Applying Biocatalysis: A Technical Forum - Pharmaceutical Technology

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Applying Biocatalysis: A Technical Forum
Scientists from DSM and Kaneka discuss various techniques in this roundtable moderated by Patricia Van Arnum.

Pharmaceutical Technology

Ammonia lyases

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).

Figure 2: Synthetic application of phenylalanine ammonia lyase. (FIGURE COURTESY OF DSM)
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.

Looking forward

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.

Section references

1. J.S. Carey et al., "Analysis of the Reactions Used for the Preparation of Drug Candidate Molecules," Org. Biomol. Chem. 4 (12), 2337–2347 (2006).

2. H.E. Schoemaker, D. Mink, and M.G. Wubbolts, "Dispelling the Myths—Biocatalysis in Industrial Synthesis," Science 299 (5613), 1694–1698 (2003).

3. P. Lorenz and J. Eck, "Screening for Novel Industrial Biocatalysts," Eng. Life Sci. 4 (6), 501–504 (2004).

4. D.E. Robertson and B.A. Steer, "Recent Progress in Biocatalyst Discovery and Optimization," Curr. Opin. Chem. Biol. 8 (2), 141–149 (2004).

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).

7. A. El-Batal, "Optimization of Reaction Conditions and Stabilization of Phenylalanine Ammonia Lyase-Containing Rhodotorula glutinis Cells During Bioconversion of Trans-Cinnamic Acid to L-Phenylalanine," Acta. Microbiol. Polonica 51 (2), 153–169 (2002).

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.


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