Yeast cell as active ingredient factory
Sanofi-Aventis uses a total biosynthesis process to produce hydrocortisone

Yeast has a reputation as a hard-working fermenter. It didn’t take long for this classic substance to make the transition from the bakery to pharmaceutical production. When it is carrying the right genes, baker’s yeast makes hydrocortisone from sugar or alcohol in a single stage. Sanofi-Aventis unveiled the first total biosyntheses of this corticoid last December at CPhI. Pilot production started last summer.

Corticoids offer relief to persons suffering from eczema. They reduce pain associated with rheumatism and kidney ailments, and combined with beta-agonists they are generally regarded as the gold standard in the field of asthma therapy. Hydrocortisone is one of the most prominent members of this class of active ingredients. It is used in many generics and as an intermediate for the production of highly effective anti-inflammatory steroids. The Sanofi-Aventis Group has developed a new, patented method of producing this active ingredient, giving it a significant competitive advantage in the corticoid market. The company developed a single-stage total biosynthesis process for hydrocortisone, and therefore it received the CPhI (Chemicals, Pharmaceuticals, Ingredients) Innovation Award last December in Brussels.

The production of hydrocortisone has a long tradition at Aventis. Back in 1953, the predecessor company, Roussel Uclaf, developed the first industrial process for this sought-after active ingredient. Starting with an acid from bovine bile, the chemists had to perform 23 chemical and biotech reaction steps to produce the final product. To enhance safety, the company switched to vegetal based production of the active ingredient at the end of the 1980s. They used phytosterols derived from soy beans to make the product in less than ten steps. When this process was introduced in the 1990s, it became clear that synthesis would face tough competition from lower cost hydrocortisone production in China. This led the French researchers to investigate alternatives. While they were looking for a microorganism which would provide them with a cost effective way of building up the basic hydrocortisone structure, they came across a proven species: saccharomyces cerevisiae, better known as baker’s or budding yeast. Yeast cells produce ergosterol to build up their plasma membranes, and this made them a potential source of corticosteroids.
Udo Hedtmann, currently Project Coordinator, recalls that research began at Roussel at the beginning of the 1990s in Romainville. Hedtmann is responsible for pharmachemical business development at the Sanofi-Aventis Global Bulk Sales Business Unit. The research effort, which was initiated by Roussel within the framework of a public-private partnership, was conducted by the CNRS (Centre National de la Recherche Scientifique) Center for Molecular Genetics and the biotech firm Transgène. The goal was to genetically modify yeast cells so that they would produce hydrocortisone instead of ergosterol. The complexity of yeast, which is eukaryotic and has a number of cell segments, was a technical impediment, and the membrane-bound reaction mechanisms also presented a challenge to the research team.
Gene transformation
The scientists initially attempted to transfer biosyntheses of hydrocortisone to the yeast, starting with cholesterol which has a structure that is similar to the ergosterol precursor ergosta-5,7-dienol, but with no success. “Initial attempts to mimic hydrocortisone synthesis in the human adrenal cortex failed, because under aerobic conditions the yeast cells did not adsorb any of the cholesterol,” explained Hedtmann. The researchers then changed their strategy. Step by step, they modified the genetic makeup of yeast in order to convert ergosta-5,7-dienol in several enzyme catalyzed stages into hydrocortisone instead of ergosterol. The crucial element in achieving success was the introduction of a vegetal gene. The scientists also had to introduce another nine mammalian genes into the yeast and deactivate three of the yeast’s own genes to achieve total biosynthesis of hydrocortisone.
While CNRS was developing molecular tools for use in genetic modification, research at Roussel and later at Hoechst Marion Roussel and Aventis was focused on completing the genetic transformation and optimizing the laboratory-scale yeast fermentation process. By 2000, researchers had developed a yeast strain which was able to produce small amounts of hydrocortisone from glucose using one-pot synthesis. For the first time, it became possible to transfer a complex, membrane-bound reaction mechanism from a mammal to a microorganism.
After that, the objective was to increase the productivity of the yeast strain and develop an industrial fermentation process. This work was carried out at the Aventis sites in Vitry and Frankfurt-Höchst with the support of the consultant Tilman Achstetter, who is now at the University of Bremen. The yield of the fermentation process has been increased significantly during the past three years, and pilot production started up in Höchst and Vitry in the summer of 2004. Hedtmann explained that “we are now implementing additional productivity optimization and developing a downstream process”. If process optimization and the drug master file run according to plan, bio-hydrocortisone will appear on the market in 2007.
One stage, many advantages
Total synthesis in a single stage offers a whole range of advantages compared to the traditional hydrocortisone production process. The initial substance is glucose, which is more readily available than phytosterols. Fewer reagents and solvents are needed for water-based fermentation. Single-stage synthesis eliminates the need for purification and storage of intermediates, and it is significantly faster, less personnel intensive and thus more cost effective. Total process yield can also be increased. Assuming a 90% yield at each stage, you will only get 8.9 kg of product from 100 kg of input material with the traditional 23-stage production process. Aventis can produce larger batches of the active ingredient with the new process at the same quality level.
Annual worldwide production of corticoids is about 250 tons. Hedtmann estimates that the price per kilogram varies between $500 and $20,000. The Sanofi-Aventis Group along with Pfizer and Schering are the leading Western producers of corticoids. However, there is increasing competition from countries with low labor costs such as China. Half of worldwide production already comes from this region. The Europeans and Americans are still the leaders in terms of turnover, because they normally produce higher-quality (e.g. halogenated) corticoids. Sanofi-Aventis primarily produces corticoids for external customers and not for in-house products. About one quarter of the approximately 100 pharmachemicals which are sold by the Global Bulk Sales Business Unit are steroids. The development of single-stage hydrocortisone synthesis gives Sanofi-Aventis a significant competitive advantage. A large number of state-of-the-art corticoid active ingredients can be synthesized using hydrocortisone as an intermediate. The yeast strain could be modified further for single-stage synthesis of corticoids that have a similar structure. The yeast cell acting as a miniature factory could be used for production of active ingredients in cases where synthesis is too complex for industrial production. “We are currently reviewing our entire corticoid synthesis program,” explained Hedtmann.