Greening Up Process Chemistry

Two industry sectors that have the most to gain by embracing green chemistry came together earlier this month to share success stories at the 3rd International Symposium on Green Processing in the Pharmaceutical & Fine Chemical Industries, which was held at the University of Massachusetts, Boston.

The discussions centered on how companies can improve the cost and environmental profiles of their chemical products and processes, with topics ranging from developing a new generation of enzyme-mediated reactions to harnessing hazardous reaction chemistry—including assessments of whether making green improvements is worth the effort.

One of the challenges pharmaceutical and fine chemicals companies face when it comes to going green is that it can be unclear where a chemical or a process lies on the green spectrum. A good barometer for chemists and chemical engineers is the “E factor,” a measure of the total amount in kilograms of solvents, reagents, and consumables used per kilogram of product made.

Small-molecule pharmaceuticals have among the highest E factors, with 25–100 kg of additional material consumed per kilogram of product, explained Roger A. Sheldon of Delft University of Technology, in the Netherlands, who introduced the concept in 1992. Fine chemicals used as drug intermediates and in flavors and fragrances aren’t far behind, with E factors of 5 to 50, he added. For comparison, petrochemicals such as polyethylene and gasoline have E factors of less than 0.1.

Big pharma and fine chemicals firms are bent on improving their E factors. The best way to do that is by improving the atom economy of reactions and reducing the number of reaction steps, Sheldon said. And the best way to do that is by devising catalytic processes in alternative solvents such as water or even in no solvent. One natural solution is enzyme-mediated processes, which were one of the conference’s focal points.

“Biocatalysis has many attractive features in the context of green chemistry,” Sheldon pointed out. Reactions are performed in large stirred-tank reactors (no specialized equipment needed) under mild conditions (ambient temperature and pressure, physiological pH) with a biodegradable catalyst (an enzyme) that is derived from renewable resources (sugar-fed microbes) and in an environmentally compatible solvent (water).

“We knew about these advantages 30 years ago,” Sheldon said. But progress in using enzymes in the pharmaceutical and fine chemicals industries has been hampered by the lack of consistent production and formulation, limited scope of substrates compared with classical chemical methods, limited stability and shelf life, and lack of easy separation and reuse, he said.

“We have had to wait until now for technology development to catch up so that enzymes are practical,” Sheldon continued. “Better genome sequencing to identify enzymes, directed-evolution strategies to repurpose them for process chemistry, recombinant DNA technology to mass produce them, and immobilization technology to formulate them have all made a big difference.”

For example, Gjalt W. Huisman of Codexis described how his company’s “custom evolved” enzymes are helping facilitate greener pharmaceutical production. Codexis scientists are using the company’s DNA-shuffling strategy guided by statistical modeling to “change the catalyst paradigm,” Huisman said. They conceptually design a desired process and then generate the biocatalysts needed to enable that process, rather than trying to develop a typically suboptimal process around an existing enzyme.

One Codexis team used this approach to create a two-step, three-enzyme process to replace a multistep chemical process for making ethyl (R)-4-cyano-3-hydroxybutyrate. This chemical, also known as hydroxynitrile, is the key chiral building block used to make atorvastatin, the active ingredient in Pfizer’s blockbuster cholesterol-lowering drug Lipitor.

Huisman and Sheldon led a study to evaluate the green benefits of the revised process (Green Chem. 2010, 12, 81). The new hydroxynitrile synthesis doesn’t require metal catalysts or chemical derivatization steps, and it is carried out at room temperature and pressure at neutral pH in water in much less time, Huisman said. Because the product of the enzymatic process is enantiomerically and chemically pure, high-vacuum distillation isn’t required as it is for the chemical synthesis, which suffered from incomplete conversion and by-product formation that diminished the yield.

The new process has an E factor of 5.8, significantly lower than any of the previous routes to hydroxynitrile. Because demand for atorvastatin is more than 100 metric tons per year, the environmental and cost savings are substantial, Huisman said.

Codexis continues to improve its directed-evolution technology, Huisman noted. For example, Codexis recently worked with Merck & Co. to develop an enzymatic process to make the chiral amine sitagliptin, which is the active ingredient in Merck’s type 2 diabetes drug Januvia. Both the atorvastatin and sitagliptin process improvements earned Presidential Green Chemistry Challenge Awards (C&EN, June 28, page 9).

Even with improved enzymes, some applications still aren’t amenable to using free enzymes—the enzymes need to be immobilized first, Sheldon noted. This is typically done by binding them to a substrate or encapsulating them in an inert matrix to improve their operational stability and provide a means to easily recover them, he said. But these strategies can be costly and in some cases still result in enzymes that have low activity, poor reproducibility, low stability, and short shelf life, as well as being cumbersome to use.

In the 1990s, the advent of cross-linked enzyme crystals, pioneered by Altus Biologics, helped solve many of these problems, Sheldon noted. But the need to crystallize the enzymes remained a disadvantage because it’s a laborious, costly procedure requiring high-purity enzymes. Sheldon reasoned that crystallization could be replaced by simply precipitating enzymes from an aqueous buffer—a process already used to purify enzymes—while also cross-linking the enzymes. With this approach, purification and immobilization are combined into a single step, starting from the crude fermentation broth.

Thus cross-linked enzyme aggregates (CLEAs) were born. CLEAs are made by precipitating enzymes and cross-linking their reactive amino groups with multifunctional molecules such as glutaraldehyde or dextran polyaldehyde, Sheldon explained. The enzymes can also be copolymerized with an alkoxysilane. The porous particles are highly reproducible, maintain their activity indefinitely, and are easier to handle than free enzymes or other types of immobilized enzymes. Combi-CLEAs containing two or more enzymes are also possible for use in multistep reactions.

“We can in principle work with any enzyme,” Sheldon said. “The key is to understand the properties of the enzyme so that the immobilization strategy can be customized.”

CLEA Technologies, a company Sheldon created, is now producing CLEAs from clients’ enzymes and commercially available enzymes. CLEAs are being used in a variety of applications: as catalysts for peptide synthesis, resolution of chiral amines, and esterification of fatty acids and in hand creams and antifouling paints, Sheldon said.

Although green chemistry provides a means to an end, it’s not possible to adhere to all or even most of green chemistry’s 12 principles in any given reaction, noted Jeffrey V. Mitten of Novasep, a company that specializes in chemical process development. Mitten addressed what he called “a green chemistry paradox” of using hazardous chemistry to develop improved production-scale processes.

Hazardous chemistry encompasses running highly exothermic reactions and handling highly reactive, mechanically unstable, or toxic compounds, Mitten explained. “Because hazardous chemistry fails the safety principles of green chemistry, synthetic routes that require hazardous chemistry typically aren’t considered green,” he said.

Green chemistry principles instead suggest that the focus should be to rework the synthesis to achieve the same benefits without the hazard. “But when the risks are contained, it’s often much greener and cost-effective to use hazardous chemistry than a safer but longer alternative route,” Mitten pointed out.

To demonstrate, Mitten presented several case studies of hazardous chemistry that Novasep has developed for its customers. In one example, he described the conversion of a hydroxyester to an aminoalcohol using diborane gas (B₂H₆).

Diborane is a versatile reagent in organic synthesis and is useful at the bench scale in solution. But at production scale, using diborane in solution requires additional solvent, and the reactions provide lower yields than those using diborane gas directly, Mitten noted. On the other hand, diborane gas is “one very hazardous reagent” that can ignite spontaneously in moist air and is hard to handle at production scale, he said.

For the aminoalcohol synthesis, the initial process required protecting the hydroxyl group on the starting substrate, followed by reducing the ester group, an amination step, and then a deprotection step. The Novasep team developed a process in which the hydroxyester is first treated with the amine to form an amide, which is then reduced by diborane gas. By devising a reactor system to generate diborane on-site and introduce the gas as needed, the process chemists and chemical engineers shortened the reaction from five steps to two steps by avoiding protection and deprotection. In addition, they used less solvent, generated less waste, improved the yield from 59% to 81%, and reduced costs by 60%.

This approach is amenable to other hazardous gaseous reagents, Mitten noted, and he presented additional case studies involving diazomethane, carbon monoxide, bromine, and ozone—each with cost and environmental benefits.

Many of the green improvements discussed at the conference were not for new processes but involved revising a synthesis or a processing step. But even when the chemistry is done, one hurdle remains when it comes to pharmaceuticals: gaining regulatory approval to implement the change.

Some companies might be reluctant to attempt improving a process to make a drug because of the misconception that it might take too long and cost too much to wend through the red tape of reapproval, Pfizer’s Peter J. Dunn said. Dunn and his colleagues decided to come up with some numbers to confirm or deny that perception by reviewing eight major process chemistry changes Pfizer made after the original regulatory approval for some of its key products.

Dunn described an improved synthesis of pregabalin, the active ingredient in Pfizer’s Lyrica, an anticonvulsant drug used to treat chronic pain. Lyrica has been on the market for about five years and now has annual sales of nearly $3 billion, Dunn noted. In 2007, Pfizer switched from a classical chemical enantiomeric resolution of pregabalin to an enzyme-based resolution engineered as a continuous process.

The improvements reduced the E factor of the process from 86 to 9, Dunn reported. In addition, Dunn calculated that through 2020 the process improvements will save Pfizer 185,000 tons of solvent (a 90% reduction), 15,000 tons of starting material (a 50% reduction), and tons of other reagents.

“It’s hard to visualize how much these savings really are,” Dunn said. “But it is well worth taking the chance on the new process.”

The trade-off was how quickly approval would come in order to start taking advantage of the benefits. As it turned out, it was pretty quick, only 100 days for Europe and 121 days for the U.S. In fact, the median time to approval for all eight processes that were reviewed came out to be 136 days, or about four-and-a-half months—“much faster than anticipated,” Dunn said.

Dunn hopes that raising awareness of the benefits of green process improvements and how easily they can be implemented will encourage others in the pharmaceutical and fine chemicals arenas—thoughts echoed by many of the chemists in Boston.