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
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.