Download Validation of an Automated High Throughput Plasma Protein

Validation of an Automated High
Throughput Plasma Protein Binding Assay
Application Note 474
1Michael
S. Shanler, 1Andrew K. Mason, 1Ronell M. Crocker,
Vardaro, 2Charles L. Crespi, 2David M. Stresser,
1Elke S. Perloff
1BD Biosciences – Woburn, MA
2Corning Gentest SM Contract Research Services – Tewksbury, MA
Dog Plasma (Male, Pooled (N≥5), EDTA K3 treated, Fasted)
Introduction
Acetonitrile with internal standard for LC/MS
The % plasma protein binding (%PPB) of a test article is a
parameter that determines which fraction of the total plasma
concentration of a compound is bound to plasma proteins and,
thus, generally considered not available for interaction with
its biological target (receptors, enzymes, transporters, etc.).
Determination of the free (%Fu) and bound (%Bound) fractions
of a test article in plasma is therefore a critical parameter, which
is routinely determined in the process of drug discovery and
development. Automated high throughput (HT) plasma protein
binding (PPB) assays by equilibrium dialysis with LC/MS quantitation allow for the screening of large numbers of compounds with
fast turnaround times.
Equipment:
2Roseann
DMSO
Methanol
Isotonic Buffer (0.1M KPO4 0.6% NaCl pH 7.4)
SM
Results from HT PPB assays performed through Corning Gentest
Contract Research Services are compared using both manual and
automated methods. A panel of compounds (1 and 10 μM) covering a range of expected protein binding (±warfarin, propranolol,
±verapamil, citalopram, and linezolid) were tested in multiple
species including human, rat, dog, and mouse. In addition, data
for a 21 compound test set was compared to literature values
using an automated HT workstation (Figure 1). %PPB was also
investigated in non-human species.
Materials
Reagents and Solutions:
Human Plasma (Male, Pooled (N≥5), EDTA K3 treated, Fasted)
Mouse Plasma (Male, Pooled (N≥5), EDTA K3 treated, Fasted)
Rat Plasma (Male, Pooled (N≥5), EDTA K3 treated, Fasted)
Incubating Microplate Shaker
Heat sealer
A
pplied Biosystems API 4000™ and API 4000 QTRAP®
LC/MS/MS systems
T
ecan® Freedom EVO® 200 Liquid Handling System w/Span-8
LiHa (liquid handling arm) and 96-channel MCA™
(Multi-channel arm) pipette heads
Consumables:
Falcon® 50 mL Conical-Bottom Tube
96 well 2 mL deep well polypropylene “Spiked Plasma”
microplate
9
6 well 1 mL deep well polypropylene “MeOH Dilution” and
“Receiver, Donor and Standards Spin” microplates
96 well PCR polypropylene “LC/MS sample” microplates
96 well 560 μL conical bottom polypropylene “Compound”
microplate
T
hermo Scientific Single-Use RED (Rapid Equilibrium Dialysis
Device) Plate with Inserts (Cat. No. 90007)
Adhesive- and Heat-sealing film
50 μL, 100 μL, and 200 μL disposable pipette tips from Tecan
Troughs for MCA™ and LiHa
Methods
110 μL of 10 mM DMSO stocks were transferred and mixed
into 90 μL of MeOH in the “MeOH Dilution plate”.
2
10 μL was then transferred from the “MeOH Dilution Plate”
to a “Spiked Plasma” deepwell plate, already containing
990 μL of Plasma.
3
300 μL /well of the spiked plasma was added to the corresponding donor well of RED plate, a polypropylene plate
preloaded with 48 equilibrium dialysis membrane inserts
(MWCO ~8,000).
4
500 μL /well of isotonic buffer was added to the receiver
well of the RED plate.
5
RED plates were sealed with adhesive film and shaken
500 RPM for 3 hours at 37°C.
Calculations
Definitions
6
“Donor Spin” plates were created by adding 100 μL Isotonic
buffer + 10 μL of blank plasma + 10 μL from each donor
chamber of the RED device
Corrections to area ratios
were applied to compensate for dilutions during
sampling
Ac: P
eak area of the
compound or test
article
7
“ Receiver Spin” plates were created by adding 20 μL of
blank plasma + 100 μL from each receiver chamber on the
RED device.
8
9
“Standards Spin” plate was created by adding 100 μL isotonic
buffer + 10 μL of spiked plasma + 10 μL blank plasma
300 μL of stop solution (2 μM Labetalol in Acetonitrile) was
added to the “Receiver Spin”, “Donor Spin” and “Standards
Spin” plates. The “spin” plates were centrifuged for 20 minutes @ 4000 RPM, 4°C.
10 T
he supernatants from these “spin” plates were transferred
into PCR plates, used for LC/MS injection.
11Samples were analyzed on LC/MS/MS systems.
Figure 1. Automated workstation for PPB assays.
Ad = (Ac/Ais)*12
Ar = (Ac/Ais)*1.2
As = (Ac/Ais)*12
Cd = (Ad/As)*Cnom
Cr = (Ar/As)*Cnom
% Bound = 100*(1-Cr/Cd)
Ais: Peak area of the
internal standard
Ad: D
onor corrected
area ratio
Ar: R
eceiver corrected
area ratio
As: S
tandard corrected
area ratio
Cd: Donor concentration
Cr: R
eceiver concentration
Cnom: N
ominal concentration of standard
12Corrections to area ratios were applied to compensate for
dilutions during sampling.
13 Plasma protein binding (% Bound) was calculated (Figure 2).
Figure 2. Plasma protein binding calculation
2
Figure 3. % Bound Comparison: Dog, Rat, Mouse, and Human
Comparison of mean % bound values across four species for a panel of compounds at 10 μM final
concentrations. Mean of replicates (N=3) are reported with standard deviation shown for error bars.
Percent Bound
Compound
Amitriptyline
Atenolol
LiteratureExperimental
95%
6-16%
95.4% (0.48%)
4%
(16.4%)
98.9% (0.19%)
Percent Bound
Compound
LiteratureExperimental
Terfenadine
97%
99.5% (0.3%)
Warfarin
99%
99.5% (0.08%)
Bumetanide
93%
Furosemide
91-99%
Carbamazepine
76%
84%
(3.85%)
Tolbutamide
96%
Erythromycin
84%
79%
(4.96%)
Verapamil
90%
Haloperidol
92% 91%(1.2%)
Thioridazine
>95%
(1.23%)
93%
(1.83%)
99.9% (0.04%)
Imipramine
89-92%
(0.89%)
Tacrine
75%
63%
(6.21%)
Ketoprofen
>99%
99.6% (0.17%)
Nadolol
30%
25%
(8.11%)
Metoprolol
12%
3.5% (32%)
Linezolid
31%
49%
(15.6%)
Naproxen
99%
95%
(1.73%)
Citalopram
80%
63%
(2.58%)
81-93%
83%
(3.95%)
Propranolol
92%
90%
98.9% (0.36%)
Table 1. Protein binding (% bound) in human plasma and comparison with literature values
Comparison of multiple compounds against reported literature binding values. Mean of replicates
(N≥4) are reported along with standard deviation listed in parentheses. Majority of literature values
were collected from RxList.
Table 2. % Bound Comparison between Automated and Manual Procedures for Human Plasma
Comparison of manual and automated methods using two compounds, each at two concentrations. Values represent the means of 48 (automated) and 24 (manual) determinations standard
deviation. % CV values listed in parentheses.
3
The methods were automated on a Tecan® Freedom EVO® 200
configured with a 96-MCA™ multi-channel arm, a LiHa (Liquid
Handling) arm with 8 fixed septum piercing tips, and a Te-Stack™
automated tip-loading system. Sample analysis was performed
using Applied Biosystems API 4000™ and API 4000 QTRAP®
LC/MS/MS systems.
Four automated liquid handling protocols performed the liquid
transfer steps covered in the methods. The deck configuration
required limited manual intervention for shaking, centrifugation,
and incubation.
Summary and Conclusions
w In this investigation, we have validated methods for determin-
ing PPB values in a high-throughput environment. Results were
obtained using automation on a Tecan Freedom EVO 200 liquid
handling workstation.
w Compounds exhibiting a broad range in percent binding to
human plasma protein were tested. Percent bound values were
consistent with available literature values (Table 1).
w Comparison of automated and manual methods showed very
similar binding values using model compounds propranolol and
warfarin at both 1 and 10 µM. CVs of less than 1% for warfarin
and 2% for propranolol give high confidence in the reproducibility in both manual and automated methods (Table 2).
w #1 Compound Preparation: (Method steps 1-2) Compounds
were prepared using 8 fixed, septum piercing tips on the LiHa
as well as 100 µL and 200 µL tips on the 96-channel MCA™
pipette head. The 100 µL tips were presented to the work deck,
using an automated stacker to maximize efficiency.
w The method was shown to be applicable to preclinical species.
A panel of 5 compounds assayed for protein binding in plasma
from human, rat, dog, and mouse species showed rank ordering to be the same across species although there were notable
differences within a species (e.g. warfarin was ~99% bound in
human and rat plasma vs. ~94% bound with mouse, representing a ~6-fold difference in fraction unbound) (Table 3).
w #2 Dialysis Plate Preparation: (Methods steps 3- 4) The 8 fixed
tips were used to transfer plasma, donor solutions, and buffer
to the assay plates.
w #3 Spin Plate Preparation: (Method steps 6-9) The 96-channel pipette head used both 5 µL and 100 µL tips. The 50 µL tips
were placed directly on the deck.
w #4 LC/MS/MS Sample Prep: (Method step 10) 100 µL tips on
the 96-channel pipette head were used to transfer samples into
plates for injection on the LC/MS/MS.
Acknowledgement
We thank Rita Vicaire, The Robot Whisperer, LLC., for assistance
with the automation.
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