Continuous Flow Processes Have a High Degree

The role of microorganisms in soy sauce production

Desmond K. O'toole , in Advances in Applied Microbiology, 2019

4.5 Use of continuous fermentation processes to produce soy sauce

Production of soy sauce on a large scale ties up large fermentation vessels for a long time. To speed up manufacturing, continuous processes and bioreactors have been studied. The first continuous method involved an enzymic process to hydrolyze the raw materials followed by lactic fermentation (Anonymous, 1982). The next step was the introduction of whole cells of T. halophilus, Z. rouxii, and C. versatilis immobilized in calcium alginate gel to ferment the moromi (Osaki et al., 1985). Following enzymic hydrolysis and passage through a column bioreactor containing immobilized T. halophilus cells for lactic fermentation, the production stream was split in two and put through either a Z. rouxii or a C. versatilis reactor. Each stream was then heated, filtered, and combined. The production time was equivalent to 2 weeks. Sauce was produced continuously for 80 days through a 280-L reactor, but it had an aroma pattern different from traditional sauce. However, the gel was unstable to heat and not resistant to a high-salt solution, so Horitsu, Maseda, and Kawai (1990) introduced a ceramic bead carrier for the whole cells and could produce satisfactory soy sauce within 8 days. Hamada, Fukushima, Hashiba, and Motai (1991) used a system based on "Chitopearl" to immobilize glutaminase and on alginate gel to immobilize the microorganisms. The koji mixture was hydrolyzed in 8.5% NaCl at 45   °C for 3 days, filtered, and then put through the glutaminase reactor followed by the microbial reactors. Although production took about 2 weeks and the system ran continuously for 100 days, the product still did not completely match the traditional product (Hamada et al., 1991). Koseko, Hisamatsu, Matsunaga, and Yamada (1993), using a Microbe Immobilized Ceramic reactor, successfully fermented non-sterilized moromi mash with the microorganisms named above and were able to produce sauce with good taste and flavor. With a residence time of 30   h for Z. rouxii, 2% ethanol and 38   mg   L^{−   1} 2-phenyl ethanol was produced, and with a residence time of 48   h for C. versatilis the same level of ethanol and 3.7   mg   L^{−   1} 4-ethyl guaiacol were produced.

This is not an exhaustive look at the work done in this area. Other relevant work includes that of Iwasaki and Ueno (1990) and Iwasaki, Nakajima, and Sasahara (1991), Iwasaki, Nakajima, Sasahara, & Watanabe, 1991, Iwasaki, Nakajima, & Sasahara, 1992). So far this apparently attractive approach to the problem of reducing the production time and thereby increasing productivity has not yet become a commercial reality.

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Synthetic Methods

V. Prakash Reddy , in Organofluorine Compounds in Biology and Medicine, 2015

6 Dediazoniation–Fluorination of Amines

The Balz–Schiemann reaction, which involves diazotization of the primary aromatic amines followed by fluorinative dediazoniation, could be achieved in preparative scale by carrying the reaction in PPHF, in which case the isolation of the otherwise explosive intermediary diazonium salts is not required (Figure 15 ). This reaction has been achieved on an industrial scale in a continuous flow reactor, using HBF ₄ instead of PPHF as the HF source. HBF₄ is a relatively safer reagent due to its ease of operation and operational safety. Using this method, large quantities of ortho-difluorobenzene could be prepared from the reaction of ortho-fluoroaniline and sodium nitrite, in 90% yield; the reaction time for the diazotization ranged from 4 to 15   s at 10–25   °C, whereas the fluorodediazoniation of the isolated diazonium salts proceeds in 1–7   min at 140–200   °C. ⁷¹

Figure 15. Balz–Schiemann fluorination reaction.

The fluorodediazoniation reaction was also achieved in high yields in ionic liquid solvents. ⁷² Further, diazotization followed by fluorinative dediazoniation of α-amino acids gives α-fluorocarboxylic acids in high yields and with high stereoselectivities (retention of configuration) (Figure 16). ^11,73,74 The corresponding 2-halo carboxylic acids are obtained when the diazotization is performed in the presence of the more nucleophilic alkaline metal halides (chlorides, bromides, and iodides). ³⁰ The retention of configuration in these reactions can be rationalized as due to the intermediacy of an α-lactone intermediate.

Figure 16. Fluorodediazoniation of α-amino acids.

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Scale-Up Production of Theranostic Nanoparticles

Dong Nyoung Heo , ... Sang Cheon Lee , in Cancer Theranostics, 2014

Scale-up Production of Iron Oxide Nanoparticles

Among the synthetic methods of iron oxide nanoparticle production, hydrothermal synthesis is probably the most effective method for process control and scalability. This method can enhance the biocompatibility of the iron oxide nanoparticles, because there are no toxic solvents or surfactants. Furthermore, the synthesis process is comparatively simple for producing iron oxide nanoparticles. As a result, many research groups have investigated the scale-up process using the hydrothermal method. The typical process for synthesis of fine iron oxide nanoparticles based on a continuous flow reactor system (described in [73] and [74]) is illustrated as Figure 24.6. In continuous hydrothermal synthesis it is easy to control the particle size and morphology by residence time, temperature, and precursor concentration. However, surface treatment of nanoparticles is required as an additional step in this method because they can form aggregated structures through hydrophobic interactions of each particle. Typically, small organic molecules and polymers are employed to passivate the surface of the iron oxide nanoparticles for protecting iron oxide aggregation.

Figure 24.6. Current scale-up process for the production of iron oxide nanoparticles by the continuous hydrothermal method.

Recently, at the lab-scale production, Park et al. prepared the metal–oleate complex by reacting metal chlorides with sodium oleate [77]. The heating of the complex at 320°C and aging for 30   min in 1-octadecene generated iron oxide nanoparticles of 40   g scale with a high yield of >95%. It was suggested that when the pilot plant is set up in parallel, several kilograms of monodisperse nanoparticles can be obtained.

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Gold nanoparticles (GNPs) as multifunctional materials for cancer treatment

A. Wei , ... J. Wang , in Biomaterials for Cancer Therapeutics, 2013

13.7.2 Synthesis and manufacturing

Many types of GNPs are easily prepared in the laboratory, but their practical manufacturing and testing as nanomedicines requires that their syntheses to be both reproducible and scalable. Multigram quantities of GNPs are needed for preclinical evaluations, including in vivo toxicological studies and ADME profiling, and batch-to-batch variability must be tightly controlled to meet safety standards. The reliable synthesis of GNPs using batch processing methods is a potential challenge for scalable production, as the nucleation and growth of GNPs are highly sensitive to reagent concentration, mixing rates, reaction volume, and heat transport (Xia et al., 2009).

Continuous-flow (CF) reactors serve as the main alternative to batch processes, and are being explored with increasing frequency for GNP synthesis. CF microreactors allow for the precise programming of mixing rates, concentration, and pressure at preset volumes, and provide tight control over variations in heat or mass transfer. An anticipated advantage of NP production by CF is the ease with which optimized conditions can be adapted for parallel processing for increased throughput. For the size-controlled synthesis of GNPs, CF microreactors can support laminar flow conditions with rapid mixing and heat transfer rates at high temperatures and pressures, and can also be equipped with multiple feed lines (Drobot, 2012). Colloidal GNPs can be prepared in the 5–50   nm range under CF conditions with polydispersities half that produced by conventional batch processes (Wagner and Köhler, 2005). Furthermore, CF microreactors can be competitive with batch reactors for the scalable manufacturing of GNPs when used in parallel, and have been shown to produce phosphine-stabilized undecagold (Au11) clusters several hundred times faster than conventional batch processing, when compared on the basis of reactor volume (Jin et al., 2010).

Long-term storage or shelf life is another important aspect in the manufacturing of nanomedicines. Like many medicaments, NP-based formulations may be prone to changes in activity or potency with age: poorly formulated GNPs have a propensity to aggregate without proper stabilization, or can leach materials into solution that compromise their biological efficacy. These issues are best addressed by optimizing the surface chemistry of GNPs, so special attention needs to be paid toward the ingredients used in their formulations.

Some of the pitfalls described above have been illustrated in the batch processing of GNRs, which are commonly prepared using micellar solutions of CTAB, a toxic surfactant that also promotes nonspecific cell uptake (Huff et al., 2007). CTAB can be removed from GNRs on a gram scale by treatment with sodium PSS as a detergent (Leonov et al., 2008). Dispersions of PSS-treated GNRs have negligible cytotoxicity when freshly prepared but become increasingly cytotoxic over time, even more so than CTAB-stabilized nanorods prior to treatment. This toxicity is not directly associated with the GNRs but is thought to be derived from a PSS-CTAB complex leached from the GNR surface, which could be removed upon subsequent filtration.

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Biofilm Formation Under In Vitro Conditions

Claudia Trappetti , Marco R. Oggioni , in Streptococcus Pneumoniae, 2015

Pneumococcal Biofilm Models

Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts, and so far much of our understanding of biofilm physiology and micro-ecology originates from experiments using in vitro biofilm models. Various in vitro models have been developed for pneumococcal biofilm growth. The first published biofilm model was a Sorbarod biofilm [11]. A Sorbarod consists of a paper sleeve containing compacted cellulose fibers placed in a silicone tube and connected via a plastic adaptor to a sterile glass tube. This simple model of biofilm was used to establish a continuous bacterial culture for over 12   h and to test its susceptibility to various antibiotics. This model system also allowed the finding that capsule-off mutations are selected in biofilms [12].

The continuous-flow reactor, established in 2004, is one of the most popular biofilm model systems. This "open" system, which allows continual replenishment of fresh nutrients, permits the formation of biofilm under a wide range of flow rates and nutrient conditions over extended periods of incubation [13,14]. This model is commonly used to study the development of mature biofilms and assess changes in the growth environment or specific genetic mutations to the structure of mature biofilms, detachment from biofilms, spatial and temporal gene expression, and, most importantly, the distribution of extracellular polymeric substances. Still, in vitro biofilms cannot be considered universally valid as an in vitro model for disease. The group of Carlos Orihuela clearly showed that using a continuous-flow-through line model, sessile bacteria were highly attenuated in experimental invasive disease models, possibly suggesting a better correlation to bacteria during colonization [15].

In addition to these systems, two static microtiter models were set up exploiting either high inocula in poor media [6,16] or low inocula in rich media [4,17] using plastic supports. This technique simplifies biofilm formation and quantification, allowing analyses of high numbers of laboratory samples. The use of plastic supports permits the study of early biofilm stages up to 24   h; however, if the growth medium is replenished daily, biofilms can also be maintained for up to 3–4 days. Moscoso et al. determined that the optimal conditions for biofilm formation of S. pneumoniae on abiotic surfaces were obtained when polyvinyl chloride plates were used [16]. In addition, they found that either a chemically defined (Cden or CDM) or semisynthetic (C) medium supported strong biofilm formation, whereas growth in a complex medium, such as CAT or Todd–Hewitt broth, resulted in weak biofilms. In C medium, the number of adherent cells reached a maximum after 8   h of incubation at 34°C. Oggioni et al. performed a biofilm time course experiment using the rich tryptic soy broth (TSB) media, where low-inoculum bacteria were grown on flat-bottom polystyrene tissue culture plates in the presence or absence of pneumococcal competence-stimulating peptide (CSP) at various concentrations (0–300   ng/mL). After the initial background attachment of a few cells upon inoculation, pneumococci attached quite abruptly to the solid support during the late exponential phase of growth. The stability of biofilm in this model was found to be dependent on a functional competence system, which, in response to exogenous CSP added to the medium, allowed for the formation of stable biofilms; this was also observed at 24   h [4]. CSP concentration for biofilm formation showed a narrow optimum condition in a range similar to that of inducing maximal competence in planktonic cells. The biofilm-competence dependence was not confirmed for microtiter models based on a more steady state of growth as continuous culture biofilm or the microtiter model in poor medium [18]. In vitro biofilm formation using microtiter plates was more recently evaluated at 6, 12, 18, and 24   h of incubation in TSB media in presence of glucose. The authors found that biofilm growth was also independent of exogenously added stimulating peptide and medium replacement. Instead, the best results were obtained after 18   h in the presence of 1% glucose. The authors proposed a correlation of the findings with the clinical biofilm disease common in hyperglycemic patients, where high concentrations of glucose in the blood can worsen systemic bacterial infection [19]. More solid are investigations that link distinct aspects of in vitro biofilms to disease and that describe lack of correlation to invasive disease potential [20,21].

Lately, Marks et al. emphasized the importance of using epithelial cells as a support for streptococcal biofilm formation, providing a better platform for bacteria to form a more mature and structured biofilm. Fixed tissue cultures are able to maintain the adhesive ability of the cells, and streptococcal biofilm formed on this substrate shares the same morphology and architecture with biofilm formed in the nasopharynx of infected mice. Moreover, biofilms formed on epithelial cells have the ability to initiate faster attachment than the glass support; after 6   h a specialized architecture matrix-like formation could be detected [22].

Selection of the appropriate in vitro biofilm model system depends not only on the preferences of the investigators and the resources available, but most importantly on the issues to be investigated. The primary advantage of the microtiter plates method is that its relatively high throughput capacity enables screens for mutants defective in attachment and evaluation of the effects of different treatments or compounds on attachment or biofilm formation. However, this method is less well suited to studies of biofilm structure or of antimicrobial resistance properties. On the other hand, flow cells are capable of creating a uniform and constant environment for in vitro purposes. Furthermore, the regulatory processes of biofilm elaboration are cyclical and dynamic, and the external stimuli, normally present in the host, trigger alterations in the expression of a subset of genes required for biofilm formation; thus, the current state of technology is still a distant representation of the dynamics of the host environment in vivo.

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Microfluidic devices for drug discovery and analysis

J.S. Kochhar , ... L. Kang , in Microfluidic Devices for Biomedical Applications, 2013

Synthesis of drug libraries

Recognition of drug targets has kept pace with the fast progress in genomic and proteomic tools. Pharmaceutical companies on the other hand are facing challenges to generate drug compounds at the fastest possible rate, in an inexpensive manner. Synthesis of drug libraries has been described as the biggest impediment in the drug discovery process (Jones et al., 2005). Improved methods in combinatorial chemistry have resulted in rapid synthesis of large number of chemical compounds, and have produced enormous drug libraries. This has been further accelerated by the improvement in the design of the microfluidic reactors. These microfluidic reactors can be classified into three types, based on the flow pattern, namely (i) flow-through type, (ii) droplet or slug type, and (iii) batch type. The most common flowthrough type enables multiple reagents to be maintained at a temperature, and be pressure driven through the channels. These reactors have been used widely in extraction procedures as well as in multiple chemical syntheses (Keng et al., 2012).

Parallel combinatorial synthesis in multiple microfluidic reactors has also been demonstrated utilizing the continuous flow of reagents in microfluidic channels. A multiple microfluidic reactor assembly was fabricated to synthesize carbamates in a multistep procedure (Sahoo et al., 2007). However, this method sacrifices the advantages of an integrated system for several reactions to be carried out on a single chip. Researchers then looked to fabricate a consolidated device with multiple layers of parallel chips. A multilayer glass chip was developed for a 2   ×   2 series synthesis in parallel (Kikutani et al., 2002). The complexity and expense of fabrication of this multi-layered device was a concern. Recently, Dexter and Parker exhibited parallel combinatorial synthesis of compounds on a single-layered microfluidic chip (Fig. 7.3a). They fabricated a single layer PDMS chip for synthesizing a 2   ×   2 series of amide formation products (Dexter and Parker, 2009).

7.3. Different types of microfluidic reactors. (a) A continuous PDMS-based microfluidic flow reactor for 2   ×   2 parallel combinatorial synthesis. The tubing has been inserted at each inlet and outlet port (Dexter and Parker, 2009). (b) Optical micrograph of a batch type microfluidic reactor with actual dimension (inset) (Lee et al., 2005).(c) Schematic of a microdroplet manipulator, including functions for (i) droplet generation, (ii) transfer of droplets to a microwell array, (iii) migration of droplets into the wells, (iv) trapping of second droplets and (v) oil change to induce droplet merging (Um et al., 2012).

However, continuous flow reactors are not suitable for multistep reactions, especially those involving sequential synthesis. A modified technique, termed batch microfluidics, in which specific microvalves control the delivery of reagents in batches, has been developed. These isolated batches can be delivered to the microfluidic reactor chamber at specific time points in a reaction cycle, exercising greater control over the reaction ( Lee et al., 2005). A fluoride radiolabelled imaging probe, in nano/microgram scale, was synthesized in five sequential processes involving fluoride concentration, water evaporation, radiofluorination, solvent exchange, and hydrolytic deprotection (Fig. 7.3b).

A newer technology, known as droplet microfluidics, has recently come to the fore. It is based on compartmentalization of each assay in a small droplet, usually in the range of 1 pL–10   nL, surrounded by an immiscible oil, which can be manipulated and processed in a high-throughput manner (Brouzes, 2012). Each of these droplets can act as a tiny microfluidic reactor, notably reducing the reagent volumes required. A mesh-grid design microwell array was fabricated by Um et al., which allows for continuous addition and trapping of picolitre single cell droplets in the microwells (Fig. 7.3c). Due to miniaturization, the device provides high-throughput screening of the droplets (Um et al., 2012), but multistep reactions using these devices are still a big challenge.

In addition, these microfluidic reactors have also been used for synthesis of biological molecules, such as DNA. Short synthetic oligonucleotides were joined under thermal cycling in a microfluidic picoArray device to form DNA constructs up to 10   kb instantaneously. The fabricated DNA construct was shown to express relevant proteins and may be used for cell free protein expression on a large scale (Zhou et al., 2004). Mei et al. developed a microfluidic array device for synthesis of chloramphenicol acetyl-transferase and luciferase, and reported the yield to be 13–22 times higher than that achieved in microcentrifuge tube, with a 5–10 times longer lasting protein expression. The device is composed of an array of units that allowed for fabrication of different proteins, protein expression and nutrient supply. The device is also capable of synthesis and analysis of proteins on a single chip, potentially eliminating the need to harvest proteins, thereby reducing wastage and increasing process efficiency (Mei et al., 2007). A droplet-based microfluidic method was recently developed for on-chip protein synthesis. Production of a water-in-oil-in-water (W/O/W) emulsion was accomplished by formation of a water-in-oil emulsion on a poly (methyl methacrylate) chip, up first, followed by complete emulsion formation on a PDMS/glass microchip. Synthesis and expression of a green fluorescent protein from a DNA template was successfully demonstrated using a microfluidic platform (Wu et al., 2011).

Most of the devices developed use PDMS as the substrate material due to its excellent optical properties as well as its mouldability. However, PDMS is incompatible with many organic solvents and adsorbs many hydrophobic compounds due to its surface properties. Keng et al. fabricated a microfluidic platform that is operated by electrowetting-on-dielectric (EWOD). The device was made from inorganic materials coated with perfluoropolymer, and offers flexibility in use with organic and hydrophobic reagents (Keng et al., 2012). The device was shown to be suitable for diverse chemical reactions with minimal consumption of reagents, with suitability for multistep procedures requiring several solvent exchange rounds.

These devices have been put to efficient use to generate drug libraries, which provide a powerful source that needs to be screened to explore new drugs. To screen these large combinatorial libraries of compounds, the pharmaceutical industry has looked at high-throughput screening (HTS) methodologies over the past two decades. Conventional screening methods were able to screen 5000–20 000 compounds over a few years, resulting in inefficient screening of only 2–20% of the compounds on the whole library. However, HTS, or newly termed ultra-high throughput screening (uHTS), methodologies aim to screen 10 000–100 000 compounds over a period of 24   h, resulting in generation of 2–18 million screening results per year (Beggs, 2001). This logarithmic increase in screening capability has given a boost to the hit-to-lead discovery process.

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Pulsed electric field (PEF) as an efficient technology for food additives and nutraceuticals development

Mahesha M. Poojary , ... Francisco J. Barba , in Pulsed Electric Fields to Obtain Healthier and Sustainable Food for Tomorrow, 2020

4.2 Principles of pulsed electric field treatment

The extraction of metabolites from vegetative cells requires diffusion of solvent into the cell and subsequent mass transfer of metabolites into the bulk of the extraction medium. The process of extraction can be accelerated by modifying the physical properties of the sample (e.g., milling, maceration, and peeling) or by application of external extraction aids (such as heat, pressure, sonication, and agitation). The PEF treatment can act as an external aid that improves the efficiency of solvent extraction by improving diffusion and mass transfer through the phenomenon called membrane "electroporation" or "electropermeabilization."

In practice, PEF-mediated membrane electroporation is achieved by applying pulses of moderate-to-high electric field to the samples placed between two electrodes in a conducting medium (Fig. 4.1A and B). In extraction applications the electric field strength may vary between 0.1 and 20 kV/cm, while the pulse duration falls in the range of nanosecond to millisecond. The instrument size may range from laboratory scale to pilot scale batch or continuous flow reactors.

Figure 4.1. Schematic representation of PEF extraction system (A), PEF induced creation of potential across the cell membrane (B), cell membrane before electroporation (C), electroporation of membrane (D), and resealing of membrane after PEF treatment (E). PEF, Pulsed electric field.

The molecular mechanism of electroporation induced by PEF is not yet well established. However, the most accepted theory is based on the "transmembrane potential (ΔΦ) breakdown model" proposed by Sale and Hamilton (1968). According to this model, the application of external PEF on a biological cell induces a transmembrane potential (ΔΦ) across its semipermeable cell membrane. If the applied field strength (E) increases the transmembrane potential beyond the physiological transmembrane potential (termed critical transmembrane voltage), the membrane loses its semipermeable property and encompasses cell lysis. The critical ΔΦ value for eukaryotic cells has been estimated to be 1   V (0.7–1.5   V), although it varies based on the treatment and sample type (Coster & Zimmermann, 1975; Sale & Hamilton, 1968). The transmembrane potential for a cell suspended in a medium can be calculated according to Eq. (4.1) based on Maxwell's equation on spherical coordinates (Maxwell, 1873) with the following assumptions: (1) the cell is spherical, (2) the radius of the cell (r) is far greater than the membrane thickness, and (3) the resistivity of the membrane is higher than those of the intracellular and the extracellular media.

(4.1) $\mathrm{\Delta }\Phi =\frac{3}{2}\mathit{rE}$

Neumann, Sowers, and Jordan (1989) later proposed a modified equation (Eq. 4.2) for the calculation of ΔΦ, based on the Schwan equation of basic electromagnetic theory (Pauly & Schwan, 1959). In this equation, θ is the angle between a locus of the cell membrane at which ΔΦ is measured and the direction of the applied electric field. The equation indicates that the maximum ΔΦ or the highest degree of electroporation occurs when θ=0° or 180° or |cosθ|=1, that is, at loci of the cell membrane facing the electrodes.

(4.2) $\mathrm{\Delta }\Phi =\frac{3}{2}\mathit{rE}·\cos \theta$

Similarly, Zimmermann, Pilwat, and Riemann (1974) and Zimmermann (1986) proposed a "dielectric breakdown" theory while investigating the disintegration of blood cells and microorganisms under PEF. According to this theory, the cell membrane can be modeled as a capacitor filled with a fluid of low dielectric constant. When a cell is present in a medium of relatively higher dielectric constant (e.g., liquid foods), the free charges will accumulate at either side of the membrane due to the difference in the dielectric constants (Fig. 4.1B). The application of external electric field increases transmembrane potential, ΔΦ (Eq. 4.1), and causes compression of the membrane due to attraction between opposite charges. When ΔΦ reaches around 1   V (critical transmembrane potential) with increasing E, the membrane loses its structural integrity by forming micropores (electroporation). A further increase in E results in irreversible electroporation due to the formation of larger pores.

Overall, the mass transfer using electroporation is a dynamic process consisting of several sequences of events (Fig. 4.1B–E). Firstly, the application of external potential induces the charging of membranes (Fig. 4.1B) followed by destabilization of its molecular conformation and formation of pores (Fig. 4.1D). This step could occur in the time scale of microseconds, however, depends largely on the nature of the cell and the strength of the applied electric field. The charging time for potato tissues was reported to be 3.0   μs when E=E0=180   V/cm (Angersbach, Heinz, & Knorr, 2000). In the second step, if the external electric field is continuously applied, the pore radii expand and additional pores are formed throughout the duration of pulses. This step lasts for several microseconds to a few milliseconds. In the last step, after the treatment duration, the pores may or may not be resealed, which is governed by the treatment parameters and the tissue type. The resealing could last for a few seconds to hours (Pataro, Ferrari, & Donsi, 2012) (Fig. 4.1E). It should, however, be noted that the formation of pores itself does not contribute to the mass transfer. The mass transfer is mediated by diffusion phenomenon, which occurs more readily when the cells are electroporated.

The PEF-induced electroporation of cell membrane can be temporary or permanent, depending largely on the process parameters and the nature of the sample. Typically, temporary or reversible electroporation occurs when the pluses are applied in several nanoseconds to microseconds range. In case the pores reseal within a time scale of seconds (Granot & Rubinsky, 2008). At this time interval the intracellular matrix can be transferred to the extracellular space and vice versa, where extracellular components can be introduced into intracellular space. A greater number of pulses, longer pulse duration or an intense electric field strength can irreversibly or permanently damage the membrane integrity and cause cell lysis, possibly due to loss of homeostasis. However, in most cases, the permanent electroporation depends on both PEF parameters and the cell type. For instance, microbial cell lysis can be achieved by PEF treatment at higher electric field strength (E=20–50   kV/cm) or moderate electric field strength (E<5   kV/cm) with varying pulse duration (10⁻⁵–10⁻³  s) (Timmermans et al., 2019; Toepfl, Heinz, & Knorr, 2007; Vorobiev & Lebovka, 2016), while electroporation of plant cells can be achieved at much lower electric field strength compared to microbial cells but generally with a longer treatment time (E=200–1000   V/cm for 10⁻⁴–10⁻¹  s) (Vorobiev & Lebovka, 2016).

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Drug Discovery Technologies

S.V. Ley , ... R.M. Myers , in Comprehensive Medicinal Chemistry II, 2007

3.35.1 Introduction

This chapter highlights the latest developments and key advances in preparative techniques that are rapidly being adopted in the modern synthesis laboratories. ¹ In particular, we describe the impact on organic synthesis by the judicious use of immobilized species to conduct reactions, quench chemical processes, scavenge by-products, and bring about the isolation of pure products. ^2–11 While the focus of the chapter is on the generation of medicinally and biologically relevant structures, broader topics, encompassing new reaction types, cleaner chemical processes, and possibilities for scale-up are also discussed. Where appropriate, we have highlighted the main opportunities and the remaining challenges in these areas, within the context of today's changing commercial, environmental, and ethical climates. Our overall aim is to present a vision for how synthesis chemistry is responding toward a more productive, efficient, and sustainable future. ^12,13

The pharmaceutical industry is without doubt a very demanding sector. Medicinal chemists are expected to make discoveries and prepare compounds at a phenomenal rate with increasing levels of structural diversity. Hence, the new methods adopted need to provide alternatives to the labor-intensive practices of the past, such as manual optimization, aqueous work-ups and extractions, difficult crystallizations or distillations, and chromatographic purifications. The desire to reduce these repetitive bottleneck operations has led to a noticeable increase in the use of automation, ¹⁴ informatics, ¹⁵ and other technology-based approaches in the laboratory. ¹⁶ Furthermore, by combining the use of supported reagents with new equipment such as microfluidic flow reactors, ^17–33 focused microwaves, ³⁴ and the practice of catch-and-release strategies, ^35–40 many new opportunities for synthesis are being recognized.

3.35.1.1 Historical Perspective

The pioneering work of Letsinger ⁴¹ and Merrifield ⁴² on solid-phase organic synthesis has become a widely used tool for rapidly constructing large compound libraries. In solid-phase synthesis, the substrate undergoing transformation is covalently attached to the support material and the reagents and/or coupling partners are present in solution. Following a transformation, the material is purified by a sequence of washing steps to elute solution-labile impurities, before a cleavage step is performed to liberate the functionalized product from the support, thereby enabling its isolation in a pure form.

While solid-phase synthesis has been an important advance, particularly for combinatorial applications, there are intrinsic difficulties associated with these methods that often outweigh the benefits, especially for syntheses of complex, biologically active compounds. Therefore, the application of solid-phase reagents, catalysts, and scavenging techniques presents an attractive alternative to linear, substrate-bound synthesis. This approach embraces many of the advantages of conventional solution-phase chemistry such as real-time reaction monitoring, convergency, and rapid optimization, but also enables purification by the simple expedient of filtration to remove the spent reagents. These techniques also readily accommodate multistep processes, parallel methods, batch-splitting, and reaction scale-up.

The first serious application of supported species was in the formation of ocadec-9-enoic acid butyl ester using a sulfonic acid resin in 1946. ⁴³ The 1950s saw further advances in the form of acidic and basic ion exchange techniques, which are still used as scavengers and buffers in water treatment plants today. ⁴⁴ In 1957 the first review of ion exchange resin catalysts was published. ⁴⁵

The 1990s, however, heralded a dramatic change in the use of supported reagents in synthesis. Underlying this was the recognition by the pharmaceutical industry as to how to better make greater numbers of compounds in response to high-throughput screening capability. This led to the concepts of combinatorial chemistry. ^46–53 Consequently, renewed interest in immobilized agents was triggered by the development of efficient agents to quench chemical reactions and scavenge out unwanted by-products. The time-saving potential of supported reagents was quickly recognized when it was discovered that clean materials could be obtained that were suitable for direct biological evaluation by a simple filtration of the reaction mixture to remove spent reagents or captured by-products. In combination with sequestering processes, solid-supported reagents also provide powerful synthetic tools for conducting multistep chemical operations. This latter topic forms the basis of a major review that summarizes relevant contributions from some 1500 papers. ⁹ This review documents early contributions and provides a significant reference source for supported reagents in chemical transformations, and details many scavenging applications. The wide-ranging use of immobilized reagents to individual processes has also been collected in a number of other articles. ^2,54–58 Further reviews of interest highlight alternative aspects of this science; for example, soluble polymer species, ^59,60 microwave methods with supported reagents, ^34,61–67 supported chiral catalysts, ^5,68–70 encapsulation and entrapment processes, ^71–90 phase-switching methods, ^91,92 natural product synthesis using supported reagents and scavengers, ^54–58 chemical library generation, ⁹³ and stop-flow and continuous-flow reactor systems. ^22,24,94,95

3.35.1.2 Suitable Supports for Immobilized Reagents

Reagents can be immobilized by tethering them to an insoluble (or semi-soluble) support material; this attachment can be via covalent or electrostatic interaction with the functionalized solid matrix. Commonly, this support is divinyl benzene (DVB) cross-linked form of polystyrene. These polystyrene resins can be either micro- or macroporous, depending on the degree of cross-linking. To date, the majority of the immobilized reagents that have been developed are polystyrene based; as polystyrene supports are not only cheap but are also easy to handle, achieve high loadings, and, importantly are relatively chemically inert. ⁸

In general, polystyrene resins swell adequately in most common organic solvents; however, this is dependent upon their cross-linking. Microreticular resins are defined by a low level of cross-linking (1–2% DVB) and swell more easily than their macroreticular counterparts (>30% DVB). Correspondingly, the bulk characteristics of the supporting polymer in terms of its physical properties and architecture can infer vastly altered reactivity and reaction kinetics for the same immobilized reagent. In this regard, a variety of other polymers such as acrylamides, polyureas, and co-polymer formulations including PEGylated polystyrenes and Jandagel ⁹⁶ have received considerable attention. With an increasing selection of synthetic polymers becoming available with new physical and functional characteristics, polymers will continue to be useful for the preparation of supported reagents.

However, while polystyrene may be the most commonly used support, it is certainly not the only material that has been used. The encapsulation of catalysts inside polymeric matrices is one class of reagent involving polymers in an innovative way. ^79,80,97 Alternative supports such as controlled pore glass, monoliths, ^98–104 cellulose, ^105,106 zeolites, ^107–109 and silicas have also been used. ^110–112 Irrespective of which support is employed, it is essential that the bound reactive species remains accessible within the support matrix to the soluble substrates.

3.35.1.3 Practical Advantages of Immobilized Reagents

The simplicity of separation allows supported reagents to be used in excess. Reagent concentrations can thus be used to force reactions to completion, which in turn leads to cleaner conversions. As discussed, another practical advantage is how the reaction mixtures can easily be manipulated; for example, by filtering off the spent supported reagent and evaporating the filtrate, the products can be isolated readily.

By contrast, solution-phase reagents often create unwanted by-products that can be difficult to remove, even with extensive aqueous work-up and conventional chromatographic purification procedures. For example, triphenylphosphine is a common, versatile, and widely used reagent in many synthetic transformations, including the Staudinger, ¹¹³ Mitsunobu ^114–115 and Wittig reactions. ¹¹⁶ Chemists readily recognize the difficulties associated with removal of the resultant phosphine oxide by-product and excess triphenylphosphine in these reactions. The utility of immobilized reagents to overcome these difficulties is illustrated by the use of a polymer-supported triphenylphosphine equivalent in a Wittig reaction. This enables facile removal of the derived phosphine oxide and excess reagent by filtration (Scheme 1). ^117–120 This example readily demonstrates the practical advantages achievable using supported reagents.

Scheme 1. Immobilized triphenylphosphine used in a Wittig reaction.

Importantly, many of the recovered spent reagents may be recycled a number of times, which is necessary to make them financially viable options. The ease of handling supported reagents is also noteworthy, particularly when dealing with expensive catalysts, since these systems are more likely to be incorporated into automated or flow processes.

Also, the process of immobilizing reagents produces reactive entities that often no longer possess the same safety or environmental concerns that their homogeneous counterparts display. Problems associated with foul stenches, extreme toxicity, and noxious build-up or other hazardous issues such as flammability or explosion are significantly reduced by immobilization. This aspect is nicely exemplified in the thionylation reaction shown in Scheme 2, whereby the malodorous sulfur by-products remain immobilized using the heterogeneous equivalent of Lawesson's reagent ¹²¹ in the conversion of amides to thioamides. ¹²² Other examples include the use of microencapsulated osmium tetroxide to perform dihydroxylation reactions to avoid the issues associated with release of volatile metallic osmium. ⁷⁵

Scheme 2. Immobilized Lawesson's reagent used in the conversion of amides to thioamides.

Supported reagents generally have slower reaction kinetics but the reactivity of supported systems can be greatly enhanced using short bursts of energy provided through focused microwave heating. ^123–125 Indeed, this is often preferred over prolonged heating using standard methods such as oil baths, which can result in partial decomposition of many of the commonly used supporting materials. For reaction systems requiring the use of poor microwave-absorbing solvents, doping with ionic liquids can provide a thermocouple for effective heating. ^126–138

3.35.1.4 Site Isolation Effects

Immobilization of a reagent facilitates both reaction work-up and product purification and consequently accelerates the synthesis overall process. This inevitably frees up more time for the creative element of synthesis planning and thereby increases overall productivity and output (Figures 1 and 2).

Figure 1. Supported reagents are able to transform substrates into new chemical products, where spent or excess reagents can be readily removed by filtration.

Figure 2. Process arrangements employing supported reagents. (a) Batch-splitting process. (b) Convergent synthesis.

The three-dimensional and steric environment of the support matrix creates a system of porelike structures with unusual topographies; they exist in isolation as a consequence of phase partitioning. Such structural characteristics can produce reagents with unique reactivities that can help overcome solvent-specific issues common to conventional solution-phase chemistry methods. ^138–143

The support material impacts on the reactivity of the attached functional groups. It can allow certain reactions to proceed at higher than usual concentrations; ones that would normally only be possible under very high dilution conditions. Macrocyclization is one example of a reaction that typically requires very high dilution. The reasoning here is that if the rate of intermolecular coupling is minimized, then it follows that there will be a concomitant increase in the intramolecular coupling reaction. It has been shown that by using an appropriate polymer-supported coupling reagent, for example an immobilized tetrakistriphenylphosphine palladium(0) reagent (Scheme 3), that controlled concentrations are of less significance. ¹⁴⁴ No dimeric or oligomeric side-products were obtained with the immobilized system when compared with the solution-phase equivalent reaction.

Scheme 3. Macrocyclization reactions immobilized tetrakistriphenylphosphine palladium(0).

These same principles also allow selective monoprotection of equivalently difunctional molecules. ^{37–39,145–152} As substrate binding is temporary for the duration of the reaction, other pendant functional groups are able to react in preference. As the reagents are anchored to the support materials they are site isolated; this also allows the simultaneous use of multiple reagents in one-pot transformations. This concept creates many new opportunities for organic synthesis.

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Microfluidic devices for radio chemical synthesis

A.Y. Lebedev , in Microfluidic Devices for Biomedical Applications, 2013

17.4.1 Main microfluidic device types reported

Reactor classification adopted in classical chemical engineering is well applicable to the microfluidic radiochemistry platforms. There are two conceivable types of reactors: flow-through and batch. Combinations of these also exist.

The flow-through reactor in its typical form is a long channel (or a vessel with a mixer) kept at a predefined conditions. Reagents are continuously infused into the reactor and then the mixture of products is continuously collected at the outlet. This type of reactor is widely used in the chemical industry where it allows for uninterrupted production of product dependent only on the continuous supply of the reagents. Early examples of the microfluidic devices applied for radiochemical synthesis followed this principle.

The first example of such a device was disclosed in 2003 in the form of patent applications by Brady et al. ¹ The patent introduced a series of novel ideas. First, this was the earliest publication that focused on microfabricated devices for radiochemical synthesis. Second, the patent application described a device integrating all aspects of radiopharmaceutical production, from processing of the cyclotron output to the quality control of the final dose. Third, the apparatus described in the published examples was designed to perform radiolabeling with a wide range of radioisotopes: ¹⁸F, ¹¹C, and ¹²⁴I. Fourth, an important innovation with this patent was the sequential connection of two identical chips for a two-step synthesis. This concept was implemented in a form of a T-mixer, followed by a long reaction channel etched on a glass substrate and covered by another layer of glass. The reagents were infused using syringe pumps, the temperature of the chip being controlled with an external heater. In the reported examples, all processes, aside from mixing and reaction, were performed in a conventional manner and decay corrected radiochemical yield (RCY) of FDG was 24%, unacceptably low by modern production standarts. ⁸ A series of more sophisticated and automated devices based on this platform was later reported by the same group of authors, but the RCY of FDG remained unimpressive. ^9–11 Substantial reduction on process time was also reported by the same group. ¹²

Another group reported a microfabricated device for radiochemical synthesis almost at the same time. ¹³ The device featured a very similar architecture: a T-shaped channel (220   μm (W) × 60 μm (D) × 14   mm (L); and total volume 0.2   μL) located at the interface of two bonded borosilicate glass layers. Two entry ports were connected to precision syringe pumps, and one exit port led to a collection reservoir (Fig. 17.1). The apparatus was used for alkylation of a carboxylic acid with [¹¹C]-MeI and [¹⁸F]-FCH₂CH₂OTos. Yield as high as 88% was reported for the former reaction, while RCY for the later one did not exceed 10%. This early prototype already demonstrated a high degree of the control microfluidic technology can provide for radio chemists. Just by changing the infusion rate, authors could modulate the reaction time with high precision and reproducibility. Soon after, another continuous flow design based on the simplest flow-through vial was published. ¹¹ In this design, the volume of the reactor was made up of a coin-shaped disk with inlets and outlet. The feasibility of very fast FDG synthesis was demonstrated using this design: 50% of radioactivity was converted to protected FDG in just 4 s. The design of a continuous flow reactor combining a series of flow-through cavities connected with capillaries was later investigated by others. ¹⁴

17.1. The simple T-shaped microfluidic reactor reported by Lu et al. Reagents enter from the two ports and the product is collected at the outlet.

(Source: Reproduced with permission from Lu, S. et al., Lab on a Chip 2004, 4, 523–5. ¹³ )

Flow-through reactors proved particularly useful for the labeling procedures involving gaseous radioactive precursors. The high liquid–gas surface achievable in a microfluidic reactor was used for carbonylation reactions involving gaseous [¹¹C]-CO. ^{4 , 15}

The high degree of reaction control, and the ability to sample process conditions in fine steps, encouraged commercialization of the flow-through microfluidic reactors. Advion currently offers a configurable microfluidic system under the 'NanoTek' brand (Fig. 17.2). ¹⁶ The central part of the machine is a reactor manufactured from fused silica and housed in a thermostated brass cartridge. One cartridge houses two-meter long 100 μm diameter tubing with total volume 15.7   μL; the cartridges can be linked together to increase the length and volume of the reactor. The key feature of the system is its ability to perform a series of experiments sampling just one parameter at a time. That is normally done even with the same batch of the ¹⁸F-K₂₂₂ complex and the same batch of the precursor solution that eliminates variability associated with trace impurities (e.g. moisture). Commercial availability of a microfluidic system stimulated a series of reports on detailed optimization of reaction conditions of several important radiochemical transformations. The system was used for labeling with both ¹⁸F⁵ and ¹¹C. ¹⁷ More than 30 compounds have been radiolabeled using this device, some of them involving complex synthetic procedures. ¹⁸ In order to enable sequential transformations, a simple T-mixer can be installed downstream from the reactor. The mixer combines the output from the first reactor with a stream of reagent needed for the next step, and feeds the mixture into the second reactor. ¹⁹ Nevertheless, it has been a trend that after careful study of the reaction with the microfluidic apparatus, authors opted for conventional synthetic modules for large scale production, and Section 17.5 will be discussing this in more detail.

17.2. Principal schematic of one of the configurations of Advion 'NanoTek' system. The system is built in the form of several independent blocks, which can be plumbed and reconfigured according to specific needs of each synthesis. Note dedicated vial for fluoride drying.

(Source: Reproduced with permission from Ravert, H. T., Holt, D. P &amp; Dannals, R. F. Radiochemical Synthesis 2012, 139–154. ¹⁶ )

As opposed to the flow reactor, the batch reactor in its classic form is a vessel that is loaded with reagents and then the product mixture is unloaded after the content has been treated under certain conditions for a predefined time.

In fact, a series of machines built upon the so-called captive-solvent method can be considered the first generation of microfluidic devices with mixed batch-flow architecture. The devices were used for preparation of [¹¹C]-labeled radiopharmaceuticals as early as 1985. ^{6 , 20 , 21} The central feature of these machines was a reactor in a form of a long tube. In some cases, the long tube was packed with an inert porous material. The inner surface of the tube was covered with a concentrated solution of the labeling precursor. Gaseous radioactive precursor was then passed through the reactor and the radioactivity was trapped in the tube due to the labeling process. A typical example ²² of the captive-solvent apparatus is illustrated in Fig. 17.3.

17.3. Schematic of a captive-solvent apparatus for ¹¹C-methylation. The reactor is coated with concentrated solution of precursor prior to the reaction, the original design of the heating device allowing for rapid changes in temperature. Reactor volume is 124 μL, and reactor volume is further decreased and surface increased by filling with acrylic yarn.

(Source: Reproduced with permission from Watkins et al., Applied Radiation and Isotopes 1988, 39, 441. ²² )

The volume of the reactor in these devices ranged from 125 to 500 μL. However, the real reaction volume was most likely considerably smaller, because the reagent captured in the reactor was only spread over a thin layer. In terms of the hardware the devices were more similar to the Advion setup, than to the later generation microfluidic batch reactors; however, the captive-solvent process is a clearly batch method, as it involves sequential loading of the reagents and only a finite amount of the product produced in one run. This early research revealed long before the 2003 Bradley patent application that radiosynthesis in a small volume benefits from low precursor consumption, easy purification due to low mass of impurities, and high labeling yield. ²⁰

There are two critical advantages of the batch reactors as compared to flow-through reactors. First, production of the radiopharmaceuticals is inherently batch process: the cyclotron produces radioactivity in batches, and the flow reactor will operate out of its optimum mode in this situation. Second, performing a multistep synthesis in a flow-through reactor is inherently difficult, due to cross-contamination between reagent streams and technical difficulties of switching from the solvent of the first reaction to a solvent suitable for the second one. Due to these considerations, batch microfluidic devices received early attention.

A fully integrated microfluidic device, performing on one chip all the synthetic operations needed for production of FDG (Fig. 17.4), was in fact one of the first reported devices. ²³ A complex pattern of channels and valves enabled all necessary processes: fluoride concentration, drying, radioactivity incorporation, hydrolysis, and evaporation. In just 14   min this circuit produced enough FDG for a mouse image. The main drawback of the device was a very low overall yield (26%), the result of interaction of free fluoride with polydimethylsiloxane PDMS, the polymer used for the device fabrication. ²³ Microfabrication technology allowed bringing together components that normally constitute separate hardware blocks, such as valves, pumps, and ion exchange column. Integration of all components on one device was the first step toward the production of cheap integrated circuits for use in the radiochemical processes. Use of an elastomer for fabrication of this complex structure was critical, as all valves in the device relied on the elastic properties of the material. A recent 'digital' batch microfluidic device, reported for optimization of protein labeling, ²⁴ also relies on PDMS for its operation.

17.4. Schematic diagrams showing main steps of FDG production in an early batch reactor (a) Concentration of dilute fluoride ion.(b) Evaporation of water from the concentrated KF solution. (c) Fluorination reaction. (d) Hydrolysis reaction. The entire device is made out of PDMS; valves are hydraulically actuated (see original publication for further explanation).

(Source: Reproduced with permission from Lee C.-C. et al. Science 2005, 310, 1793–6. ²³ )

The latest development in batch microfluidic radiochemistry is an application of electro-wetting-on-dielectric (EWOD) method for control of liquid movement. In the previous example, segregation of the chip into distinct process zones is achieved with the mechanical valves. The EWOD device (Fig. 17.5) uses an electromagnetic field to control movement of independent droplets, and thus different zones of the reactor are separated not by valves, but by gas surrounding droplets. The first EWOD synthesis of radiopharmaceutical was reported as part of a conference presentation ²⁵ and recently disclosed with full details. ²⁶ Several reported advances include, but are not limited to, use of the same electrodes to change the temperature and receive feedback, absence of mechanical parts, and use of a Cherenkov camera to monitor radioactivity.

17.5. (a) EWOD microchip with four concentric heaters (dashed circle) with a maximum volume of 17 μL. Inset shows the magnified area of the heater with four concentric individually controlled resistive heating rings. (b) Schematic side view of the EWOD chip sandwiching a reaction droplet between two plates coated with ITO electrodes, a dielectric layer, and a hydrophobic layer.

(Source: Reproduced with permission from Keng, P Y et al. PNAS 2012, 109, 690–5. ²⁶ )

Use of microfluidic reactors recently brought microfluidic technology to an important milestone: the first clinical PET image obtained with microfluidically produced radiopharmaceutical. The synthesis of the tracer was performed with P-IV machine, a late stage prototype developed by Siemens Molecular Imaging (Fig. 17.6). ²⁷ This fully integrated system includes all necessary parts for successful clinical production of a radiopharmaceutical, starting from concentration of fluoride and up to the optional reformulation of the final product. The machine is based around a 50 μL reactor, made up of a PEEK chip and transparent polydicyclopentadiene (PDCPD) lid. The reactor utilizes on-chip pneumatically actuated valves for liquid control. Demonstration of clinical capabilities is admittedly simplistic: [¹⁸F]-Fallypride production performed in one step, followed by a specially designed purification procedure eliminating reformulation. The capability of the system to perform multistep complex reactions was demonstrated separately. ²⁸

17.6. Main parts of the reactor assembly used in clinical production of ¹⁸F-Fallypride. Left: Schematic cross-section of the reactor cavity, lid, and heater; Right: 3D rendering of the same parts. ²⁷

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