ORIGINAL ARTICLESA Fully Integrated Assay Panel for Early Drug
Metabolism and Pharmacokinetics Profiling
Johan Wernevik,1 Fredrik Bergström,2 Anna Novén,1Johan Hulthe,1 Linda Fredlund,1 Dan Addison,3 Jan Holmgren,4
Per-Erik Strömstedt,1 Erika Rehnström,5 and Thomas Lundbäck1
1

Mechanistic Biology & Profiling, Discovery Sciences, R&D,
AstraZeneca, Gothenburg, Sweden.
2
DMPK, Early CVRM, BioPharmaceuticals R&D, AstraZeneca,
Gothenburg, Sweden.
3
Sample Management, Discovery Sciences, R&D, AstraZeneca,
Cambridge, United Kingdom.
4
Sample Management, Discovery Sciences, R&D, AstraZeneca,
Gothenburg, Sweden.
5
Clinical Sampling & Alliances, Precision Medicine, AstraZeneca,
Gothenburg, Sweden.

ABSTRACT
Evaluation and optimization of physicochemical and metabolic
properties of compounds are a crucial component of the drug
development process. Continuous access to this information
during the design-make-test-analysis cycle enables identificationof chemical entities with suitable properties for efficient projectprogression. In this study, we describe an integrated and automated assay panel (DMPK Wave 1) that informs weekly on lipophilicity, solubility, human plasma protein binding, andmetabolic stability in rat hepatocytes and human liver microsomes. All assays are running in 96-well format with ultraperformance liquid chromatography–mass spectrometry (MS)/MS
as read-out. A streamlined overall workflow has been developedby optimizing all parts of the process, including shipping ofcompounds between sites, use of fit-for-purpose equipment andinformation systems, and technology for compound requesting,data analysis, and reporting. As a result, lead times can beachieved that well match project demands across sites independently of where compounds are synthesized. This robust
screening strategy is run on a weekly basis and enables optimization of structure-activity relationships in parallel with DMPK
properties to allow efficient and informed decision making.
Keywords: mass spectrometry, automation, DMPK, profiling,
assay panels

INTRODUCTION

R

eduction of attrition rates in clinical studies is a criticalobjective for improving R&D efficiency.1 Hence, significant investments are going into the optimizationof compounds toward candidate drugs, such that theirproperties allow adequate testing of clinical hypotheses. This isachieved through iterative design-make-test-analyze (DMTA)
cycles, where compounds are characterized for their impact on
the pharmacology of interest as well as aspects that control
drug metabolism and pharmacokinetic (DMPK) properties.2–4
Historically DMPK assays were run on selected compounds
as dictated by low-throughput assays. As these assays weretransferred to 96-well formats the capacity grew to support
bespoke orders placed by DMPK project leads, with waves of
assay panels of increasing complexity supporting projects
based on maturity and needs.5 In 2012, AstraZeneca initiateda strategic initiative, in which part of the synthetic chemistry
and DMPK work was outsourced, with careful efforts to ensure
concordance between tests and results between sites.6
This on-going research exchange includes the DMPK Wave
1 panel of assays, which report on physicochemical properties
such as lipophilicity (logD7.4) and solubility (Sol), plasma
protein binding (PPB), and two metabolic stability assays inhuman liver microsomes (HLMs) (Mics) and rat hepatocytes(Heps), respectively. As of today, this panel is run on a weekly
basis on all synthesized compounds across AstraZeneca toensure maximal integration of DMPK aspects already at earlystages of the DMTA cycle.
Some of the individual assays within the DMPK Wave 1 panel
have been previously described,7,8 while extensive efforts havesubsequently been completed to integrate the assays in a fully
automated workflow. In addition, these assays have over time
been consolidated to one of our R&D facilities in Gothenburg anda partner site (Pharmaron). The assay workflow is schematicallyillustrated in Figure 1 and includes compound requests, shippingof compounds to Gothenburg if not already available, establishment of mass spectrometry (MS)-based analysis methods forthe individual compounds, completion of the five assays, andreporting through Genedata Screener to our internal database.

ª Johan Wernevik et al. 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1089/adt.2020.970

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Fig. 1. Schematic illustration of the integrated process for the DMPK Wave 1 panel in Gothenburg. Assay requests are made automaticallyor manually by DMPK or Medicinal Chemistry leads (1), triggering provisioning of samples via Sample Management to Gothenburg (2).
Shipping complies with a 48 h time frame from the United Kingdom and a 72 h time frame from the United States. Once available,
compounds are ordered to test, with one set of plates for the panel assays (3) and another set of plates for establishment of analyticalmethods (4). Ordering is integrated with compound pooling scripts based on their monoisotopic mass to reduce analysis time on the MS
instrumentation (5). Each assay also includes dedicated assay performance controls (6). LC/MS/MS methods are created over the weekend(7) and stored and shared between instruments. The assays start weekly and run between 1 and 2 days (8), with identification andintegration of chromatographic peaks in dedicated software (9). Integrated data are imported into GeneData Screener for further quality
control and calculations (10) and finally made available globally by submission into an internal database (11). DMPK, drug metabolism andpharmacokinetic; LC, liquid chromatography; MS, mass spectrometry. Color images are available online.

In this study, we describe the integrated workflow, including detailed descriptions of test methods and reporting
routines. Finally, we provide assay metrics data after 6 yearsof operation and detail a case study where the assay panel had
a significant impact on compound optimization.

MATERIALS AND METHODS
Reagents and Consumables
All compounds and quality controls (QCs) were obtainedinternally from Sample Management facilities as 10 mM stock
solutions in dimethyl sulfoxide (DMSO). DMSO was from
Sigma-Aldrich (D2650), formic acid (FA) was of p.a. quality(e.g., Merck, Germany), acetonitrile (AcN) was of high performance liquid chromatography (HPLC) grade (e.g., Rathburn, Scotland), water was from a Milli-Q system (Millipore),and 1-Octanol was from Sigma-Aldrich (293245). Pipette tips
for the Hamilton STARplus liquid handling robot were ZT-

158 ASSAY and Drug Development Technologies MAY/JUNE 2020

100-L-R (301-12-101; Genuine Axygen) and ZT-200-L-R(301-12-251; Genuine Axygen). Microplates and accessories
were Nunc 2 mL polypropylene (PP)-based deep well plate(278752; Thermo Fisher Scientific), Nunc 1 mL 96-deep well PP
Plate Natural (260252; Thermo Fisher Scientific), conical Nunc
plates 0.45 mL/well (249944; Thermo Fisher Scientific), Nunc
microplate lids (263339; Thermo Fisher Scientific), Nunc 96well cap natural (276002; Thermo Fisher Scientific).
Assay Automation
Three Hamilton Microlab STARplus robots (Hamilton Robotics, USA) controlled with Hamilton’s Venus Software havebeen setup in an identical configuration to support all five
DMPK Wave 1 assays. The setup of the robots consists of a 96well pipetting head, an eight-channel pipetting arm, both
capable of moving plates or lids. Several carriers that are used
for the different assays are available and interchangeable

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between robots. Each robot is equipped with three Hamilton
heated shakers that are temperature controlled and have acustom holder for the plate types used for the reaction plates in
the metabolic stability assays. This minimizes potential edge
effects due to temperature differences between ambient andincubation temperature. In addition, there are 10 temperaturecontrolled positions (water bath) with custom adapters for theplates.
Ultraperformance Liquid Chromatography/MS
Sensitive, selective, and rapid methods for the analysis ofsamples from the assays were based on ultraperformance liquid chromatography (UPLC-MS)/MS. The chromatographic
separations are performed on a Waters Acquity UPLC HSS T3
column packed with 1.8 mm particles, 2.1 · 30 mm, with a shortgradient elution for samples from metabolic incubations. Here,the mobile phases consist of A (water with 0.1% FA) and B(AcN with 0.1% FA). The liquid chromatography (LC) gradientprofile is as follows: 0.2% B during 0 to 0.1 min, a linear increase to 95% B during 0.1 to 0.7 min, hold at 95% B during 0.7
to 1.0 min, and then back to 0.2% B from 1.01 to 1.1 min.
Analysis of samples from the logD7.4, Sol, and PPB assays,
where up to 13 different analytes are analyzed, was based on a
Waters Acquity UPLC HSS T3 column packed with 1.8 mm
particles, 2.1 · 50 mm, and a 2 min gradient elution.
The LC gradient profile for these assays is as follows: 0.2% B
during 0 to 0.3 min, a linear increase to 95% B during 0.3 to
1.3 min, hold at 95% B during 1.3 to 1.8 min, and then back to
0.2% B from 1.8 to 1.81 min. The flow rate is 1 mL/min with a
total run time of 1.5 and 2.9 min, respectively, per sample. Detection is achieved by the multiple reactions monitoring (MRM)of the transitions determined in the optimization process by using
QuanOptimize (Waters, Manchester, United Kingdom). LC-MS iscarried out on a Waters Xevo TQS triple quadrupole equippedwith a Waters Acquity UPLC interface, allowing switching between positive and negative ionization modes (Waters).
MS/MS Optimization
All compounds to be screened are delivered from in-house
Sample Management as 10 mM DMSO solutions in 96-wellformat. The compounds are diluted in a 1:2 AcN/water (0.1%
FA) solution. We consider 1 mM optimal for optimization on
the Waters Xevo TQS instrument and 5 mM for using the Waters TQD instrument. The QuanOptimize software (Waters) isused as a high-throughput tool for automated method development and batch processing of quantitative bioassays. The
QuanOptimize routines can ramp and optimize cone voltage
and collision energy. The cone voltage is set to 35 eV for allcompounds and only the collision energy is optimized and

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ramped both in positive and negative mode between 10 and
50 eV in steps of six.
The software identifies the most intense fragment and thecorresponding collision energy and creates an MRM transition, which is stored in an MS/MS library. The MS/MS libraryis shared between the LC-MS/MS instruments. QuanOptimize
is also used for creating the sample list by generating MS/MS
files from the MS/MS library combining up to 13 different
MRM transitions, LC file, MS/MS tune file, and creating
quantification methods used for peak integration, calibration,and quantification.
Pooling
Pooling of compounds is used as a strategy to increase assay
capacity and reduce analysis time in the mass spectrometers.
All assays have dedicated excel-based macros to enable optimal
pooling. In the logD7.4 and PPB assays, pooling of compounds
occurs at assay start, whereas in the Sol, Mics, and Heps assays
pooling is achieved after the experimental procedures, but
before analysis. We recognize that there is a risk of interactions
and/or interferences between compounds in the same pools,
especially in the logD7.4 and PPB assays as pooling is done
already in the physically/biologically relevant assay step. These
concerns are addressed in the main text for each of these assays,
each of which is qualified by the illustration of test results fromtwo separate test occasions (and thus different pools) and between AstraZeneca and the Contract Research Organization(CRO) (also representing different pools).
Up to 13 compounds are pooled, depending on the assay. To
avoid coelution, the pooling is designed by sorting the compounds based on molecular weight and then the compoundwith lowest mass will be placed in pool 1 and the second
lowest in pool 2, and after 10–13 pools, there is enough difference to avoid mass conflicts in the first quadrupole of themass spectrometer. Fast scanning triple quadrupole mass
spectrometers are used to get ten data points over the chromatographic peak that is about 1.5 s wide at baseline. Positive
and negative MS/MS methods are often combined. UPLC is
used to increase selectivity and sensitivity.
Statistical Analysis
All assays include three or more QCs in each run and two
statistical tools are used to monitor performance of the control
compounds. First, the in-house developed ‘‘Manhattan tool’’,
which allows monitoring of the performance of the control
compounds by plotting the data against the date of the run.6
Acceptance criteria for control compounds are defined foreach control in all the assays and a statistical change in theperformance of any control is visualized in a way that allows

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the experimenter to pass or fail a run. Second, the ‘‘Minimum
Discriminatory Difference/Ratio’’ (MDD/MDR) is monitored asa measure of intra- and intersite assay variability, estimating
whether a difference in the data generated for two compounds
is likely to be a real difference or not.6 It does so by defining
the statistically significant difference/ratio threshold at the
95% confidence level.

RESULTS
Integration of Assay Workflows
The consideration of DMPK properties was historicallyachieved within our organization through ordering of bespoke assays by DMPK leads as motivated by project needs.
The throughput of these assays varied considerably, ranging from tens of compounds per week to several hundreds,
often necessitating prioritization between projects. To improve data coverage and strengthen our abilities to predict
DMPK properties, we implemented the DMPK Wave 1 panelof assays.6 This panel provides weekly delivery of lipophilicity, solubility, PPB, and metabolic stability in rat hepatocytes and HLMs on all compounds synthesized globally
within AstraZeneca. The integration into a combined panel
includes a common ordering and delivery process and analytical method development that is shared between the fiveassays (Fig. 1).
Orders to the panel triggers a provisioning and shipping ofsamples (113 mL of a 10 mM stock solution in DMSO) whenrequired to our R&D facility in Gothenburg. Service Level
Agreement for these shipments is at 48 h for samples from theUnited Kingdom and 72 h for United States. Samples are collected during the week and an order for plating is submitted on
Friday mornings, with deliveries before lunch of four sets ofplates to support method development and the individual assays. These sets are complemented with plates holding compounds separately ordered to the respective assays. Analyticalmethod development is initiated from one of the four sets of 96well plates, with somewhere in the range of 150–300 compounds/week, using QuanOptimize (Waters) to generate MRM
methods. This process runs over the weekend and involves
ramping of collision energy and switching between positive andnegative ionization modes.9,10 Established methods are saved ina MS/MS method library (see Materials and Methods section for
details) for use with each assay.
This generic approach is successful in establishing methods
for >95% of submitted compounds. Our process for failed
compounds, for example, multicharged or in-source fragmented compounds, include assessment of identity and purity
and subsequent full scans in an appropriate mass range(commonly 100–1,100 g/mol) to identify responses that can

160 ASSAY and Drug Development Technologies MAY/JUNE 2020

be traced back to the parent molecules. In a year, <3% of
submitted compounds fail these routines and are investigatedseparately as motivated. Next all compounds are combined inpools of 2–10, ensuring maximal mass distribution, using inhouse scripting routines (Microsoft Excel). These routines simultaneously generate run methods for all liquid handlingand MS analysis steps. Finally, the utility of the established
MRM methods is corroborated by means of chromatographic
separation, investigating peak shape and area to generate
UPLC-MS/MS methods. Finally, all samples are matrix matched and differ only in compound concentrations, except forany metabolites or other products formed.
Panel testing is achieved based on the three remaining
plate sets (logD, Sol, and PPB share source plates) using acommon liquid handling platform as detailed below for eachassay. Peak identity and size are evaluated using TargetLynx (Waters) and subsequent evaluations of linearity in response and verifications of pool and run performance throughQCs is achieved in Genedata Screener according to businessrules. Importantly, retention time warnings are integrated into
the evaluation templates, thus eliminating the risk that a peakfrom a potential metabolite, with the same molecular weightand MS fragment as a compound within the pool, is mistakenly integrated. Finally, qualified results are published in our
database between Tuesday and Thursday of each week.
Lipophilicity (logD7.4)
Background description. A critical physicochemical property that
affects the availability of compounds in biological test systems,
animal models of disease, and man is the lipophilicity. Describedas one of the components of the Lipinski rule of five for druglike molecules, this property reflects the distribution of compound between 1-octanol and an aqueous solution reported as alogP value (referred to as logD7.4 when the distribution extends
to include also charged species).11,12 The original Lipinski rule
pointed at the need for logP values below 5, while later refinements argues for a more narrow distribution with a desirable
range for drug-like compounds between 1 and 3.13
Practically, these measurements can be achieved using one
of two basic principles: (1) by sampling from octanol andbuffer samples after thorough shaking and equilibration(shake-flask method)14 or (2) by a chromatographic method,in which the retention of compounds on a hydrophobic C18
column is measured and used as a proxy of the distribution bycomparisons with standards with established logD7.4 values.15,16 The logD7.4 assay within our DMPK Wave 1 panel isconducted in a 96-deep well format at controlled pH (7.4) andrepresents a fully automated variant of the shake-flask
methodology.7

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Assay workflow. The logD7.4 assay workflow is schematically
depicted in Figure 2 and outlined in detail in Table 1. The assay
starts with transfer of individual compound solutions into
pools of 10 compounds, alongside two pool QCs, using a
Hamilton STARplus liquid handling workstation. This poolingis achieved using unique run lists for each week, generated
through in-house scripts based on delivery notes from Sample
Management. This step combines 4 mL each of 10 mM compound solutions to a total of 48 mL in an intermediate plate(pool QCs are at 5 mM each). These and subsequent transfers as
outlined below involve fresh pipetting tips to eliminate risksof cross-contamination.
The next assay step involves transfer of 500 mL octanol froma tray to an empty deep well plate, to which 10 mL of each poolis subsequently transferred and thoroughly mixed by repetitive aspiration and dispensing. An octanol-saturated phosphate buffer at pH 7.4 is then added (500 mL) followed bysealing of the content with a PP-based lid. Vigorous shaking

Fig. 2. (A) Schematic illustration of the workflow for the logD7.4 assay. (a) Up to four compound plates with 93 mL in each well are
obtained from Sample Management (the same source plates are used for the solubility and PPB assays). (b) Compound pools are createdin an intermediate dilution plate through cherry-picking on a Hamilton STARplus. (c) Each pool is transferred to a deep well platecontaining 1-octanol, mixed and complemented with aqueous buffer. (d) Samples are vortexed, equilibrated and centrifuged. (e) Samplingand serial dilution of both octanol and buffer phases is achieved on the same liquid handling station, with subsequent detection performed
for the combined pools using UPLC-MS. (B) Correlation between data in the automated 96-well logD7.4 assay versus literature values for
44 commercially available compounds. The slope between average values from up to five independent measurements with previously
reported data is 0.992 (r2 = 0.976). Dotted lines represent a 95% CI of 0.943–1.04. (C) Correlation between two independent experiments
submitted from this assay to our internal database. When more than two independent replicates were available, we included the first two
instances. The slope based on testing of 1,856 compounds comes out at 0.992 (r2 = 0.970) with a 95% CI of 0.984–1.00. (D) Correlation
between harmonized assays at AstraZeneca Gothenburg (y-axis) and at Pharmaron (x-axis). The comparison is based on 1,682 compoundswith a slope of 0.985 (r2 = 0.916) and a 95% CI of 0.970–0.999. (E) Manhattan plot following 6 years of operation from June 2013 to April
2019—nicardipine (brown filled circle), cyclobenzaprine (green filled triangle), and caffeine (black filled diamond). CI, confidence interval;
PPB, plasma protein binding; UPLC, ultraperformance liquid chromatography. Color images are available online.

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Table 1. Protocol for the Shake-Flask logD7.4 Assay
Step

Parameter

Value

1

Add octanol

500 mL2

Add compounds and controls

10 mL

Description

10 test compounds (at 10 mM in
DMSO) + two QCs (nicardipine andcyclobenzaprine at 5 mM each) arepooled3

Add buffer

500 mL

4

Vigorous shaking

1 min

5

Equilibration shaking

3hRoom temperature

6

Centrifugation

30 min, 3,000 · g

Room temperature

7

Octanol sampling

5 mL

8

Removal of Octanol

495 mL9

Buffer sampling

20 mL

10

Sample dilution series

1:10

Phosphate buffer pH 7.4

Buffer samples are serially diluted(1:10) in acetonitrile/water (1:2,
0.1% FA, 2 nM verapamil—IS
solution) in four steps and octanol
samples in five steps

11

Plate sealing

12

Assay readout

Peak area

LC/MS, TargetLynx

13

Data evaluation

logD7.4

Genedata Screener

Step Notes
1–3. Octanol, compounds and buffer are added to a 2 mL PP-based deep well plate (ProductNo.: 278752; Thermo Fisher Scientific) using a Hamilton STARplus. A PP-based microtiterplate (Product No.: 249944; Thermo Fisher Scientific) is used as an intermediate plate forthe compound pooling.
4–5. The 2 mL plate is lidded (Product No.: 276002; Thermo Fisher Scientific) and shaken
vigorously followed by equilibration under shaking for 3 h.
6. The phases are separated by centrifugation of the 2 mL plate in an Eppendorf 5810R(Eppendorf).
7–10. Sampling and removal of octanol is carried out on the Hamilton STARplus.
11. Plates are manually sealed with a 96-well cap natural seal (Product No.: 276002; Thermo
Fisher Scientific).
12. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS.
Chromatograms are evaluated using Waters TargetLynx software.
13. Data evaluation is achieved through Genedata Screener and approved results are reported
to our internal database D360.
AcN, acetonitrile; DMSO, dimethyl sulfoxide; FA, formic acid; IS, internal standard; LC/MS, liquid
chromatography/mass spectrometry; PP, polypropylene; QC, quality control.

of the content is achieved through vortexing for 30 s andequilibration of compound between the two phases is further
established through a 3 h incubation on a plate shaker.
Following equilibration, the deep well plate is centrifuged
to ensure separation between phases and the plate is next

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moved back to the Hamilton platform for samplingof 5 mL from the octanol phase. The sample isplaced in a first dilution plate together with 495 mLof an AcN:H2O mix (1:2) containing FA (0.1%) andverapamil as internal standard (IS). The acidified
and IS containing AcN solution, which is common
for the logD7.4 and Sol assays, is referred to as the
IS solution. The content is thoroughly mixed andnext serially diluted to four additional plates with
10-fold dilutions to maximize the ability to detect
weakly concentrated solutions and afford investigations of linearity in response.
After sampling, the remaining octanol is removed
from the deep well plate through careful pipetting,followed by sampling from the aqueous buffer. Thispipetting step is preceded by the prior sampling of a
5 mL plug of the AcN:H2O solution to avoid octanol
contamination, and the pipette tips are additionallywashed (see Assay validation and concordance testing section) before delivery of 20 mL samples from theaqueous solutions to the dilution plates. Also thesesamples are correspondingly diluted, and all plates
are sealed with rubber lids that are compatible with
the injection needles in the LC-MS instrumentation.
The plates are next brought to the MS instrumentation (Xevo TQS; Waters) and read out using theestablished UPLC-MS/MS methods. Run lists fromthe in-house scripts are imported into MassLynx tocombine MRM methods and peak integration methods for all compounds within a pool using QuanOptimize. Data from each run are first processed in
MassLynx and peak identities and integrated peakareas are next confirmed in TargetLynx. Confirmed
data are exported to a raw data server for final
evaluation in Genedata Screener. All aspects of theassays are documented in electronic laboratory notes,
including compound identities, method descriptions,chromatograms, and a Genedata Screener report.
Calculated logD values are exported into our internal
database ensuring global availability to users.
Choice of validation test sets, QC compounds, and IS. A

set of 44 commercially available compounds covering a logD7.4 range from -1.4 to 6.1 were selected for experimental validation of the automated method(Supplementary Table S1). We also included controls allowing
for assessment of reproducibility between runs and between
pools of compounds within each run (Table 2). These controls
were chosen to reflect polar compounds with low logD7.4

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Table 2. Quality Controls and Internal Standards
Name

Used inMonoisotopic
mass (g/mol)MRM
methodConeVoltage (V)Collisionenergy (V)Caffeine

logD7.4

194.1(+) 195.1–138.0

35

16

Cyclobenzaprine

logD7.4

275.2(+) 276.0–216.0

35

22

Nicardipine

logD7.4

479.2(+) 480.2–315.0

35

22Verapamil

logD7.4, Sol, Mics, Heps

454.3(+) 455.3–165.07028

Astemizole

Sol

458.2

(+) 459.3–135.1

35

40

Phenytoin

Sol

252.1

(+) 253.1–182.1

35

16

Chlorpromazine

Sol

318.1

(+) 319.1–86.0

35

22

Warfarin

PPB

308.1

(+) 309.1–162.9

5

16Propranolol

PPB

259.2

(+) 260.1–116.0

33

16Metoprolol

PPB, Mics

267.2(+) 268.2–116.3

35

16

5,5-diethyl-1,3-diphenyl-

PPB, Mics, Heps

335.2(+) 336.2–195.0

46

28

Diclofenac

Mics

295.0

(+) 296.0–214.2

3540

Imipramine

Mics

280.2

(+) 281.4–86.1

35

16

Phenacetine

Mics

179.1

(+) 180.1–110.2

35

22

Benzydamine

Mics

309.2

(+) 310.2–86.1

35

16

Bosentan

Heps

551.2

(+) 552.2–202.0

35

28

Dofetilide

Heps

441.1

(+) 442.1–197.9

528

Indapamide

Heps

365.1

(+) 366.1–132.0

22

16

Lorazepam

Heps

320.0

(+) 321.0–274.9

35

22

Terfenadine

Heps

471.3

(+) 472.3–436.2

35

282-iminobarbituric acid

Heps, assay for metabolic stability in rat hepatocytes; Mics, assay for metabolic stability in human liver microsomes; PPB, plasma protein binding; Sol, solubility.

values (caffeine at 0.117), intermediate values (cyclobenzaprine at 2.88,18), and more lipophilic compounds with highlogD7.4 values (nicardipine at 4.5—in-house data).
In our setup, caffeine is included randomly as a control
between runs. Nicardipine is a good indicator of octanol
contamination when sampling the buffer side and is therefore
included in each pool and used to fail affected pools when thelogD7.4 is lower than expected. Each pool also includes cyclobenzaprine with an intermediate logD7.4 to report on
consistency between pools. To verify consistency between
injection volumes, all samples contain 2 nM of verapamil (IS).
Assay validation and concordance testing. The key challenges

associated with automation of the shake-flask assay concern

MARY ANN LIEBERT, INC.  VOL. 18(1) equilibration between phases in a microplate format; (2)octanol contamination of the aqueous phase when sampling;and (3) the large difference in concentrations between phases,
which is especially pronounced for highly lipophilic compounds. The first challenge was solved through capping of the
deep well plates and vigorous mixing of the two phases byvortexing followed by a 3 h equilibration period on a plateshaker. We compared the output of this approach with reference values taken from the literature17 as shown in Figure 2B,
demonstrating that conditions closely resembling equilibrium
was reached using this protocol and time period.
Automation of sampling from the aqueous phase presents
significant challenges. Prior removal of the viscous octanol
was achieved through a series of slow aspiration steps close to

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the buffer surface, with final aspiration achieved from within
the aqueous solution. As already mentioned, contaminationof any residual octanol, when sampling 50 mL of the buffer
sample, is further prevented through the introduction of a plugin the tip before entering the aqueous phase.
We next discard 20 mL of this solution and wash the outsideof the tips extensively by serial dipping into four independent
AcN solutions. The sample is finally introduced into the dilution plate and the remainder of the solution discarded. The thirdchallenge was addressed by automated 10-fold serial dilutions
in multiple steps following sampling and assessment of linearity in response between dilutions, enabling measurements oflogD values at either end of the scale from about -1 to 4. Thiswas considered enough for a Wave 1 screening assay.
Establishment of the automated logD assay entailed testingof reproducibility as illustrated in Figure 2C, which compares
data for all compounds that have been tested at a minimum of
two different occasions in the assay (the first two instanceswere used for the illustration in case data are available for
additional test occasions). Agreement between test occasions
is excellent with few outliers (95% of compounds differing<0.3 between runs).
To ensure rapid availability of DMPK Wave 1 data for compounds synthesized at our external partner Pharmaron, efforts
were put in place to fully harmonize this assay panel.6 A review
of data in our database now includes a total of 898 compounds
that have been tested both internally and at Pharmaron, with acomparison of results illustrated in Figure 2D. The data show
acceptable agreement with 87% of compounds within 0.3 between sites. As described in detail below these assays have beenrunning since 2013 and the performance of the QC compounds
over a 6-year period at AstraZeneca R&D in Gothenburg is illustrated in Figure 2E. Except for a few occasions, the assay has
been stably reporting expected values for these compounds withthe largest variability observed for nicardipine, reflecting thechallenges associated with octanol contamination when sampling the aqueous buffer.
Solubility (Sol)
Background description. Solubility in aqueous solution is aparameter that determines whether a biological test system ororganism is appropriately exposed to the compound. It also
affects our ability to create suitable formulations for preclinical and clinical studies.19,20 For most test assays in earlydrug discovery, we report values based on nominal concentrations, that is, based on what is added to the test system,
rather than performing actual measurements of these concentrations. This can be visible in concentration responses
both in biochemical and cellular assay formats, where in-

164 ASSAY and Drug Development Technologies MAY/JUNE 2020

complete saturation or apparent bell-shaped responses can beobserved for poorly soluble compounds simply because theyfall out of solution.
Early solubility assessments are therefore critical for accurate interpretation of these responses and for understandingof structure-activity relationship (SAR). Solubility measurements can be achieved in several different ways and generally
starts from known amounts of solid compound (lowthroughput assays) or from DMSO stock solutions with orwithout removal of solvent.21,22 The solubility assay within
the DMPK Wave 1 panel is based on the dried DMSO principle,
is conducted in glass vials placed in a 96-vial rack holder, andrepresents a further miniaturized variant of previously published assays from AstraZeneca.7,23
Assay workflow. The solubility workflow is illustrated in Fig-ure 3, with a detailed description of each assay step in Table 3.
Unlike the logD7.4 assay compounds are not pooled, but instead 50 mL of individual DMSO stock solutions are transferred
to glass vials kept in a 3D printed rack that facilitates subsequent liquid transfers using a 96-well pipetting head on the
Hamilton STARplus platform. There is no pool QC in the Sol
workflow, instead this is replaced with the inclusion of three
QCs, the positioning of which is randomized for each week.
Solvent is evaporated and the dried compounds are resolubilized in 500 mL of aqueous phosphate buffer at pH 7.4.
Individual Stir Stixs are added to each glass vial using adrop dispenser from V&P Scientific, Inc., a rubber mat is used
to seal the vials, and samples are then equilibrated under
mixing for 20–24 h. The following day starts with removal of
the Stir Stixs and subsequent centrifugation to pellet insolublecompound, before 200 mL of the supernatants are transferred
to new glass vials on the Hamilton STARplus platform.
The process of centrifugation and transfer is repeated to remove any remaining debris, except that the 20 mL supernatanttransfer combines sampling and pooling of 10 samples to a poolplate with 200 mL of an IS solution (same as in logD7.4). As
mentioned previously, this pooling is achieved using unique
run lists for each week. The liquid handling workflow is finished with transfer of 40 mL of the pooled samples to an analysis
plate, where the samples are diluted fivefold in IS solution(160 mL). Standard curves are generated for each pool by
combining and diluting 5 mL of each compound DMSO stock
solution in the IS solution. The pooled samples are seriallydiluted in a four-point series to accommodate the span from 1
to 1,000 mM. Finally, sample and standard plates are sealed andread out, analyzed, and qualified as described for the logD7.4
assay. Calculated solubility values (in mM) are exported into ourinternal database ensuring global availability to users.

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Fig. 3. (A) Schematic illustration of the workflow for the Sol assay. (a) Samples are provided from the same source plates as those used
for the logD7.4 and PPB assays. (b) Individual compound solutions are transferred to glass vials in 96-well racks. (c) DMSO is evaporated
and dry compounds are solubilized in aqueous buffer and stirring bars are added to each vial. (d) Samples are equilibrated at 25C withshaking for 20–24 h. Plates are centrifuged and cleared supernatants are transferred to fresh glass vials and centrifuged again. (e) Clearedsupernatants are pooled (up to 10 samples/pool) and serially diluted in 50% acidified AcN:H2O with IS. Standard samples are treatedsimilarly in a parallel workflow, with subsequent detection performed using UPLC-MS. (B) Correlation between two independent experiments submitted to the database. When more than two independent replicates were available, we included the first two instances. The
slope based on testing of 1,219 compounds comes out at 0.911 (r2 = 0.814) with a 95% CI of 0.887–0.936. (C) Correlation between
harmonized assays at AstraZeneca Gothenburg (y-axis) and at Pharmaron (x-axis). The comparison is based on 1,200 compounds with aslope of 0.847 (r2 = 0.749) and a 95% CI of 0.819–0.875. (D) Manhattan plot following 6 years of operation from June 2013 to April 2019—astemizole (black filled diamond), phenytoin (green filled triangle), and chlorpromazine (brown filled circle). AcN, acetonitrile; DMSO,
dimethyl sulfoxide; IS, internal standard; Sol, solubility. Color images are available online.

Choice of QC compounds and IS. While the performance ofprevious versions of our miniaturized dried DMSO solubility
assay was validated through comparisons with a classicalthermodynamic solubility test,7,23 in this study, we describe
the inclusion of multiple QC compounds for assessment of
assay performance between runs (Table 2). These were chosen
to reflect the different solubility tiering and include astemi-zole at 2.9 mM, phenytoin at 86 mM, and chlorpromazine at
0.45 mM in published dried DMSO assays.23 These threecompounds are included in each run with their glass vial
positioning randomized to avoid any systematic bias. As forthe logD7.4 assay, all samples also contain 2 nM of verapamil(IS) to verify consistency between injection volumes during
analysis.

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Table 3. Protocol for the Dried Dimethyl Sulfoxide Solubility Assay
Step

Parameter

Value

Description

1

Transfer compounds to glass vials

50 mL

10 mM DMSO solution

2

Evaporate DMSO solvent

1h

Evaporate solvent in a vacuum centrifuge at 40C3

Add buffer

500 mL4

Add stir sticks

1/well5

Equilibrate samples

20–24 hIncubation at 25C under stirring

6

Centrifugation

30 min, 2,000 · g

Room temperature, Eppendorf 5810R

7

Sample transfer

200 mL

Transfer cleared supernatants

8

Centrifugation

30 min, 2,000 · g

As in 69

Sampling and pooling

20 mL

Sampling and pooling of 10 samples + twofold dilution of pool

10

Pooling of standards

5 mL

Pooling of 10 standards +10-fold dilution of pool

11

Establish dilution series of samples and standards

1:5

Serial dilution (1:5) in four steps

12

Plate sealing

13

Assay readout

Peak area

LC/MS, TargetLynx

14

Data evaluation

mM

Genedata Screener

Step Notes
1. Compound stock solution is added to 1 mL Glass Flat Bottom Vials (Product No.: 4100FB-930VL; JG Finneran, ScanTec) situated in a 96-well rackusing a Hamilton STARplus.
2. Solvent DMSO is evaporated in a Genevac HT-4 evaporator (Igenevac, Ipswich, Great Britain).
3. Phosphate buffer at pH 7.4 (I = 0.1 M) is added to the glass tubes containing dried compounds on a Hamilton STARplus.
4. Stir Stix from V&P Scientific (Product No.: VP 735-2).
5. Samples are incubated in an Eppendorf plate shaker (Eppendorf).
7. Supernatants are transferred to fresh glass tubes using a Hamilton STARplus.
9. Sampling and pooling of cleared buffer samples, including a twofold dilution in IS solution using a Hamilton STARplus.
10. Sampling and pooling of standards from DMSO stock solutions, including a tenfold dilution in IS solution using a Hamilton STARplus.
11. Samples and standards are further diluted 50-fold and then serially diluted in IS solution on a Hamilton STARplus.
12. Plates are manually sealed with a 96-well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).
13. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.
14. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.

Assay validation and concordance testing. As described in theliterature, there are several significant challenges associatedwith aqueous high-throughput solubility assays.21,22 Many ofthese are practical, such as achieving enough mixing in a glassvial format to establish equilibrium within a reasonable timeframe (here 20–24 h), pelleting of insoluble compound andsampling of the clarified supernatant without contamination from nonpelleted microaggregates or flocculating compounds. Others are physicochemical in nature, for example,
the potential formation of different crystalline forms withdifferent dissolution rates in the dried DMSO assays, adding tovariability in results between test occasions.
Results are also affected by the purity of each compound solution and this can differ depending on time of storage. In our

166 ASSAY and Drug Development Technologies MAY/JUNE 2020

semiautomated asthe practical challenges are mitigated by anovernight incubation under constant stirring to facilitate establishment of equilibrium as well as two cycles of centrifugation and aspiration to new containers to reduce contaminations
from nonsoluble material. Even so, challenges remain for compounds that form microaggregates and flocculate and hence theautomated assay, where visual inspection of individual samples
is not included in our routines, produces valuable tiered Wave 1
results rather than precise measurements (Fig. 3B).
Illustrations of assay performance include reproducibility
between separate test occasions as illustrated in Figure 3B,
which compares data for all compounds that have been tested
at least twice in the assay. Agreement between test occasions
is considered reasonable (slope between occasions is at 0.91)

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given the significant challenges in miniaturized dried DMSO
solubility assays, although it is immediately apparent from
Figure 3 that this method is associated with a larger variability
than the other panel assays. A slightly weaker correlation(slope at 0.85) is observed for data generated in Gothenburg
and at the CRO (Fig. 3C), which at the time of writing allowed
comparisons across 1,200 compounds.
The larger variability for this assay is also apparent from the
Manhattan plots monitoring performance of the QC compounds over a 6-year period (Fig. 3D), although at the sametime it illustrates how solubilities can be tiered as low(<10 mM—availability in biological test assays can be severely
limited), intermediate (<100 mM—likely available at screen
concentration of 10 mM), and high (up to 1 mM—readily
available in most test assays). Apart from the variability, whichis especially pronounced for the poorly soluble astemizole, theassay has delivered stable results over this time period.
Human PPB
Background description. PPB reflects the degree to which acompound is bound to proteins within blood, primarily toserum albumin (*60%) and a1-acid glycoprotein, and to alesser degree to lipoproteins and a, b, and g globulins. Suchbinding directly impacts the exposure of relevant tissue as
only unbound compound is available to interact with extracellular targets or partition over cell membranes to an intracellular site of action.24,25 Consequently, plasma proteins act
as a reservoir from which bound compound is continuously
released when unbound compound is taken up by cells, metabolized, or excreted. The parameter naturally affects also
compound availability in cellular assays, which commonly
include serum as a component of the cell medium.26
Measurements of PPB is therefore essential for accurateinterpretation of cellular SAR, although there is still an ongoing debate as to whether this parameter should be optimized.27,28 PPB measurements can be achieved using different
principles, with equilibrium dialysis over a semipermeable
membrane separating a plasma and a buffer-containing
chamber as the most widely accepted method.29–31 Other approaches include ultrafiltration and ultracentrifugation
methods as well as binding assays based on immobilized
protein.32 The PPB assay within our panel is based on equilibrium dialysis and represents our implementation of a previously published method.33
Assay workflow. The semiautomated PPB assay workflow isschematically depicted in Figure 4 and outlined in detail in
Table 4. Compound pooling, including warfarin as QC in eachpool, is achieved on the Hamilton liquid handling platformusing unique run lists. Pooled compound solutions in DMSO
are then diluted 140-fold to 7 mM in human plasma from
BioIVT. This plasma is recovered from whole blood donations(50 donors, equal number of males and females), pooled, aliquoted, and stored at -20C until use.
This intermediate solution is used for two purposes: (1) toestablish standard curves in plasma; and (2) for further dilution to 5 mM before addition of samples to the inner chamberof the rapid equilibrium dialysis (RED) devices. A phosphatebuffer at pH 7.4 is added to the surrounding chamber to allow
equilibrium dialysis over a semipermeable membrane with amolecular weight cutoff at 8 kDa. The filled RED devices are
incubated on an orbital shaker for 18 h. Besides addition to the
RED devices, the 5 mM solutions are also used to establish
compound stability in plasma and recovery (mass balance) in
the RED devices, with sampling performed at both the 0 and
18 h timepoints.
While the 18 h samples are incubated alongside the RED
Devices, the 0 h samples are immediately quenched throughaddition to cold acidified AcN, containing 10 nM 5,5-Diethyl1,3-diphenyl-2-iminobarbituric acid as IS, to precipitate proteins and prevent degradation. To ensure matching of sample
matrices in these crash plates, a corresponding volume ofphosphate buffer—in case of sampling plasma—or plasma—incase of sampling buffer—is added alongside AcN. A sevenpoint calibration curve in plasma, ranging from 1.4 nM to
7 mM, is also prepared for each pool through serial dilution of
the 7 mM solution on the Hamilton liquid handling platform.
These samples are also transferred to the crash plates and kept
at -20C until further processed for analysis the following day.
Following equilibration, all samples are brought to room
temperature before sampling. This process includes centrifugation of the thawed crash plates before lids are removed.
Sampling of 50 mL from the RED devices and the Stability &Recovery plate into crash plates is handled through manual
pipetting, followed by lidding and vortexing of the crashplates to ensure adequate mixing. The assay procedure is finalized with a centrifugation step and transfer and twofold
dilution of 75 mL of the supernatants in acidified H2O (0.2%
FA) in a first set of high concentration analysis plates. A
further 10-fold dilution is performed in acidified AcN:H2O(1:3; 0.1% FA) in a low concentration set of analysis plates,
which are used primarily for the analysis to avoid signal
saturation (the high concentration plates remain as backups
should the signals be inadequate).
Analysis plates are finally sealed and read out, analyzed,
and qualified as described above to enable export of calculated fu, stability, and recovery values (all in %) to our internal
database. If the recovery deviates significantly from 100%, it

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Fig. 4. (A) Schematic illustration of the workflow for the PPB assay. (a) Samples are provided from the same source plates as those used
for the logD7.4 and Sol assays. (b) Compounds are pooled in a pool plate on a Hamilton STARplus. (c) Compound pools are transferred toplasma-containing deep well plates (referred to as standard plates), mixed by shaking and centrifuged. Part of content is further diluted in
an intermediate plate, while remaining content is serially diluted to create standard curves (top). Content in the intermediate plate is mixed
by shaking and centrifuged before transfer of samples and buffer to the inward and outward chambers of Red Devices, respectively(bottom). (d) Time zero samples and standards are transferred from the intermediate plate to crash plates containing AcN with IS andmatrix matched samples. The crash plate is kept in the freezer overnight. Incubated buffer and plasma samples from the Red Devices aretransferred to the crash plate alongside plasma stability samples (T18) from the intermediate plate. The crash plate is thoroughly shaken
and centrifuged cold. (e) Samples are diluted in acidified H2O and subsequently in acidified 37% AcN to and detected using UPLC-MS/MS.
(B) Correlation between data in the automated PPB assay versus literature data for 47 drugs. The slope between average values from aminimum of three independent measurements with previously reported data is 0.964 (r2 = 0.834). Dotted lines represent the 95% CI 0.835–
1.09. (C) Correlation between two independent experiments submitted to the database. When more than two independent replicates wereavailable, we included the first two instances. The slope based on testing of 624 compounds comes out at 0.982 (r2 = 0.960) with a 95% CI
of 0.966–0.997. (D) Correlation between harmonized assays at AstraZeneca Gothenburg (y-axis) and at Pharmaron (x-axis). The comparison
is based on 801 compounds with a slope of 0.977 (r2 = 0.938) and a 95% CI of 0.960–0.994. (E) Manhattan plot following 6 years ofoperation from May 2013 to June 2019—warfarin (brown filled circle), metoprolol (green filled triangle), and propranolol (black filled
diamond). Color images are available online.

indicates binding to the dialysis equipment or solubility issues. If the compound is unstable in plasma under the described assay conditions, it complicates interpretation of theassay data. The fu values are therefore only reported as aggregated data when both recovery and stability are >50%.
Choice of validation test sets, QC compounds, and IS. While a set of

72 internal compounds covering a fu range from 0.06% to

168 ASSAY and Drug Development Technologies MAY/JUNE 2020100% were originally used for experimental validation of theherein described automated method, here we examined ourinternal database for experimental values of approved drugs
with well-established literature values and identified 50
drugs31,33–35 (detailed in Supplementary Table S1). As with theother panel assays, we include multiple controls allowing forassessment of reproducibility between runs and between poolsof compounds within each run (Table 2). While propranolol

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Table 4. Protocol for the Human Plasma Protein Binding Assay
Step

Parameter

Value

Description

1

Thaw human plasma

3 min, 750 rpm

Thawing and centrifugation at room temperature

2

Pooling compounds

3 mL

Nine test compounds + one QC (warfarin)

3

Dilute pools in plasma

5.6 mL

Add pooled compounds to 792 mL plasma (to 7 mM)

4

Sample pool aliquots and dilute

400 mL

400 mL diluted pools +160 mL of predispensed plasma

to start concentration
5

Sampling (t = 0 h)

50 mL

Take t = 0 h control samples from the 5 mM deep well plate for the stability
and recovery studies

6

Add samples to RED devices

300 mL

Place in RED device plasma chambers

7

Add phosphate buffer at pH 7.4

500 mL

Place in surrounding buffer chamber

8

Sealing9

Equilibrate samples

10

Generate standard curves

11Store in freezer

18 h, -20C

Crash plates

12

Adjust samples to RT

15 minRED devices, Stability & Recovery and crash plate

13

Centrifugation

1 min, 1,000 · g

Stability & Recovery and crash plate at RT, Eppendorf 5810R

14

Buffer sampling

50 mL

From RED devices buffer chambers to crash plates

15

Plasma sampling

50 mL

From RED devices plasma chambers to crash plates

16

Sampling (t = 18 h)

50 mL

From Stability & Recovery plate to crash plate17

Lidding and vigorous shaking

30 s

Silicone Cap Mats

18

Centrifugation

20 min, 3,000 · g

4C, Sigma 6–16K (SIGMA Laborzentrifugen GmbH)

19

Dilution and transfer

1:2 + 1:10

Crash plate content is diluted and transferred to analysis plates (H2O + 0.2% FA)

20

Plate sealing

21

Assay readout

Peak area

LC/MS, TargetLynx

22

Data evaluationfu, recovery, stability (%)

Genedata Screener

Mounting of breathable seal on RED Devices
18 h, 500 rpm

Incubation with shaking at 37C and 5% CO2
Serial dilution of 7 mM samples

Step Notes
1–4. Compound pooling is achieved in a PP-based microtiter plate (Product No.: 249944; Thermo Fisher Scientific). Pools are mixed and then diluted in plasma
predispensed to deep well plates (Product No.: 260252; Thermo Fisher Scientific). A portion of the diluted pools are further diluted to 5 mM in the same deep wellplate type (herein referred to as the Stability & Recovery plate), while the remaining content is used for generation of standard curves (see further down in thisTable footnote).
5. A t = 0 h control sample for the Stability & Recovery studies is sampled to a crash plate already containing 400 mL of cold IS solution.
6–9. Plasma samples and buffer are added to the RED devices, sealed (Product No.: BEM-1, Diversified Biotech) and incubated on an orbital shaker (Eppendorf) for 18 h
in a Galaxy R incubator (Richmond Scientific) alongside the Stability & Recovery plate.
10. Samples (at 7 mM) from Step 2 are serially diluted in plasma to generate a standard curve from 1.4 nM to 7 mM on the Hamilton STARplus. 50 mL of these samples
are immediately transferred to the crash plate (see Step 5).
11. Crash plates are stored at -20C overnight.
12. The Stability & Recovery plate is vortexed before the centrifugation step.
14–16. Sampling is carried out manually with an 8-channel pipette to crash plates already containing 400 mL of cold IS solution and either 50 mL buffer or plasma toensure matrix matching between samples.
17. Crash plates are lidded using silicone cap mats (Product No.: 276002; Thermo Fisher Scientific) and vortexed.
19. Crash plate content is diluted in two steps, starting with transfer and mixing of 75–75 mL of acidified water in a PP-based microtiter plate (Product No.: 249944;
Thermo Fisher Scientific) on the Hamilton STARplus liquid handler. This content is further diluted 20–180 mL of acidified AcN:H2O (1:3, 0.1% FA).
20. Plates are manually sealed with a 96-well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).
21. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.
22. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.
PP, polypropylene.

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reflects compounds with an intermediate fu in the 10%–30%
range,31,36 metoprolol validates assay performance for compounds with limited PPB (fu *85%).36
During the validation work, it was observed in some poolsthat extensively bound compounds showed higher fraction
unbound than expected. Closer inspection of these poolssuggested that the reason was leakage of plasma proteins into
the buffer side as previously reported.37 To control for potential leakage of the RED Device insert membrane, warfarinwith an fu of *1%33 is included in each pool and used to failaffected pools when the measured fu is >2%. To achieve this, it isincluded in each pool and used to fail affected pools when themeasured fu is >2%. All samples also contain 50 nM of 5,5diethyl-1,3-diphenyl-2-iminobarbituric acid (IS) to allow fornormalization between small variations in injection volume in
the UPLC-MS/MS instrumentation.
Assay validation and concordance testing. The herein described

PPB assay represents a further development of an in-houseprotocol31 as implemented with commercially available RED
devices dialysis chambers.33 The weekly flow-through ofcompounds is accommodated at an acceptable cost by pooling,31 such that 10 compounds are dialyzed in parallel in each
RED device. While the miniaturized assay does carry a risk ofnonspecific binding to semipermeable membranes and plastic
surfaces in the dialysis chambers, it is carefully controlled inour setup through parallel measurements of compound stability and recovery. There is also a potential risk of interference between compounds in their competition for available
binding sites to plasma proteins.
To put this in perspective, each compound is available at
5 mM concentration (50 mM total compound in each pool),while the availability of binding sites on albumin is>500 mM.38 For human alpha-1-acid glycoprotein, the estimated plasma concentration is around 15–30 mM,39 such thatthe risk of interference is larger should more than one compound in the pool bind extensively to this protein. As illustrated below, the agreement between data from different test
occasions and between test sites, which in practice translates
to different pools, suggest that this problem is limited but
remains a consideration when interpreting data. The assay
also reports back on the 18 h plasma stability of each compound through parallel incubations. Assay performance aswell as regular testing and acceptance of new batches of human plasma is achieved through a validation set, the composition of which has been continuously updated to reflect thechemical space of on-going drug discovery projects.
A comparison with literature data is shown in Figure 4B,
demonstrating excellent agreement, especially considering the

170 ASSAY and Drug Development Technologies MAY/JUNE 2020

known variability between batches of plasma from different
donors. Internal assay performance is further illustrated by acomparison of data from different test occasions in Figure 4C,
where a slope close to unity confirms agreement between datafrom independent test runs. The strong correlation between dataholds true when comparing data from the harmonized panels at
AstraZeneca and the CRO (Fig. 4D), for which changes in plasmabatches are synchronized. As shown in the Manhattan plot in
Figure 4E, the assay has reported stable values for the QCs over a
6-year interval.
Human Liver Microsomal Stability
and Rat Hepatocyte Stability
Background description. Exposure of compounds in animalmodels of disease and man is dependent on uptake through thechosen route of administration, distribution, metabolism, andexcretion, with metabolism reflecting the liability (tendency)of the drug to biochemical transformation.40 Metabolic stability is therefore an essential parameter in compound optimization,41,42 and it can be studied using suitable in vitro
model systems. Most commonly, these are based on metabolically active hepatocytes or isolated liver microsomes,43
both of which can be obtained from different species. Comparisons across these model systems are required for prediction of exposure and reliable interpretation of data fromin vivo studies in animal models of disease.
Comparison of stabilities in these two types of model systems
can also help differentiate between phase I and phase II biotransformation.43 While phase I transformation (microsomes
and hepatocytes) modifies molecules by introducing functionalgroups through oxidation, reduction, and hydrolysis, thus
making compounds more polar and easily excreted, phase IItransformations (hepatocytes) incorporate endogenous molecules such as glucuronic acid, sulfuric acid, and acetic acidthrough conjugation. It is important to consider that stability
measurements are influenced by compound availability in thetest system, thus requiring suitable measurements and compensations to accurately reflect intrinsic metabolic stability.44
Metabolic stability measurements are commonly conductedin a kinetic mode, where the degradation of molecules isfollowed over time after addition to the metabolically active
system and there are several published variants of screencompatible formats.45,46 The Mics and Heps assay in our
DMPK Wave 1 panel are fully automated assays conducted in
96-well format that represent further developments of previously published assays.7
Assay workflows. In line with previous descriptions, theworkflow for the HLM assay is schematically depicted in

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DMPK WAVE 1 ASSAY PANEL

Fig. 5. (A) Schematic illustration of the workflow for the Mics assay. (a) Up to three compound plates with 1 mL in each well are obtained
from Sample Management. (b) The compounds are diluted in 50% AcN. Human liver microsomes diluted in NADPH containing PBS buffer
are dispensed into a matching number of deep well plates. (c) The plates are heated to 37C and incubations are subsequently started byaddition of diluted compounds. Sampling, pooling and quenching is achieved at different time points on the Hamilton STARplus platformby adding aliquots to crash plates already holding AcN with IS. A blank plate with diluted liver microsomes is prepared separately. (d)Following incubations all plates are centrifuged to pellet the microsomes. (e) Final sampling of cleared supernatants is combined withadditional pooling of plate content from the upper and lower parts of each plate into one half of a single analysis plate, that is, eachanalysis plate holds the content of two crash plates. These samples are diluted in predispensed H2O and subsequently detected using
UPLC-MS/MS. (B) Illustration of recent data (2019) from a validation of a new batch of cryopreserved human liver microsomes. The slope
based on testing of 46 compounds comes out at 0.980 (r2 = 0.951) with a 95% CI of 0.913–1.05. (C) Correlation between two independent
experiments submitted to the database. When more than two independent replicates were available, we included the first two instances.
The slope based on testing of 1,122 compounds comes out at 0.935 (r2 = 0.894) with a 95% CI of 0.916–0.954. (D) Correlation between
harmonized assays at AstraZeneca Gothenburg (y axis) and at Pharmaron (x axis). The comparison is based on 1,313 compounds with aslope of 0.977 (r2 = 0.882) and a 95% CI of 0.957–0.996. (E) Manhattan plot following 6 years of operation from June 2013 to April 2019—
diclofenac (black filled diamond), verapamil (green filled triangle), imipramine (brown filled circle), phenacetine (purple unfilled square),benzydamine (blue filled triangle), and metoprolol (gray unfilled circle). Mics, assay for metabolic stability in human liver microsomes.
Color images are available online.

Figure 5 and further detailed in Table 5. The first assay step
involves preparation of a homogeneous solution of HLMs,
starting with thawing of commercial preparations from
BioIVT at 37C on a water bath and subsequent dilution in
buffer with NADPH added as cofactor. This working solution
is transferred to a set of up to three 96 deep well plates, which
are placed on heated positions with shaking on the Hamilton
STARplus liquid handler.

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The next step involves dilution of test compounds and QCs to
50 mM, while the HLM working solutions are preincubated toreach 37C. Reactions are started by adding the diluted compounds to the preincubations, and following mixing, by repetitive aspiration and dispensing, sampling is continuously
achieved from the same reaction mixtures under a 30 min time
period. Sampled volumes are immediately quenched in predispensed IS solution in ‘‘quench plates,’’ where the contents from

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Table 5. Protocol for the Human Liver Microsome Stability Assay
Step

Parameter

Value

Description1

Add liver microsomes

245 mL

Prepare and add working solution

2

Preincubation£3

37C and continuous shaking

3

Dilute compounds and controls

1 mL

Test compounds + six QCs (diclofenac, imipramine, metoprolol,
benzydamine, phenacetin and verapamil)

4Start reactions

5 mL

5

Continuous sampling, pooling and quenching

20 mL

Aliquots taken at 0.5, 5, 10, 15, 20, and 30 min

6Centrifugation

20 min, 3,000 · g

4C, Sigma 6–16K (SIGMA Laborzentrifugen GmbH)

7

Sampling and dilution

2 · 40 mL

Supernatants are further pooled and diluted with deionized water

8

Plate sealing

9

Assay readout

Peak area

LC/MS, TargetLynx

10

Data evaluation

Clint

Genedata Screener

Step Notes
1. HLMs are thawed at 37C and diluted at room temperature with phosphate buffer at pH 7.4 alongside freshly prepared NADPH to reach a concentration of 1 mg
protein/mL and 1 mM NADPH. 245 mL is distributed to wells of up to three 1 mL PP deep well plates (Product No.: 278752; Thermo Fisher Scientific). Plates are placedon a shaker and heating position on the Hamilton STARplus platform (kept at 37C while shaking at 800 rpm).
2. A maximal number of three plates are placed on a Hamilton STARplus platform and preincubated under continuous shaking until all wells are at 37C (*15 min).
3. Compound and control stock solutions (1 mL at 10 mM) are diluted to 50 mM by adding 199 mL of AcN:H2O (1:2) using a Hamilton STARplus. The six QCs are used tomonitor specific human P450 isoform activities: diclofenac (2C9), imipramine (2C19), metoprolol (2D6), benzydamine (flavin-containing monooxygenases), phenacetin(1A2), and verapamil (3A4).
4. Reactions are started by transfer of a 5 mL aliquot from the 50 mM solutions to preheated microsomes at 37C on the Hamilton STARplus platform. The diluted
microsomes remain on the shaker throughout the experiment.
5. Samples are aliquoted from reaction plates 1–3 at different time points and pooled (1–3 compounds depending on number of reaction plates) in cooled AcN with
20 nM 5,5-Diethyl-1,3-diphenyl-2-iminobarbituric acid to quench the reactions in a PP-based microtiter plate (Product No.: 249944; Thermo Fisher Scientific). The AcN
volume is matched with the number of incubation plates, that is, from 80 mL (1 plate) to 240 mL (3 plates). This step generates one quench plate per time point, all ofwhich are lidded with a microplate lid (Product No.: 263339; Thermo Fisher Scientific).
7. The content in wells A1 through to D12 are pooled with the content in wells E1 through to H12 (top half with bottom half) for each quench plate (time point) anddiluted twofold with water (80 mL in each well is first placed in each well) in PP-based microtiter plates (Product No.: 249944; Thermo Fisher Scientific).
8. Plates are manually sealed with a 96 well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).
9. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.
10. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.
HLM, human liver microsome.

up to three reaction plates are pooled. This procedure contrasts from other published automated HLM assays, whereseparate reaction plates are used for each time point, with theprocedure as described herein allowing testing of up to 282
compounds (+6 QCs) across three deep well plates and sevenquench plates.
Precipitated proteins in the IS solution are pelleted throughcentrifugation and clarified supernatants are further pooled
by a factor of two by combining the upper and lower content
of the 96-well plates in analysis plates (Fig. 5A). These aresealed and read out as described for the other assays, withquantification of peak areas in TargetLynx and further analysis using dedicated templates within Genedata Screener tocalculate intrinsic clearance rates (Clint).

172 ASSAY and Drug Development Technologies MAY/JUNE 2020

The assay procedure for the rat hepatocyte stability assay
closely resembles that of the Mics assay as described in detail in
Figure 6A and Table 6. Small differences between these assays
remain for historical reasons, but the main essential differences
are the introduction of cryopreserved rat hepatocytes instead of
the HLMs and the use of a different set of QCs and validation sets
to reflect the different metabolism. The sampling from the same
deep well plate over a 2 h time period puts significant demands
on continuous mixing to retain the hepatocytes in suspension,
which is achieved through plate shaker positions on the Hamilton STARplus station.
Choice of validation test sets, QC compounds, and IS. Compound

validation sets, representing a range of different structural

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Fig. 6. (A) Schematic illustration of the workflow for the Heps assay. (a) Up to three compound plates with 1 mL in each well are obtained
from Sample Management. (b) The compounds are diluted in 50% AcN. A homogeneous suspension of rat hepatocytes is dispensed into amatching number of deep well plates. (c) The incubations follow the same procedure as the Mics assay except for the exact timings andchoice of QCs. (d) Following incubations all plates are centrifuged to pellet the hepatocytes. (e) Sampling, pooling, dilution and detection is
performed as described for the Mics assay. (B) Illustration of recent data (2019) from a validation of a new batch of cryopreserved rat
hepatocytes. The slope based on testing of 24 compounds comes out at 0.985 (r2 = 0.996) with a 95% CI of 0.958–1.01. (C) Correlation
between two independent experiments submitted to the database. When more than two independent replicates were available, we
included the first two instances. The slope based on testing of 1,825 compounds comes out at 0.954 (r2 = 0.906) with a 95% CI of 0.940–
0.968. (D) Correlation between harmonized assays at AstraZeneca Gothenburg (y axis) and at Pharmaron (x axis). The comparison is based
on 1,679 compounds with a slope of 0.929 (r2 = 0.881) and a 95% CI of 0.913–0.946. (E) Manhattan plot following 6 years of operation from
October 2012 to April 2019—bosentan (black filled diamond), dofetilide (green filled triangle), indapamide (brown filled circle), lorazepam(purple unfilled square), terfenadine (blue filled triangle), and verapamil (gray unfilled circle). Heps, assay for metabolic stability in rat
hepatocytes; QC, quality control. Color images are available online.

classes and metabolic stabilities (1 £ Clint £ 300), are in use forregular introduction of new batches of HLM and cryopreserved hepatocytes. The compositions of these test sets aredetailed in Supplementary Table S1. Agreement is required for
adoption of new batches, with the cutoff set at a maximal
twofold average difference across the compounds in the validation sets. About one in four new batches of rat hepatocytes
fail to comply with this criterion, such that it is returned to
the vendor, whereas the failure rate is smaller for the HLM
batches.
Multiple controls are included in the assays to reflect metabolism through different human P450 isoforms in the Mics

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QC set and with focus on CYP 3A4 metabolism and phase IIelimination in the Heps set (detailed in Table 2). These werealso chosen to reflect fast, intermediate, and slow metabolism
such that the test runs can be failed when results for the QCs
fall outside of norm. As for the PPB assay, all samples contain
20 nM of 5,5-diethyl-1,3-diphenyl-2-iminobarbituric acid as
IS to allow for normalization of injection volumes.
Assay validation and concordance testing. While absolute com-

parisons with published metabolic stabilities are challenging
given their dependence on the input material, especially in thecase of the primary rat hepatocytes as this depends on strains

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ASSAY and Drug Development Technologies 173

WERNEVIK ET AL.

Table 6. Protocol for the Rat Hepatocyte Stability Assay
Step

Parameter

Value

Description
6

1

Add rat hepatocyte suspension

245 mL

10 cells/mL and viability >80%2Preincubation£3

37C and continuous shaking

3

Dilute compounds and controls

1 mL

Test compounds + six QCs (bosentan, dofetilide, indapamide, terfenadine,verapamil, and lorazepam)

4

Start reactions

5 mL

5

Continuous sampling, pooling and quenching

15 mL

Aliquots taken at 0.5, 5, 15, 30, 45, 60, 80, 100, and 120 min

6Centrifugation

20 min, 3,000 · g

4C, Sigma 6–16K (SIGMA Laborzentrifugen GmbH)

7

Sampling and dilution

2 · 40 mL

Supernatants are further pooled and diluted with deionized water

8

Plate sealing

9

Assay readout

Peak area

LC/MS, TargetLynx

10

Data evaluation

Clint

Genedata Screener

Step Notes
1. Cryopreserved male Wistar han rat hepatocytes are rapidly thawed in a water bath at 37C and washed in 50 mL prewarmed (37C) Leibovitz medium pH 7.4.58 The cellsuspension is centrifuged for 3 min at 50 rpm, supernatant is removed, and pellet is resuspended (repeated twice). After automated cell counting the suspension isdiluted to a final concentration of 106 cells/mL in Leibovitz medium and 245 mL of the cell suspension is manually transferred to 1 mL PP deep well plates (Product No.:278752; Thermo Fisher Scientific) using a multipipette.
2. A maximal number of three plates are placed on a Hamilton STARplus platform and preincubated under continuous shaking until all wells are at 37C (*15 min).
3. Compound and control stock solutions (1 mL at 10 mM) are diluted to 50 mM by adding 199 mL of AcN:H2O (1:2) using a Hamilton STARplus. Five of the QCs were
inherited from previous protocols and reflect important CYP 3A4 metabolism in man: bosentan, dofetilide, indapamide, terfenadine, and verapamil, while lorazepam
controls for phase II metabolism (UGT2B15).
4. Reactions are started by transfer of a 5 mL aliquot from the 50 mM solutions to preheated hepatocyte suspensions. The reaction mixtures remain on the shaker
throughout the experiment.
5. Samples are aliquoted from reaction plates 1–3 at different time points and pooled (1–3 compounds depending on number of reaction plates) in cooled acidified AcN(0.1% FA) with 20 nM 5,5-Diethyl-1,3-diphenyl-2-iminobarbituric acid to quench the reactions in a PP-based microtiter plate (Product No.: 249944; Thermo Fisher
Scientific). The AcN volume is matched with the number of incubation plates, that is, from 60 mL (1 plate) to 180 mL (3 plates). This step generates one quench plate pertime point, all of which are lidded with a microplate lid (Product No.: 263339; Thermo Fisher Scientific).
7. The content in wells A1 through to D12 are pooled with the content in wells E1 through to H12 (top half with bottom half) for each quench plate (time point) anddiluted twofold with water (80 mL in each well is first placed in each well) in PP-based microtiter plates (Product No.: 249944; Thermo Fisher Scientific).
8. Plates are manually sealed with a 96 well cap natural seal (Product No.: 276002; Thermo Fisher Scientific).
9. The samples are analyzed by using a Waters iClass Acquity and Waters Xevo TQS. Chromatograms are evaluated using Waters TargetLynx software.
10. Data evaluation is achieved through Genedata Screener and approved results are reported to our internal database D360.

and gender, it is important that the test results allow comparisons over an extended time interval. This can only beachieved through careful selection and control of input material, that is, through comparisons of assay data between
different batches of HLM and rat hepatocytes. To illustratesuch assay concordance, we include the results from the lastbatch validations in 2019, which are provided in Figures 5B
and 6B, respectively, where data obtained with the new reagents are compared with current production batches. Excellent agreement is observed in both cases with slopes close
to unity and with much smaller deviation from the accepted
average two-fold differences.
Further illustrations of assay performance over the 6-year
period of operation can be seen by the comparison of test

174 ASSAY and Drug Development Technologies MAY/JUNE 2020

results from two independent test occasions as illustrated in
Figures 5C and 6C for the Mics and Heps assays, respectively,
and correspondingly between sites in Figures 5D and 6D.
Performance over the full-time period can also be evaluated through the Manhattan plots in Figures 5E and 6E,
both of which demonstrate excellent assay stability overtime. Such performance has allowed our organization tobuild reliable prediction models and to compare resultsover time.
Assay panel metrics and examples of project impact. Delivery oftimely and harmonized data across a global R&D organization
requires optimization of all steps, from registration andshipping of physical samples to operation of the assay panel

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ASSAY and Drug Development Technologies 175

Fig. 7. (A) Schematic illustration of internal metrics designed to measure times from registration of samples to the appearance of experimental data in internal databases (SLA).
Measurements include the full ‘‘End to End Metric’’—starting with sample registration, ‘‘Sample Availability to End Metric’’—starting from when a physical sample becomes available
at a global sample management facility (probing shipping efficiency and assay performance) and ‘‘Plating Request to End Metric’’—from ordering of locally available compounds topublication of assay results (probing assay performance). (B) Top—Histograms of panel results since the start of operations in 2013 through to May 2019. Mid–pie charts illustrating
number of days from samples being physically available at a Sample Management site to published data in database (combined statistics from 2018 and 2019). Green <10 days,orange <15 days and red if outside of SLA. Bottom—corresponding pie charts for the number of days from plating request to published data. Green <5 days, orange <10 days and red
if outside of SLA. SLA, Service Level Agreement. Color images are available online.

WERNEVIK ET AL.

itself. Measures are in place to probe the performance of eachof these steps (Fig. 7A), that is, from original registration of
new samples at any site, their physical arrival at each local
sample management facility, subsequent shipping and delivery, local sample solubilization and plating to assays, andfinally operations of all assays through to publication ofquality-controlled data. Here, we focus this analysis to thelatter steps, which probes timings from physical availability at
any global sample management facility and from when
compounds are ordered to the panel test, respectively, as these
are important metrics for internal DMTA testing.
Following 6 years of operation, there is now a total of229,354 data points reported from the assay panel (not including data from the harmonized panel at Pharmaron),approximately evenly spread between the five assays (dataretrieved on May 14, 2019). Historical data are presented in
the form of histograms in Figure 7B, while an example of
how these properties are optimized throughout a project
lifetime is provided below. Below each histogram are pie
charts where samples that meet timelines are given as green
if qualified results are published within 5 days from orderingof compounds to test and 10 days from physical sample
availability at a sample management facility, orange if
within 10 or 15 days, respectively, and red if outside of thesetimelines. Given excellent historical performance of internal
shipping and the DMPK Wave 1 panel, our current focus is
toward further improving the flow of samples from internaland external chemistry organizations to Sample Management.
The value of the panel data for the optimization of internal
prediction models has already been described,47 tools that arecontinuously applied for characterization of virtual compound sets and associated decision-making for compound
synthesis. Here, we additionally illustrate the role of panel
assay results in the optimization of inhibitors of the Ataxia
Telangiectasia Mutated (ATM) kinase (Fig. 8). While the initialstarting point showed mM potency on the target protein with ahigh logD7.4 value of 3.5, low mM solubility, and high intrinsic clearance, a published intermediate compound had
improved considerably on all these aspects.48 As illustrated in
Figure 8, further optimization yielded a published candidate
drug49 with sub-nM potency and significant improvements inall DMPK Wave 1 measures, especially with regard to aqueous
solubility that exceeded the assay limit of 1 mM.
What is noticeable from these comparisons is that later
optimization efforts toward a candidate drug with improved
availability in CNS showed a return in the logD value to 2.7,
while retaining solubility, metabolic stability, and potency forthe target.50,51 Collectively, these data demonstrate how the

176 ASSAY and Drug Development Technologies MAY/JUNE 2020

availability of high-throughput quality DMPK assays for every round in the DMTA cycle allows identification of leads
with excellent properties that can be progressed to candidate
drugs.

DISCUSSION
The DMTA cycle is central to drug discovery, serving to
generate hypotheses that can be experimentally tested throughiterative compound synthesis and testing.52 While progress on
the biology of interest is monitored through biochemical andcellular SAR profiling assays, such optimization also includes
physicochemical and metabolic properties that affect availability in biological test systems and in man. Given the importance of early consideration of these aspects, AstraZeneca
took a strategic decision in 2012 to implement a DMPK Wave 1
panel of assays, with the intent to harmonize and broaden thedelivery of relevant, high-quality early DMPK data.
Each of the panel assays were carefully selected to provide afingerprint that enables ranking of compounds and identification of liabilities within a compound series. Assays for lipophilicity, solubility, human PPB, and metabolic stability inboth rat hepatocytes and HLMs were included, as data fromthese assays constitute the basis for estimating human pharmacokinetic parameters and oral bioavailability, and incombination with target potency can provide early humandose predictions (eD2M).53
Lipophilicity is described as a partition coefficient, logD7.4,
which is defined as the ratio of compound concentration atequilibrium between 1-octanol and an aqueous phosphatebuffer at pH7.4. Lipophilicity correlates with many otherproperties and is the main descriptor for prediction of several
parameters related to metabolism, absorption, distribution,and potency.13 One of these is the aqueous solubility, aproperty that influences the oral bioavailability of candidate
drugs. It is challenging to develop formulations for poorly
soluble compounds,54,55 such that early identification of solubility limitations in a compound series enables design ofcompounds with greater likelihood for good bioavailability inclinical settings. Another parameter that affects drug disposition is the PPB, which is used together with metabolic stability data to predict hepatic clearance. Although human PPB
is an important property to measure, it is generally not a
property that should be optimized.24,26–28
Various methods investigating in vitro metabolic stability
are applied in drug discovery to predict the in vivo metabolismof compounds.42,56 The most commonly used approaches are
incubations in HLMs, which mainly contain enzymes responsible for oxidative metabolism such as cytochrome P450s(CYPs).57 Since most marketed drugs are predominantly

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DMPK WAVE 1 ASSAY PANEL

Fig. 8. (A) Illustration of data obtained in the ATM inhibitor project, separated on neutral compounds (gray filled circle) and bases (brownfilled triangle). (A) Potency on primary target reported as the negative logarithm of apparent IC50 values. (B) logD7.4. (C) Logarithm of theaqueous solubility (mM). (D) Logarithm of the fraction unbound (fu) obtained in the PPB assay. (E) Logarithm of the intrinsic clearance
values in human liver microsomes. (F) Logarithm of the intrinsic clearance values in primary rat hepatocytes. The lines in each graphrepresent the moving average for the compounds (each period defined by 10 compounds). The chemical structures of the publishedchemical starting point (brown unfilled circle), an optimized lead compound (purple unfilled square) and two candidate drugs (green
unfilled triangle, blue unfilled diamond) are provided below the graphs. ATM, Ataxia Telangiectasia Mutated; PPB, plasma protein binding.
Color images are available online.

cleared by hepatic CYP-mediated metabolism, HLMs are ideal
for studying in vitro intrinsic clearance and to predict humanclearance. Metabolic stability in rat hepatocytes is primarily
screened to additionally inform about the ability to predict thein vivo pharmacokinetics from in vitro data for a compound
series.
A good in vitro–in vivo correlation in rats and other preclinical species will increase the confidence in the predictionof the human pharmacokinetic profile from in vitro data. In
addition to oxidative metabolism that can be studied in microsomal incubations, measurements in rat hepatocytes include contributions of other enzymes and cofactors forconjugation to the metabolic processing of compounds.

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In conclusion, this work describes an integrated andsemiautomated workflow that takes experimental consideration of the above-mentioned properties. This five-assay
DMPK Wave 1 panel ensures global availability and capacity to routinely accommodate all newly synthesizedcompounds within AstraZeneca and through CROs.59 As
illustrated through detailed experimental descriptions, accompanied by illustrations of accumulated data following 6
years operations, the panel delivers quality data on physicochemical properties and metabolic stabilities concurrent
with SAR-profiling assays, allowing for appropriate consideration of these aspects in a smooth and efficient DMTA
process.

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ASSAY and Drug Development Technologies 177

WERNEVIK ET AL.

ACKNOWLEDGMENT
We are grateful to Stephanie Ashenden and Ola Engkvist at
AstraZeneca for valuable input to the model generation andillustration of the impact of the DMPK Wave 1 data.

DISCLOSURE STATEMENT
No competing financial interests exist. All authors areeither current or former employees of AstraZeneca.

FUNDING INFORMATIONThis work was performed as part of the AstraZeneca R&D
operations and funded by AstraZeneca.

SUPPLEMENTARY MATERIAL
Supplementary Table S1

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Address correspondence to:
Johan Wernevik, BSc
Mechanistic Biology & Profiling, Discovery Sciences, R&D
AstraZeneca
SE 43183 Mölndal
Gothenburg
SwedenE-mail: johan.wernevik@astrazeneca.com
Thomas Lundbäck, PhD
Mechanistic Biology & Profiling, Discovery Sciences, R&D
AstraZeneca
SE 43183 Mölndal
Gothenburg
SwedenE-mail: thomas.lundback@astrazeneca.com
Abbreviations Used
AcN ¼ acetonitrile
ATM ¼ Ataxia Telangiectasia mutated
CI ¼ confidence interval
Clint ¼ intrinsic clearance rates
CRO ¼ contract research organization
CYP ¼ cytochrome P450
DMPK ¼ drug metabolism and pharmacokinetic
DMSO ¼ dimethyl sulfoxide
DMTA ¼ design-make-test-analyze
FA ¼ formic acid
Heps ¼ assay for metabolic stability in rat hepatocytes
HLM ¼ human liver microsomes
HPLC ¼ high performance liquid chromatography
IS ¼ internal standard
logD7.4 ¼ lipophilicity assay
Mics ¼ assay for metabolic stability in human liver microsomes
MRM ¼ multiple reactions monitoring
MS ¼ mass spectrometry
PP ¼ polypropylene
PPB ¼ plasma protein binding
QC ¼ quality control
RED ¼ rapid equilibrium dialysis
SAR ¼ structure-activity relationship
SLA ¼ Service Level Agreement
Sol ¼ solubility assay
UPLC ¼ ultraperformance liquid chromatography

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ASSAY and Drug Development Technologies 179