Applying Biocatalysis: A Technical Forum

Scientists from DSM and Kaneka discuss various techniques in this roundtable moderated by Patricia Van Arnum.
Sep 01, 2009
Volume 2009 Supplement, Issue 5

This article is part of PharmTech's supplement "API Synthesis and Formulation 2009."

PharmePLE (DSM) is a nonanimal-derived, pure enzyme used in the synthesis of optically pure compounds. (IMAGE COURTESY OF DSM)
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.

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

Figure 1: Opportunities for enzymatic processes due to advances in biocatalysis. (FIGURE COURTESY OF DSM)
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.

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.

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