSM (5). This system delivers nonanimal-derived PLE isoforms (PharmaPLEs, DSM) at a large scale for pharmaceutical applications.
The PharmaPLE-based production process replaced an established chemical process. The overall productivity of the process increased more than 50%. Waste production was significantly reduced by avoiding the double-resolution steps in the first-generation chemical process, which generated a large amount of organic and inorganic waste. A life-cycle analysis showed a 50% reduction of greenhouse gas emission for the enzymatic process.
Ammonia lyases are another biocatalyst type that can be applied on an industrial scale for producing amino acids and their derivatives, which are important building blocks of APIs. One prerequisite for the industrial application of ammonia lyases is high activity and operational stability under extreme conditions such as high ammonia concentrations (> 10% weight) and basic conditions (> pH 10), which is required to push the equilibrium toward the synthesis of the amino acid (6, 7).
DSM used one of the most studied phenylalanine ammonia lyases (PALs), one that is derived from parsley and the yeast Rhodotorula glutinis, for producing substituted phenylalanine derivatives (8). Although DSM successfully synthesized several target compounds at a multigram scale, severe substrate inhibition of the PALs was observed.
To deal with this problem, a screening and optimization program revealed several PALs that met the required selectivity, activity, and stability at industrially relevant conditions. One of these enzymes was cloned into the Escherichia coli-based PluGbug production system, resulting in very high expression levels at laboratory and industrial scales (9).
The single enzymatic step resulted in an approach that produced the desired amino acid at high yield (> 90%) and high enantiomeric excess (> 99%). The biocatalytic approach replaced a process based on the Fischer indole synthesis followed by a classical resolution. Based on this enzyme (phenylalanine ammonia lyase), an industrial process for producing a phenylalanine derivative was developed and recently implemented on an industrial scale (see Figure 2). The first-generation chemical process required seven steps, but the second-generation process using biocatalysis consisted of only two steps, thereby offering significant economic and ecological benefits.
Figure 2: Synthetic application of phenylalanine ammonia lyase. (FIGURE COURTESY OF DSM)
The benefits in sustainability for the two reactions previously described are quite typical for biocatalysis. Based on the growing number of enzymatic processes (e.g., more than 30 different enzymatic processes have been introduced at DSM), there is greater agreement about the true value of biocatalysis. The primary benefits are: the reduction of steps in a synthesis; increased yield; less waste per step by providing higher selectivities; and allowing the use of cheaper raw materials by enabling different chemistries and routes. The next step forward in biocatalysis would be to implement the technology earlier (e.g., in the supply campaign of clinical trial materials). This change would allow us to learn from the initial delivery of campaigns and avoid risk by changing to lower-cost enzymatic processes at later phases. This transformation is only possible if the speed, cost, and versatility of biocatalysis is competitive with all other available solutions in organic synthesis. Having access to a broad and versatile enzyme toolbox provides a high chance of technical as well as commercial success. Being able to rapidly identify the right enzymes, produce them, and apply them for sample preparation within weeks is another requirement for implementing biocatalysis. This vision is possible in the very near future. Those companies that are able to introduce the most sustainable processes as early as possible in API manufacturing without compromising speed and development cost will capture the benefits that modern biocatalysis offers.
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2. H.E. Schoemaker, D. Mink, and M.G. Wubbolts, "Dispelling the Myths—Biocatalysis in Industrial Synthesis," Science 299 (5613), 1694–1698 (2003).
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5. M. Hermann et al., "Alternative Pig Liver Esterase (APLE)—Cloning, Identification and Functional Expression in Pichia pastoris of a Versatile New Biocatalyst," J. Biotechnol. 133 (3), 301–310 (2008).
6. A. Gloge et al., "Phenylalanine Ammonia Lyase: The Use of its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis Synthesis of L-Arylalanines," Chem. Eur. J. 6 (18), 3386–3390 (2000).
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8. A.H.M. de Vries et al., "Process for the Preparation of Enantiomerically Enriched Indoline-2-Carboxylic Acids Via Cyclization of Phenylalanine Derivatives," PCT Int. Appl., WO 2006069799, 2006.
9. F.B.J. Van Assema and N. Sereinig, "Production of Optically Active Phenylalanine Compounds for Cinnamic Acid Derivatives Employing a Phenylalanine Ammonia Lyase Derived from Idiomarina loihiensis," PCT Int. Appl., WO 2008031578, 2008.
Biocatalyst-driven Chiral Amines
By Masahiko Yamada, senior researcher, Frontier Biochemical & Medical Research Laboratories, a research division of Kaneka
Chiral amines, chiral amino acids, and chiral alcohols are key pharmaceutical intermediates. Kaneka, a producer of chiral alcohols (1-4), recently implemented a systems-biotechnology approach using protein engineering and molecular biology to introduce the chiral amine group into desired compounds.
Nonnatural L-amino acids
Reductive amination. Nonnatural amino acids are increasingly becoming critical building blocks for drug manufacturers, in particular for the synthesis of protease inhibitors. Kaneka has been investigating the production of such nonnatural amino acids by the reductive amination of ketoacids (see Figure 3). The method developed by Kaneka combines an amino-acid dehydrogenase and a coenzyme recycling system in Escherichia coli. By selecting the appropriate dehydrogenases, both aliphatic and aromatic L-amino acids can be produced. Phenylalanine dehydrogenases provide a chiral aromatic amino-acid series and leucine dehydrogenases a chiral aliphatic amino-acid series.
Figure 3: Nonnatural L-amino-acid production by reductive amination. (FIGURE COURTESY OF KANEKA)
Because the dehydrogenase requires NADH ( -nicotinamide adenine dinucleotide) as a coenzyme, the recycling of the coenzyme is critical to the reaction. Kaneka has succeeded in regenerating NADH to accelerate the reductive amination. This successful recycling is accomplished by using durable formate dehydrogenases (FDH) isolated from a specific soil-based microorganism, in which FDH survived even under high-substrate concentrations, even with electrophiles, including halogenated hydrocarbons (5).
The harmonization of the reductase library and coenzyme regeneration crafted in E. coli allows the system to produce nonnatural L-amino acids in high concentrations, sometimes as much as 100 g/L. Essentially no undesired products, except carbon dioxide from formate, are produced, and enantiomeric excesses of more than 99% are common.
One-pot enzymatic deracemization. Kaneka produces nonnatural L-amino acids from readily available racemic unprotected amino acids. Kaneka's technology uses four enzymes: a D-amino-acid oxidase and an L-amino-acid dehydrogenase for the actual reaction steps and two other enzymes, including coenzyme recycling. The mechanism is an expansion of the reductive amination technology previously described. In the oxido-reduction sequence, the oxidation of the undesired D-amino acid to the a-ketoacid or to the imino derivative takes place in concert with the reductive amination (see Figure 4). The system was scaled, giving yields of greater than 90% and enantiomeric excess of more than 99% (6, 7).
Figure 4: One-pot enzymatic deracemization for nonnatural L-amino acids. (FIGURE COURTESY OF KANEKA)
Chiral amines are the remaining frontier of chiral synthesis. About 70% of central-nervous-system drugs possess amine moieties because neuronal receptors are usually triggered by amines. To date, however, no single technology stands out as being the best to make chiral amines at commercial scale. Catalytic hydrogenation has not been successful so far because of its high-pressure requirements, and optical resolution has not been universally applicable. Enzymatic synthesis using a transaminase is certainly a promising concept, although a long-standing issue has been its poor conversion rate. The transamination reaction is reversible, which makes it quite difficult to isolate the desired amines from the complex reaction mixture, including the three other chemical species: the substrate ketone, the amino donor, and the resultant ketone.
Kaneka has developed a practical transaminase system by creating a transaminase-containing E. coli transfectant and introducing a special module that efficiently drives the system (see Figure 5). The system produces chiral amines in high yield and high enantiopurity from the corresponding ketones. The key is the special module, which shifts the equilibrium to the desired amines (8–10). This whole system accommodates a variety of ketone substrates, including aliphatic, aromatic, and cyclic ketones, and produces both the R and S isomers through the selection of robust transaminase libraries.
Figure 5: Multienzyme harmonic systems of transaminase. (FIGURE COURTESY OF KANEKA)
By implementing a systems-biotechnology approach, Kaneka has introduced industrialized processes to manufacture nonnatural L-amino acids using reductive amination or deracemization technology. Depending on the availability of the substrates, the best arrangements can be selected. These harmonic systems are effective in surpassing other methodologies, particularly in the case of L-t-leucine. In addition, the company's chiral-amine transaminase systems produces a wide range of amines, aromatic, aliphatic, and cyclic compounds for projects in development and at commercial scale. The industrialization of these compounds is supported by large-scale capabilities in genetically modified organisms, synthetic technology, and process-engineering technology, including amino-acid–peptide purification systems.
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2. J. Hasegawa et al., Large-Scale Asymmetric Catalysis (Wiley-VCH, Weinheim, Germany, in press in 2009).
3. The Commendation for Science and Technology in the Development Category awarded by Japan's Minister of Education, Culture, Sports, Science and Technology, Tokyo, May 2008.
4. The Japan Chemical Industry Association Technology Award Grand Prize, Tokyo, June 2008.
5. H. Nanba et al., "Purification and Characterization of an Alpha-Haloketone-Resistant Formate Dehydrogenase from Thiobacillus sp. strain KNK65MA and Cloning of the Gene," Biosci. Biotechnol. Biochem. 67 (10), 2145–2153 (2003).
6. H. Kanamaru et al., "D-Amino Acid Oxidase and Method for Production of L-Amino Acid, 2-Oxo Acid or Cylic Imine," EP 1918375, May 2008.
7. T. Ohishi et al., "Integrated Solutions of Unnatural α-Amino Acids" in Asymmetric Synthesis and Application of α-Amino Acids, V.A. Soloshonok and K. Izawa, Eds. (Oxford University Press, Oxford, UK, 2009), p. 337.
8. A. Iwasaki et al., "Microbial Synthesis of Chiral Amines by (R)-Specific Transamination with Arthrobacter sp KNK168," Appl. Microbiol. Biotechnol. 69 (5), 499–505 (2006).
9. A. Iwasaki et al., "Microbial Synthesis of (R)- and (S)-3,4-Dimethoxyamphetamines through Stereoslective Transamination, Biotechnol. Lett. 25 (21), 1843–1846 (2003).
10. S. Kawano et al., "Method for Production of Optically Active Amine Compound, Recombinant Vector and Transformant Carrying the Vector," EP 2022852 , Feb. 2009.