In 2009, Metrics opened a state-of-the-art dedicated potent and cytotoxic facility. The facility, which can accommodate Phase I through Phase III solid-oral dosage form projects up to 10 kg in size, provides containment for processes with hard-wall isolation technologies, specifically a custom-engineered system of isolators to contain solid oral dosage form processes that can be assembled to create a desired equipment train. 

In developing its facility, Metrics used many design concepts from the International Society for Pharmaceutical Engineering's (ISPE's) 

Baseline Pharmaceutical Engineering Guide for New and Renovated Facilities,

 with particular emphasis on allowing for one-way flow of materials, personnel, and equipment. As the industry continues to define what is meant by "potent compound facilities," Metrics made capital investments in best-practice solutions that would offer a defensible position on the matter. 

On the basis of its involvement with ISPE's community of practice with regard to isolation, Metrics recognized that there are several ways to address containment and many options were considered. During the project's planning phase, it was determined that although local ventilation and laminar–down-flow technologies provided some benefits, including versatility and comfort, they were heavily dependent on work practice. Spills or unanticipated discharges of powders can result in acute exposures, and although flexible containment solutions are practical and achieve good containment performance, they pose challenges from a cleaning standpoint or, if disposable, a cost of goods standpoint. Moreover, because a contract organization is likely to have a large variety of compounds moving through its facility, the management and control of these disposable "bags" can be complex. 

Finally, knowing that a contract organization would be subject to scrutiny from regulatory agencies and client quality-assurance units, the company determined that hard-wall isolators designed for good manufacturing practice (GMP) applications would be the most robust and defensible containment technology. It was essential to quantify the degree of containment to substantiate any containment claim and then to develop and implement policy to ensure acceptable performance from both operator exposure and cross-contamination standpoints. The containment verification testing activities were performed in a manner that was consistent with the best industrial hygiene practices and in accordance with the guidance published in "Section II: Sampling, Measurement, Methods, and Instruments" of the 

Federal Occupational Safety and Health Administration 

 and the ISPE SMEPAC Committee document, "Assessing the Particulate Containment Performance of Pharmaceutical Equipment: A Guide," (1, 2). 

Merely achieving the desired technical claims would be insufficient to allow for a smooth transition from traditional personal protection equipment to isolation. The only way to frame isolation technology as something to be embraced rather than imposed was to vest employees with design responsibility. A specialized formulation development team was formed, organized under a manager reporting to the vice-president of pharmaceutical development, and charged with everything from conceptualization and design of the facilities to policy and management of its operation. The team successfully led the entire endeavor. Detailed job knowledge in the context of Metrics' overall operation was essential to a successful launch of these services.  

Dedicating staff at the inception of the project provided the added benefits of limiting access to the facility, ensuring better work-practice resulting from routine experience, and instituting more centralized training in addition to the standard GMP curriculum. This team functions as a small department integrated with supporting groups from other departments such as quality assurance, employee health and safety, and analytical development.  

The design effort required frequent, usually daily, meetings with architects, engineers, and other involved disciplines, including safety and equipment vendors.  The formulation team (that is, the end user) had the benefit of working with a variety of talented collaborators who became increasingly comfortable with this arrangement as all parties progressed through the learning curve. 

Two significant challenges were the limited available physical footprint for the renovation and how to maximize throughput of the area given all involved processes needing containment. A 1:1 ratio of equipment-to-isolators was not practical, so a series of hard-wall isolators each with three to five multioperation docking ports was constructed.  

The system needed to be more sophisticated than simply placing equipment in a "box," because such an approach would result in poor ergonomics and contaminate mechanical and electrical non-process surfaces of the equipment. A baffle or plate was needed to separate these areas of equipment. If this plate could be standardized about its periphery, it could be installed or interchanged at multiple locations on any given isolator within the facility. 

In addition to the standardization of the docking locations, each piece of processing equipment had to penetrate each standardized plate in a manner that placed the processing surfaces in an ergonomically viable space within the isolator. Once the required equipment was installed, the plates formed the rear wall of the isolator. The isolator was then sealed and negatively pressurized. Each was also fitted with a variety of utilities inside including electric, water, compressed air, and vacuum. 

It was essential that long process trains such as milling, screening, granulating, fluid-bed drying, sizing, blending, compressing, and film coating  could be constructed in one room simultaneously. Getinge LaCalhene (Rochester, NY) was selected to build the system. Familiar brands of processing equipment were chosen for the integration to mitigate any potential client apprehension. 

Shipping the necessary processing equipment (mills, blenders, granulators, and a roller compactor) to Getinge's facility and then building a full-sized mock-up facility proved to be a challenge. However, a full-sized mock-up facility is necessary to determine that each piece of equipment is properly positioned within the isolator when installed. 

After fabrication, the system was installed and validated. Several clinical supplies have been successfully manufactured within these isolators and the feedback from the team,  now working in the area, has been very positive. In the future, as long as a given piece of equipment can penetrate a vertical 3-ft

 plate with the product contact surfaces on one side, it can be adapted to work within the system. Or if bench-top space is needed, blank plates can be installed. The limitations of the system are primarily related to size, as certain pieces of equipment will not work through a vertical plate. Separate isolators integrated with our tablet press and fluid bed unit were also designed and installed. 

Isolated equipment includes: a Fluid Energy Jet-O-Mizer micronizer, Fitzpatrick Fitz L1A, Globe Pharma Mexi-Blend (0.5 to 16 quart), Vector TFC Lab micro roller compactor, Fluid Air Pharm X High Shear Granulator with 1L, 2L, 4L, and 8L bowls, Vector FLM1 fluid-bed dryer/top-spray granulator, Quardro Comil U-5 model, Riva 10-station instrumented Picolla tablet press, capsule filling devices, and tablet film coater (see Figures 1–3). 

Figure 1: An isolated fluid-bed dryer that was specially designed for the new potent products facility. (IMAGE COURTESY OF THE AUTHOR) 

Because of the hard-wall isolation technology and resulting containment verification, Metrics eliminated any need for the use of bulky suits with powered air purifying respirators. Processing rooms are essentially clean while in-process. The improved comfort and cleanliness has been a popular milestone for the team members. 

Figure 2: The custom multiuse, hard-wall isolator was engineered to house up to three pieces of processing equipment.  (IMAGE COURTESY OF THE AUTHOR) 

Several policy instruments have been enacted or altered as a result of this new capacity. If the compound is deemed highly potent at a projects' inception?that is, if the operator exposure limit is determined to be less than one micron per cubic meter of breathing zone air?then all processing operations are conducted in a the new facility. 

Figure 3: An integrated tablet press for the potent-products facility. (IMAGE COURTESY OF THE AUTHOR) 

More conservative cleaning verification policies pertaining to the new capacity were implemented. After every batch or campaign, a comprehensive health-based cleaning verification of shared surface areas is conducted. This new procedure requires high-performance liquid chromatography recovery of the active pharmaceutical ingredient  below a residual threshold after every batch or campaign of batches, regardless of what a subsequent activity may be. One-hundred-percent cleaning verification supplants a cleaning validation program. As a result, there is an analytical record that proves every active is removed properly after every processing event. 

 is director of potent pharmaceutical development at Metrics, 

 1.  Occupational Safety &  Health Administration, US Department of Labor, 

Assessing the Particulate Containment Performance of Pharmaceutical Equipment 

http://www.ispe.org/cs/ispe_good_practice_guides_section/ispe_good_practice_guides

Articles

The author describes the approach taken to develop a facility dedicated to handling potent and cytotoxic drug substances.

Design and Construction of a Potent Pharmaceutical CGMP Suite

Drug development is an arduous, financially risky process that has been estimated to cost pharmaceutical firms an average of $802 million for each therapeutic gaining marketing approval (1). To maximize return on the initial investment, companies look to strengthen their market position and extend product life cycles through reformulations, new routes of administration, and use of the latest technology. 

For injectable therapeutics, one way to accomplish this goal is to move a product's presentation from a vial to a prefilled syringe. A prefilled syringe helps to increase dosing accuracy, convenience, and safety; enhance patient quality of life; and reduce patient time in the clinic. While the introduction of prefilled syringe presentations varies among drug classes and therapeutic categories, the overall market for prefilled syringes is expected to grow by 12.8% per year (see Table I) (2).  

Table I:  Partial list of injectable therapeutics  and vaccines available in prefilled syringes in the US.  

This article presents a project manager's perspective on the rationale and timeline for moving an injectable therapeutic to a prefilled-syringe format. The author also discusses a regulatory strategy that can facilitate a smooth transition to the new dosage form. 

The natural first question is, "Why change a drug's presentation to a prefilled syringe?" For many companies, the decision to change to a prefilled-sryinge format is strategic because it meets the demands of physicians and patients looking for easier modes of administration and it helps further differentiate the product from competing drugs in the same therapeutic category. 

Data from Frost and Sullivan, a global market-research firm, demonstrate the importance of product presentation to physicians and patients. Three of the top five factors influencing a physician's choice of a drug-delivery type—ease of use by patients (16%), convenience (11%), and comfort (9%)—are affected by the presentation of the product. Physicians also commonly view patient satisfaction (14%) and a minimum of side effects (9%) as important factors in their choice of drug-delivery methods (3).  

When selecting a drug-delivery device for their patients, 46% of physicians take into account whether it easily enables self-administration (3). Patients who have a choice between drug-delivery devices also judge the ease of self-administration (37%) and whether the product has been recommended by the physician (24%) (4). 

Prefilled syringes also present economic advantages for pharmaceutical companies marketing injectable therapeutics. Because the devices meet customer demands for increased safety and convenience, companies often are rewarded with premium pricing for prefilled syringes compared with vials (5). Moreover,  prefilled syringes help increase the saleable yield of active pharmaceutical ingredient (API). Filling API in prefilled syringes enables the required dose to be delivered precisely. Consequently, only trace amounts of API remain in the needle of the prefilled syringe after injection. In contrast, single- or multi-use vials require overfilling the API to ensure that an accurate dose is pulled into the syringe each time.   

The question of when to move an injectable therapeutic into a new presentation is as significant as the rationale for why. Speed to market is a critical factor for the development of new pharmaceuticals, as well as for producing reformulations or improved delivery devices. Injectable drugs typically are introduced in a vial, because vials enable a faster pathway to regulatory approval, particularly when individual patient dosing varies according to factors such as age or weight.  

Once the product is on the market, a reasonable timeline for developing improved packaging depends on whether the initial product presentation is liquid or lyophilized (see Table II) (6). Moving from a liquid vial to a prefilled syringe can be accomplished within 6 to 18 months in most cases. 

Table II:  Differentiation of an injectable  therapeutic throughout its life cycle.   

The timeline is a bit longer for molecules introduced to the market as lyophilized powder. As a consequence, movement to a prefilled syringe from a dry vial typically is considered a mid-term life-cycle strategy. The process of developing a stable liquid formulation and gaining regulatory clearance for the formulation and enhanced packaging may require 18–36 months, and occasionally longer depending on clinical-trial results. 

Teva Pharmaceuticals (Petach Tikva, Israel) employed a mid-term strategy in 2002, when it changed the lyophilized vial presentation of Copaxone (glatiramer acetate) into a stable liquid offered in a prefilled syringe. Before reformulation, market share of Copaxone was declining. Yet, the new presentation achieved rapid acceptance in the market; 64% of patients switched to the prefilled syringe version within the first three months of availability. The remainder switched within six months of the new product's launch (6).  

The Copaxone prefilled syringe had measurable advantages for patients on chronic therapy for multiple sclerosis, particularly the amount of time required for self-administration. The average time a patient spent preparing for a Copaxone injection was reduced from the 235 s it took to reconstitute the product and draw it into a syringe to 38 s with a prefilled syringe. Consequently, the reformulated version saved a typical patient more than 20 hours over the course of a year. For Teva, increased patient convenience was rewarded with premium pricing, compared with the original formulation. In 2002, the premium started at 5% and rose to 48.6% by 2005 (6). 

Similarly, in 2004, Amgen (Thousand Oaks, CA) changed the presentation of Enbrel (entanercept), a treatment for rheumatoid arthritis, from a dry-vial prefilled diluent-syringe kit to a single-use prefilled syringe. In 2006, the company launched Enbrel in an autoinjector, which further differentiated the product and added value. Although unit sales of Enbrel have remained stable, sales revenue has increased because of the price premium associated with the prefilled syringe and autoinjector formats (see Figures 1, 2) (6). 

Figure 1: Unit sales of Enbrel following movement from a dry-vial prefilled diluent-syringe kit to a prefilled syringe and autoinjector (5). (FIGURE COURTESY OF THE AUTHOR) 

For most companies, the process of moving an injectable therapeutic to a prefilled syringe can be fairly complex because of sterility and stability issues associated with small and large molecules in addition to challenges related to the size and structure of biologic molecules. To facilitate a smooth path to regulatory approval, companies must establish a strategy that carefully evaluates proposed changes in manufacturing, packaging, and shipping processes and that ensures the company can validate the effect of these changes on the therapeutic molecule. 

Figure 2: Growth in worldwide and projected sales for Enbrel following introduction of a prefilled syringe and autoinjector format in 2004 and 2007, respectively (5). ROW is rest of world. (FIGURE COURTESY OF THE AUTHOR) 

The US Food and Drug Administration requires that any change in the manufacture of a drug product, whether major or minor, be put into place only after the license holder assesses the effect of the change on the identity, strength, quality, purity, and potency of the molecule, because these factors will influence the safety and effectiveness of the pharmaceutical (7). 

In addition, FDA has stringent requirements for changes that may affect parenteral drug product. These requirements pertain to moving a therapeutic to a prefilled syringe from another container–closure system; silicone treatments in the closure systems such as in elastomeric closures or the syringe barrels; and changes in the size or shape of a container that holds a sterile drug product (8). Any such change is considered to be "major" by FDA and must be documented in a prior approval supplement.   

Stability and clinical testing for parenterals depend on a multitude of factors, including the formulation, indication, mode of administration, size and functionality of packaging, and the introduction of new materials. Any potential need for clinical or stability data must be noted and planned for at the outset, and included in the stability protocol.  

 The choice of a syringe system should be a critical part of the regulatory strategy and must be considered at the outset, perhaps even with product reformulation. The syringe system is important because a packaging system that is acceptable for one therapeutic will not necessarily work for another because of differences in dosing ranges, patient populations, and in how individual molecules react to surrounding materials and conditions. Evaluation of the proposed syringe system must consider these factors, and regulatory submissions must support the suitability of the proposed packaging and storage conditions.  

An analysis of proposed syringe systems should take into account their appropriateness, functionality, and materials. First, the dosing device has to be suitable, and its graduations appropriate, for the intended patient population. At a basic level, the syringe must deliver the dose in the amount and rate stipulated in the package insert. In addition, the physical characteristics of the liquid drug formulation must be appropriate for the dosing device to ensure accurate dispersion of the drug through the needle. 

Second, the proposed system must function correctly throughout its shelf life. The results of coating integrity testing, effect of viscosity, and syringe-ability must be considered. Moreover, scale intervals on the syringe must represent the labeled dosages, and gradations in dose must be easily read. The type should be embossed or printed and legible throughout the shelf life of the product. These markings cannot fade or become dislocated during the use or life of the product. In addition, stopper movement during use, as well as the effect of shipping on stopper movement, must be considered to ensure sterility of the product and to avoid particulate contamination. 

Third, materials in the proposed syringe must be compared with those of the current vial. The analysis should consider whether:   

The material in the proposed syringe, including in the container, plunger device, and closure system, protects the drug product

The syringe system introduces a new material 

The system will require any changes to the drug product formulation.

Choosing a container and closure system made of the same materials as the vial will eliminate the need for additional extractable and leachable studies. Any new materials, including those in the hub or needle, will need to be evaluated in stability testing. Extractable studies also must include proposed labels, adhesive, and ink if the barrel of the syringe is not made of glass.  

 FDA rules governing current good manufacturing practice (GMP) stipulate that companies develop and thoroughly document a testing program to assess the stability characteristics of the drug product (9). Consequently, a company must define stability testing requirements related to the container–closure system. These requirements include how temperature, humidity, and light influence the shelf life of the drug and help to determine appropriate storage conditions and expiration dates for the product. A stability protocol that is specific to the molecule and the proposed drug-delivery device must be written, implemented, and evaluated to assess the stability characteristics of the molecule in the container–closure system (9). 

Although some extractable and leachable studies related to the dry or liquid vial presentation may be transferable to the new format, some new stability testing is required when moving an injectable therapeutic to a prefilled syringe (see Table III). The long, narrow dimensions of a syringe increase the molecule-to-surface-area ratio from the previous vial presentation. As a consequence, accelerated and long-term stability studies are necessary to determine whether the change affects the extractable and leachable parameters and to understand the impurity and degradation profile of the molecule in the container–closure system. 

Table III:  Filing requirements for prefilled syringes. 

The stability protocol should detail the sample size, test intervals, storage conditions, validated analytical methods to be performed, the container–closure system, test specifications, and number of batches required. In addition, reconstitution studies will be required for parenteral products offered in a kit with a prefilled diluent syringe or in a dual-chamber prefilled lyophilized syringe. Tests and specifications must be based on knowledge of the degradation pathway for the particular molecule, use validated and stability-indicating methods, and specify limits appropriately. 

   Clinical studies are required when there is a change in indication or new route of administration for the drug. However, movement from a dry or liquid vial to a prefilled syringe also necessitates clinical testing if the reformulated drug's degradation or impurities profile change. 

Scenario one: liquid vial to liquid prefilled syringe

A company markets a contrast imaging agent used in the imaging of major organs. Originally available in 50-mL, 100-mL, and 200-mL single-use vials for intravenous and intra-arterial administration, the company seeks to make the product available in 10-mL and 50-mL prefilled syringes for intrathecal administration for imaging the spinal cord and nervous system. The 50-mL syringe also would be used in the previously approved routes of administration and indication.  

In this situation, it is important to consider which factors change and which remain the same. The container–closure system are new, meaning a new size and dimension of container and new functionality of the device are involved. Because the device would enable administration directly into the spinal cord, the route of administration is new, as is the indication. The formulation and long-term storage conditions remain the same, however, because the contrast imaging agent was previously available in a liquid vial. 

In selecting a syringe system, the company chooses a device with components similar to those used in the single-use vials. Because the glass and rubber formulation are identical between vial and syringe, previous extractable and leachable studies will apply. However, the company must compare all the materials in the container–closure system because some new materials may be introduced in the components. In addition, the syringe may require additional siliconization to improve the function of the plunger.  

To support the regulatory filing, the company will need to submit three months of comparative accelerated stability data. Long-term data on at least one batch, possibly three, will be needed at the time of submission. Moreover, the company will need to commit to placing the first of three production batches on long-term stability and to placing another batch on long-term stability each year. Additional clinical data also will be needed to support the new indication and the new intrathecal route of administration.  

Scenario two: lyophilized vial to prefilled syringe

A company producing a lyophilized product packaged with reconstitution fluid for intramuscular injection considers marketing a prefilled syringe for the same indication, in the management of rheumatoid arthritis. The company must reformulate the molecule to a stable liquid. The route of administration will remain the same.  

The container–closure device, however, will change, which means that some materials and the functionality of the system will be different. In addition, long-term storage conditions may change because of the new formulation. 

When evaluating a syringe system, the company must choose one with components that are similar to those used in the vial and reconstitution device to best leverage previously conducted extractable and leachable studies. As with other prefilled syringes, additional siliconization may be necessary. Clinical studies will be required as well if the degradation or impurity profile changes. 

Upon submission, FDA will require three months of comparative accelerated data and long-term data on three batches of drug product. The stability tests will need to focus on appearance, color, clarity, sterility, pyrogens, syringe functionality, container– closure integrity, impurities, and particulates. The company also will need to commit to placing the first three production batches and annual batches on long-term stability tests. 

The process of moving an injectable therapeutic from a vial presentation into a prefilled syringe or, perhaps, into an autoinjector format, can be complex, regardless of whether the original product is in a liquid or lyophilized formulation. Companies pursing a new dosage form must carefully document the sterility and purity of the product and ensure the stability of the molecule in the new container–closure system. Complexity of the process multiplies when the dosage form is to be marketed on a global scale. Countries differ in the interpretation of regulatory guidelines established through the International Conference on Harmonization (10). 

To ensure a smooth and speedy transition to a new dosage form, most companies assign responsibility for the change- management process to a project manager, who coordinates roles and responsibilities of the team involved in the project, establishes processes and procedures, and manages regulatory submission timelines. Working with team members, the project manager develops and implements the regulatory strategy to control and properly validate the effect of new processes and materials on the therapeutic molecule.  

Moving from Vial to Prefilled Syringe

Organic chemists usually spend a substantial amount of time on the scale-up of synthetic routes to new materials. The best pathways need to be chosen, and reaction conditions must be optimized. Once a product is successful and demand for larger quantities is growing, the whole synthesis process needs to be revised again and readjusted for larger batch sizes. 

In sharp contrast to the batch mode, chemical synthesis becomes a time-resolved process in flow chemistry. Reagent streams are continuously pumped into a flow reactor, where they are mixed and allowed to react. The product instantly leaves the reactor as a continuous stream. Only flow rate and operation time determine the scale of the synthesis. The same reactor with an inner volume of less than a milliliter will produce kilogram quantities of material.   

Parameters other than size determine the performance of a microreactor and whether it will offer superior properties compared with a conventional batch reactor. The material that a reactor is made of—typically metal, glass, or silicon—is one such parameter. Each material offers advantages and disadvantages with regard to price, compatibility with reagents, and heat conductivity. 

Figure 1: Heat distribution in a batch-synthesis reactor. (FIGURE COURTESY OF SAFC) 

Because of their small surface-to-volume ratio, microreactors can absorb heat created from a reaction much more efficiently than a batch reactor can. Figure 1 shows the initial heat distribution for a neutralization-model reaction in a simulated 5-m

  batch reactor stirred at 500 rpm. The batch reactor is heated by an exothermic reaction. Cooling only takes place at the surface of the reactor. As a result, there is a strong temperature gradient of about 10 °C from the surface of the reactor to its center. In a microreactor, the heat created by mixing the two reagents is also detectable, but the temperature gradient is much smaller, only 3 °C (see Figure 2). Additionally, it only takes a few millimeters of path length for the reagent stream to cool down again to the temperature of the outside-cooling medium. The formation of hot spots or the accumulation of reaction heat may favor undesirable side reactions or fragmentation (see Figure 3). Microreactors, with their superior heat-exchange efficiency, present a perfect solution. Precise temperature control affords suppression of undesired byproducts. 

Figure 2: Heat distribution in a microreactor. L is path length, and y is inner-channel diameter. (FIGURE COURTESY OF SAFC) 

The following examples describe the use of microreactors in the industrial production of chemicals. In some cases, not all process details (e.g., catalysts, reagents, and structures) are described, but each example highlights specific advantages of microreactor technology and flow chemistry over batch-synthesis protocols. 

Figure 3: Temperature control in a microreactor (MR) enhances product quality by suppressing side reactions. A is the reagent, B is the product, C is the undesired byproduct from a side reaction, and B and C are intermediate stages. (FIGURE COURTESY OF SAFC) 

Precise control of the reaction temperature. 

The production of certain ionic liquids in high purity is a special challenge. The reaction between imidazole derivatives and sulfonates is extremely exothermic (see Figure 4). Conventional batch-synthesis procedures fail to yield products with purities higher than about 96%. Even with very slow dosing times and careful cooling, batch reactors are heated by the reaction. Investigations at Sigma Aldrich (Buchs, St. Gallen, Switzerland) showed that severe brown impurities are formed if the temperature rises above 20 °C in any part of the batch reactor, which leads to a miscolored product. Subsequent purification is troublesome and can only be achieved with huge efforts. Microreactors provide an ideal way out of this dilemma. Their superior heat-exchange capabilities make it possible to carry out the synthesis with neat reagents at an external cooling of only –3 °C. Under these conditions, the temperature inside the microreactor in the reaction zone is kept at a constant value of 8 °C, thus allowing ionic liquids to be produced with a purity exceeding 99% with only traces of undesired materials. Because neat reagents can be used without any solvent, the output of this procedure is very high. 

Figure 4: Production of ionic liquids.  (FIGURE COURTESY OF SAFC) 

There is much discussion whether ionic liquids can generally be called "green." Although the answer to this question may be debated, it is quite obvious that the production of ionic liquids in a microreactor is as green as chemical synthesis can be. All reagents are quantitatively converted into the final product. No solvents are needed for the synthesis or purification. Absolutely no waste is generated.  

Ring closure in synthesizing exomethylenecyclopentane

The cyclization of 1,5-hexadiene to exomethylenecyclopentane poses multiple challenges. The batch reaction affords an inseparable mixture of the endo

 and exo- cyclization product (see Figure 5). The desired kinetically favored product isomerizes under reaction conditions in the presence of the catalyst. Separation of the two isomers is difficult because both have virtually the same boiling point. In addition, the transition-metal catalyst used for this transformation requires a minimum working temperature of 55 °C. At this temperature, the starting material evaporates. 

Figure 5: Production of exomethylenecyclopentane in a microreactor. DIBAH is diisobutylaluminium hydride.  (FIGURE COURTESY OF SAFC) 

Microreactor technology solved all these problems. By inserting a simple pressure valve into the product channel, the reaction was performed at an elevated pressure of 2 bar. This higher pressure raised the boiling point of the reactant sufficiently to 58 °C, a perfect working temperature for the catalyst, and the reaction proceeded without gas formation. Isomerization of the desired product was completely suppressed, and a highly pure product (> 99%) was formed at a multikilogram scale.  

Efficient phase-transfer catalysis, safe and scale-independent handling of explosive intermediates in small hold-up volumes with instant conversion. 

Organic azides are high-energy compounds. Substrates with multiple azide moieties and low-carbon content are potentially explosive. For example, the azide intermediate shown in Figure 6 has a potential energy of 3.8 kJ/g at 114 °C, as measured by differential scanning calorimetry. 

Figure 6: Two-stage microreactor assembly for the safe production and immediate conversion of organic azides.  (FIGURE COURTESY OF SAFC) 

A two-stage microreactor with a continuous-flow system allows for the safe handling of organic azides in small hold-up volumes with immediate conversion in the second microreactor. In the first microreactor, the azide intermediate is formed by nucleophilic substitution. The azide intermediate undergoes Staudinger hydration with triphenylphosphine in a subsequent second microreactor. Notably, it is possible to work with biphasic systems in microreactors. A phase-transfer catalyst moderates the initial reaction with efficient mixing by the microreactor. 

-xylylenediamine in two subsequent glass microreactors exceeds 1 kg/day with an overall yield of 60%. The work-up and isolation of the potentially hazardous intermediate is completely avoided between reaction steps, thereby saving time and money while offering the utmost scale-independent safety level. 

Accelerated and economic process development for large-scale synthesis. 

Microreactors have simple and pragmatic advantages over traditional batch vessels. They take up less space on the factory floor, but can be used to make large quantities of product. 2-Benzoyl pyridine is an important building block and has annual demand of about 15 metric tons. It is synthesized through a Grignard reaction (see Figure 7). In a microreactor, this reaction takes less than one minute. The Grignard microreaction is followed by two on-line quench modules. Precise reaction control leads to a highly pure crude product, thereby making a distillation step unnecessary, as would be the case for purification of the lower quality batch product. 

Figure 7: Grignard microreaction with two subsequent quench modules. (FIGURE COURTESY OF SAFC) 

This reaction was performed in a stainless-steel plate reactor (ART, Alfa Laval, Lund, Sweden). The reactor took up only 30 X 50 cm of space on the bench. Rapid flow rates were achieved with this reactor, thus allowing the continuous production of 200–300 kg of 2-benzoyl pyridine per day.  

These reactions show the versatility and utility of microreactor technology and flow chemistry in chemical synthesis. Such an approach offers many advantages over traditional batch-mode manufacturing. Depending on the reaction, improved process economics, greater reaction efficiency, and waste reduction may be achieved. Problems inherent in scale-up are eliminated or reduced, making microreactor technology a viable tool in the synthesis of APIs. 

, is global business director of SAFC Pharma, Industriestrasse 25, 9470 Buchs, Switzerland, tel. 41 81 755 2405, fax 41 81 755 2584, 

, is product manager of chemistry at Sigma-Aldrich.  

*To whom all correspondence should be addressed. 

 1. T. Schwalbe, V. Autze, and G. Wille, "Chemical Synthesis in Microreactors,"

 2. T. Schwalbe et al., "Novel Innovation Systems for a Cellular Approach to Continuous Process Chemistry from Discovery to Market," 

The authors discuss the advantages of microreactors and flow chemistry for various reaction types in achieving improved process economics and reaction efficiency.

Using Microreactors in Chemical Synthesis: Batch Process versus Continuous Flow

Biosimilars, also known as follow-on biologics, generic biologics, and biogenerics, remain somewhat controversial, but their potential to yield cost savings is inspiring pro-biosimilar legislation in developed pharmaceutical markets such as Europe and the United States. Key patents for several first-generation biologics are set to expire, or have already done so, in the seven major markets (i.e., France, Germany, Italy, Japan, Spain, the United Kingdom, and the United States). Although these drugs represent the low-hanging fruit of the biologics world in terms of ease of development and commercialization, they are unlikely to provide biopharmaceutical manufacturers with the monetary gains that complex biologics bring. First-generation biosimilars will likely provide indirect benefits such as allowing the key biosimilars players to establish themselves within the industry. Europe, Germany in particular, is emerging as the testing ground for biosimilars, but the US market will prove to be the real prize for biosimilars manufacturers.  

To date, three biosimilars have entered the European market: somatropin, epoetin alpha, and granulocyte-colony stimula-ting factor. These products have yet to have a significant effect on the market, however. Despite this slow start, biosimilars are expected to garner peak sales of $2 billion in 2014 in the seven major markets (1). Given that major-market sales of branded epoetin market totaled $9.9 billion in 2008, it is not surprising that biosimilar epoetin alpha is forecast to dominate the biosimilars market. Biosimilar insulin-glargine is expected to be the second-largest product. Yet, with the exception of insulin-glargine, the biosimilars uptake is forecast to decline after 2014, primarily because second-generation biosimilars will enter the market at that time (see Figure 1). 

Figure 1: Prediction of biosimilar sales by product in seven major markets (i.e., France, Germany, Italy, Japan, Spain, the United Kingdom, and the United States) during 2008â2019. HGH is human growth hormone. IFN is interferon. A hyphen indicates that a product was not launched. Sources: 2008 sales data are from MIDAS, IMS Health, March 2009; forecast for 2009â2019 is from Datamonitor.  (FIGURE COURTESY OF THE AUTHOR) 

One of the major advantages of epoetin alpha is that it is a small, simple biologic (it contains 165 amino acids), with a relatively inexpensive and well-established manufacturing process. These considerations facilitate molecule development and characterization and therefore reduce perceived risk.  

In contrast, standard human insulin is a poor commercial prospect for biosimilars because new insulin analogs are available. But human insulin biosimilars may provide an attractive market entry point for companies that plan to build a reputation in diabetes treatment as part of a long-term strategy. Standard human insulin does not have patent protection and, as one of the simplest and best-characterized biologics, it is simple and inexpensive to manufacture. In fact, numerous companies in the emerging markets of Eastern Europe, India, and China already market generic human insulin. In 2008, the insulin-analog market was worth $7.2 billion and accounted for 77% of all insulin sales in the seven major markets (2). This market is a lucrative opportunity for biosimilars companies. sanofi aventis's (Paris) long-acting Lantus (insulin-glargine), which represented 38% of all seven major markets' sales of insulin analogs in 2008, is the most lucrative product in this class. Datamonitor anticipates that biosimilars for this product will enter the market first in the US and Germany in 2014. Adoption of biosimilars in these markets and in the UK is predicted to result in peak sales of $530 million by 2019. 

The US market represents the greatest opportunity for the emerging biosimilars industry and is forecast to constitute nearly 90% (or $1.75 billion) of the biosimilars market in 2014 (1). The size of the US market and the generic erosion that characterizes it make it an attractive prospect for would-be biosimilars manufacturers. Success in this market, however, is contingent on the establishment of a biosimilars approval pathway. Datamonitor anticipates a pathway to be in place by 2010 and expects the first biosimilar to enter the US market in 2013.  

In July 2009, an approval pathway came one step closer to reality when the House Energy and Commerce Committee overwhelmingly approved the America's Affordable Health Choices Act. This act would guarantee manufacturers of branded biologics an exclusivity period of 12 years. These terms are similar to those attached to a bill passed by the Senate's Health, Education, Labor, and Pensions (HELP) committee, on July 13 (4). 

The historically low generic drug use in France, Italy, and Spain will contribute to slower and more limited biosimilar adoption in these markets, which are collectively forecast to constitute a maximum of 25% of biosimilar sales in the seven major markets in 2012. High brand loyalty and physicians' greater discretion in prescribing mean that marketing will be critical to promoting biosimilar uptake. Therefore, only the large and established companies with high brand recognition are anticipated to succeed in those markets. 

Although guidance for biosimilar approval was issued in Japan in 2009, this market is unlikely to experience significant biosimilar sales. It is expected to contribute at most 2% to sales in the seven major markets in 2012. The slow acceptance of conventional small-molecule generics in Japan suggests that biosimilars will face an uphill struggle there. The key stakeholders' distrust of the quality and efficacy of generic drugs has hindered uptake to date (see Figure 2). 

Figure 2: Sales of biosimilars by country in the seven major markets during 2008â2019. US is United States. UK is United Kingdom. Sources: 2008 sales data are from MIDAS, IMS Health, March 2009; forecast for 2009â2019 is from Datamonitor. (FIGURE COURTESY OF THE AUTHOR) 

Although the growth of the biosimilars market has been slow, particularly in the US, global pharmaceutical trends, combined with increasing healthcare expenditures, have created momentum that could make the biosimilars market even more lucrative than the generics market. Figure 3 outlines some of the major reasons for and against the acceptance of biosimilars. 

Figure 3: Causes for (orange) and obstacles to (blue) the uptake of biosimilars. (FIGURE COURTESY OF THE AUTHOR) 

The high cost of biologics will spur payers to look to bio-similars for cost savings. Specialty drugs are often priced at a premium to recoup development costs from a limited market size, and the absence of generic-drug competition has given biologics manufacturers freedom to set prices as they see fit. Specialty biologics are among the most expensive medicines, ranging in price from $5000 to more than $300,000 per year. Annual consumer expenditure for specialty biologics has grown by 12–15%, which is double the rate of nonspecialty drugs in the US (3). The high price of branded biologics and the expectation that their use will overtake that of small-molecule drugs provide a significant reason for biosimilars adoption. 

The number and use of biologics are expected to grow, thus expanding the potential biosimilars market. Specialty biologic drugs are used by 3% of the US population. This proportion will increase as more biologics are approved and the indications for those already on the market are expanded (3). Demographic trends are also increasing the use of biologics. As the global population ages, the incidence of complex and chronic diseases that require specialty biologic treatments grows accordingly. The number of approved or soon-to-be-approved biologics in the US has more than doubled every five years since 1990. More than 800 biologic drugs are in the pipeline, and the number of biologics on the market will continue to climb (3).  

The need to contain rising healthcare costs is the primary motive for regulatory and legislative reform aimed at promoting the use of biosimilars. Datamonitor expects that biosimilars uptake will mirror that of generic drugs and occur more rapidly in the mature generic-drug markets (i.e., Germany, the UK, and the US) but face obstacles in France, Italy, Japan, and Spain. 

Providing sufficient demonstration of biosimilarity to alleviate fears and promote acceptance remains one of the major issues facing biosimilars manufacturers. Demonstration of biosimilarity will increase physicians' willingness to prescribe biosimilars and reduce patients' resistance to these products. In principle, biosimilarity should be relatively easy to prove for the simple and well-characterized biologics such as insulin, epoetin, and human growth hormone. The larger and more complex biologics such as monoclonal antibodies (mAbs), however, present a challenge. These biologics tend to be the most lucrative and attractive prospects for biosimilar manufacturers, who must establish sophisticated development processes to realize their potential. 

Rebates and service agreements between branded manufacturers and healthcare providers or insurers are likely to be a considerable deterrent to the adoption of biosimilars. Biosimilars manufacturers must either offer better incentives than the branded companies do or offset them through competitive pricing. Datamonitor anticipates that branded companies will defend their market share aggressively against biosimilars, probably through competitive pricing. Biosimilar manufacturers must therefore contend with the frequently longstanding relationships between branded companies, healthcare providers, and patients, and find a way to mitigate them. The strategy may be straightforward for biosimilar manufacturers that are affiliated with branded companies. But the traditional generic-drug company that is not accustomed to the marketing and promotion that will almost certainly be required in the emerging biosimilars market faces a substantial challenge. Moreover, these companies may lack the logistical and financial power to offer the same level of service and support that branded companies routinely offer. In an environment such as this, a collaboration that unites the technical know-how of small companies with the marketing prowess of larger companies will be critical. 

As the first region to introduce guidelines for the approval of biosimilars, Europe has emerged as the testing ground for these products. In 2008, the European Medicines Agency (EMEA) said that a biosimilar approved through the pathway is as safe and effective as any other EMEA-approved drug (5). Not surprisingly, all of the biosimilars were first launched in Germany, the largest generic-drug market in Europe. Although a European regulatory pathway now exists, general acceptance and a path to market—in terms of pricing, pricing, reimbursement, and distribution systems—remain to be established (6). Creating demand among the key stakeholders is critical and requires information that counters branded companies' promotional efforts. 

Lessons learned from Europe's hospital and retail markets should also be considered. Biologics in Europe tend to be dispensed in hospitals, although there is considerable regional variation for molecules such as epoetin and filgrastim. In a hospital, pharmacy managers and payers are likely to influence physicians' prescribing behavior, making this setting an attractive one for biosimilars manufacturers. Moreover, anecdotal evidence suggests that patients are often unwilling to switch drugs after leaving the hospital (1). 

But drug acquisition through the tender process may make it difficult for biosimilars manufacturers—who may be less familiar with the process or lack the established relationships—to sell to hospitals. Acquisition in the retail sector, while more fragmented, is far more transparent, and competition based on price is a real possibility. This conclusion is partly borne out by biosimilar epoetin alpha sales in 2008 in Germany, where uptake was almost eight times faster in the retail sector than in hospitals (2). 

Although Japan is the second market to establish an approval pathway for biosimilars, the products are not expected to enter the Japanese market until 2012 at the earliest. Biosimilars in Japan will be subject to the same approval process as branded drugs, with the concomitant delays to which branded manufacturers have grown accustomed. The median approval time for new drugs in Japan was 34.3 months in 2007 (7). Therefore, Datamonitor assumes that biosimilar approvals in Japan will take at least three years. Only one biosimilar, epoetin, is known to have been submitted for approval, and market entry is likely to occur significantly later than in the other markets. 

Because the development and marketing of biosimilar drugs is much more demanding than it is for traditional generic drugs, the number and types of players entering the market will be limited.  

 In 2007, Sandoz (Holzkirchen, Germany) was the first company to launch a biosimilar—Omnitrope (human growth hormone)—in Europe. Although Omnitrope failed to gain significant market share, the move enabled Sandoz to establish itself as a major biosimilars player in an industry where gaining physicians' trust is as important as price. The company reinforced its position by being the first to launch biosimilar epoetin in 2007. Sandoz's parent company Novartis (Basel) lends it the marketing experience and infrastructure of a branded organization. 

 It is unsurprising that Teva (Petach Tikva, Israel), one of the largest generic-drug companies in the world, is looking to establish itself in the biosimilar arena. Although the company is initially concentrating on Europe, it perceives the US biosimilars market as a genuine, albeit tougher, opportunity and expects it to boom in the middle of the next decade (8). Teva has three biologic manufacturing facilities and sells biosimilar interferon alpha 2b in some of the less-regulated markets such as China and Lithuania. In partnership with Savient Pharmaceuticals (East Brunswick, NJ), Teva markets Tev-Tropin (recombinant human growth hormone) in the US. Many view the product as a biosimilar despite the fact it was granted full approval. 

 Some branded biologic companies have begun to produce biosimilars in hopes of maintaining their market share. Manufacturing biosimilars would be relatively quick and easy for most branded companies. In December 2008, Merck & Co. (Whitehouse Station, NJ), AstraZeneca (London), and Eli Lilly (Indianapolis, IN) announced their intention to enter the biosimilars arena. Merck & Co. plans to launch six biosimilars between 2012 and 2017 (9). 

All three companies are using the biotech expertise they gained through acquisitions. Merck & Co. acquired GlycoFi in 2006, whose technology enables control of glycosylation during biopharmaceutical production. Merck bought the biosimilars portfolio of Insmed, which includes drugs for patients undergoing chemotherapy, in February 2009. AstraZeneca acquired MedImmune, whose products include the FluMist influenza vaccine, in 2007, and Eli Lilly acquired ImClone, maker of the Erbitux cancer therapy, in 2008. 

The importance of an established marketing presence has particular significance for biosimilars because companies need to assure physicians that their products are as safe and efficacious as branded products. Initial adoption will greatly depend on physicians' opinions. Companies that already have relationships with physicians will have an edge over smaller enterprises focused on small-molecule generic drugs for whom marketing is less important. As a result, branded pharmaceutical companies will be able to take advantage of their marketing infrastructure and their reputation to promote acceptance. 

 Although Glaxo-SmithKline (London) produces biologics and aims to establish a biopharmaceuticals operation, the company decided that biosimilars are a high-risk venture that requires too much investment for too little return, at least for the present (10). Johnson & Johnson (New Brunswick, NJ) has taken a similar line, stating that the considerable investment required for conducting clinical trials would drive up costs excessively (11). 

Cutting healthcare spending by promoting generic drugs and biosimilars is at the heart of President Obama's healthcare agenda (12). One of the biggest hurdles to a biosimilars pathway, however is determining the marketing exclusivity period for branded biologics. Branded biologics manufacturers state that 14 years is required to recoup costs, stimulate innovation, and attract future investors. The Generic Pharmaceutical Association strongly opposes this view and instead favors an exclusivity period of three to five years (13). Obama has voiced support for a seven-year exclusivity period. 

On March 17, 2009, Rep. Anna G. Eshoo (D-CA) introduced a biosimilars bill titled the Pathway for Biosimilars Act (HR 1548) in the House of Representatives. The bill's goal was to provide the US Food and Drug Administration with a process to review and approve biosimilars. The issues of interchangeability and market exclusivity were highly contentious. The Eshoo bill provides 12 years of data exclusivity for a new biologic and extends that period to 14 years if the biologic is approved for a new indication during the first eight years after its original approval. The bill includes a six-month exclusivity period for pediatric use. The Eshoo bill also requires new clinical trials comparing the immunogenicity of the biosimilar to that of the branded biologic to be submitted in the biosimilar approval application. 

The Eshoo bill, which was favored by small- and large-molecule innovator companies, gained strong bipartisan support in the House. The bill gained further momentum when the Senate HELP Committee approved an amendment that incorporated the Eshoo bill's key principles, including a minimum of 12 years' data exclusivity for biologics. In July 2009, the House included the Eshoo bill in its healthcare-reform legislation, marking another step forward toward a US regulatory pathway for biosimilars (4). 

Some believe that the ability of pharmacists to substitute a biosimilar for a branded biological is vital to promoting biosimilars and creating a viable market. Given the novelty, the inherent molecular complexity, and the associated safety concerns about biosimilar drugs, automatic substitution or interchangeability will not occur in the short term. Sufficient clinical data may be generated to make interchangeability a possibility in the long term. All of the five major European markets (France, Germany, Italy, Spain, and the UK) have banned automatic biosimilar substitution. Physicians in the UK are encouraged to prescribe brand-name biologics. The Eshoo bill does not permit outright interchangeability and recommends that FDA issue guidance on this topic. 

The advent of the biosimilars market has spawned a flurry of ill-defined terms to describe the relationship between bio-similars and their reference products. Although EMEA focuses on comparability and not interchangeability, the US bills make a distinction between the two. FDA defines 

 as "the situation where scientific data convincingly demonstrate that two products with very similar molecular compositions or active ingredient(s) can be safely substituted for one another and have the same biologic response and not create adverse health outcomes" (14). Therefore, automatic substitution, which has spurred the growth of the generics market, is unlikely to have a prominent role in biosimilars uptake in Europe or the US. 

The first biosimilars introduced to the market represented the low-hanging fruit of the biologics world. They were relatively simple molecules with established safety profiles, compared with mAbs, which have considerably higher barriers to entry. Although mAbs are currently the most profitable biologics, biosimilar mAbs are still a long way off. In addition to their complexity and the hurdles to their manufacture, mAbs still enjoy patent protection because they were introduced fairly recently (15). 

At least three Indian companies have launched or are developing mAbs. Dr. Reddy's (Hyderabad, India) and Biocon (Bangalore, India) both brought biosimilar versions of Roche's (Basel) Rituximab to market. Specialty generic-drug company Zenotech Laboratories (Hyderabad, India) claims to be developing a range of at least six biosimilar versions of mAbs already, including Rituximab (which is ready to enter Phase III) and Herceptin (trastuzumab) (16). The degree to which these monoclonals are similar to their respective reference products is unclear, and the products would warrant considerably more clinical testing before approval in any of the major markets. 

Biologics such as Amgen's (Thousand Oaks, CA) rheumatoid arthritis drug Enbrel (etanercept) are expected to lose sales to biosimilars earlier than will mAbs. The market for biosimilar Enbrel is potentially more lucrative than that for first-generation biosimilars, and its high cost is already prompting payers to limit drug use in markets such as that in the UK. A biosimilar could therefore erode not only sales of branded Enbrel, but also sales of newer branded competitors (17). 

Undoubtedly, the level of biosimilars adoption will have as much to do with the outcome of a public relations battle as with practical matters such as biosimilars' price and performance compared with those of biologics. Competition from biosimilars manufacturers will prompt innovator companies to modify their strategies to sustain revenue growth. Attaining biotech expertise through partnering and acquisitions will be one technique for success, but some branded companies will solidify their own presence in the biologics space. Regardless of the approach, product-positioning strategies will be imperative in a market where building reputation and trust among physicians, patients, and payers will be key to acceptance in the short term. 

, is the director of strategic development for healthcare at Datamonitor, 111 Devonshire St., Boston, MA 02109, tel. 617.722.4606, fax 617.523.6993, 

The author reviews the major biopharmaceutical markets' activity and predicts how the markets may evolve.

Market Outlook for Biosimilars

Achieving high yields with desired stereoselectivity is an ongoing goal in synthesizing active pharmaceutical ingredients (APIs), and biocatalysis is an important tool in this regard. To gain a perspective, 

's Senior Editor Patricia Van Arnum asked leading experts to share recent applications of biocatalysis. Hans Kierkels, senior scientist, and Oliver May, corporate scientist of biocatalysis, both with DSM (Geleen, The Netherlands) discuss the development of a nonanimal pig-liver esterase used in the synthesis of the API aliskiren and the application of ammonia lyases for producing amino acids and derivatives. Masahiko Yamada, senior researcher with Frontier Biochemical & Medical Research Laboratories, a research division of Kaneka (Osaka, Japan), provides various biocatalytic approaches to making chiral amines. 

PharmePLE (DSM) is a nonanimal-derived, pure enzyme used in the synthesis of optically pure compounds. (IMAGE COURTESY OF DSM) 

By Hans Kierkels, senior scientist, and Oliver May, corporate scientist of biocatalysis, DSM Pharmaceutical Products

A special challenge in manufacturing pharmaceuticals is the increasing complexity that requires many steps in the synthesis of a given molecule. On average, eight steps are required for the synthesis of an API, according to a recent analysis (1). The dynamics of drug development are challenging as well. The high attrition rate of drug candidates usually does not justify extensive route scouting and process development in early-development phases. In these phases, the focus is on speed of delivery rather than on manufacturing cost-efficiencies. This limited focus often leads to suboptimal routes and poorly developed processes for manufacturing clinical trial material.  

Given the limited number of rapidly accessible biocatalysts and the prohibitive timelines and costs for developing new or improved enzymes, biocatalysis had been a niche technology. But much has changed. During the last decade, breakthroughs in molecular biology, analytics, bioinformatics, and gene synthesis brought about an increasing spectrum of readily available biocatalysts faster and cheaper than ever before (1–4). It now takes only a few mouse clicks to identify new enzymes in exponentially growing databases using bioinformatic tools, and another mouse click to order genes or even mutant libraries in custom-made expression systems. Within a few weeks, those enzymes are shipped to laboratories where they are rapidly produced. The PluGbug expression system (DSM) is an example of a system that uses these tools. Such activities previously took several years, but now they take only several weeks. As a result, the enzyme toolbox is growing rapidly. DSM, for example, increased its enzyme collection from several hundred enzymes to more than 2000 enzymes during the last decade. Access to a rapidly growing spectrum of readily available biocatalysts, increasing cost pressures, and changing mindsets, will increase opportunities for biocatalysis. This change will happen throughout all phases of drug development, including preclinical and early clinical phases, where first-time-right processes using enzymes (see Figure 1) will be used in these shorter timelines. 

Figure 1: Opportunities for enzymatic processes due to advances in biocatalysis. (FIGURE COURTESY OF DSM) 

The mindset of scientists involved in route scouting and process research and development is changing slowly, and only a few pharmaceutical companies have captured those developments. Flexible and open partnering approaches with enzyme-service providers and contract manufacturers that provide access to a broad enzyme toolbox and offer interdisciplinary route-scouting expertise are just emerging. The main impact of biocatalysis is still in developing second-generation processes for late clinical phase or launched APIs. Two recently introduced processes highlight the benefit of biocatalysis.   

An excellent example of a successful process substitution was recently reported for producing an intermediate used in the synthesis of aliskiren, a renin inhibitor used to treat hypertension. A key step in the synthesis of aliskiren is an enzymatic resolution catalyzed by pig-liver esterase (PLE). PLE is a versatile biocatalyst for organic chemists because it has a broad substrate spectrum and excellent enantio- and regio-selectivity.  

The commercially available PLE is animal derived. Its quality can vary significantly from batch to batch, and it is therefore not safe or suitable for pharmaceutical applications. To address this problem, DSM and its collaboration partner, the Graz University of Technology in Austria, identified different isoforms of PLE. Using capabilities in enzyme development and production, a highly efficient and patented microbial expression system and fermentation process was developed for different isoforms of PLE that runs at a 25,000-L scale at DSM (5). This system delivers nonanimal-derived PLE isoforms (PharmaPLEs, DSM) at a large scale for pharmaceutical applications.  

The PharmaPLE-based production process replaced an established chemical process. The overall productivity of the process increased more than 50%. Waste production was significantly reduced by avoiding the double-resolution steps in the first-generation chemical process, which generated a large amount of organic and inorganic waste. A life-cycle analysis showed a 50% reduction of greenhouse gas emission for the enzymatic process. 

Ammonia lyases are another biocatalyst type that can be applied on an industrial scale for producing amino acids and their derivatives, which are important building blocks of APIs. One prerequisite for the industrial application of ammonia lyases is high activity and operational stability under extreme conditions such as high ammonia concentrations (> 10% weight) and basic conditions (>  pH 10), which is required to push the equilibrium toward the synthesis of the amino acid (6, 7).   

DSM used one of the most studied phenylalanine ammonia lyases (PALs), one that is derived from parsley and the yeast 

, for producing substituted phenylalanine derivatives (8). Although DSM successfully synthesized several target compounds at a multigram scale, severe substrate inhibition of the PALs was observed.  

To deal with this problem, a screening and optimization program revealed several PALs that met the required selectivity, activity, and stability at industrially relevant conditions. One of these enzymes was cloned into the 

based PluGbug production system, resulting in very high expression levels at laboratory and industrial scales (9). 

The single enzymatic step resulted in an approach that produced the desired amino acid at high yield (> 90%) and high enantiomeric excess (> 99%). The biocatalytic approach replaced a process based on the Fischer indole synthesis followed by a classical resolution. Based on this enzyme (phenylalanine ammonia lyase), an industrial process for producing a phenylalanine derivative was developed and recently implemented on an industrial scale (see Figure 2). The first-generation chemical process required seven steps, but the second-generation process using biocatalysis consisted of only two steps, thereby offering significant economic and ecological benefits. 

Figure 2: Synthetic application of phenylalanine ammonia lyase. (FIGURE COURTESY OF DSM) 

The benefits in sustainability for the two reactions previously described are quite typical for biocatalysis. Based on the growing number of enzymatic processes (e.g., more than 30 different enzymatic processes have been introduced at DSM), there is greater agreement about the true value of biocatalysis. The primary benefits are: the reduction of steps in a synthesis; increased yield; less waste per step by providing higher selectivities; and allowing the use of cheaper raw materials by enabling different chemistries and routes. The next step forward in biocatalysis would be to implement the technology earlier (e.g., in the  supply campaign of clinical trial materials). This change would allow us to learn from the initial delivery of campaigns and avoid risk by changing to lower-cost enzymatic processes at later phases. This transformation is only possible if the speed, cost, and versatility of biocatalysis is competitive with all other available solutions in organic synthesis. Having access to a broad and versatile enzyme toolbox provides a high chance of technical as well as commercial success. Being able to rapidly identify the right enzymes, produce them, and apply them for sample preparation within weeks is another requirement for implementing biocatalysis. This vision is possible in the very near future. Those companies that are able to introduce the most sustainable processes as early as possible in API manufacturing without compromising speed and development cost will capture the benefits that modern biocatalysis offers. 

 1.  J.S. Carey et al., "Analysis of the Reactions Used for the Preparation of Drug Candidate Molecules," 

 2. H.E. Schoemaker, D. Mink, and M.G. Wubbolts, "Dispelling the Myths—Biocatalysis in Industrial Synthesis," 

 3. P. Lorenz and J. Eck, "Screening for Novel Industrial Biocatalysts," 

 4. D.E. Robertson and B.A. Steer, "Recent Progress in Biocatalyst Discovery and Optimization," 

 5. M. Hermann et al., "Alternative Pig Liver Esterase (APLE)—Cloning, Identification and Functional Expression in 

 6. A. Gloge et al., "Phenylalanine Ammonia Lyase: The Use of its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis Synthesis of L-Arylalanines," 

   7. A. El-Batal, "Optimization of Reaction Conditions and Stabilization of Phenylalanine Ammonia Lyase-Containing 

 8. A.H.M. de Vries et al., "Process for the Preparation of Enantiomerically Enriched Indoline-2-Carboxylic Acids Via Cyclization of Phenylalanine Derivatives," 

  9.  F.B.J. Van Assema and N. Sereinig, "Production of Optically Active Phenylalanine Compounds for Cinnamic Acid Derivatives Employing a Phenylalanine Ammonia Lyase Derived from 

By Masahiko Yamada, senior researcher, Frontier Biochemical & Medical Research Laboratories, a research division of Kaneka

Chiral amines, chiral amino acids, and chiral alcohols are key pharmaceutical intermediates. Kaneka, a producer of chiral alcohols (1-4), recently implemented a systems-biotechnology approach using protein engineering and molecular biology to introduce the chiral amine group into desired compounds.   

 Nonnatural amino acids are increasingly becoming critical building blocks for drug manufacturers, in particular for the synthesis of protease inhibitors. Kaneka has been investigating the production of such nonnatural amino acids by the reductive amination of ketoacids (see Figure 3). The method developed by Kaneka combines an amino-acid dehydrogenase and a coenzyme recycling system in 

. By selecting the appropriate dehydrogenases, both aliphatic and aromatic L-amino acids can be produced. Phenylalanine dehydrogenases provide a chiral aromatic amino-acid series and leucine dehydrogenases a chiral aliphatic amino-acid series.  

Figure 3: Nonnatural L-amino-acid production by reductive amination. (FIGURE COURTESY OF KANEKA) 

Because the dehydrogenase requires NADH ( -nicotinamide adenine dinucleotide) as a coenzyme, the recycling of the coenzyme is critical to the reaction. Kaneka has succeeded in regenerating NADH to accelerate the reductive amination. This successful recycling is accomplished by using durable formate dehydrogenases (FDH) isolated from a specific soil-based microorganism, in which FDH survived even under high-substrate concentrations, even with electrophiles, including halogenated hydrocarbons (5). 

The harmonization of the reductase library and coenzyme regeneration crafted in 

 allows the system to produce nonnatural L-amino acids in high concentrations, sometimes as much as 100 g/L. Essentially no undesired products, except carbon dioxide from formate, are produced, and enantiomeric excesses of more than 99% are common.    

Kaneka produces nonnatural L-amino acids from readily available racemic unprotected amino acids. Kaneka's technology uses four enzymes: a D-amino-acid oxidase and an L-amino-acid dehydrogenase for the actual reaction steps and two other enzymes, including coenzyme recycling. The mechanism is an expansion of the reductive amination technology previously described. In the oxido-reduction sequence, the oxidation of the undesired D-amino acid to the a-ketoacid or to the imino derivative takes place in concert with the reductive amination (see Figure 4). The system was scaled, giving yields of greater than 90% and enantiomeric excess of more than 99% (6, 7). 

Figure 4: One-pot enzymatic deracemization for nonnatural L-amino acids. (FIGURE COURTESY OF KANEKA) 

Chiral amines are the remaining frontier of chiral synthesis. About 70% of central-nervous-system drugs possess amine moieties because neuronal receptors are usually triggered by amines. To date, however, no single technology stands out as being the best to make chiral amines at commercial scale. Catalytic hydrogenation has not been successful so far because of its high-pressure requirements, and optical resolution has not been universally applicable. Enzymatic synthesis using a transaminase is certainly a promising concept, although a long-standing issue has been its poor conversion rate. The transamination reaction is reversible, which makes it quite difficult to isolate the desired amines from the complex reaction mixture, including the three other chemical species: the substrate ketone, the amino donor, and the resultant ketone. 

Kaneka has developed a practical transaminase system by creating a transaminase-containing 

 transfectant and introducing a special module that efficiently drives the system (see Figure 5). The system produces chiral amines in high yield and high enantiopurity from the corresponding ketones. The key is the special module, which shifts the equilibrium to the desired amines (8–10). This whole system accommodates a variety of ketone substrates, including aliphatic, aromatic, and cyclic ketones, and produces both the 

 isomers through the selection of robust transaminase libraries. 

Figure 5: Multienzyme harmonic systems of transaminase. (FIGURE COURTESY OF KANEKA) 

By implementing a systems-biotechnology approach, Kaneka has introduced industrialized processes to manufacture nonnatural L-amino acids using reductive amination or deracemization technology. Depending on the availability of the substrates, the best arrangements can be selected. These harmonic systems are effective in surpassing other methodologies, particularly in the case of L-

-leucine. In addition, the company's chiral-amine transaminase systems produces a wide range of amines, aromatic, aliphatic, and cyclic compounds for projects in development and at commercial scale. The industrialization of these compounds is supported by large-scale capabilities in genetically modified organisms, synthetic technology, and process-engineering technology, including amino-acid–peptide purification systems. 

1.  H. Nanba et al., "Bioreactor Systems for the Production of Optically Active Amino Acids and Alcohols," O

Scientists from DSM and Kaneka discuss various techniques in this roundtable moderated by Patricia Van Arnum.

Pharmaceutical Technology-09-01-2009

Design and Construction of a Potent Pharmaceutical CGMP Suite

Moving from Vial to Prefilled Syringe

Using Microreactors in Chemical Synthesis: Batch Process versus Continuous Flow

Market Outlook for Biosimilars

Applying Biocatalysis: A Technical Forum

Orally Disintegrating Tablets: The Effect of Recent FDA Guidance on ODT Technologies and Applications