COVER STORY MERRY-GO-ROUND Radleys Discovery Technologies' modular Starfish heating and stirring workstations hold standard glassware for flexible setups.
SPEEDING UP PROCESS DEVELOPMENT Parallel experimentation tools help process chemists optimize manufacturing routes ANN M. THAYER, C&EN HOUSTON
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H E T H E R D R U G DISCOV-
ery has benefited fully from combinatorial chemistry and high-throughput screening methods is open to debate. But high-throughput experimentation and parallel approaches for exploring synthetic routes and optimizing safe and cost-effective reaction conditions are gaining favor among some process chemists. The benefits include higher productivity, wider coverage of experimental space, increased information, and—every so often—unanticipated insights into the chemistry taking place. Parallel reactor equipment available to the process chemist ranges from the relatively simple, such as multivessel stir-plate carousels, to very sophisticated highthroughput systems. In turn, prices range from a few thousand dollars into the millions, depending on the complexity of the reactor setup and the associated hardware, software, and automation capabilities. The 54
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mental data being generated and to characterize materials on a very fast basis." Symyx has collaborated with Merck since late 2000. Initial projects focused on developing workflows—a combination of specific Symyx Discovery Tools equipment and software to complete a desired task—for solubility testing and for identifying polymorphic forms of lead compounds. In 2003, the companies began work on process optimization workflows. Merck has used a Symyx parallel pressure reactor (PPR) to screen and optimize catalytic high-pressure reactions, including homogeneous asymmetric hydrogénation, heterogeneous hydrogénation, and reductive cyclization reactions. The system has 48 reactor cells of 2 to 6 mL each operating at up to 500 psi and 200 °C under inert conditions. It also integrates experimental design, preparation, control, and analytical analysis and results viewing. In one instance, the goal was to come up with an economically viable process for the optimized synthesis of 2-substituted indoles for Merck drug compounds in development, says D. Richard Sidler, who is a senior research fellow in process research at Merck Research Laboratories. Known conditions reported in the literature required relatively high catalyst loadings, temperatures, and pressures.
THE LAB SCREENED different palladium market also is competitive, users say, with sources, phosphine and phenanthroline at least a few manufacturers occupying ligands, catalyst loadings, catalyst-solvent each niche and many others offering a combinations, and catalyst-ligand ratios in range of products. several PPR runs, taking about a day each. 'Automated experimentation can be as iC We were able to go from 5 to 10 mol % palsimple as a robot doing one experiment," ladium and 4 0 0 to 500 psi CO says Scott G. Sheffer, vice presiat 120 °C down to 0.1% catalyst dent ofmarketing and operations loading, 15 psi, and 70 °C and get for Symyx Technologies. "Parala better yield," Sidler says. The lel chemistry is a nice way of doresult is a reaction he calls "truing multiple experiments, but if ly catalytic and economically viyou don't increase throughput on able." Without high-throughthe analytical side, you are just put methods, the optimization doing more experiments, and it process might have taken one takes just as long to characterize full-time chemist three to six those experiments. months to complete, he esti"From our perspective, highPINË mates, whereas the PPR screenthroughput experimentation is CHEMICALS ing took about a week to 10 days. where you actually achieve the "The ability to run anywhere from a few greatest productivity and operational efdozen to hundreds of experiments in parficiency gains," he continues. "It's a comallel is giving us a huge advantage," Sidler bination of parallel experimentation with says. "We apply it anywhere we can in a backbone of hardware and software that process research." His group, for example, allows you to keep up with the experiWWW.CEN-0NLINE.ORG
has unearthed important solvent effects that might otherwise have been missed. In the past, "you might look at 10 reactions, and when none ofthem seem to work, you'd just move on to a new approach," he ex plains. "Now, you can cover more ground, and you'll often find a particular combi nation of solvent, catalyst, and ligand that gives unique benefits." The Merck lab has added a Symyx highpressure reactor system to allow for more rapid reaction screening in finding initial leads. It has a 96 -well plate format with re action volumes of 100 to 1,000 μ ι using 1 to 5 mg of substrate per well and oper ates between -10 and 2 0 0 °C at up to 1,500 psi. According to Symyx, when the system is combined with other workflow components, chemists can run and ana lyze up to 384 reactions per day in an au tomated manner. Material usage is an advantage of the 96-well format, Sidler says, because the amounts needed add up to a reasonable
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MULTIDIMENSIONAL Product yield from low (small yellow spheres) to high (big blue spheres) is shown as a function of catalyst, solvent» and additive for hydrogénation reactions of an aliphatic nitro compound. [Reprinted from Org. Process Res. Dev. 2004,8,469. ©2004 American Chemical Society.] quantity of compound to obtain and consume early in process R&D. "We use an initial screen to hone down to a particular set of conditions that we want to explore more thoroughly" he explains. Once that's done, "we'll scale those up in a little bit larger reactors—maybe 2 to 20 mL, using anywhere from 10 mg to a few hundred milligrams per reaction—to look at fewer variables." If the results are consistent, conditions are then optimized by using the best leads in even fewer experiments at even larger volumes.
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COVER STORY long drug circulation. Both the protein drugs and PEG reagents are expensive, van Aken explains, and as much as 50% of the drug can be lost during the coupling reaction. Reaction times are also very long (100 hours). But with parallel experimentation, 100 to 200 PEGylation reactions using very small MULTITASKING amounts of material can be Different equipment meets the needs of various process development stages screened and then optiPROCESS CHEMISTRY ROUTE SCOUTING & mized for the correct solOPTIMIZATION PROCESS SCALE-UP SCREENING OPTIMIZATION vent-reagent combination, Process development Process development, Medicinal chemistry, Process research, Function coupling chemistry, yield, process development process support process support process research and impurity profile in a Stirred tanks Reactor type Microtiter array Parallel Jacketed matter of weeks. Number of 2-4 1 >100 vessels 10-48 High-throughput exper100-250 mL 1-3L 2-40 mL Vesselvolume t Γ 0-^^J
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PLAN AHEAD Lonza researchers used statistical experimental design to optimize a two-step oxidative cleavage and cyclization reaction. Water in the first step is normalized for the 100-mmol scale. Instrument makers and users admit that the technology brings an increased burden in data handling and analysis. "Clearly when you start to run many experiments, the anal ysis and trying to make sense of the results take a lot more time," Merck's Sidler says. "Probably one of the biggest drawbacks we find in high-throughput and parallel screen ing is the analysis time." Appropriate infor matics tools are crucial, he and others agree, for collecting, processing, storing, sharing, and reporting the data. "The bottleneck very quickly becomes analysis," adds ChristopherJ. Welch, who heads the analysis and preparative sepa rations group within process research at
Merck. For example, even using a rela tively fast, 10-minute HPLC assay for an alyzing a run of 100 experiments adds up to a significant amount of time. Welch's group has been working with Sidler's to develop more rapid analytical methods to ease the bottleneck. Working in their favor is the fact that the analysis can be more qualitative when screening reactions, Welch explains. "Ύοιι don't need to know whether an individual well gives 95% or 98% —you can find that out in a follow-up experiment. Initially you just need to know whether the results are good or bad." In addition to faster assays, the Merck researchers are also looking at parallel analysis to complement parallel re action screening. 'Ύοη have two routes in trying to go for high-throughput analysis," Welch says, "^bu can do very, very short assay times, or you can do massively parallel assays, keep ing traditional assay times but doing eight or 24 at a time." The best of both worlds, he adds, will be doing assays that are both fast and in parallel. Merck is evaluating parallel microfluidic chromatography equipment, including an eight-channel HPLC instrument that it has developed with Eksigent Technologies and a 24-channel system from Nanostream. In stead of taking a day or more, a 96 -well re action plate can be analyzed with an eightchannel system in under an hour. In the meantime, supporters of highthroughput experimentation say it allows them to design a range of experiments and explore conditions at a rate that otherwise would not have been possible. Statistical
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tools such as chemometrics and design ofex periments (DOE) have emerged to go hand in hand with experimentation (C&EN J u ly 14, 2003, page 37). Some researchers, such as at Pfizer, are combining process op timization with microreactor technology to explore new reactions (see page 43). Dominique M. Roberge, project leader at Lonza, advocates combining reaction calorimetry, automated reactors, and D O E as a central part of process development {Org. Process Res. Dev. 2004,8,1049). Lon za scientists have used this approach to study a two-step reaction: the rutheniumcatalyzed oxidative cleavage of a cyclic α,βunsaturated ketone followed by cyclization to a lactam {Org. Process Res. Dev. 2004, 8, 1036). Understanding and optimizing the reaction parameters led to a techni cally feasible process, subsequently scaled up for production.
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Scaling up to kilogram or ton quantities may be problematic after finding a result at a microliter scale, Avantium's van Aken warns. Thus, Avantium emphasizes ra tional experimental approaches based on statistical techniques, rather than simply automating hundreds or thousands ofvery small-scale experiments every time. For example, after finding a lead in highthroughput mode, the company has a sys tem for doing 192 targeted D O E reactions, testing the important parameters on a mil liliter reaction scale. Reaction screening is helping the process development lab meet workload demands, originating from discovery re search, and rapidly zero in on areas most likely to produce scalable reactions. De velopment timelines are shrinking, Roche's Guinn says. "There is such a rush to get compounds into the clinic that the old par adigm of being able to develop each step through a one-experiment-per-day ap proach is really not going to fit with the realities of our current timeline. "Management in industry recognizes that we need to be more innovative in our process development and use the tools that are available to get data we hadn't been get ting in the past," he continues. "We have less flexibility to have failures. We need to be successful the first time." •
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