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On-Site Therapeutic Drug Monitoring

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DOI: 10.1016/j.tibtech.2020.03.001

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 TIBTEC 1906 No. of Pages 16

Trends in Biotechnology

Review

On-Site Therapeutic Drug Monitoring

H. Ceren Ates,1,2 Jason A. Roberts,3,4,5,6,7 Jeffrey Lipman,3,4,7 Anthony E.G. Cass,8
Gerald A. Urban,1,9 and Can Dincer1,2,*,@

Recent technological advances have stimulated efforts to bring personalized Highlights

medicine into practice. Yet, traditional application fields like therapeutic drug On-site therapeutic drug monitoring has
monitoring (TDM) have remained rather under-appreciated. Owing to clear the potential to improve patient out-
comes and drastically reduce healthcare
dose-response relationships, TDM could improve patient outcomes and reduce
costs.
healthcare costs. While chromatography-based routine practices are restricted
due to high costs and turnaround times, biosensors overcome these limitations Despite being on the radar of the scien-
by offering on-site analysis. Nevertheless, sensor-based approaches have yet to tific community for almost two decades,
sensor-based approaches have yet to
break through for clinical TDM applications, due to the gap between scientific
break through and support the clinical
and clinical communities. We provide a critical overview of current TDM practices, application of therapeutic drug monitor-
followed by a TDM guideline to establish a common ground across disciplines. ing, potentially due to the gap between
Finally, we discuss how the translation of sensor systems for TDM can be facili- scientific and clinical communities.

tated, by highlighting the challenges and opportunities. Chromatography as a routine practice

is limited due to its lack of standardiza-
tion, high turnaround-times and instru-
Translational Challenges for Therapeutic Drug Monitoring (TDM) mentation costs, and labored sample
Developments, particularly in the past 50 years (Box 1), have made it clear that the concentration of preparation.
a drug in blood is correlated to the pharmacological activity, so concentration is a better candidate
Sensors offer a low-cost, easy-to-use,
than dosage for quantifying efficacy or toxicity. TDM is the clinical practice of measuring this drug
and on-site analysis method to explore
concentration in blood or plasma, or in other biological fluids that can be linked to blood drug con- the full potential of therapeutic drug mon-
centrations. This measured drug concentration is then used to adjust the drug dosing regimen by itoring, overcoming these limitations.
targeting a predefined concentration or exposure interval, called a therapeutic range. Hence, the
The success of individualized dosing
reliability of TDM is strongly related to the specificity and sensitivity of the analysis method. In the
strongly relies on two factors: how PK/
contemporary context of clinical TDM, these tests are performed by using either chromatographic PD studies are integrated with therapeu-
methods coupled with special detectors (often mass spectrometers) or immunoassays. Neverthe- tic drug monitoring and how the mea-
less, these traditional methods have some practical limitations for the envisioned large-scale, dis- surement process is managed.

tributed TDM practice, such as lack of standardization in workflows, long turnaround times, and
high instrumentation costs with complex sample preparation. In this regard, new developments
in sensing technologies offer a unique opportunity to overcome these limitations and to explore 1
Freiburg Centre for Interactive Materials
the full potential of TDM. Recent advances and the current capabilities of such applications have and Bioinspired Technologies – FIT,
University of Freiburg, 79110 Freiburg,
been well addressed in several recent reviews [1–4]. Nonetheless, these novel technologies are fail- Germany
ing to diffuse rapidly into clinical practice, unlike their predecessors from the 1960s and 1970s. In 2
Department of Microsystems
order to understand the reasons behind this translation problem, which is the focus of this review, Engineering – IMTEK, Laboratory for
Sensors, University of Freiburg, 79110
we first explain different notions of drug monitoring and discuss current trends by providing an Freiburg, Germany
overview of recent TDM studies in both clinical and scientific contexts, particularly focusing on stud- 3
Centre of Clinical Research, Faculty of
ies that have worked with human samples in the past 5 years. Then, we present a guideline on how Medicine, The University of Queensland,
4072, Brisbane, Queensland, Australia
to establish a complete TDM practice, followed by a discussion on the conceptual differences be- 4
Department of Intensive Care Medicine,
tween frontline players (clinicians) and technology or model developers (the scientific community) Royal Brisbane and Women's Hospital,
and how this difference is reflected in their modus operandi (Figure 1, Key Figure). 4029, Brisbane, Queensland, Australia
5
Department of Pharmacy, Royal
Brisbane and Women's Hospital, 4029,
Current TDM Practice and Recent Advances Brisbane, Queensland, Australia
6
Chromatographic Methods Centre for Translational Anti-infective
Pharmacodynamics, School of Pharmacy,
Although chromatography (Box 2) has been successfully implemented in clinical studies, there are The University of Queensland, 4102,
still many challenges that are needed to be addressed in liquid chromatography with tandem Brisbane, Queensland, Australia

Trends in Biotechnology, Month 2020, Vol. xx, No. xx https://doi.org/10.1016/j.tibtech.2020.03.001 1

© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Trends in Biotechnology

7
mass spectrometry (LC-MS/MS) (see Glossary) methods. Despite its high specificity, matrix interfer- Division of Anaesthesiology Critical Care
Emergency and Pain Medicine, University
ence may lead to falsely low or high results; that is, the matrix and co-eluting compounds can interfere of Montpellier, Nîmes University Hospital,
with the ionization process in MS (via ion suppression/enhancement) [5]. Furthermore, the throughput 34090, Nîmes, France
8
of LC-MS/MS is lower than that of the conventional immunoassay platforms. Recent studies are ei- Department of Chemistry and Institute of
Biomedical Engineering, Imperial College
ther addressing these issues [6,7] or exploiting or improving this method’s inherent advantages [8,9]. London, SW7 2AZ, London, UK
9
Freiburg Materials Research Centre –
Accordingly, there has been a significant effort to increase the throughput of chromatographic FMF, University of Freiburg, 79104
Freiburg, Germany
methods [10]. Pioneering multiplex approaches have been reported for antiretroviral agents (ARVs)
[11], antifungals [12], antineoplastics [13], antibiotics [6,7,14], antidepressants [15], and immunosup-
pressive drugs [16] in the past decade. More recently, ultra-performance liquid chromatography *Correspondence:
(UPLC)-MS/MS has been used for simultaneous quantification of antibiotics [17] and ARVs from dincer@imtek.de (C. Dincer).
@
plasma [11] and breast milk samples [18]. Another focus is the extension of TDM studies toward un- Twitter: @ImtekSensors
conventional samples and sampling. Several LC-MS/MS methods have been developed and applied
for hair [11,18], dried blood spots [19], urine [20], sweat [21], saliva [22,23], and tissue biopsies [24].

Box 1. Historical Perspectives on TDM

The notion of personalized medicine and on-site treatment is as old as human civilization. Even hunter-gatherer microsocieties were aware of the fauna and flora sur-
rounding them and healers offered the best medicine in their arsenal to those seeking help [75]. The earliest examples of drug therapy from antiquity include Sumerian
clay tablets (2000 BCE), the Ebers Papyrus (1550 BCE), the Sushruta Samhita (600 BCE), De materia medica (50–70 CE), Shennong Bencao Jing (200 CE), and many
others. The basic principle of treatment remained more or less the same for thousands of years until the 19th century, the age of synthetic chemistry. As scientists de-
veloped systematic ways to design the structure of organic substances at will, local apothecaries offering personalized remedies were replaced by industrialized ‘one-
size-fits-all’ mass production. Nevertheless, this paradigmatic idea is now being challenged with a revolution arising from developments in electronics, data science,
manufacturing technology, and process control over the past century.

The critical role of dosage has been long known for patients hovering between life (efficacy) and death (either toxicity or subtherapeutic exposure) since antiquity. As
Paracelsus put it, ‘All things are poison and nothing is without poison; only the dose makes a thing not a poison’. The ability to monitor and correlate this transition, how-
ever, was first demonstrated in 1932 by Widmark [76] (Figure I). In the following decades, concerns about the ‘one-size-fits-all’ approach began to appear in scientific
communities, supported by blood concentration measurements for several drugs. In the 1960s, the first PK study was published and the importance of PK was
established [77]. Another historically important landmark was a paper from 1965, the first structured review on the importance of ‘monitoring of drugs’ [78]. These in-
vestigations gained further momentum with developments in chromatographic techniques. The following years were a golden age of drug monitoring. Concentrations of
various drugs were measured by using first gas chromatography (GC) then HPLC, and mass spectrometry (MS). Another substantial milestone was the introduction of
immunoassays, which revolutionized the concept by increasing feasibility of performing assays [79]. Simultaneously, previously proposed notions, such as dose-toxicity
relationships, PK, and drug–protein binding, were being investigated extensively, which eventually led to optimization of sampling strategies and analytical workflows.
The 1990s onwards saw the introduction of more sophisticated chromatographic techniques, software packages to design dosage regimens, the concept of noninva-
sive and minimally invasive methods, wearable sensors, and feedback-controlled smart drug delivery systems.

Trends in Biotechnology

Figure I. Historical Timeline of Key Events in Therapeutic Drug Monitoring. Abbreviations: ISF, interstitial fluid; TDM, therapeutic drug monitoring. See
[54,73,74,76–93].

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Key Figure Glossary

Overview of Therapeutic Drug Monitoring (TDM) Coefficient of variation (CV): the ratio
of the standard deviation to the mean.
Dried blood spot (DBS): a bio-
sampling technique where blood
samples are dropped and dried on a
filter paper for further analysis.
Free drug concentration: drugs tend
to bind serum proteins in different
degrees; free drug concentration
provides information about metabolically
active portion of the drug.
IMM: the ‘interacting multiple model’,
an improved Bayesian approach that
can change the patient’s model
parameters with time.
Interstitial fluid (ISF): a fluid between
blood vessels and cells containing a
wealth of biomarkers and analytes,
including sugars, hormones, fatty acids,
amino acids, salts, coenzymes, white
blood cells, and waste-products of cells.
LC-MS/MS: liquid chromatography
coupled to tandem mass spectrometry,
an analytical chemistry technique that
combines liquid chromatography with
mass spectrometry to obtain high
physical separation and mass analysis
capabilities.
Localized surface plasmon
resonance (LSPR): unlike SPR, LSPR
utilizes noble metal nanoparticles as the
solid state for immobilization, which
decreases background intervention and
enables colorimetric readout by the
Trends in Biotechnology naked eye.
Microneedles: small needles
Figure 1. TDM should include the active management of free drug concentrations in human body fluids using either specifically designed to penetrate only
chromatographic or immunoassay-based methods or on-site solutions (sensors and wearables) for the optimum benefit of the outer layer of skin without puncturing
each individual patient. the deeper layers of the epidermis and
dermis (nerves and blood vessels),
allowing a painless sampling procedure
for ISF.
One-size-fits-all: a drug prescribing
HPLC methods are also employed in combination with simpler detectors such as UV, flame ion- process based on predefined and
ization detection, (FID), or diode-array detection (DAD) in order to alleviate the disadvantages of widely accepted therapeutic ranges.
LC-MS/MS, such as matrix effects, expensive equipment costs, time-consuming optimization re- Orbitrap: a new type of high-resolution
mass analyzer that includes a barrel-like
quirements, and the necessity for well-trained personnel. In such cases, however, overlapping
electrode outer layer and a spindle-like
peaks between analytes with similar retention times may be observed, which can be even electrode inside, which traps ions
more amplified by the presence of unknown components in complex sample matrices. One rem- around the spindle in an orbital motion.
edy for this problem is the use of chemometric algorithms, which enable the elimination of unex- Pharmacodynamic (PD): relating to
the relationship between the
pected signal interferences from the total signal via mathematical modelling [25,26]. concentration of a substance at time of
action and its effect on a living organism;
Immunoassays ‘what the drug does to the body’.
Immunoassays have been used extensively for drug monitoring in clinical laboratories for more Pharmacokinetic (PK): relating to the
movement of a substance into (liberation
than half a century due to their high affinity, low sample volume requirement, simplicity, adaptabil-
and absorption), through (distribution
ity to high-throughput, and low cost. The basic principle depends on detecting an analyte via and metabolism), and out of living
binding to analyte-specific antibodies. The assay design can be classified into competitive and organisms (excretion); ‘what the body
sandwich modes, which are mainly used for analytes with small or large molecular weights, does to the drug’.

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Box 2. Chromatography PK/PD model: a modelling approach

The basic principle of all chromatographic methods is the separation of unknown components within a mobile phase by that combines pharmacokinetic and
hindering the relative mobility of each molecule by interactions between the eluent and the stationary phase. The mobile pharmacodynamic properties to
phase can be a gas, supercritical fluid, organic solvent, or ionic liquid. As the eluents leave the separation column at describe time-course and factors
their respective retention times, compounds are identified using a variety of techniques with different complexities, such controlling drug effects on the body.
as UV/Vis spectrophotometry, refractive index, DAD, FID, and MS. The selection of mobile and stationary phases is the Surface-enhanced Raman
most critical parameter as they determine the success rate of adequate resolution of individual compound peaks. spectroscopy: a very powerful
technique to detect even a single
The very first junction in the decision-making process is the choice between GC and LC. Although GC ensures high res- molecule by enhancing Raman
olution, it requires the compounds to be gaseous or intrinsically volatile, or have a volatile derivative. Therefore, only about scattering via metal nanostructures or
20% of the organic compounds are separable via GC. Conversely, LC can be used for a wide range of compounds without nanoparticles.
the need for derivatization, which makes it amenable for at least 80% of the candidate compounds. This is why LC Surface plasmon resonance (SPR):
methods such as HPLC are usually the methods of choice in clinical laboratories, except for the few cases where LC is an optical technique based on
not suitable. The second decision is the type of detector coupled with the chromatography method. A variety of detectors monitoring changes in refractive index,
are utilized in different fields, yet LC-MS/MS has been preferred in both reference and clinical laboratories for more than allowing label-free and real-time analysis
two decades due to its superior specificity and sensitivity over conventional immunoassays, the possibility of processing of analytes in complex matrices.
multiple analytes, and smaller sample volume requirements [6,94]. Therapeutic index: the ratio between
doses causing unacceptable toxicity
There are also studies focusing on improving MS-based approaches for TDM measurements. One emergent strategy is and doses producing a therapeutic
the replacement of conventional mass analyzer (triple quadrupoles) by Orbitrap technology, allowing high mass resolution effect. A narrow therapeutic index
measurements over wider concentration ranges [8]. This high-resolution mass spectrometry approach has been means that even small dosage changes
implemented and tested for anticancer drugs [9,95], antivirals [96], antifungals [97], antibiotics [98,99], and psychoactive could result in toxic effects, so
drugs [8]. monitoring is of utmost importance.
Total drug concentration: gives the
sum of bound and free drug
concentration.
respectively. Most TDM assays use a competitive format, in which small-sized target drug mole- Ultra-performance liquid
cules in the specimen compete with labelled competitors for a limited number of binding sites. In chromatography (UPLC): a liquid
other words, the analyte concentration in the tested sample becomes inversely proportional to chromatography technique utilized for
the separation of different constituents of
the signal produced. With the rise in biopharmaceuticals and biosimilars, however, the noncom- an analyte using high pressure.
petitive format has begun to play a larger role [27].

Unfortunately, the major drawback of immunoassays comes from this fundamental working prin-
ciple, meaning there may be a lack of analytical specificity due to the presence of crossreactants,
endogenous human antibodies, and other components in a specimen such as hemoglobin. In the
past couple of years, great efforts have been dedicated to overcome the specificity issues by
performing multicenter evaluation studies [3,28], developing new reagents, and immobilization
strategies [29] and signal enhancement approaches. An in-depth review of reported interferences
has been presented for digoxin, immunosuppressant, anticonvulsant, and tricyclic antidepres-
sant immunoassays elsewhere [3]. Recently published review papers have discussed immunoas-
say methods used for different TDM applications for detailed comparisons, particularly for
immunosuppressive agents [5,30,31]. There are also studies comparing the performance of
commercially available immunoassays by benchmarking their performance with those of LC–
MS/MS [32,33].

Biosensors
A biosensor is an analytical device that converts a biological response into a quantifiable signal via
molecular recognition involving bioreceptors. These recognition elements, which can either be
natural (such as antibodies, enzymes, membranes) or artificial (such as molecularly imprinted
polymers or aptamers), have a high binding affinity toward a specific analyte. This analyte–
bioreceptor interaction creates a change in local physicochemical properties, which can be trans-
duced into an interpretable signal. In principle, this interaction can be used to monitor both the
drug and biomarker (metabolite or protein) concentrations.

Biosensors can be classified in several different categories: according to their detection method
(optical, electrochemical, thermometric, magnetic, or mechanical), sensing mechanism (direct/

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label-free or indirect/labelled), functionality (single- or multi-use), or degree of invasiveness. In this

section, we will present the most recent biosensor-based TDM practices (Figures 2 and 3) and
then elaborate on some wearable biosensor applications (Box 3). Recent review articles give
more detailed information on material selection, recognition elements, signal detection tech-
niques, amplification methods, and different application areas of sensors [4,34]. Another particu-
larly interesting perspective is the utilization of no-wash biosensors for real-time monitoring of
small molecules [35,36].

Optical sensing is one of the most commonly employed approaches for therapeutic monitoring of
drugs. These sensors can operate at different spectral bands between the UV and near IR regions
and can be integrated with powerful analytical techniques such as NMR and Raman

Trends in Biotechnology

Figure 2. Sensor-Based Systems for Therapeutic Drug Monitoring. (A) Aptamer-based electrochemical sensors for monitoring and feedback-controlled delivery of
drugs. Applicability of the proposed system was demonstrated using antibiotics (vancomycin) as a model drug in a rat animal model. Reproduced, with permission, from
[56]. (B) Portable transmission-localized surface plasmon resonance device coupled with DNA aptamers for label-free quantification of antibiotics (tobramycin) in filtered,
undiluted serum samples. Reproduced, with permission, from [40]. (C) An antibody-free electrochemical biosensor for sensitive and low-cost monitoring of ß-lactams
(piperacillin, cefuroxime, and cefazolin) from undiluted human plasma. Reproduced, with permission, from [53,58]. (D) Electrochemical microfluidic multiplexed
biosensor for simultaneous and on-site quantification of two different antibiotics (tetracycline and pristinamycin) from undiluted plasma samples. Reproduced, with
permission, from [54]. (E) Paper-based surface-enhanced Raman spectroscopy (SERS) device for barbiturate (phenobarbital) detection from artificial lacrimal fluid.
Reproduced, with permission, from [48]. (F) Paper-based SERS sensor for monitoring antifungal (flucytosine) levels from untreated serum samples. Reproduced, with
permission, from [70]. Abbreviations: BSA, bovine serum albumin; CE, counter electrodes, CMOS, complementary metal oxide semiconductor; PCB, printed circuit
board; RE, reference electrodes; WE, working electrodes.

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Figure 3. Minimally Invasive Wearable Biosensors for Therapeutic Drug Monitoring. (A) Hydrogel-forming microneedle arrays for methylxanthine (theophylline)
monitoring from interstitial fluid (ISF). Reproduced, with permission, from [71]. (B) Wearable electrochemical sweat sensor (s-band) for the surveillance of methylxanthines
(caffeine). Reproduced, with permission, from [72]. (C) Microneedle array wearable biosensor for continuous therapeutic monitoring of ß-lactam antibiotic
(phenoxymethylpenicillin). Reproduced, with permission, from [73]. (D) An integrated wearable device for TDM of antibiotics from ISF. Vancomycin is detected by
receptors immobilized on gold-coated hollow microneedle and quantified with an optofluidic sensing unit. Reproduced, with permission, from [74]. Abbreviations: HRP,
horseradish peroxidase; TMB, tetramethylbenzidine.

spectroscopy. Surface plasmon resonance (SPR) and localized surface plasmon

resonance (LSPR) are powerful technologies utilized within optical sensors that depend on
changes in the local refractive indices (RIs) of metal films or nanoparticles, respectively. Since mol-
ecules of interest in TDM applications are usually small, their influence on local RI can be minus-
cule, depending on the size of the analyte. This is why indirect methods, such as those including
nanoparticles, have been used in recent SPR studies for signal enhancement. These advanced
techniques have been implemented in biosensor-based TDM studies for anticancer drugs
[37,38], antibiotics [39–42], and therapeutic drug antibodies [43,44]. SPR has also been com-
bined with lateral flow assays for therapeutic immunogenicity monitoring of infliximab [45].
Surface-enhanced Raman spectroscopy is another advanced optical technique based on

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Box 3. Wearable Biosensing Technology

Traditional biosensors have been designed for use in in vitro diagnostics, yet their inherent advantages such as low costs,
fast turnaround-times, low power and sample requirements, and portability have paved the way for new opportunities in
wearable biosensors. Current applications enable noninvasive or minimally invasive chemical analysis of samples, includ-
ing tears, saliva, sweat, or interstitial fluid (ISF) rather than blood. There are many promising examples, where the working
principle of the sensor is based on electrochemical [100,101], colorimetric [102,103], or piezoelectric [104] methods.

ISF, which holds the potential to provide reliable correlations between the blood and ISF concentrations, is the most com-
monly used matrix for TDM applications. ISF analytes can be extracted noninvasively (via iontophoresis or sonophoresis) or
with minimal invasion (using microdialysis or microneedles) and can be processed off-device or on-device [62].
Microneedles are particularly interesting as they can handle very small volumes (b1 nl) and eliminate the need to transfer
the sample for in vitro analysis [73]. This aspect can be of particular value for neonates or small children [71]. Microneedles
have been combined with sensors for the detection of antibiotics [73,74,93] and glucose [105].

However, there are still fundamental challenges in wearable sensing technologies that need to be overcome before com-
mercial realization: establishing reliable correlations between the analyte blood concentrations; stability; eliminating the
need for constant (re)calibration; identifying new biomarkers; supplying the necessary power for sensing, data processing,
storage, and transmission; multiplexing; and security and privacy issues [61]. Regarding analyte-blood concentrations, the
quantity of interest is the analyte concentration at the site of action as this is where the correlation with pharmacological
activity should be strongest. In practice, however, free blood concentration is commonly accepted to be an indicator of
that quantity. In this manuscript, it is also referred as blood concentration, to be consistent with the recent literature.

Raman spectroscopy, which has been used for anticancer drugs [46], antibiotics [47], as well as
antiepileptic drugs [48]. Although optical sensors can provide short turnaround times and high
sensitivity, they often suffer from significant background signals, attenuation of analyte signal
through the matrix, high instrument cost, and potentially low specificity due to nonspecific binding
of bioreceptors.

Electrochemical sensing is another commonly used method for TDM. The major advantages of
electrochemical sensors are their simplicity, low cost, high sensitivity, multiplexing capability,
low detection limits and capability of working with small sample volumes, and miniaturization
allowing on-site monitoring [49]. Electrochemical biosensors coupled with C-nanotubes [50],
nanoparticles/doped-electrodes [51], and various bioreceptors such as DNA [52], antibodies
[53,54], membranes [55], and aptamers [56,57] have recently been used for the detection of an-
ticancer drugs [52], antibiotics [53,54,56–58], and antifungals. Similar to optical sensors, the
issue of nonspecific binding still abides in electrochemical sensors due to the inherent nature of
bioreceptors and hence needs to be addressed.

Guidelines for TDM

Traditional TDM has a well-established success history spreading over half a century, yet the
practice has been limited to a small fraction of drugs analyzed in specialized, central laboratories.
With the recent advances in micro/nano-scaled sensing technologies, however, cheap, simple,
rapid, and even on-site measurement of drug concentrations in bodily fluids is increasingly pos-
sible, creating new opportunities for patient healthcare in general. In order to highlight the gap be-
tween the clinical practice and innovative solutions in sensing technology discussed in the
previous chapters, we give guidelines describing the flow of a typical TDM practice, which
could be useful to comprehend the steps required before broad clinical use can be realized.

Drugs Requiring TDM

TDM is fundamentally a dynamic dosing process with a feedback loop that necessitates a control
variable: precise measurement of a specific drug concentration in a relevant biological matrix at
the right time. This measured concentration is then interpreted to adjust the transient dosing reg-
imen, completing the feedback loop. The very first step to confirm the potential value of TDM is to

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determine the suitability of the candidate drug for TDM. From a clinical point of view, candidate
drugs must satisfy the following criteria. First, there should exist no other readily measurable end-
points of pharmacological effectiveness, such as blood pressure for antihypertensive drugs.
Second, there must be a reliable correlation between the analyte concentration and the clinical
effect. Third, the difference between the therapeutic concentration must be close to the noneffec-
tive and toxic concentration values (low therapeutic index). Fourth, the drug dosing regimen–
blood concentration relationship must be poorly predictable, with high interindividual variability.
Fifth, the duration of the therapy should be sufficiently long to benefit from TDM. Sixth, and finally,
it should be clear whether serious consequences are expected or not in the case of under- or
overdosing. Satisfying the majority of those conditions does not necessarily make a drug suitable
for TDM. In particular, if a drug acts intracellularly or its metabolite is the active substance, then
blood concentrations may become too poorly correlated to measure other biological matrices.
Other cases can be listed as drugs for which individuals may have developed tolerance
(e.g., pain medications) and those for which the effect lasts longer (e.g., chemotherapy). Therefore,
it is recommended to first consider those aspects for the candidate analyte before applying TDM.

Clinical TDM currently targets various drugs, including anticonvulsants (phenytoin, carbamaze-
pine phenobarbital, primidone, valproic acid, clonazepam), cardioactive drugs (digoxin, procain-
amide, quinidine), respiratory-acting drugs (theophylline, caffeine), psychotropic medications
(clozapine), mood active drugs (lithium, nortriptyline, doxepin), immunosuppressants (cyclospor-
ine, tacrolimus, mycophenolate, sirolimus, everolimus), antineoplastic (methotrexate), and anti-
infectives (amikacin, gentamicin, tobramycin, vancomycin, piperacillin, cefuroxime, voriconazole,
ganciclovir, meropenem, flucloxacillin) [59].

Matrix Selection
After selecting the candidate drug or metabolite for TDM, the second step is to decide which
sample or matrix is to be collected for the analysis. Current clinical practice is mainly based on
plasma samples, except for immunosuppressant drugs, which are usually measured in whole
blood [60]. This is also true for scientific community: plasma has been the most commonly stud-
ied matrix over the past couple of decades. Accordingly, the accumulated knowledge on drug
concentration and therapeutic response dynamics has been mostly established for whole
blood and plasma-based TDM. However, there are two major handicaps related to blood-
related matrices: first, blood sampling is relatively invasive, which might be impractical and even
unethical for certain patient populations, and second, costs associated with collecting,
transporting, and processing the blood are significant. Recent developments regarding sampling
matrix selection focus on ways to alleviate those issues by either deploying minimally invasive
microsampling strategies for plasma or blood, or utilizing alternative matrices within the frame
of minimally invasive or noninvasive sampling. Alternative matrices also have the potential to re-
place blood-based analysis for cases where blood concentrations may not accurately represent
the drug concentration at the sites of action.

Dried blood spot (DBS) analysis has been studied as a method to alleviate large-volume venous
sampling. The basic approach is to collect a finite amount of blood on a filter paper, dry it under
ambient conditions, and send it to a central laboratory via regular mailing services. Its simple de-
sign brings the opportunity for multiple sampling at any time of the day and hence allows home-
based TDM performed by the patient. Since samples can be collected and transported without
the need for centrifuging or refrigerated storage, DBS also has the potential to accelerate the dif-
fusion of TDM into resource-limited areas. Although many analytes are considered to be stable,
the influence of temperature, humidity, and sunlight exposure must be assessed during the prod-
uct development. Patients or care-givers must receive instruction on how to minimize the

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contamination of the sample. Since therapeutic ranges are usually determined for plasma
samples, further validation studies must be performed for DBS, which brings additional bias for
analytes that have binding tendencies for plasma proteins or red blood cells. Interpatient variation
in hematocrit concentrations is another concern, as it leads to sample nonhomogeneity
(spreading, size, and thickness of the sample). In other words, additional correlations or correc-
tion factors must be introduced in order to use pre-existing therapeutic ranges.

Several alternative matrices have been studied to replace blood-based approaches, yet only epi-
dermal [sweat or interstitial fluid (ISF)] and oral fluids (saliva) seem to have the potential to re-
place blood as a TDM sampling matrix in the near future. In principle, these matrices reflect the
free (i.e., not protein-bound) concentrations in blood and hence do not need additional steps
to determine the clinically relevant free drug concentrations. The common disadvantage of
these matrices, however, is the fact that they must be coupled with sensitive analytical methods
as the expected analyte concentrations are quite low. Both matrices also require further studies
to correlate drug levels with blood and plasma concentrations.

Sweat-based TDM is the oldest approach, dating back to the 19th century [60]. Even though it
has the most viable sampling area, sample collection suffers from several issues. First, sweat
glands are unevenly distributed, so analyte concentration profiles may be location-dependent.
Second, the analyte needs to be collected by stimulating sweat glands, such as by exercise or
thermal heating, or being induced by stress or drugs like carbachol. Third, analytes are
transported via both passive and active mechanisms, which complicates quantitative analysis.
Fourth, the concentration of a collected analyte is a function of many parameters, from emotional
status to environmental and physiological parameters like temperature, humidity, and pH. Fifth,
there are concerns regarding drug stability and concentration accuracy with sweat evaporation
and contamination during the sampling process. Sixth, and finally, there can be high inter- and
intraindividual variability. Despite some progress toward overcoming those issues [61], current
practice seems to be limited to qualitative fitness monitoring.

The other promising epidermal matrix is ISF. The function of ISF is to provide a medium between
vasculature and cells, so it is rich in candidate signaling molecules and metabolites targeted in
TDM. Nevertheless, the use of ISF in drug monitoring is investigated in only a limited number of
studies [62], where the main focus is usually on glucose sensing. The major challenge with ISF is
to extract a reasonable amount of analytes to the skin surface for downstream analysis, which
has been studied extensively within the frame of wearable biosensing technology (Box 3). In any
case, reliable correlations between ISF-blood concentrations are needed before ISF can replace
blood-based analysis.

Similar to ISF, saliva also reflects free analyte concentrations. Unlike epidermal matrices, saliva
can be collected easily even without patient stimulation. However, correlations between the
drug concentrations in saliva and blood are still being debated [63]. Drug concentrations in sa-
liva are a function of the sample collection method (whether or how it is stimulated), molecular
weight, dissociation constant, lipophilicity, pH, ionizability, and protein binding affinity, as the
transport mechanism through epithelial cells relies on both passive and active mechanisms
[60]. This complex dependency further increases the interpatient variability. As a result, it is
not always easy to develop correlations for free drug concentration. Another issue is the timing
of sampling, as equilibrium of analyte transport between the blood and saliva must be present.
Furthermore, contamination of saliva due to food, drink, or smoking, or bias from oral intake,
has to be minimized. Another contamination source is the sampling method itself, which is
usually not sterile.

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Other matrices can be utilized within the scope of TDM, which have their own unique traits making
them suitable for specialized applications. Tear fluid contains many proteins, peptides, lipids,
metabolites, and electrolytes [64], for which free blood concentration relationships can be derived
directly. More importantly, it is an ideal medium for the diagnosis of ocular diseases. The disad-
vantages of using tear fluid include the limited sample volume, unsuitability for self-sampling,
high inter- and intraindividual variability, potential evaporation, and concentration dependency
on the collection method. Ocular wearable sensors can avoid those limitations as they offer a di-
rect, continuous contact with basal tears. Currently, those wearable biosensors are being de-
signed in the form of contact lenses to exploit the well-correlated tear glucose concentrations
[65]. Nevertheless, the same approach, at least in theory, can be used to process proteins like
albumin that are correlated with different diseases.

Another example is hair, which is a sample of interest in forensics and toxicology. Unlike other ma-
trices, hair has the capability of providing a history of data as it ‘records’ the history of drug intake,
cessation, wrong dosage, and even drug abuse in line with the timing of hair growth. As a me-
dium, hair is stable, easy to transport, and hygienic. Nevertheless, the sample has to be proc-
essed to remove all external contamination and divided into sections of ‘known time intervals’.
The growth of each hair is not uniform, even for the same individual, and depends on each
hair’s individual life cycle. Hence, hair sectioning before analysis has inherent bias. More impor-
tantly, there is no existing correlation between hair and free blood concentrations for drugs rele-
vant to TDM [66].

There are also other readily available bodily fluids for TDM such as exhaled breath or urine. In the
case of exhaled breath, drugs have to pass from the blood stream to aerosol particles through
capillary walls, interstitial space, and epithelial cells. Although it is easily accessible, only small li-
pophilic molecules can pass through all of these barriers. Because of these mass transfer limita-
tions, concentrations in the aerosol particles are expected to be extremely low (on the order of
picograms to nanograms per liter), requiring very sensitive analytical methods. Urine samples
go through an even more complicated cycle, which composes a very rich sample at the end. Ac-
cordingly, urine can be processed easily for a variety of targets but for qualitative purposes only.
Here, the challenge is to handle the wide variation between and within the patients.

In short, alternative matrices offer a great potential for a wide range of future (especially for on-site)
TDM applications, yet there are two major prerequisites for alternative matrices: reliable correla-
tions linking the measured analyte levels to free blood concentrations and intensive clinical valida-
tion studies. Blood-based TDM, on the other hand, needs to evolve in order to minimize the
degree of invasiveness and the sample volumes required for the analysis.

Interpretation of the Results

The third and last stage of TDM is interpreting the measured analyte concentrations for a given
matrix, which occurs in clinical practice. After the initial dosage, a sample (for instance, blood)
is collected and sent to a certified laboratory. After determining the analyte concentration, this in-
formation is delivered to an expert who can interpret the result and adjust the dosing regimen for
the patient.

The first important consideration in this process is to decide when and how to collect the sample,
as errors in sampling timing are one of the major reasons for misinterpreting the measured data. For
a typical drug, the blood concentration is expected to reach a maximum value, which is followed by
a decay to a minimum value. Special care may be needed if there are concerns about toxicity, the
drug has a long half-life, or the patient suffers from impaired organ function leading to slower

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elimination (such as a kidney failure). Information regarding the time of sampling, current dosage,
and the time of drug intake should be reported together with patient clinical and demographic
data. Furthermore, storage and delivery limitations must also be taken into account while designing
the sampling frequency schedule [67].

The second and equally critical step starts when the measured data reach the physician who in-
terprets the drug concentration. The result is interpreted via dosing adaptation algorithms based
on the pharmacokinetic (PK) characteristics of the analyte of interest. The algorithms are typ-
ically developed using PK models, ideally built from large-scale studies that characterize PK var-
iability according to anthropometric and genetic factors, physiological and pathological history,
dietary habit, and possible drug–drug interactions (including herbal or nutritional supplements).
The model might be based on a large population, a subgroup of similar patients, or one single in-
dividual. The quality of the database can determine how successful the dose adjustment may be.
This model must also be ‘calibrated’ with respect to the analytical technique, that is, whether the
measured concentration reflects the unbounded (free) or total drug concentrations. With the
available information, the clinician decides whether dosage adjustments are required and deter-
mines a process for future monitoring.

When the entire cycle of TDM is considered, it is seen that the success of TDM depends not only
on the measurement accuracy of drug concentrations, but also adequate sampling, the quality
dose adaptation algorithm, and the extent of adjustments made to include the unique clinical
state of the individual. In this regard, the biggest challenge in TDM is the fact that reference interval
databases are mainly based on a limited number of healthy populations, while the borders of TDM
lie in between the efficacy and toxicity levels in special patient populations. Furthermore, individ-
uals are expected to have different PK and response dynamics, especially in the case of com-
bined therapies, which makes adjusting therapy highly challenging and requires expert clinical
judgment rather than strict adherence to PK algorithms only.

Translation Problems in On-Site TDM

Despite being studied thoroughly for more than two decades in the scientific community, sensor-
based approaches still have not achieved their potential for clinical application. In order to under-
stand what hampers the transition of these new technologies to the routine practice, we need to
revisit the definition of TDM and how it is understood by different practitioners. The common no-
tion of TDM indicates a decision-making process consisting of two steps: measurement of drug
or metabolite blood concentrations and clinical judgment based on the analyte concentration
ranges derived from previous clinical studies. Therefore, the ideal TDM application must be capa-
ble of providing precise, rapid, and accurate data in the measurement step and must be inte-
grated with an adaptive decision-making strategy.

The first criterion implies that all candidate methods, including biosensors, must ensure a certain
degree of uncertainty appropriate for the clinical decision. The analytical precision of a given mea-
surement is a function of random errors and can be quantified by the coefficient of variation
(CV). Although CVs less than 15% are accepted as a standard precision level for bioanalytical as-
says [68], narrow therapeutic ranges necessitate further improvements. The other source of error
is systematic error, which is usually encountered in the form of bias. Unlike random errors, sys-
tematic errors cannot be estimated statistically and can only be alleviated via proper calibration
of the instrument with standardized reference materials. Matrix effects and probe degradation
are two well-known sources of bias. In fact, systematic error is one of the greatest challenges
in TDM as only few standardized samples are available for manufacturers to assess possible sys-
tematic errors in their products. Measurements are usually standardized via quality control

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Trends in Biotechnology

Figure 4. Feedback-Controlled Therapeutic Drug Monitoring. The success of individualized dosing strongly relies on
two factors: how pharmacokinetic/pharmacodynamic (PK/PD) studies are integrated with TDM (PK databases) and how the
measurement process is managed (therapeutic drug monitoring cycle). PK/PD models consist of differential equations
describing how drugs are distributed, accumulated, and excreted [68,69]. By solving this initial value problem, physicians
can estimate the total amount of drug at a given time using the relationships between dosage regimen, metabolic activity,
and drug excretion. Accordingly, they can design an optimized dosing strategy to produce a desired serum concentration,
which is the main goal of TDM. Nevertheless, these model equations rely on some parameters (like decay or equilibrium
constants), which have been defined by observing the drug behavior in certain populations. This is why such models
cannot accurately predict the transient drug concentrations, even for the individuals from which these averaged fitting
parameters were derived. In other words, if these general PK models are directly used in any TDM scheme, it will be
practically impossible to reach the serum concentration targets set by the clinician. Accordingly, treatment cannot be
optimized for the patient. Therefore, a feedback mechanism similar to the one described above has to be integrated into
TDM practice. Abbreviation: IMM, interacting multiple model.

programs and proficiency tests performed among alternative technologies. In this regard, LC-MS
is accepted as a reference analytical method, while immunoassays are treated as routine
methods in clinical practice, where it is common to find benchmark studies comparing the pre-
dictions of immunoassays with those of LC-MS. Bias is sometimes observed across different
certified laboratories for the same analyte, even when similar LC-MS configurations are utilized
[68].

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As quantifying the uncertainty is of utmost importance for the clinical decision-making process, Outstanding Questions
any candidate technology proposed as an alternative to the current practice must be validated What are the challenges that possibly
in terms of accuracy and precision. When sensor-based studies are examined through this per- hinder the transition of sensor system
in the clinic for therapeutic drug
spective, the focus is on quantifying detection capabilities, usually in spiked simple reference
monitoring (TDM)?
media. Regrettably, no reported study has demonstrated the sensor-to-sensor variability, preci-
sion, and accuracy of the proposed approach with real samples collected from patients by quan- Which drugs are eligible for TDM? How
tifying the bias of the developed sensor (i.e., benchmarking the predictions with respect to can one judge its eligibility?
clinically accepted methods, LC-MS/MS). In our opinion, this is the main reason why sensor- What are the limitations of noninvasive
based technologies have yet to achieve a breakthrough in practice despite their inherent advan- matrices within the frame of TDM?
tages (low-cost, easy-to-use, fast and easily accessible, capability to measure unbound concen-
What are the practical consequences
trations), which offer an ideal remedy to alleviate expensive, centralized chromatographic
of the way we interpret the ‘M’ of
methods and to enable on-site applications. Therefore, translation of sensor-based technologies TDM: do we seek monitoring or
demands active collaboration between certified laboratories, sensor developers, and clinicians. In management?
this context, validation and translation of immunoassays into clinical practice can be studied as a
What should be the expected end-
process model during the development stage of sensor-based technologies in order to answer
product cost? Would it be affordable,
the practical needs of the clinical community. particularly for many patients in
resource-limited settings?
Technical realization with respect to accurate and precise measurement of the analyte concentra-
Could artificial intelligence play a role in
tion with sensor-based technologies is only one important part of the TDM process. Perhaps a future applications of TDM?
bigger translation challenge is interpreting the result in the context of therapeutic target concen-
trations. For any drug, the magnitude of both desired and undesired effects increases with in-
creasing drug serum concentration. Nevertheless, the rate of change in response to drug
concentration is different for therapeutic and toxic effects, so there exist concentration ranges
where the benefit from a dose increase is much greater than the risks posed by the resultant
higher exposure. These ‘operational windows’ are called the therapeutic range [69]. In a typical
TDM practice, clinical laboratories determine the serum concentrations and report it back with
typical therapeutic ranges without seeing the patient or evaluating the individual response to
the therapy. Therefore, the patients’ serum concentrations are categorized as either subthera-
peutic, therapeutic, or toxic, according to statistically accepted ranges derived from small trials
or healthy populations. At best, new dosage regimens are recommended to adjust the serum
concentrations.

In this regard, TDM is an extension of a ‘one-size-fits-all’ approach. In an ideal TDM program,

however, the dynamics of the treatment process must be tailored in advance according to the re-
quirements of each individual by integrating other quantitative tools. This can be achieved by de-
signing an individualized feedback control loop (Figure 4). Unfortunately, this loop brings a much
greater burden to the clinical laboratory: many more samples are needed to be processed even
more frequently to tailor both the PK/pharmacodynamic (PD) model and the therapeutic tar-
get for the individual patient. This burden, necessary for TDM to fulfil its raison d'être, obviously
cannot be left to centralized laboratories or expensive spectroscopic methods for a large-scale
implementation of TDM. This profound demand is what makes biosensor technology a unique
and inevitable partner for personalized on-site TDM. The scientific community should, therefore,
be aware of these practical needs of the clinical community and develop technologies in this
context.

Concluding Remarks and Future Perspectives

The concept of TDM has evolved from passive monitoring of what has been achieved via previous
dosage regimens to the active management of free drug concentrations in blood for the optimum
benefit of each individual patient. In order to respond to the needs of the clinical community, the
complex and dynamic nature of the personalized treatment process needs to be understood (see

Trends in Biotechnology, Month 2020, Vol. xx, No. xx 13

Trends in Biotechnology

Outstanding Questions). Despite the extensive effort and numerous publications in the field of
biosensing and other novel technologies, none have broadly diffused into TDM practice like
their predecessors, such as the commonly used immunoassays.

In our opinion, there are three major reasons behind this gap between the clinical and scientific
communities. The first and most obvious challenge is the lack of standardization and validation
of these novel technologies. This is not just a technical challenge but also an economic and
practice-based challenge, as this technical realization step necessitates a strong collaboration
between academic, industrial, and medical partners. In this regard, the ideal time for developing
a TDM protocol could be the drug development stages, where it can be coupled with the devel-
opment of PK databases. This collaboration can follow in the successful footsteps of prior analyt-
ical methods like immunoassays. The success of this step is strongly related to the second
challenge: awareness of the dynamic nature of TDM. The TDM approach is not a linear process;
the ultimate goal is not to determine drug concentrations and it definitely does not end with this
measurement step. On the contrary, it is the beginning of a complex feedback control process,
which has to be tuned for each individual during therapy. This dynamic nature has profound re-
lationships with the behavior of the drug in each individual (PK), so it necessitates a number of
measurements to optimize the drug regimen for the patient compared with a simple linear pro-
cess (where knowing the drug concentration level would be sufficient to make a clinical judg-
ment). This underappreciated issue implies two practical outcomes: the technique has to be
simple, fast, and economical enough to make TDM feasible, and the measurement step has to
be decentralized and performed on-site in order to rapidly respond to this dynamic nature. There-
fore, proposed technical solutions have to be developed with these limitations as the design con-
straints. This development perspective is also related to the third challenge: the right sensing
technology must be developed at the very beginning for the right drug, which is collected in the
right matrix with the adequate sampling scheme and with the proper sample and data transfer
solutions.

Acknowledgements
C.D. and G.U. would like to thank the German Research Foundation (DFG) for partially funding this work under grant num-
bers UR 70/15-1 and DI-2345/3. J.A.R. would like to acknowledge funding from the Australian National Health and Medical
Research Council for a Centre of Research Excellence (APP1099452) and a Practitioner Fellowship (APP1117065). A.E.G.
C. would like to acknowledge that his research was partly funded by the National Institute for Health Research (NIHR)’s In-
vention for Innovation (i4i) Programme (Grant Reference Number II-LA-0313-20004). The views expressed are those of the
author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

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