Biocatalysis for Efficient Ultrasonic Manufacturing Rheometer Possible PAT Candidate SM Pharma Chemicals (DPC, Sit- and biotransformations at DPC. As an tard, the Netherlands) and Diversa example, Wubbolts explains how An innovative viscosity measurement DCorporation (San Diego, CA) have enzymes can be used to selectively mod- technology developed at Pacific North- signed an agreement for the discovery ify a macrolide, a complex antibiotic west National Laboratory (PNNL, Rich- and development of biocatalysts, with that may contain various similar func- land, WA) could potentially help the aim of simplifying and lowering the tional groups. “If you want to modify advance the pharmaceutical industry’s cost of chemical transformations. Under one specific alcohol functionality PAT-driven push toward real-time the agreement, DPC, a division of DSM among five others to get a new product, process monitoring and control systems. Pharmaceutical Products, will identify you can do that with an appropriate the targeted chemical conversions and enzyme, because of its selectivity. With Diversa will develop the appropriate bio- organic chemistry, you would have to catalysts. DSM will then scale up the re-synthesize the whole molecule.” process to manufacture pharmaceutical Enzymes can have three types of selec- intermediates and active ingredients. tivity—chemoselectivity, regioselectivity, Biocatalysis, in which enzymes are and stereoselectivity. Chemoselectivity used to drive chemical reactions, can be means that enzymes, unlike some chemi- used in pharmaceutical manufacturing cal catalysts, are capable of distinguishing as an alternative to standard organic between chemical structures such as an chemistry. The key advantage of biocat- amino group and hydroxyl group. Regio- PNNL’s prototype ultrasonic rheometer. alysts is that their use can often simplify selectivity, seen in the macrolide exam- the synthesis of complex molecules, ple, means that enzymes can distinguish The “ultrasonic rheometer” combines reducing the number of steps required between identical groups in different ultrasonic and pressure-sensor data and the related costs, particularly when locations. For example, if a ring structure using sophisticated signal processing chiral molecules are involved. The selec- contains two hydroxyl groups, an enzyme algorithms to determine the viscosity of tivity of enzymes in particular can make can selectively modify one of them, based material flowing in a pipeline in real certain chemical conversions more com- on its position, whereas a chemical cata- time. Since viscosity is often a quality- mercially attractive. lyst would modify both, because they are control parameter in blending opera- “The selectivity of enzymes is phe- chemically equivalent. The third type of tions, the ability to collect measurement nomenal,”says Marcel Wubbolts, PhD, selectivity is stereoselectivity, which data without interrupting production competence manager for biocatalysis BIOCATALYSIS continued on page 20 processes for off-line batch testing could provide pharmaceutical producers sig- nificant cost and time savings. As noted by David Pfund, PNNL senior research engineer, the need for off-line viscosity measurements to either confirm specifications or ensure quality control inevitably increases production schedules as well as the amount of stor- age capacity required. “And, if it turns out that the product is off-specification, it will have to either be re-blended or scrapped, increasing waste.” PNNL first began work on the tech- nology in the late 90s with funding from the Department of Energy, which was Figure 1: DPC uses an R-hydroxynitrile lyase (R-HNL) to produce an R-mandelic acid derivative for a pharmaceutical intermediate. RHEOMETER continued on page 22

Pharmaceutical Technology MARCH 2004 17 In the Field NIR Spectroscopy Bioerodable Eye Brings PAT into Focus Implant May Help When FDA set forth its process analyti- Treat Macular Edema cal technologies (PAT) initiative, there was little doubt that it would foster new A bioerodable sustained-release eye collaborations among the agency, the implant shows promise as an effective pharmaceutical industry, and equip- method for treating eye diseases such as ment manufacturers. And, the industry’s macular edema (swelling of the retina first innovations specifically targeted at that causes decreased vision). Data from meeting PAT objectives are indeed now a six-month Phase II clinical trials study making their debut on the marketplace. revealed that patients with macular Especially notable are those incorporat- edema experienced a significant increase ing near infrared (NIR) spectroscopy. in vision after being surgically implanted Already a familiar laboratory tech- Figure 1: L.B. Bohle’s integrated NIR system with the investigational technology. nique for raw material identification for real-time monitoring of blend homogeneity. and quantitative assessment, NIR tech- nology is expected to move on to the venting “pockets” where raw material next logical step of being implemented may be trapped; a flush interior that on the manufacturing process line. won’t restrict powder flow; and, most For example, late last year, L.B. Bohle important, the ability to maintain con- (Bristol, PA)unveiled its integrated NIR ditions inside the blender that will allow system for monitoring blend homogene- stable readings with the NIR probe. ity in real time (Figure 1). Designed for Although operating and maintaining laboratory- and full-scale production, the the “correct” conditions is essential for instrument combines the company’s project management, understanding the The Posurdex eye implant is bioerodable and blending technology with NIR spec- process is equally, if not more, important. provides sustained drug release. troscopy and 21 CFR Part 11–compliant Says Sievert, “We have customers ask us software. Data are obtained by shooting How do you scale up? How do you scale NIR light through a window mounted on down? With what speed should we The "Posurdex" implant from Aller- the bin blender and results are sent to an blend? If we transfer from a V-blender to gan, Inc. (Irvine, CA), delivers dexam- on-line or remote-access computer. a bin blender, how fast do we have to ethasone (a corticosteroid) in small Because sampling doesn’t involve rotate the bin? How about filling levels? amounts over an extended period of physically opening the bin, the material But what’s really important here is not time, which allows the drug to reduce is always in containment, which is espe- establishing these individual parameters, the swelling of the retina while curtail- cially beneficial to manufacturers of but rather achieving blend homogeneity ing possible side effects. Recent data potent materials. Also, bringing the and then understanding how these indi- from other studies has provided evi- analysis on-line saves time because test vidual factors impact that homogeneity.” dence that corticosteroids can be useful results are immediate. As Reinhard Siev- Establishing such correlations among for treating several retinal diseases, ert, general manager at L.B. Bohle ob- manufacturing processes and how they specifically macular edema. However, serves, “It’s much faster than the tradi- affect product quality is a goal of the the main side effects of steroid use in tional method of taking a sample out of PAT initiative. As observed by Robbe the eye are glaucoma (increased pres- the blender, going to the lab, and analyz- Lyon, deputy director of FDA’s Division sure in the eye) or cataracts. ing the result to find out, only then, if of Product Quality Research, PAT According to Scott Whitcup, MD, you have blend homogeneity.” “requires a level of scientific under- vice-president and head of the ophthal- Integrating an analytical system with standing of how formulation and man- mology therapeutic area at Allergan, the a complex manufacturing process was ufacturing process factors affect product sustained-release aspect of Posurdex was not an easy task. “You can’t just take quality and performance.” one of the main factors of its success in NIR technology, install it onto a blend- Although PAT can essentially incor- the Phase II clinical trials. "The thought ing system, and expect it to work,”says porate several technologies, NIR is prov- is that if a patient receives smaller Sievert. “It’s a combination of the hard- ing to be a preferred method among amounts of steroid over time, as ware, the optics, the electronics, and the analysts. In part, this is because during opposed to big boluses of steroids, then software with the blending system that the past 10–15 years, the implementa- maybe you can minimize the complica- makes it perform well.”Sievert notes at tion of infrared cameras, array detec- tions but maximize the treatment least three major attributes that had to tors, digitally tunable infrared optical effects," he says. be incorporated into the design: pre- NIR continued on page 224 IMPLANT continued on page 223

18 Pharmaceutical Technology MARCH 2004 www.pharmtech.com

In the Field

BIOCATALYSIS continued from page 17 In many cases, the use of biocatalysts biocatalyst toolbox more quickly. means that enzymes can be used to pro- makes the process more cost effective. In “What DPC is looking for from us are duce different stereoisomers of chiral one application, DPC uses an R-hydroxy- unique activities where the known molecules. nitrile lyase (R-HNL) to produce an R- enzymes just don’t perform well,”says “Enzymes are really one of the best mandelic acid derivative for a pharma- Mark Burk, PhD, vice-president of tools to address chirality in manufactur- ceutical intermediate (see Figure 1), a research and development at Diversa. ing,”says Wubbolts. Chiral molecules 100% maximal yield route. “One of the “Our technologies, which stem from a exist in two or more different forms, or traditional prejudices against biocataly- genomics approach to enzyme discov- stereoisomers, which have the same sis is that even when you can make ery, allow us to do that.” something, it’s frequently Diversa starts its enzyme-identifica- the wrong stereoisomer,” tion process by sampling the entire notes Wubbolts. “Unlike global environment, particularly chemical catalysis you extreme conditions of temperature and typically cannot simply pH, to find organisms that produce use the stereochemical unique enzymes. They then extract and opposite catalyst. How- process the DNA from the organisms Figure 2: Examples of left-handed (S) and right-handed ever, in this case we used contained in those samples, and clone (R) stereoisomers of a generic amino acid. the R-enzyme, but we also the DNA into a host such as Escherichia have an S-enzyme avail- coli.“In any given environmental sample chemical composition but different con- able, so we can address both the S-enan- there are probably 5–10,000 different figurations that are not superimposable tiomer and the R-enantiomer.” organisms, generally bacteria and fungi, on each other. For example, most amino Biocatalysts, in addition to being used most of which have never been observed acids exist in two forms, depending on in manufacturing chiral compounds, or characterized before,”notes Burk. how the various component groups are can also simplify other complex synthe- Each E. coli clone, which contains arranged around the central carbon sis processes. “We have examples where unique DNA, then becomes part of a atom. In the right-handed (R) stereoiso- we have been able to replace an eight- genomic library that the company can mer, the amino group is on the right, step organic synthesis route with a two- screen for a specific kind of activity. whereas in the left-handed (S) configu- step process,”says Ronald Gebhard, Through this process, Diversa has cre- ration, the amino group is on the left R&D director of DPC. Gebhard also ated more than 2500 genomic libraries, (see Figure 2). Pairs of stereoisomers are notes that biocatalysis generally uses each containing 106–108 unique clones referred to as enantiomers. very mild reaction conditions, because that can be screened. The pharmaceutical importance of many reactions can be performed in If the screening process identifies an chirality was first discovered in the 1960s water instead of organic solvents. This enzyme that isn’t exactly what is needed, with the tragic case of thalidomide, means that sensitive functionalities on Diversa uses directed evolution tech- which was given to pregnant women for organic molecules can be left unpro- nologies to mutate the genetic informa- morning sickness. However, although tected during the process, which can tion to be able to produce an enzyme one of the stereoisomers of the drug had also save steps. “Nature is efficient, and with the desired characteristics. the desired sedative effect, the other form sometimes it allows us to shortcut clas- Through this process, they create new was a teratogen, causing birth defects. As sical organic chemistry,”Gebhard says. libraries of evolved enzyme variants, a consequence, FDA now requires that Because biocatalysts can simplify which again are screened using their chiral molecules be tested to determine chemical conversions and reduce costs, high-throughput screening techniques. the behavior of the enantiomers in the they can make the production of certain “This combination of enzyme discovery human body. In most cases, the function- molecules, not easily produced using and evolution usually takes us where we ality is not the same, which means that chemical catalyts, commercially viable. want to be, with an enzyme suitable for the final pharmaceutical product must be “There are products in pharmaceutical commercialization” says Burk. enantiopure, containing only the correct pipelines that never made it beyond the Once the new biocatalysts are identi- isomer. If a racemic mixture is produced discovery phase because nobody could fied, DPC will scale them up for com- (i.e., one that contains both enantio- synthesize them to scale in an economi- mercial production of pharmaceutical mers), the stereoisomers must be sepa- cally attractive way,”says Wubbolts. actives and intermediates. “Scale up is a rated, which can be done with selective “Using biocatalysts gives you new routes challenge,”comments DPC’s Gebhard. co-crystallization, chromatography, or to new products.” “Not only do you have to use the enzy- biocatalysis. In this case the maximal DSM currently has more than 25 bio- matic reaction on a large scale, but you production yield will be 50%. Alternately, catalysis processes for life sciences appli- also must be able to scale up the pro- other chiral production methods, which cations, about 15 of which are used in duction of the enzyme.” again include biocatalysis, can be used, pharmaceutical manufacturing. Through Wubbolts estimates that across the which will produce an enantiopure com- the partnership with Diversa, the com- industry, approximately 10% of chiral pound with 100% maximal yield. pany expects to be able to increase its BIOCATALYSIS continued on page 224

20 Pharmaceutical Technology MARCH 2004 www.pharmtech.com In the Field

RHEOMETER continued from page 17 multiple data sources. Velocity profile lower frequency doppler returns will looking for a way to continuously moni- information is obtained using an ultra- need to be averaged over a longer period tor transfer lines at a radioactive waste sonic doppler velocimeter. A series of of time to provide adequate resolution. tank farm. Subsequently, the laboratory ultrasonic pulses are transmitted into the “Individual pulse sequence data sets may has been looking at applying the tech- pipe and the backscatter echo signal be collected every 100th of a second, but nology to process-control/blending returned (corresponding to the struc- with correlation of multiple scans and applications in the cosmetic and food tural properties of the material) is col- processing time etc., you may be looking industries. So far, the group hasn’t had a lected. Since the pulses and return echoes at time increments in the range of 10 sec- pharmaceutical industry sponsor, but is are time-gated, the relative range of the onds,”says Pfund. anxious to explore that possibility. Says returns can be calculated. Then, since Clearly, the data correlation, automa- Pfund, “What we currently have is a based on whether the return signal was tion and reporting software supporting technique, a (3rd-generation) prototype travelling away from or toward the trans- the instrument is an important compo- instrument, and some proof-of-concept nent of its capa- measurements.” bility. The PNNL development Principle effort included The basic principle behind the ultra- input and exper- sonic rheometer is that by measuring tise from a num- the velocity of a fluid passing through a ber of different pipe, and correlating these measure- groups within the ments with pressure-drop data, the vis- laboratory cosity of the material can be determined including fluid- as a function of shear rate. dynamics, elec- As explained by Pfund, the velocity tronic systems field in a pipeline flow is indicative of and non-destruc- the rheological behavior of the material. tive testing. Writ- Viscosity is expressed as the ratio of ten in C, the soft- shear stress (force/unit area) to the shear ware is fully-inte- rate (rate change of shear strain). grated with the For any type of pipeflow, the flow instrument. velocity will be zero at the walls of the Completed rheogram based on ultrasonic doppler velocimeter (UDV) As is the case pipe and maximum at the center line. results plotted against results from a conventional rheometer. with many poten- Many materials (such as water) exhibit a tial in-line process constant (shear-independent) viscosity ducer (transmitter/receiver), it will have a measurement technologies being consid- profile and are referred to as “Newton- distinctive doppler-shift characteristic ered for pharmaceutical production appli- ian” fluids. In these cases, fluid velocity proportional to the velocity of the fluid. cations, additional analytical groundwork will vary quadratically with relative dis- To complete the viscosity calcula- will likely need to be done before an actual tance from the centerline of the pipe, tions, pressure-drop measurements are implementation can be accomplished. As defining a parabola-shaped profile. taken over long sections of pipe to pointed out by Pfund,“It’s not always Other materials, however, are known as obtain pressure-drop/unit length data. clear (to users) what kind of data will be “shear-thinning” fluids. These include The pressure drop measurements indi- required to do this operation. Many exist- one common example (ketchup) but cate the shear-stress at the pipe wall and, ing quality control, or product-approval also a host of important polymers and since shear-stress (amount of force/unit specifications don’t directly relate to vis- colloidal substances. area on a layer of moving fluid in a cosity, but are only indicative of it.”As an With shear thinning fluids, the higher pipe) varies linearly with distance from example, he notes that a customary test in the rate of shear (velocity/height), the the pipe wall, and the shear stress at the the food industry to determine if a batch lower will be the viscosity. The maxi- center is zero, it’s possible to determine of tomato paste is on spec is to put the mum velocity in these cases will persist shear stress throughout the pipe. The product behind a small “dam” and mea- for some distance away from the center- ratio of shear stress to shear rate is the sure how fast it takes to “slump” when the line of the pipe, and fluids will tend to viscosity, and by plotting this, viscosity dam is removed.“The point is that there’s flow through the pipe with a central versus shear rate can be obtained. not always a documented quantitative “plug.”The shape of the velocity profile According to Pfund, the rate at which relationship between observables and vis- will therefore reflect the shear-thinning measurements can be taken varies cosity, and detailed measurements will behavior of the fluid. according to a number of factors, but the often need to be taken up-front to estab- principle consideration is the fluid flow lish specific acceptable viscosity parame- Instrument rate—lower flow rates require relatively ters,”says Pfund. The ultrasonic rheometer makes use of longer measurement times since the John Haystead

22 Pharmaceutical Technology MARCH 2004 www.pharmtech.com IMPLANT continued from page 18 mates that the market potential for a The study, which monitored the technology such as Posurdex is probably effects of the steroids on macular edema $500 million to $1 billion in peak sales. and how the implants were tolerated, Allergan hopes to use the Posurdex followed 306 patients who had been implant with additional drugs to treat diagnosed with macular edema associ- other retinal diseases such as age-related ated with diabetes, retinal vein occlu- macular degeneration, a condition that sions, uveitis, and postcataract surgery. affects approximately 12 million people The patients were given one of three in the United States. The company cur- treatments: a single implant with either rently has an antiangiogenic program of a 350-g or 700-g dose of dexametha- drugs that inhibits new blood vessel sone or observation without drug ther- growth in the retina, countering the apy. The results showed that patients overproduction of growth factors, who had been implanted with the 700- g dose had the greatest improvement in vision—significantly more of these patients exhibited a three-line increase (relating to an eye chart) in visual acuity compared to the control group. The bioerodable Posurdex is con- structed of a mixture of poly lactic and poly glycolic acid. These polymers are Allergan has developed an applicator with a also mixed with the drug. Therefore, 22-gauge needle for inserting Posurdex in an when the body begins to break the office-based surgery setting. implant down, it results in a controlled drug release (which could be over a which can destroy the retina and cause period of approximately six weeks, blindness. "Retinal disease is one of the according to data from animal studies). biggest unmet medical needs in the After the drug has been absorbed by the ophthalmology field, and we felt that it body, all that is left of the implant is was important to not only have novel essentially carbon dioxide and water, compounds but also have a novel way to which pose no risk to the patient. deliver them to the back of the eye," Data from the study also showed a Whitcup says. low incidence of complications, no Patients in the Phase II study were increase in the presence of cataracts or required to go to a medical operating eye infection, and a glaucoma rate room to be surgically implanted with potentially less than what has been Posurdex, however, Allergan has now reported in other investigational meth- developed an applicator with a 22-gauge ods of delivering steroids to the eye. needle for inserting Posurdex into the Although improved visual acuity is eye that can be used in an office-based the type of dose response and efficacy surgery setting such as the ophthalmol- that Allergan hopes to present to FDA ogist's office, thereby making the and other regulatory bodies to receive process more comfortable and conve- approval to market the technology com- nient for the patient. mercially, the technology must still also Whitcup notes that even with the perform favorably in follow-on Phase large number of people diagnosed with III clinical trials (which are planned to macular degeneration, there’s still only begin in the second quarter of 2004). one approved therapy on the market to Currently, Posurdex is positioned to be treat their illness. "Clearly, there are the first approved treatment for macular patients who still need help, so we're edema. If Phase III trials are successful, excited to be working on this for them," Allergan anticipates marketing Posurdex he says. in 2006 or 2007. Posurdex was initially developed by With approximately 750,000 people Oculex Pharmaceuticals, Inc., which was in the United States and 2.5 million peo- acquired by Allergan in November 2003. ple worldwide suffering from some Ronelle Russell form of macular edema, Whitcup esti- Circle/eINFO 182 In the Field

NIR continued from page 18 With this objective in mind, FDA recently entered into a cooperative research and development agreement with Pfizer to investigate the application of NIR chemical imaging in process manufacturing. According to Lyon, the current status of that project is that “a prototype process imaging system is ready for deployment and initial testing, and a study plan has been developed and protocols are being finalized.” Spectral Dimensions is also planning to introduce an NIR system for the process line. According to Lewis, the company “recently produced an instru- ment designed exclusively for process applications.”Meanwhile, the company continues to offer and improve upon its off-the-shelf NIR chemical imaging equipment and software that can be incorporated at or near the process line. According to Lewis, this is possible Figure 2: Each pixel on the 2-D image is because the instruments have no moving represented by a spectrum. A single-channel parts. “You can effectively take the equip- image at a particular wavelength provides ment, put it into a NEMA enclosure, and chemical and spatial information. take it right to the factory floor.”Chemi- cal imaging then becomes not just a way dient blending, particle-size distribu- to detect root-cause problems in the tions, agglomeration of component par- tablet, but also a way to monitor what’s ticles, and the presence of polymorphs, happening during manufacture. Says hydrates, and other trace contaminants.” Lewis, “What and how much we may be Thus when implemented on or near able to learn about the complexities of the process line, NIR imaging can con- pharmaceutical manufacturing from NIR tribute significantly to the understanding chemical imaging is still relatively of manufacturing processes. As Neil Lewis unknown, but early indications suggest CEO of Spectral Dimensions, Inc. (Olney, that it might be a great deal.” Figure 3: NIR chemical image of a tablet MD) observes,“There’s nothing new Maribel Rios showing distinctive agglomerations of three about the technology. What is new is that active pharmaceutical ingredients: acetamino- FDA is pushing to modernize the indus- BIOCATALYSIS continued from page 20 phen (red), aspirin (green), and caffeine (blue). try such that NIR and the PAT initiative have really begun to take off.”He adds, pharmaceutical intermediate manu- filters, improved computer memory, “The PAT initiative is really about under- facturing is performed using biocata- and faster data processing speeds have standing the complex dosage form, and lysts. Few large pharmaceutical compa- made NIR chemical imaging possible. chemical imaging provides a tremendous nies use biocatalysis in drug discovery Information that could once only be handle on that because it allows you to and lead development because there is represented as a series of spectra is now get at not only the chemical species, but not enough time to develop suitable in the form of a two-dimensional image the spatial relationships, which has a biocatalysts and because most chemists (see Figures 2 and 3). bearing on factors such as dissolution.” are more comfortable with classical As FDA’s Lyon observes, “The advan- High-resoution images provide spa- organic chemistry methods. tage of chemical imaging compared to tial and chemical information about the As a result, DPC sometimes finds that other sensor technologies is the ability structure of a product’s matrix (the dis- they must switch technologies for scale to analyze the spatial distribution of the tribution and particle size of the active up. “If biocatalysis were used all along, component materials in blends, gran- and the excipients). Knowledge of this that would save in development time ules, or finished dosage forms. This matrix can be used to predict character- over the entire route,”notes Wubbolts. approach has the potential to monitor istics such as ease of manufacture and Laura Bush processes to reveal the extent of ingre- performance during quality testing.

224 Pharmaceutical Technology MARCH 2004 www.pharmtech.com