enzymatic microreactors utilizing non-aqueous media

54 Chimica Oggi - Chemistry Today - vol. 32(1) January/February 2014

KEYWORDS: Biocatalysis, enzymes, microfluidics, liquid-liquid two phase systems, ionic liquids, non-aqueous media.

Abstract Implementation of biocatalytic reactions in chemical processes is often hampered by poor solubility of organic compounds in water, as well as with low biocatalyst stability and/or near-equilibrium reactions,

preventing high final yields. The use of non-aqueous solvents can substantially improve the applicability of biocatalysts for organic synthesis by offering substantially higher substrate and product solubility along with the possibility for in situ product removal in two-liquid phase systems, among others. Miniaturization and continuous-flow processing is gaining importance also in biocatalytic processes, especially when mass transport across phase boundaries is included. This review gives an insight into enzymatic microreactors utilizing either dissolved catalysts within various two-liquid phase systems, or immobilized enzymes employing non-aqueous media, namely ionic liquids and organic solvents. Benefits and drawbacks of parallel and segmented liquid-liquid flows within microfluidic systems, as well as of packed bed mezzo- or microscale reactors utilizing non-aqueous media for biocatalytic reactions are highlighted.

Enzymatic microreactors utilizing non-aqueous media

INTRODUCTION

Several evidences on the advantages of microreactor technology, such as substantially improved heat and mass transfer, better process control and safety, lower consumption of chemicals and time during process development stages, the possibility to perform reactions not feasible in conventional systems, as well as conceptually different approach to increase the capacity by numbering up instead of tedious scale-up have gradually changed the paradigm of chemical engineering (1, 2). Various classes of chemical reactions have already been performed in microchannels offering also more control over selectivity and suppression of by-product formation. Microfluidic-based continuous processing nowadays represents an excellent complement to traditional batch reactors in fine chemicals and pharma industry including drug discovery and development (3-6). There are evidences about approximately 50 companies which approached or entered industrial productions based on microreactor technology (3 and refs therein). The motivation for industrial application of microstructured devices is mainly related to safety and apparatus costs, as well as to process intensification and faster process development. (3, 7-10). On the other hand, miniaturization of microbioreactors for cell culturing is still a matter of academic research and significant technological improvement is still required to provide automated solutions that can speed upstream process development (11-12).

Advantages of microreactor technology have been successfully combined with biocatalytic processes in several analytical and diagnostic devices, as well as in synthesis/degradation lab-scale reactions, mainly as tools of biocatalytic process research (13-22). In contrast, an industrial application of microreactors in biotransformations has not yet been reported, although there is a growing interest to involve these tools in process development stages in order to shorten time and costs for development of an optimized biocatalytic process (13-22). Since biocatalyst productivity and process intensity are frequently too low to bring about an economically feasible process, process intensification is also of a great concern in biocatalysis, gaining increased importance in sustainable processing. Therefore it is believed that microreaction technology would help the biocatalysis to reach its full potential in pharmaceutical and fine chemicals industry (21). Implementation of biocatalytic reactions in industrial processes is often hampered by poor solubility of organic compounds in water, as well as with low biocatalyst stability and/or near-equilibrium reactions, preventing high final yields. The use of non-aqueous solvents or two-phase systems can substantially improve the applicability of biocatalysts for organic synthesis due to several advantages vs. aqueous media such as substantially higher substrate and/or product solubility leading to chemical intensification (3); the ability to use enzymes synthetically rather than degradatively; the ability to modify native selectivity by tailoring the reaction

POLONA ZNIDARŠIC-PLAZLUniversity of Ljubljana, Faculty of Chemistry and Chemical Technology,

Aškerceva 5, 1000, Ljubljana, Slovenia

Polona Znidarsic-Plazl

ENZYMATIC MICROREACTORS

55Chimica Oggi - Chemistry Today - vol. 32(1) January/February 2014

Enzymatic microreactors with liquid-liquid parallel flowEnzymatic microreactors comprising parallel flow of aqueous phase either with an organic solvent (37, 39-41) or a hydrophobic ionic liquid (38) are chronologically listed in Table 1. Parallel flow of immiscible fluids within microchannels (Figure 2 a,b) offers the possibility to separate phases at the exit of the microchannel. As evident from Table 1, most of the processes performed in microchannels with Y- or Y-shaped inlets and outlets (Figs. 1 a and 1b, respectively) took this advantage. In order to establish phase separation, either one of the hydrophilic exit channels was chemically modified to become hydrophobic (37), or the flow rate ratio was adjusted in order to obtain stable interface at the position of outlet channels junctures (38, 40, 41). Recently, the correlation for the flow rate ratio required for positioning the interface in the middle of the microchannel was proposed, where the flow rate ratio of two liquids was related to the relative dynamic viscosity (34). In all examples included in Table 1, Reynolds number indicated laminar flow conditions and was far below the critical values for transition to turbulent flow, reported to be above 1100 for

media rather than the enzyme; the possibility to improve enzyme activity and stability; often substantially improved thermostability of enzymes in dehydrated media; the possibility to shift reaction equilibrium towards product synthesis by in situ product removal and to prevent enzyme inactivation by transferring inhibitor in the second phase of a two-phase solvent system; and easier downstream processing for product recovery (22-26).While organic solvents have been applied in enzyme-catalyzed reactions for over three decades, recent attention has been focused also on other non-aqueous solvents with lower environmental impact, including supercritical fluids (27 and refs therein). Ionic liquids seemed to be a “green” alternative in the beginning of this century, but current studies on their toxicity and non-degradability gave some doubt in this anticipation, raising a huge interest in their biocompatible/degradable analogues. Several enzymatic processes were shown to benefit of using these liquid salts owning huge potential related to their molecular structure ability to be tailored according to application requirements (27-30). Latest trends in the search for environmentally benign solvents revealed deep eutectic solvents as very promising media, also for biocatalytic processes (31). Intensively growing knowledge of protein structures, functions and engineering along with other biocatalyst modifications aiming at increased enzyme activity and stability in non-aqueous environments give further prospective for their implementation in organic synthesis.In quest of high-throughput tools for screening biocatalyst/solvent systems, enabling high productivities that would justify large-scale process realization, miniaturization and continuous-flow processing offer a huge potential. This review gives an insight into examples of enzymatic microreactors using either dissolved biocatalysts within various two-liquid phase systems, or immobilized enzymes employing non-aqueous media. Benefits and drawbacks of parallel and segmented liquid-liquid flows within microfluidic systems, as well as of packed bed mezzo- or microscale reactors utilizing non-aqueous media for biocatalytic reactions will be highlighted.

MICROREACTORS WITH DISSOLVED ENZYMES USING TWO-LIQUID PHASE SYSTEMS

The flow pattern in microscale channels with non-miscible liquids is a function of operational conditions, such as flow rates, phase ratio and properties of the fluids, as well as of the geometry of the inlet channels (Figure 1), channel diameter or aspect ratio for cylindrical and rectangular channels, respectively, and the channel wall roughness and wettability. There are several studies considering flow dynamics and transport phenomena in microfluidic systems, which deal also with liquid-liquid two-phase systems (32-35 and refs therein). Typical liquid-liquid flow patterns obtained within microfluidic systems include parallel flow of immiscible fluids in two layers with one interfacial area (Figure 2a) or in three layers with two interfaces (Figure 2b), droplet flow (Figure 2c), Taylor flow, referred also as slug or plug flow (Figure 2d), annular flow (Figure 2e) and a dispersed flow (Figure 2f). A specific flow pattern resembling an irregular annular flow mixed with a dispersed flow of n-heptane droplets in a hydrophilic ionic liquid was presented by Pohar et al. (36), where lipase B dissolved in an ionic liquid acted as a surfactant (Figure 3).

Figure 1. Flow regimes in liquid-liquid systems depend also on inlet channel geometry: a) Y-shaped, b) Y-shaped, c) T-shaped and d) X-shaped microchannel inlets, the last two usually used to obtain segmented flows.

Figure 2. Typical flow regimes of multiphase flow within microchannels: a) parallel flow of two immiscible fluids; b) immiscible fluids in three parallel flows with two interfaces; c) droplet flow; d) Taylor flow; e) annular flow and f) dispersed flow (the latter from (36)).


microchannels have led to enhanced volumetric productivities in microfluidic devices as compared to conventional reactors. Almost 70% conversion of 0.1 mM p-chlorophenol was achieved within the residence time of only 2 s when using parallel-flow microchannel for laccase-catalyzed dechlorination, where up to 50 times better conversion per aqueous-organic interface area and time than in a screw-cap bottle with gentle shaking was reported (37). A significant reduction in residence times needed to accomplish comparable conversions as in batch process at similar substrate concentrations was observed also for lipase B-catalyzed isoamyl acetate synthesis in an aqueous/n-hexane system (40) and for cholesterol oxidation

microchannels with aspect ratio up to 4 (32), which was also the case in all studies presented. However, at very low flow rates (usually below 3 ml/min), an unstable flow with nonuniform droplet formation was reported (38, 39) or observed (40, 41).Another benefit of a parallel flow of immiscible liquids within microchannels is a defined interfacial area, where most of reactions included in Table 1 took place. Enzymes were introduced in an aqueous or hydrophilic phase and were in most cases adsorbed to the liquid-liquid interface, while substrates mostly entered the channels in organic phase. Specific interfacial areas in presented studies ranged from 2.0 × 103 to 1.4×104 m-1, which is a few orders of magnitude higher than in batch reactors with stirring (37). Known reaction area enabled process description either by a simple model assuming immediate substrate conversion at the interface and ignoring velocity profiles (37), or by more accurate modeling including ping-pong bi-bi kinetics for lipase-catalyzed esterification at the interfacial area and velocity profiles of both liquids (40).In most cases shown in Table 1, enlargement of the liquid-liquid interfacial area for reaction and in situ product removal together with substantially improved mass transfer due to short diffusion paths within

Table 1. Enzyme-catalyzed reactions within microfluidic systems comprising parallel liquid-liquid flow.a Data taken from refs (37-39) or calculated from original data; b Data taken from ref. (37) or calculated from data on microchannels’ geometries and number of flows in the channel; c Data taken from graphs or text in refs (37-39,41) or calculated from flow rates and microchannel volumes; IL: ionic liquid; [hmim][PF6]: 1-n-hexyl-3-methylimidazolium hexafluorophosphate; PDMS: polydimethylsiloxane; CRL: lipase from Candida rugosa; PPL: lipase from porcine pancreas; CaLB: lipase B from Candida antarctica.

Figure 3. Liquid-liquid flow pattern developed at various positions from the beginning of the glass microchannel comprising Y-shaped inlet, with length increasing from a) to c); d) a flow pattern within the major part of the microreactor consisted of long droplets of n-heptane with concave tail surrounded with small droplets of this phase within the hydrophilic ionic liquid [bmpyr][dca] (36).


shifted the equilibrium towards the hydrolysis, so the highest conversion obtained within the microchannel at specified conditions was 35% (40).

Enzymatic microreactors with non-parallel liquid-liquid flowAdditional improvement of mass transfer between the phases within microfluidic systems as a consequence of internal vortex flow in segments caused by shearing motion and enhanced surface area to volume ratio as compared to the parallel flow could be obtained by contacting immiscible fluids in the form of segmented or droplet flow (32, 33). Table 2 lists reactions catalyzed with free enzymes within microfluidic devices comprising multiphase flow either in the form of undefined segments (42), Taylor flow (Figure 2d) (44-47), droplet flow (Figure 2c) (48) or mixed flow (Figures 2f and 3) (36). In order to obtain such flow regimes, Y-shaped (Figure 1a) (43, 47), Y-shaped (Figure 1b) (36), T-shaped (Figure 1c) (45, 46), X-shaped (Figure 1d) (48) or tapered microchannels (44) were used. On the other hand, microscale continuous separation of two immiscible phases in the form of segments is not so straight-forward as in the case of parallel-flow and needs additional techniques such as flow focusing, manipulation of surface wetting characteristics, application of capillary forces, or using specific geometries, guides, or surface patterning at the end of a microreactor (22 and refs therein). In most cases of enzymatic microreactors, shown in Table 2, phase separation was achieved outside the microchannels by means of sedimentation or centrifugation (42-43, 45-47). An exception was reported by Mohr et al. (44) who developed a closed microreactor system employing recirculation of two

performed in an aqueous/n-heptane parallel flow (41). Further optimization of the latter process in a microreactor, catalyzed by cholesterol-oxidase, based on enzyme recycle and by-product removal by integration with a plug-flow reactor containing immobilized catalase aiming at the reduction of hydrogen peroxide formed in enzymatic microreactor in order to avoid biocatalyst deactivation (42). Furthermore, intensification of enantioselective separation of (S)-ibuprofen from a racemic mixture using a thin film of [hmim][PF6] ionic liquid between both aqueous phases with two different lipases was reached by applying a microchannel with a parallel flow of three streams (Figure 1b and 2b) enabling phase separation at the outlet of a Y-shaped microreactor (38). One of the attractive features of such microfluidic device was the ability to precisely control the thickness of the ionic liquid film with the flow rate adjustment (38). Beside, a severe reduction of reagents when compared to conventional methods was reported in the study aiming at obtaining kinetic data for lipase-B-catalyzed esterification (39).On the other hand, very short residence times in tested microfluidic systems, which were below 250 s, as well as thermodynamics of reactions prevented complete conversions of substrates in all studies shown in Table 1. In case of lipase B-catalyzed isoamyl acetate synthesis in ionic liquid/n-heptane solvent system with parallel flow, no conversion was observed due to extremely short residence time of n-heptane resulted from high flow rates needed to establish a parallel flow within a Y-shaped microchannel (36). Furthermore, the presence of water together with n-hexane in lipase B-catalyzed isoamyl acetate synthesis

Table 2. Enzyme-catalyzed reactions within microfluidic systems comprising non-parallel liquid-liquid flow.

MTBE: methyl tert-butyl ether; PC: polycarbonate; PTFE: polytetrafluoroethylene; CaLB: lipase B from Candida antarctica; TADH: thermophilic alcohol dehydrogenase; FDH: formate dehydrogenase; [bmpyr][dca]: 1-butyl-3-methylpyridinium dicyanamide.


Two-phase solvent systems comprised aqueous (usually buffered) phase with immiscible organic solvent (43-47) or hydrophilic ionic liquid with organic solvent (36, 48). Enzymes were in all cases introduced in a hydrophilic phase, while reactants of the primary reaction were dissolved in an organic phase, which usually enabled the use of higher substrate concentrations as opposed to aqueous media and also served as a sink for products. Efficient mass transfer of substrates to the enzyme in the aqueous phase or adsorbed on the interphase, as well as product transfer back to the organic phase could thus significantly affect the reaction rate. Transport intensification as a way to process intensification (3) might be therefore achieved by implementing microreactors with reduced diffusion length and with increased interfacial area. As determined by Mohr et al. (44), the surface to volume ratio of the organic to aqueous phase in the microreactor with Taylor flow was approximately 10 times higher than in sealed vials on the bench. Furthermore, segment lengths in Taylor flow could be lowered by increasing the fluid flow rate or varying the flow rate ratio of both phases (44-47). Besides, very long residence times could be achieved in microchannels with segmented flow simply by adding long tubular holding

phases with miniaturized membrane pumps and the use of electrostatic phase separation using static charge on the flowing droplets. Furthermore, a membrane-based microseparator was recently integrated with an enzymatic microreactor system containing droplets of n-heptane in ionic liquid, aiming at continuous separation of organic solvent with the product from the ionic liquid phase with lipase B and thereby enabling enzyme and ionic liquid recycling (48). This example opens prospective for bioprocess design intensification which addresses issues such as reduction of process complexity, integration of unit operations, and unified utility in modular plants (3). It could be implemented in 1-heptanol isolation from organic phase, where distillation was used as a downstream process (46). Very broad range of enzymes was employed for reactions within microreactors utilizing two-phase solvent systems shown in Table 2, from hydroxynitrile lyase (43), pentaerythritol tetranitrate reductase (44), various alcohol dehydrogenases (46, 47), and lipase B from Candida antarctica (36, 48). In case of tetranitrate reductase (44) and thermophilic alcohol dehydrogenase (46), in situ cofactor regeneration was accomplished by utilizing various dehydrogenases and corresponding substrates in the aqueous phase.

Table 3. Enzyme-catalyzed reactions within microfluidic systems comprising immobilized catalysts within non-aqueous media.

THF: tetrahydrofuran; MTBE: methyl tert-butyl ether; PTFE: polytetrafluoroethylene; d: diameter; L: length; W: width; D: depth; V: total volume; VL: liquid volume; Lipozyme TL: immobilized lipase from Thermomyces lanuginosus; Lypozyme TM: lipase B from Mucor miehei immobilized on weak anion exchange resins; CaLB: lipase B from Candida antarctica; Novozym 435: CaLB immobilized on acrylic resin; Lipase AK: lipase from Pseudomonas fluorescens; Lipase PS: lipase from from Pseudomonas cepacia; CRL: lipase from Candida rugosa; TADH: thermophilic alcohol dehydrogenase; [bmpyr][Tf2N]: 1-butyl-3-methylpyridinium bis(trifluoromethanesulfonyl)imide; [hepdmim][Tf2N]: 1-heptyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide.


fundamentals and applications of immobilized enzymes within microfluidic devices (14, 18, 20, 49-51). Table 3 lists mezzo- and microreactors with immobilized enzymes employing organic solvents (53) or their mixtures (52, 54, 55), ionic liquids (56, 57), aqueous/organic segmented flow (46) or a solvent-free system with liquid organic substrates (58). Most of the studies employed commercially available immobilized enzyme preparations from Novozymes (various lipases), while sol-gel entrapped lipases of various origins (53, 55) and lipase immobilized on the surface of ceramic particles (52) or alcohol dehydrogenase in resin beads (46) were also used. Except for the case where beads with an enzyme were used in a segmented flow (46), other researchers employed tubular packed bed reactors of mezzo-scale dimensions (52-55, 58) or rectangular microreactors with channel depth below 0.5 mm containing one layer of enzyme beads with volumes usually below 1 ml (56, 57). Enzyme load in packed bed reactors varied from 208 to 539 mg/ml of immobilized preparation (calculated from data in refs. 52-58), which was substantially higher than in counterpart reactions performed in batch mode, where loads of below or equal to 10 mg/ml (52, 54), 12.5 mg/ml (56), 31 mg/ml (58), 33 mg/ml (57) and 44 mg/ml (55) were calculated. Much higher enzyme loading was also among major contributes to higher volumetric productivity reported in most reactions shown in Table 3. However, the importance of shorter diffusion paths and efficient control of operating conditions in milliliter-scale devices should also be considered. In the case of isoamyl acetate synthesis, the highest volumetric productivity reported so far was achieved by employing packed bed reactor with only one layer of immobilized lipase B from Candida antarctica in imidazolium-based hydrophobic ionic liquid enabling very efficient mass transfer to/from enzymes in the outer layer of porous beads (57). Although the calculated specific biocatalyst productivity was higher in batch operation, the high enzyme loads obtained in packed bed reactors could not be achieved within the batch reactor, preventing high ester yields within the given time and

sections. In studies summarized in Table 2, residence times were up to 2 orders of magnitude higher as compared to parallel flow (Table 1), enabling also better conversions at the exit of the microchannels.All stated properties had beneficial influence on the performance of microscale continuous processing when compared to the conventional batch counterparts. Significant improvement in volumetric productivity when compared to vigorously stirred batch system and to literature data was reported for isoamyl acetate synthesis in ionic liquid/n-heptane mixed (36) or droplet flow (48), for reduction of trans-2-(2-nitrovinyl)thiophene, ketoisophorone, trans-cinnamaldehyde and 2-methylmaleimide (44), as well as for hexanal production (47). Furthermore, 1-heptanol production (46) and enantioselective synthesis of cyanohydrins (43) in segmented flow microscale system eliminated emulsion formation and thus significantly reduced downstream costs and efforts. Beside, a severe reduction of reagents when compared to conventional methods was reported in the study aiming at obtaining kinetic data for hydroxynitrile lyase-catalyzed reaction (43), or for the evaluation of enzyme stability and its improvement either by addition of surfactant or by catalyst immobilization (45, 46). The latter study also offers very useful strategy to analyze specific process boundaries of flow processing in microreactors, based on evaluation of the Damköhler number, the enzyme effectiveness factor, and process productivity (46).

MICROREACTORS WITH IMMOBILIZED ENZYMES USING NON-AQUEOUS MEDIA

Enzyme immobilization offers several advantages over the use of dissolved catalysts such as alleviated separation from the product and thereby elimination of product contamination and simplification of downstream operations, as well as the possibility to reuse costly enzymes and enhance their stability. Furthermore, in context of this review, immobilization might substantially improve enzyme activity and stability in non-aqueous media (23). There are several review papers covering

Table 4. Benefits of implementation of enzymatic microreactors using non-aqueous media for bioprocess intensification and development.


space (57). Detailed mathematical descriptions of lipase-catalyzed transesterifications in packed bed miniaturized reactors were recently reported (56, 58), which enable better understanding and prediction of bioreactor performance, as well as process optimization.Another benefit of using mezzo-scale reactor for testing immobilized enzyme stability in non-aqueous media with small reagent consumption was shown for alcohol dehydrogenase, tested in a segmented flow reactor (46), for lipase B from Candida antarctica in ionic liquids (56, 57), and for evaluating the activity of various lipases modified by molecular imprinting during sol-gel immobilization aiming at enhanced enzyme activity in organic solvents (55). Very stable long-term operation of enzymatic mezzo- or microscale reactors was found in these studies.Considering product isolation from the solvent, integrated system of isoamyl acetate removal by evaporation from an ionic liquid in a micro-scale device was developed by Pohar et al (56). This again shows the possibility for process design intensification.

CONCLUSIONS

Reports on enzymatic reactions in microreactors performed in organic solvents, ionic liquids or various two-liquid phase systems revealed the benefit of using microscale devices for bioprocess studies (kinetics, biocatalyst activity and stability in non-aqueous media), needing lower amounts of chemicals and time when compared to their batch counterparts, as well as for process intensification through transport, chemical and process design intensification (summarized in Table 4). The variety of liquid-liquid flow patterns possible in microchannels could lead to increased and controlled specific surface area and thereby mass transfer among the phases, where implementation of non-aqueous solvents permits to use higher substrate concentrations as opposed to aqueous media and also serve as a sink for products enabling in situ removal and thereby increased product yield. Furthermore, the use of microfluidic devices prevents formation of stable emulsions, usually present in batch processes with two-liquid phase systems, and hence significantly reduces downstream costs and efforts. The use of immobilized enzymes in milliliter-scale continuous flow systems enabled much higher enzyme loading, shorter diffusion paths and efficient control of operating conditions, which again resulted in higher volumetric productivities as compared to batch processing. Very high stability of enzymes in miniaturized packed bed reactors with non-aqueous solvents and the possibility to test various process conditions and solvents in a short time with low material consumption again confirm reasonableness for their industrial implementation. For this purpose, development of

plug-n-play modular microfluidic tools providing also automated in-line analysis is needed. Two EC funded projects (BIOINTENSE and EUROMBR) are currently aiming at developing such platforms.

ACKNOWLEDGEMENT

The financial support of the Ministry of Education, Science and Sport of the Republic of Slovenia (Grant No. P2-0191) and the European Union 7th Framework Programme support through the project BIOINTENSE (Grant Agreement No. 312148) is gratefully acknowledged. Author is thankful to U. Novak for providing photographs.

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