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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5344_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 4.2. A selected list of important immobilized enzymes and their industrial applications.
Immobilized enzyme Applications
β-Galactosidase Splitting of lacrosse to glucose and galactose.
Ribonuclease Synthesis of nucleotides from RNA.
Penicillin acylase Commercial production of semi-synthetic penicillins.
Nitrilase Production of acrylamide from acrylonitrile.
Invertase Splitting of sucrose to glucose and fructose.
Histidine ammonia lyase Production of urocanic acid from histidine.
Glucose isomerase Production of high-fructose syrup from glucose (or starch).
Fumarase Synthesis of malic acid from fumaric acid.
Aspartase Production of aspartic acid from fumaric acid.
Amylase Production of glucose from starch.
Aminocyclase Production of l-amino acids from d, l-acyl amino acids.
4.9.1 Production of l-amino acids
l-Amino acids (and not d-amino acids) are very important for use in food and feed
supplements and therapeutic applications [49, 51]. The chemical methods used for
their production result in a racemic mixture of d- and l-amino acids. They can be
acylated to yield d, l-acyl amino acids. The immobilized enzyme aminocyclase
(frequently immobilized on DEAE-sephadex) can selectively hydrolyze d, l-acyl
amino acids to produce l-amino acids.
The free l-amino acids can be separated from the unhydrolysed d-acyl amino
acids. The latter can be racemized to d, l-acyl amino acids and recycled via an
enzyme reactor containing immobilized amnocyclase. Large amounts of l-methionine, l-phenylalanine, l-tryptophan and l-valine are produced worldwide using this
method.
4.9.2 Production of high-fructose syrup
Among the monosaccharaides, fructose is the sweetest, and has doubled the
sweetening strength of sucrose. Glucose is about 75% as sweet as sucrose. Thus,
glucose (the most abundant monosaccharide) cannot be a good replacement for
sucrose for sweetening. Therefore, there is a great demand for fructose which is very
sweet, but has a similar calorific value to that of glucose or sucrose. High-fructose
syrup (HFS) contains about equivalent amounts of glucose and fructose. From the
nutritional point of view high-fructose syrup is similar to sucrose [52]. High-fructose
syrup is a good replacement for sugar in the manufacturing of soft drinks, processed
foods and baking. High-fructose syrup can be manufactured from glucose by using
an immobilized glucose isomerase. The starch-containing raw materials (wheat,
potato, corn) are exposed to hydrolysis to produce glucose. Glucose isomerase then
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Figure 4.11. The production of high-fructose syrup from starch with glucose isomerase as the immobilized
enzymes.
isomerises glucose to fructose (figure 4.11). The ultimate product formed is highfructose syrup comprising 50% fructose.
4.9.2.1 Glucose isomerase
Glucose isomerase is an intracellular enzyme synthesized by several microorganisms.
Species of Arthrobacter, Bacillus and Streptomyces are the preferred sources. As it is
an intracellular enzyme, the separation of glucose isomerase without the loss of
biological activity involves specialized and expensive methods. Often, whole or
partly broken cells are immobilized and used [17].
4.9.3 Immobilized enzyme and cell analytical applications
4.9.3.1 In biochemical analysis
Immobilized enzymes (or cells) can be employed for the advancement of precise and
specific analytical methods for the determination of several compounds. The main
principle of analytical assays involves the action of the enzyme on the substrate. A
decrease in the substrate concentration, an increase in the product or an alteration in
the co-factor concentration can be used for the assay. Two types of detectors are
commonly employed, thermistors and electrode devices.
Thermistors are heat measuring devices which can analyze the heat produced in
an enzyme-calibrated reaction. Electrode devices are employed for determining
potential differences in the reaction systems. In figure 4.12 an enzyme thermistor,
enzyme electrode and a urease electrode are shown.
4.9.3.2 Affinity chromatography and purification
Proteins purification by traditional methods is often tedious and partial, and the yields
are often low. Earlier isolation of enzyme is limited to certain concepts such as:
• its specific biological property;
• its reversible association capability with specific substrates;
• its reversible association capability with specific inhibitors.
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Figure 4.12. Immobilized enzymes or cells in analytical biochemistry. (a) Enzyme thermistor. (b) Enzyme
electrode. (c) Urease electrode.
During affinity chromatography, the desired enzyme considered for the purification is allowed to pass through a column which usually contains a cross-linked
polymer or gel. This specific competitive enzyme is covalently attached with polymer
or gel present in the column. Enzymes without considerable affinity for the bound
inhibitor present in the polymeric or gel matrix will pass directly or spontaneously
through the column, whereas those that have substantial affinity, recognize the
inhibitor and form in a complex way and are eventually retained by the matrix.
Elution of complex or bound enzymes can be achieved by altering certain factors
such as salt concentration or pH, or by addition of a competitive inhibitor in
solution. Generally, the matrix is usually inert in nature, i.e., it shows minimal
interaction with proteins, both before and after coupling to the specific binding
group such as substrate or inhibitors. For affinity chromatography, the adsorbent
must have a number of promising characteristics. A loose and porous matrix or gel
allows easy entry and exit of macromolecules. Additionally, it also retains
suitable flow properties during use. The chemical nature of the matrix must allow
the suitable and extensive attachment of the particular ligand under mild conditions.
This will only happen with chemical bonds that are stable to the conditions of
adsorption and elution.
Affinity chromatography can be employed in several applications such as
purification of nucleic acid and protein from cell free extracts and blood. Based
on the property of affinity it is possible to purify several compounds e.g. antigens,
antibodies and co-factors.
4.10 Immobilized enzymes for biomedical applications
The investigation of immobilized enzymes for biomedical purposes was initiated
during the 1960s. The primary goal was to improve the stability of enzymes, lower
their immunogenicity and toxicity, and increase their time in circulation in living
organisms to be used clinically [53]. Since then, various approaches have been
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utilized in enzyme treatment. These methodologies have been employed for several
objectives, encompassing the detection of bioactive compounds for disease diagnosis, and managing certain ailments. Fixing innate metabolic problems, heart
problems, cancer, intestinal problems, and managing alcoholism are some examples
of these kinds of conditions. There are two different ways that enzymes have been
immobilized. The initial technique involves the immobilization of the enzyme
through cross-linking or covalent attachment to a scaffold, commonly known as
the process of immobility by attaching [54]. The second methodology is around the
confinement of the enzyme within a matrix, commonly referred to as immobilization
via inclusion. Polymer conjugation has emerged as a leading method for immobilization via binding, attracting a lot of attention in recent years. Enzyme immobilization is a process that could be accomplished with a wide variety of polymers. It is
important to keep in mind, though, that the strict standards needed for a biological
application make it very hard to test a lot of polymers. For the enzyme immobilization process, the polymers used should be completely biocompatible and biodegradable. They should also be very pure and have a regular distribution of molecular
weights. Furthermore, it is crucial to consider that the covalent bonding of enzymes
to polymers might result in alterations in molecular structure and a significant
decrease in enzymatic functionality [55]. Polyethylene glycol (PEG) is currently a
highly favored polymer for the purpose of modifying peptides and proteins that
possess therapeutic promise PEG possesses several advantageous characteristics,
including its cost-effectiveness, biocompatibility, non-toxicity, and existing approval
by drug regulatory bodies. Pegylation can enhance the characteristics of enzymes
without compromising their enzymatic performance, rendering them suitable for
applications as protein-based pharmaceuticals or as catalysts in bioreactors.
Currently, several enzyme/PEG conjugates are employed in clinical settings.
Villard provides a comprehensive list of these conjugates in a recent review [56],
which includes recombinant human tissue-plasminogen activator, adenosine deaminase, arginine deaminase, and asparaginase [57]. The methods of immobilization by
inclusion offer several advantages compared to those employed for immobilization
by binding. Firstly, there is no requirement for enzyme derivatization. A photobioreactor system powered by solar energy is depicted in figure 4.13.
Additionally, these systems afford a greater level of protection against enzymatic
degradation and other detrimental factors. Furthermore, they enable higher drug
loads to be accommodated. Finally, enzymes could be fixed in a more stable manner
inside various systems, such as multi-enzyme networks, groups of enzymes, or the
cells responsible for producing a particular enzyme [58].
Nevertheless, the implementation of inclusion methods is accompanied by certain
drawbacks associated with the utilization of synthetic materials. However, these
concerns can be addressed by employing biodegradable polymers, Examples of
polymers and co-polymers that can be generated from lactic and glycolic acid,
alginate, and chitosan are commonly utilized in many academic and scientific
applications. In addition, the application of biodegradable anchoring transporters,
like liposomes or red blood cells, presents an acceptable replacement method.
Liposomes have been widely investigated as particle drug carriers and have been
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Figure 4.13. A diagram illustrating a photobioreactor system powered by solar energy. This system includes an
internal light source, where optical fibers are excited by sunlight collected through a system, as well as a multiLED light source powered by electricity generated from solar panels and a wind power generator.
found to possess highly favorable features for the encapsulation of polypeptides.
Liposomes are nano-sized vesicles that consist of phospholipid bilayers resembling
cell membranes, which enclose compartments filled with water-based solutions [59].
These substances exhibit biological inertness biocompatibility and induce little toxic
or antigenic responses. Liposomal encapsulation of enzymes can increase their
in vivo circulation through the bloodstream.
Additionally, liposomes can effectively conceal the antigen determinants of
enzymes, hence mitigating the occurrence of undesirable immunological reactions.
Controlled-release systems composed of biodegradable polymers, such as nanoparticles, present a viable alternative to liposomes [60]. Micro/nanoparticles refer to
polymeric colloidal systems with sizes ranging from 10 nm to micrometers, wherein
the drug is dissolved, entrapped, encapsulated, or adsorbed. Nanoparticles possess
various desirable characteristics, including their ability to be formulated, sustained
release capabilities, subcellular size, and compatibility with tissue and cells. These
attributes make nanoparticles a potentially advantageous solution for immobilizing
enzymes and protein medicines. Asparaginase, an enzyme that has garnered significant
attention from researchers, has been immobilized on several supports. The enzyme
L-asparaginase facilitates hydrolyzing L-asparagine, classified as an essential amino
acid, into L-aspartic acid and ammonia. Leukemic cells that do not possess asparagine
synthase cannot sustain their life [61]. L-asparaginase administration results in a fast
reduction of extracellular asparagine, hence inducing targeted apoptosis of leukemic
cells. Cells possessing the capacity to synthesize asparagine intracellularly exhibit
heightened resistance to the cytotoxic effects of L-asparaginase.
In the treatment of leukemia, L-asparaginase therapy is often used. This treatment is generally administered with vincristine, daunomycin, methotrexate, or
cyclophosphamide. The treatment effectively lowers the level in the blood of the
amino acids, hence impeding the method of protein formation [62]. In normal use,
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L-asparaginase is often created by Escherichia coli or Erwinia chrysanthemums.
Asparaginase has shown promise in the therapy of lymphoblastic lymphoma and
other lymphoid tumors in children, according to the results of many recent clinical
studies. Despite its usage, L-asparaginase for treating acute lymphoblastic leukemia
(ALL) is not as successful as other options [63]. Because of the short duration of its
half-life in vivo, it must be administered by many injections before the desired
therapeutic enzyme levels may be achieved. When L-asparaginase is administered,
immunological adverse effects are a possibility as well. These can range from mild
allergic responses to anaphylactic shock. These side effects happen because the
enzyme is a foreign protein in people. This is especially true when given repeatedly to
reach therapeutic enzyme levels. As a result, a significant amount of research has
been undertaken in recent years to investigate alternative medicines or prospective
substitutes for L-asparaginase. One of the ways investigated in clinical settings,
which has demonstrated favorable outcomes in terms of reduced immunogenicity
and extended half-life, involves the utilization of PEG for binding purposes. This
technique has led to the developing of a novel molecule known as pegaspargase [64].
The technique has been approved by the US Food and Drug Administration (FDA)
and is commercially available under the trade name Oncaspar. This alteration leads
to a reduced delivery frequency and yields a less immunogenic response. However,
several authors caution against the systematic replacement of L-asparaginase with
pegaspargase due to a potential increase in pancreatitis linked to pegaspargase [65].
4.11 Detecting biomass with immobilized cells via bioluminescence
and other biosensor uses
4.11.1 Bioluminescence
The incident of bioluminescence was initially documented over one hundred years
ago, with its original observation being made in fireflies. The enzyme responsible for
facilitating the oxidative reaction that produces light was subsequently identifi ed and
dubbed luciferase:
luciferase
+⎯→⎯+++Luciferin O Oxyluciferin CO AMP light
2
Subsequently, many more bioluminescent creatures were discovered, encompassing
many taxa such as fish and microbes. The reaction ‘luciferase’, as depicted in the
above equation, employs adenosine triphosphate (ATP) as a substrate for light
production. However, in the case of marine luminous bacteria, reduced flavin is
utilized instead of ATP [66]. Bioluminescent bacteria are naturally occurring
microorganisms that inhabit many ecological niches, encompassing marine and
terrestrial ecosystems. In addition to the utilization of naturally occurring microorganisms, genetically modified microbes are also employed in the development of
bioluminescence-based biosensors. The process of mapping, isolating, and cloning the
enzymes responsible for gene coding in bioluminescence was undertaken.
Advancements in genetic technology have facilitated the transmission of bioluminescent properties to a diverse range of microbes. The utilization of genetically modified
strains containing bioluminescent reporter plasmids is becoming significant in
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advancing microbial luminescent biosensors [67]. The initial documentation of the
analytical utilization of bioluminescence emerged about a century ago, as Beijerinck
elucidated the identification of oxygen produced during photosynthesis in leaf extract
by employing luminescent bacteria. The utilization of bioluminescence as a basis for
developing several biosensor approaches, both bioluminescent and chemiluminescent,
has been seen. The practical uses of bioluminescence primarily lie in the advancement
of instrumental approaches within the field.
1. The quantification of microbial and other cellular entities by assessing ATP
levels [68].
2. The determination of active immobilized biomass, which encompasses the
measurement of biologically active microorganisms attached to surfaces or
within a matrix, critical for assessing the effectiveness of biofilm-based
treatment systems
3. The primary focus of bioluminescence-based biosensors lies in their application within the fi eld of environmental studies.
4.11.2 The measurement of biomass using bioluminescence-based techniques
The basis of bioluminescence has been expanded to encompass the detection of
naturally nonluminescent species and fluorescent cells. The utilization of intracellular ATP, and perhaps other nucleotides, can facilitate luminescence production
after introducing light-generating enzymes into the biological system. The initial
studies focused on the quantification of biomass utilizing the principle of bioluminescence, and these investigations have been conducted for over three decades
[69]. A comprehensive investigation examined the intracellular ATP levels in 19
distinct E. coli species. The results revealed a variation in ATP content, ranging from
0.29 to 8.7 × 11–10 μg of ATP per organism. Cell detection is often performed
in vitro, relying on effectively extracting intracellular ATP while minimizing ATP
hydrolysis. Following this, a light-generating system of luciferin and luciferase is
introduced, and the resulting emitted light is quantified. Various substances have
been examined for their efficacy in extracting ATP [70]. The range of substances
includes solvents made from organic materials, inorganic acidic substances, and
surfactants. Hence, examining ATP can be employed to identify microbial cells at
exceedingly low concentration levels. The approach was modified to facilitate
discerning various cellular classifications, encompassing bacterial constituents in
liquids, microorganisms’ mycobacteria, yeast cells, neoplastic cells, and other
biological structures. The necessary components for the assay, namely luciferin
and luciferase, can be readily obtained from commercial sources.
Additionally, commercial luminometers or other instruments capable of detecting
light are also available for use in this context [71]. A recent study was undertaken to
examine publicly accessible luminometers and imaging equipment specifically
developed to conduct low-light level measurements. Other methods include using
chromosome lux AB and GFP genes, which express bacterial luciferase and green
fluorescent protein, accordingly, to observe cell number and metabolic performance
directly. The relationship between bioluminescence output and cellular metabolic
activity is attributed to the energy requirements of the metabolism [72].
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4.11.3 Bioluminescence analysis for biomass captured in a microfluidic device
Estimating active immobilized biomass concentration using the luminometric
approach is predicated on measuring ATP content within the cells. ATP functions
as a vital chemical energy carrier within cells, wherein the energy is saved within the
chemical bonds connecting the terminal phosphate groups. Following cellular
demise, the quantity of ATP experiences a swift decline [73]. The ATP concentration
relies on the quantity of active biomass, making it a reliable indication of cell
viability. The determination of ATP concentration can be achieved by utilizing the
bioluminescence method, as previously stated. The proper measurement necessitates
the essential extraction of intracellular ATP. Various extraction procedures exist,
and selecting an appropriate extractant is contingent upon the specific microbe
under consideration [74]. The extraction protocol must effectively facilitate the
complete release of nucleotides in a quantifiable manner while also ensuring the
deactivation of any nucleotide changing enzymes that might be present within
the halted cells. Ninety percent of the living cells that have been immobilized
through entrapment within gel beads are concentrated within a 141 μm dense
external layer of a 1.1 mm blob. Furthermore, it has been observed that these cells
maintain over 91% of their activity following the immobilization process.
Consequently, the efficacy of ATP extraction from immobilized cells has been
demonstrated [75]. While there is extensive literature on measuring free biomass using
bioluminometry, a limited number of studies focus on estimating immobilized biomass
with bioluminescence-based methods. The immobilized Benecken natriegens cells
on silica particles were subjected to bioluminometry analysis to assess their content,
which was then compared to the viability of the biomass in its free form. The
determination of yeast biomass that has been immobilized in various ionotropic
hydrogels has been documented in a published study. The experimental section
comprehensively explains this specific procedure, serving as an informative scenario [76].
4.11.4 Biosensors relying on bioluminescence
The utilization of biosensor approaches that rely on bioluminescence and chemiluminescence has recently gained significant traction, mostly due to
notable advancements in light sensing technology. The utilization of advanced
photomultipliers and charge-coupled device (CCD) cameras enables highly efficient
light detection. Furthermore, compared to other methodologies, contemporary
instruments possess the advantageous characteristics of compactness, portability,
and relative affordability. Luminometry has certain advantages compared to
alternative optical techniques, namely its exceptional sensitivity and extensive
dynamic range [77]. Luminometry exhibits a significantly higher level of sensitivity,
reaching up to 105 times greater than absorption spectrometry. From a theoretical
standpoint, detecting a single photon in a photomultiplier allows for potentially
detecting a particular molecule of analyte or a separate cell. In practical applications, the efficiency of light detection may be limited.
Nevertheless, bioluminescent and chemiluminescent approaches have demonstrated the ability to detect minute quantities of analytes or organisms. The primary
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area of implementation for bioluminescence biosensors, which utilize immobilized
intact cells, is the monitoring of the environment [78]. The detailed evaluation
conducted by researchers focused on the applications of luminescence biosensors in
environmental monitoring. Luminescence-based biosensor approaches have been
widely employed in the surveillance of diverse organic and inorganic chemical
pollutants. Numerous hazardous chemicals and environmental pollutants have the
potential to disrupt energy metabolism and the electron transfer system in a general
sense. The substances generate substrates that facilitate bioluminescence; hence, any
disruption leads to observable consequences in the form of less luminescent activity
[79]. The utilization of immobilized cells in luminescence biosensors has been the
subject of recent publications. Given that bioluminescence relies on intracellular
metabolism, it is reasonable to expect any alterations in bioluminescence to arise
because of exposure to harmful substances that can enter the cell. The association
between concentrations of active compounds is often unsatisfactory due to transport
processes, and this correlation is typically highly particular to each system involving
microorganisms and harmful compounds [80].
The immobilization procedures employed for bioluminescent cells are typically
moderate approaches utilized in several other domains, which aim to maintain the
vitality of the arrested cells. Another essential criterion is the optical characteristic of
an immobilization substance, which should lead to a minimum reduction in
bioluminescence intensity. Multiple immobilization materials and procedures were
documented in the study, including alginate, agar, porous sol and gel glass,
immobility on a gold surface, and various instances of immobilization on polyamide
membranes. The utilization of bioluminescence biosensors holds significant promise
in environmental monitoring due to its inherent simplicity and capacity to identify
hazardous pollutants that pose risks to human health effectively. The bioluminescence approaches provide several key characteristics that render them highly
appealing. The implementation is simple, and it can be used in portable field
equipment. In contrast to other approaches, this one has an exceptionally high
sensitivity and speed [81].
4.12 Bioluminescence-based microbial biosensors
Numerous methodologies have been devised in recent decades, establishing a
foundation for the replacement of tissues and organs and the regulated and sustained
delivery of therapeutic substances to the recipient. The process of encapsulating live
cells refers to the technique of confining cells inside encapsulation systems that
possess semipermeable properties [82]. These systems are fabricated using either
naturally occurring or synthetic polymers and are specially designed to mitigate
immune resistance. This biotechnology approach possesses two significant therapeutic potentials. Firstly, it involves the extended transplantation of bioactive
compounds that can boost or revive normal tissue function. Secondly, it entails
advancing and refining innovative drug delivery systems that enable the sustained
release of therapeutic products over an extended period. Both approaches have a
substantial influence in terms of therapy and economics. The utilization of a
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technologically optimized encapsulation system would effectively hinder the infiltration and subsequent destruction of encapsulated cells by antibodies and other
immune cells [83]. Consequently, the continuous administration of immunosuppressant drugs could be minimized, leading to an improved quality of life for patients
undergoing this therapeutic intervention. The first documented instances of cell
transplantation within polymer matrices may be traced back to 1933, when Bisceglie
conducted an experiment involving the introduction of tumor cells into a polymer
framework, which was subsequently transplanted into a pig’s abdominal cavity.
During the 1960s, the term ‘artificial cell’ was used to describe the process of
immuno-isolating cells and enzymes within microcapsules possessing semipermeable
properties. Since that time, significant endeavors have been undertaken globally to
enhance education in the fields of genetics, biology, science of polymers, and
medicine [82]. Enhanced microcapsules with advanced technological features have
been developed with the aim of enhancing the survivability and usefulness of
encapsulated cells, while also improving their resistance to handling, transplantation, and stress within the host organism. Furthermore, a wide range of cell sources
have been immobilized and evaluated as prospective drug-secreting entities. Based
on the findings of many investigations, it has become evident that not all cells
possess the necessary characteristics to be deemed acceptable for microencapsulation
[82]. The proliferation of cells after encapsulation has the potential to completely
occupy the capsular space, resulting in reduced effectiveness of therapeutic diffusion.
This, in turn, may jeopardize the sustained viability of immobilized cells in the long
run. In contrast, cell types that exhibit limited proliferation following encapsulation,
such as myoblasts, possess the capacity to sustain the release of therapeutic agents
over extended durations. It is worth noting that the application of the process of
immobilizing transgenic cells from islets within alginate polylysine small capsules for
the purpose of glucose control in diabetes has evolved beyond its initial implementation in the 1980s [84]. This biotechnological strategy has now been employed for
various therapeutic purposes. Indeed, the efficacy of its medication delivery system
and allogeneic transplantation has been effectively established in animal models for
several diseases including cancer, haemophilia, and renal failure.
4.12.1 The microencapsulation process involves the utilization of polymers and cells
The effectiveness of the technology of cell microencapsulation relies heavily on the
appropriate mix of its two primary components, namely cells and polymers.
Numerous natural and synthetical polymers have been evaluated for their suitability
as capsule matrices and outer membranes [85]. It is imperative that all these
materials exhibit biocompatibility, ensuring that they do not disrupt the internal
cellular balance or trigger an immune response from the host organism that could
compromise the sustained effectiveness of the transplanted microcapsules. In
addition, it is imperative that the microcapsules produced using these polymers
have sufficient stability to withstand both mechanical stress and osmotic stress upon
implantation [86]. Hence, it is imperative to meticulously choose the components
that will comprise the ultimate composition of the immobilization apparatus.
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