Issue Date: May 28, 2012
It says a lot about the maturity of biocatalysis as a synthetic tool that four major drugs—Pfizer’s Lipitor and Lyrica, and Merck & Co.’s Singulair and Januvia—are manufactured using enzymatic steps.
“From a technology point of view, it is now fully recognized that biotransformations can be commercialized,” says Junhua (Alex) Tao, a chemist who once headed a Pfizer biotransformations group and now runs two enzyme-related firms in China.
Although many drug and fine chemicals companies have developed biocatalytic syntheses, California-based Codexis has been a leader when it comes to major drugs. This year, the firm and its long-term partner Merck published a route using an enzyme to make the new hepatitis C drug Victrelis (J. Am. Chem. Soc., DOI: 10.1021/ja3010495).
In 2011, Codexis sold a record $49 million in enzymes and other products, most of which went to drug and fine chemicals companies. According to Codexis, its manufacturing partners produced more than 10,000 kg of its enzymes for making pharmaceutical intermediates.
From its origins as a biotech start-up a decade ago, the company has blazed trails in its work with big pharma to create new biocatalytic routes. However, other enzyme suppliers have not enjoyed the same success, and many have gone out of business.
Knowing now that biocatalysis technology is sound, a new crop of enzyme developers is emerging. For these small firms to compete, they will need to hone business plans incorporating lessons about working with pharma that Codexis had to learn the hard way.
“In the mid-90s, we thought we could change the industry, but it was impossible because the enzymes were just not good enough,” says Peter Seufer-Wasserthal, Codexis’ senior vice president for pharmaceuticals, about his own 20 years of experience as a student and with Codexis and other firms.
When Codexis emerged in 2002, it had a new directed-evolution method for modifying and optimizing enzymes for specific reactions and process conditions. But it still had to persuade drug companies and their process chemists to use them. To prove its technology worked reliably at large scale, the company began producing intermediates and generic drugs.
The facts appeared to be in Codexis’ favor. Enzymes are superbly enantio-, chemo-, and regioselective and can be highly efficient. They perform under mild pH, temperature, and pressure. Enzymes can also offer environmental benefits, which is why biocatalysis is viewed as a promising green chemistry tool (see page 20).
“The ability to generate enzymes is the most powerful technique for providing industrial catalysis today,” says David Dodds, a private consultant who previously directed fermentation and biocatalysis development at Bristol-Myers Squibb. Prior to that, Dodds led Schering-Plough’s biotransformations group.
Despite their attractive features, enzymes have proven difficult to turn into a viable business, in part because of resistance among pharmaceutical scientists. Biocatalysis must also compete against more familiar chemical synthetic routes. Even today, some drug companies are only beginning to adopt the technology.
In the early days of biocatalysis, recalls Ian Fotheringham, “process chemists regarded enzymes as a necessary evil or a last resort. If no other chemistry worked, you tried the enzymes.” Fotheringham is president of Ingenza, a Scottish bioprocess development firm that was founded in 2002 with a focus on enzymes for making chiral amines and nonproteinogenic amino acids.
At the time, chemists were unfamiliar with handling or screening enzymes as reagents and with conducting enzymatic reactions. And many were dissatisfied with the time and cost it took to engineer or optimize biocatalysts.
Since then, discovery and development work to find an enzyme, determine its gene sequence, and express it for production has matured in many respects, Fotheringham says. Enabling technologies have become faster and cheaper. They include enzyme evolution methods, microbial genome sequencing, protein engineering, DNA synthesis, bioinformatics, and robotic screening.
The field has moved from individual variants toward carefully selected and evolved libraries of many diverse specificities and optimized activities within a single enzyme class. Although many aspects of industrial biocatalysis make the discipline still largely empirical, Fotheringham points out, the utility of some enzyme classes is relatively predictable because their workable substrates and target products have been well characterized.
For example, lipases, esterases, and hydrolases find widespread use to resolve chiral compounds. In synthesis, ketoreductases, which transform ketones into chiral alcohols, are the best-established tools, according to enzyme developers. And transaminases, at least for producing primary amines, are a close second.
Now that these enzyme classes have become mainstays, enzyme suppliers are working on generating new ones. They include enzymes for oxidation chemistry, halogenation, more complex amine production, carbon-carbon coupling, and carbon double-bond reductions. Farther in the future will be enzymes designed with nonnatural catalytic activities.
But offering highly productive or even unique catalysts hasn’t been enough to attract a lot of customers. “In the early days, Codexis may have underestimated the adoption barrier, or how difficult it would be to actually get biocatalysis adopted by a large group of people and at a commercial scale,” says Gjalt Huisman, Codexis’ vice president for product planning.
The company began to gain traction after adjusting its business model to make greater inroads with big pharma. About five years ago, he says, “we started thinking about how we could get our technology in the hands of process chemists.” In 2007, the company offered the first of its 96-well enzyme panels, which allow customers to broadly screen their own substrates, rather than having to bring targets to Codexis. Codexis can then optimize and scale up hits.
In late 2010, Codexis launched even easier-to-use kits of 24 dried enzymes packaged in standard vials. It also changed its licensing policy. Previously, Codexis wanted exclusive licensing and development deals and at one time sought royalties on drug sales. Now a user agrees to a license by breaking the kit’s shrink-wrap. If a hit is found, the user owns the rights to use that enzyme in a process to synthesize a specified compound.
The changes came in response to what customers always wanted, Seufer-Wasserthal says. More important, he adds, neither Codexis nor the customer can tie up or block broader use of the technology. The change in licensing policy “was a breakthrough,” he says. “Before we did it, we had about 10 customers using our research enzymes, and now it is more than 50.”
Codexis also decided to make its enzymes available to other pharmaceutical service providers for route scouting and process development. “We got comfortable with the idea that we never will see all the opportunities that contract manufacturers will see,” Seufer-Wasserthal says. Today it has enzyme supply agreements with Ampac Fine Chemicals, Dishman Pharmaceuticals & Chemicals, Lonza, and DSM.
As Codexis found its footing, other pharmaceutical chemical suppliers expanded their in-house biocatalysis capabilities for producing intermediates and active pharmaceutical ingredients (APIs). They included BASF, Kaneka, Almac Sciences, Novasep, and Fabbrica Italiana Sintetici. India’s Piramal Healthcare got biocatalysis-related assets when it purchased an Avecia business in 2005, as did Dr. Reddy’s Laboratories when it bought part of Dowpharma in 2008.
Acquisitions by contract manufacturers have contributed to the consolidation of the enzyme business over the past seven years. Cambrex acquired the German firm IEP, and PCAS bought its Biosolution joint venture partner, Protéus. Known for its metal catalysts, Johnson Matthey added enzymes when it purchased X-Zyme. And Codexis absorbed Jülich Fine Chemicals, Enzis, and BioCatalytics.
Industry watchers say the desire of custom manufacturers to own these tools is a sign of enzymes’ acceptance, but they find it difficult to gauge how widely the manufacturers actually use them.
At the same time, new enzyme developers, many of which are university spin-offs, have emerged and hope to exploit newer technologies for discovering, developing, and producing enzymes. They are trying to show more business savvy than enzyme developers that have come and gone, in part by broadening their scope beyond the pharmaceutical industry.
One example is Eucodis Bioscience. Founded in 2007, it focuses on enzyme engineering and production. Headquartered in Vienna, the company also has a site at Germany’s Martin Luther University of Halle-Wittenberg. Initially, Eucodis offered in vivo recombination technology as a service to improve enzymes, R&D Head Jan Modregger explains. “In recent years, this evolved into developing more of our own enzymes and supplying them to customers.”
Business is growing steadily, he says. The firm supplies biocatalysts for drug synthesis and enzymes for environmental monitoring and for quality control in antibiotics production. It has in-house purification capabilities and fermentation capacity of up to 1,000 L for making kilograms of enzymes. Outside partnerships allow it to also produce at larger scales.
About half of Eucodis’ business is related to the drug industry. Although the firm can operate under the current Good Manufacturing Practices required by drug regulators, it is also enjoying a non-cGMP market niche. “Many companies in the development stage of a project don’t need cGMP-produced protein,” Modregger says. “They don’t want to pay the price at this stage, but they want to be able to transfer the process later on.”
Another niche for Eucodis is improved lipases, and it has developed at least 70. “There’s the impression that you can basically catalyze every reaction you want to do with CALB, which is sort of the Swiss Army knife of lipases,” Modregger says, referring to Candida antarctica lipase B. “It can be tough to convince people to change, but after screening our kit they can always identify one or another lipase that actually works better.” The company has collaborated on enzyme development with Northern Ireland-based Almac and has inked a product distribution deal with Germany’s Evocatal.
Evocatal, another young company developing enzymes, is a 2006 spin-off of the Institute for Molecular Enzyme Technology at Heinrich Heine University Düsseldorf. It provides biocatalysts for synthesizing APIs and intermediates, as well as for producing and processing textiles, adhesives, detergents, and cosmetics. In addition to selling ready-to-use enzymes, it develops customized enzymes and conducts biocatalytic process development for customers.
In late 2011, Evocatal launched a kit of new thiamine pyrophosphate-dependent enzymes, developed with researchers at German-government-supported Jülich Research Center and the University of Freiburg. “We have made the first seven commercially available,” says Michael Puls, managing director of Evocatal.
These new C–C coupling enzymes can produce a range of substituted enantiopure 2-hydroxyketones. A chemical route to the same product would not only cost more, but it would yield an undesirable mix of products because the stereochemistry is hard to control, Puls says.
Evocatal also sells chiral intermediates. In February, it and Switzerland’s RohnerChem, which has large-scale production capabilities, announced a collaboration to provide customers with their combined expertise in chemical and biocatalysis process development. The partners have developed a route involving an enzyme to a key chiral building block for aprepitant, a Merck antiemetic that will soon lose patent protection.
Another company trying to build a biocatalysis business is Enzymicals. It is a 2009 spin-off from the research group of Uwe T. Bornscheuer at the University of Greifswald in Germany. Initially, the small company sold specialized enzymes and developed processes for pharma and chemical applications, Chief Executive Officer Ulf Menyes says. It recently moved into making fine chemicals at up to the kilogram scale, and this year it partnered with Herbrand PharmaChemicals in Gengenbach, Germany, for large-scale cGMP production.
Enzymicals’ focus has been on esterases, monooxygenases, and transaminases, Menyes says. The company sells a screening kit of transaminases that it licensed for lab-scale use from Lonza, a collaborator of Bornscheuer’s. “We have found a new way to make (R)-transaminases usable at the industrial scale,” Menyes adds.
Making transaminases and other biocatalysts available to chemists isn’t enough; chemists have to want to use them. Chemists should consider all synthetic options available, consultant Dodds suggests. “But you can only put so many things on the shelf, and if they don’t work, what do you do?” he says. If frustrated with enzymes, they may turn to more familiar chemistry or rearrange the process, because doing so is faster than developing new enzymes.
The process development considerations for using a biocatalyst are largely the same as for a chemical catalyst. The challenge for biocatalysts is generating enzymes for a specific purpose when they are needed. “We have the technology, but as development timelines become compressed and demands increase, we lose the time to develop new reagents,” Dodds says. “No one likes to have to back up and go build a new reagent for one step.”
In addition to time, cost is a factor. “There is a limited price that customers can pay for an enzyme,” Seufer-Wasserthal says. Economies of scale kick in: The more enzymes you develop in a certain class, the better the collection becomes, he says, both because the likelihood for hits increases and because the enzymes may be available for production or already made at large scale.
“We see more hits directly out of the kits and people saying, ‘that is good enough’ and ordering more enzyme,” he explains. In early development, customers have become more willing to forgo enzyme optimization and use large quantities in their reactions because doing that may be cost-effective.
If a project advances, a few rounds of optimization can reduce the amount of enzyme needed. To get to an enzyme that will be satisfactory at large scales “becomes about three or four months’ work, not a two-year program,” Seufer-Wasserthal says. This proof-of-concept approach has led to an increase in “pipeline” products, which for Codexis means selling at least a kilogram of enzyme or the amount needed to make drugs for clinical trials.
As Codexis did, other companies trying to find a competitive niche in enzymes have had to alter their business plans. Based at Northumbria University in Newcastle, England, Nzomics Biocatalysis was founded in 2006 by a chemist and a biologist. The partners thought they would sell enzymes such as nitrile hydratases that were useful but not widely available, but they have shifted to offering consulting and services in the areas of custom enzymes and libraries. Finding success in this, Nzomics lists major drug firms and contract manufacturers among its customers.
One challenge in setting up a business was finding out “what products people actually wanted,” says Justin Perry, the firm’s commercial director. Other enzyme suppliers express similar frustrations with drug industry customers that don’t share information on their molecules or provide feedback on how the enzymes perform in actual processes. And even if customers do share information, confidentiality agreements can restrict suppliers from talking about products under development.
In addition to responding to feedback, Codexis tries to anticipate customers’ needs, Huisman says. It combines information that emerges on drug structures with its knowledge about enzymes to proactively develop new biocatalysts, he adds. It also makes customers aware of the capabilities of different enzymes on the chance that one might have an interest in potential new reactions. However, he suggests, the field still faces hurdles to bring together the technology with people who believe it can improve their processes.
Some customers’ needs are unpredictable—but biocatalysis companies see this as a good sign. “We get some unusual requests that aren’t for run-of-the-mill biocatalysis enzymes,” says Gary Black, Nzomics’ technical director. “That is quite encouraging because it shows that pharma is starting to embrace biocatalysis a bit more and use it in areas that maybe they hadn’t thought of before.”
Responding to these requests has become easier because of the wealth of available microbial genomic data, which Black says is growing faster than the industry can make enzymes. “It essentially allows us to produce a range of related enzymes with subtly different activities,” he says.
If a customer likes a particular enzyme, “we can quite easily survey the sequence space around that enzyme and create another lot of enzymes similar to it that might be more optimal for their application,” he adds. “This becomes quite a powerful tool.”
U.K.-based Prozomix is also taking advantage of genomics data and related bioinformatics tools. Created in 2008, it mines data to find gene families and select sequences likely to have desirable characteristics. Using a high-throughput in vitro cloning method that it developed, called GRASP (genomics-based related-activity screening protocol), the company generates libraries of natural enzymes.
To offer a good chance of finding a novel enzyme, the company avoids known enzyme sequences, Managing Director Simon Charnock explains. And, in contrast to methods that make variants with only small changes in amino acids, “none of our enzymes are more than 80% identical to any other,” he says. “At that level, you get gross changes in the active sites and radically different substrate specificities.”
Although Prozomix initially focused on biofuels and related products, like many other companies, it quickly rebalanced. Within 18 months of launching, the majority of its business shifted to developing biocatalysts for pharmaceuticals. Last year, the firm debuted enzyme tool kits designed for synthetic chemists. Provided free of charge for screening, the kits include a panel of enzymes, along with sequence data. Expression plasmids are also available.
Prozomix came up with its tool kit idea after customers said they didn’t like paying large amounts of money for enzymes when they might get only one or a few hits. With the Prozomix kit, a user pays a fee of about $3,000 for the first hit and less for subsequent ones, Charnock explains. Instead of negotiating contracts or licensing fees, Prozomix offers per-enzyme pricing for most of its services.
Because customers receive the sequence information, he says, “once they have paid the hit fee, they are free to go produce the enzyme themselves or use a third party.” Alternatively, users can return to Prozomix for further enzyme development and manufacturing, which it can provide up to the ton scale via partners.
Nzomics has a similar business model, in which customers get the clone to make the enzyme as part of an initial agreement or later purchase. It’s not unusual that drug companies want to own the microbial strain to ensure a secure supply of the enzyme, rather than depend on perhaps only one supplier, Black explains.
Although large-scale enzyme production “is not our expertise,” Black says, “we have found that if we do a good job, they come back to us for more enzyme, because they have more important things to do with their fermenters than produce enzymes.”
Clearly, enzyme suppliers have created different plans for success. On the basis of his experience, Codexis’ Seufer-Wasserthal believes that a company won’t get rich just selling enzymes but needs to offer biocatalyst optimization, application, and supply as well.
Similarly, as he sets up operations in China, Tao believes that a company needs to show potential customers that it can support them. After developing a key step and demonstrating it at small scale, he says, “the ideal situation is being able to say that you can produce the catalysts to meet their demand when they want to take the process from kilograms to 100 tons.”
Last year, EnzymeWorks, one of the two companies Tao helped establish in China, built what he says is the largest modern enzyme manufacturing facility in the country, capable of producing at up to the ton scale. Located in Zhangjiagang, the contract manufacturing company integrates chemical and biological transformations. Nearby, the second firm, called Metabomics, offers contract services in enzyme discovery, expression, and engineering for use in drug discovery and synthesis.
Because China is a center for drug intermediate manufacturing, Tao says his firms receive pressure from customers to produce chiral intermediates. But he calls the practice a “distraction” for biocatalysis providers. He focuses instead on opportunities around making generic drugs and working with U.S. and European drug companies setting up operations in China.
Evocatal, for example, is working with Lanxess on methods to make rubber precursors. Even Codexis has created a significant business in biofuels and biobased chemicals. “Although the numbers in pharma look bigger, my advice to a number of companies has been to go make industrial chemicals,” consultant Dodds says.
Noticing interest from a broadening customer base, Ingenza underwent such a transition a few years ago, Fotheringham explains. The company now boasts long-term partnerships with major firms in the biopharma, food, fuel, and natural products sectors and develops routes to make building blocks for industrial polymers.
“Despite all the talk about how biocatalysis was going to increasingly impact drug manufacture, our experience is that there are still a relatively limited number of opportunities,” he says. In addition to competition from other enzyme providers, biocatalysis companies face challenges from chemical routes, including those carried out by providers in low-cost countries.
A small company can’t support every enzyme class, which limits the chemistry it can conduct, Fotheringham says. What’s more, there are no guarantees that its enzymes will work for synthesizing a particular molecule. These factors make biocatalysis for pharmaceuticals “a risky business,” he says.
But Fotheringham remains upbeat about the pharmaceutical opportunities that do exist. “Any drug company developing a molecule will consider a biocatalytic route,” he says, “because it can be extremely competitive.”
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