Bioanalytical Method Development Strategy for Therapeutic

Bioanalytical Method Development Strategy
for Therapeutic Peptides
The Tools
Sam p l e P re pa rat ion
Oasis® SPE sorbents are unique in their combination of purity, reproducibility, stability, and retention
characteristics. T he use of the extraction selectivity of two of the Oasis mixed-mode ion-exchange
sorbents, in combination with a simple extraction protocol, is a key contributor to the success rate of
this generic method development approach.
Format
T he innovative Oasis µElution plate format allows for up to a 15× sample concentration, increasing
the possibility of reaching the required sensitivity levels for bioanalytical assays. The low (25 µL) elution
volume eliminates the need for evaporation and reconstitution significantly reducing the potential
analyte loss due to absorption to the walls of the collection plate and/or chemical instability.
Column
BEH Technology™ is Waters patented second generation hybrid particle that combines the efficiency
and structural integrity of silica particles with the pH stability of polymeric resins. T he 300Å C18
version of this particle delivers excellent peak shape for a wide range of peptide molecular weights
and is ideally suited for this generic approach.
LC
ACQUITY UPLC® Technology is now firmly established as the LC platform of choice for bioanalytical
assays. Holistically designed to maximize the potential of the BEH sub-2 µm particle technology, the
resultant chromatographic resolution, sensitivity and sample throughput are fundamental contributing
factors to the accuracy and reproducibility of pharmacodynamic data.
Mass Sp ec t romet ry
Xevo™ TQ MS is a highly advanced tandem quadrupole mass spectrometer. Its unique collision cell
technology and extended capabilities allow simultaneous and multifunctional data acquisition within
a timescale compatible with UPLC® Technology. T he 2000 amu range of the first resolving quadrupole
represents a critical element for the generic applicability of this approach.
T he combination of these unique, novel technologies facilitates the generation of
method development solutions for extremely complex analytical challenges.
Waters Xevo System with Peptide Separation Technology Consumables
Creating a Universal Approach to a Complex Problem
T he desire to commercialize peptides as therapeutic drug candidates creates a new series of challenges for the bioanalytical chemist in developing
simple, robust, and sensitive quantitative assays in support of clinical trial activities.
Desmo p ressin Ac etat e
OH
A successful generic bioanalytical assay needs to be:
O
HN
■ Applicable to a diverse range of therapeutic peptides
HN
O
■ Selective and free from significant matrix interferences
H2N
S
S
H
N
O
■ Linear over a wide dynamic range
N
H HN
NH2
N
N
H
O
O
O
H HN
N
N
H
O
O
O
O
NH2
O
NH2
■ Capable of supporting high throughput assays.
O
SCH2CH2C-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2•CH3COOH•3H2 0
100
MRM of 1 Channel ES+
1.28e3
%
Blank Plasma
0
MRM of 1 Channel ES+
1.28e3
1.09
%
20 pg/mL (21.4 fmol/mL)
Desmopressin in human plasma
104% SPE Recovery
Matrix Effects < 5%
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4 min
A sensitive LC/MS/MS method for the extraction and analysis of Desmopressin from human plasma was developed utilizing a simple universal method development strategy. T his method
could easily support an LLOQ of 20 pg/mL and was demonstrated to have a linear response over 4 orders of magnitude.
The Detection Challenge
Bioanalytical laboratories are extremely familiar with the use of triple quadrupole mass spectrometers for the routine analysis of biological
samples. However, as the molecular weight of the analytes increases, there are several characteristics of the particular mass spectrometer
chosen for this application that are critical to success.
Ident if ying t he P recu rsor
T herapeutic peptides, when analyzed by mass spectrometry, can exist in multiple charge states such as 2+, 3+ and 4+. As the triple quadrupole
mass spectrometer detects analytes by their mass to charge ratio [m/z], this does allow for the detection of peptides with a molecular weight
greater than the mass range of the spectrometer. A successful, highly sensitive generic MS method for the analysis of peptides relies upon the
detection of the major precursor and may be limited by the operating range of the first resolving quadrupole.
100
Scan ES+
100
100
Scan ES+
Scan ES+
9.91e7
9.91e7
1498.32Enfuvirtide
1498.32
100
Scan ES+
mw 4492
1498.64
1498.64
1498.01
1498.01
1498.89
%
1498.89
%
%
1499.34
1499.34
%
0
m/z
1494
m/z
0
400
600
800
1000
1200
1400
1600
1800
m/z
0
400
600
800
1000
1200
1400
1600
1800
1495
1496
1497
1498
1499
1500
1501
1502
1503
Enfuvirtide, mw 4492, has possible precursor m/z values of 1498 for 3+ charge state
and 1124 0
for 4+ charge state. Under the analytical conditions used, the 3+ was the most
1494
1495
1496
1498 1499 1500
1501
intense charge state
present,
very little
of the 4+1497
was observed.
1502
Opt imizing for F ragmentat ion
Bivalirudin
mw 2180
T he ultimate sensitivity of the triple quadrupole MS detection method
depends upon the ability to optimize for, and detect the most intense
fragment(s). T he operating range of the second quadrupole should be
able to accommodate large fragment ions, as seen in the example
here, and fragment m/z values which are higher than the precursor.
100
650
Doubly charged precursor
Singly charged fragments
1091
%
1530
MRM Transitions identified:
1091 > 650
1091 > 1530
Bivalirudin, mw 2180, MS scan shows 2+ precursor present at m/z 1091.
After performing MSMS of m/z 1091 from 100 to 1900, major fragments are observed
at m/z 650 and m/z 1530. Precursor appears at lower m/z even though mw is higher.
0
Note: If need be, area counts from several MRM transitions can be summed to
increase sensitivity.
400
600
800
1000
1200
1400
1600
m/z
C h romatogra phic P erformanc e
A key parameter in the success of a generic method development approach is the correct choice of LC instrumentation and associated column chemistry.
T he combination of ACQUITY UPLC and the 1.7 µm BEH 300Å C18 PST column results in excellent peak shape and chromatographic resolution for a
wide range of peptide molecular weights. T he generic 3.5 minute gradient chromatographic method generates peak widths of less than 3 seconds
at base, even for peptides with mw > 4000. MS data acquisition of > 12 points across each peak ensures accurate and reproducible quantitation.
1 2 3
100
4
5
MRM of 5 Channels ES+
Peak Width
(seconds)
MS Data Points
Across Peak
1. Vasopressin
1084
1.8
15
2. Angiotensin II
1046
2.2
15
3. Desmopressin
1069
2.2
18
4. Bivalirudin
2180
2.4
18
5. Enfuvirtide
4492
2.1
16
ACQUITY UPLC BEH300 C18 2.1 X 50 mm, 1.7 µm Peptide Separation
Technology Column, Mobile phase A = 0.1% formic acid, Mobile phase B
= acetonitrile, Flow rate = 0.4 mL/min 5% B to 75% B over 2 minutes,
Total run time 3.5 minutes.
%
0
MW
Analyte
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8 min
Sample Preparation Challenge
T he selective extraction of peptides from other endogenous biological components is probably
one of the most challenging sample preparation tasks. Approaches such as ELISA/Ligand
Binding Assays are effective but are time consuming to develop and are not ideally suited
to routine bioanalytical DMPK studies. T he technique of protein precipitation or conventional
reversed-phase SPE, although very common for this type of assay, suffers from significant
ion suppression due to matrix effects thus impacting the achievable limits of detection.
Sel ec t iv e Sam p l e E x t rac t ion
The Oasis family of SPE products are based upon a polymeric, water-wettable reversed-phase sorbent with a usable pH range of 0–14. After extensive
analytical method development studies with a diverse range of therapeutic peptides, Oasis WCX (weak cation exchanger) and Oasis MAX (mixed-mode
anion exchanger) showed the greatest utility for the selective extraction from human plasma in terms of % recovery and reduced matrix effects.
A simple generic extraction protocol has been developed for these sorbents which, when applied to the target list of peptides, achieved > 80% recovery
for 9 of the 12 compounds. With further stepwise minor modifications to the protocol, acceptable recoveries were achieved for all 12 compounds.
pl
8.6
9.1
9.3
3.87
12
7.3
4.06
7.51
7.35
8.93
10.4
9.1
MW
1069
1084
1019
2180
3464
1270
4492
1296
1046
1673
1638
4118
120
100
% Recovery
80
Oasis MAX
60
Oasis WCX
40
20
Te
r
ip
ar
at
ta
id
e
tin
in
at
os
te
m
So
sin
eu
ro
N
en
ns
II
I
ot
gi
An
An
gi
ot
vi
en
rti
sin
de
in
fu
En
er
el
P
os
BN
ud
lir
va
G
in
e
id
Bi
ct
re
ot
es
O
pr
so
Va
De
sm
op
re
ss
sin
in
0
The diverse range of therapeutic peptides detailed above were extracted from human plasma using a generic extraction
protocol, the Oasis MAX and WCX sorbents and the µElution 96-well plate format. With no modifications of the protocol,
all extracts (with the exception of BNP, Enfuvirtide, and Somatostatin) exhibited > 80% recovery and < 11% matrix
effects. Analysis was carried out using the PST T herapeudic Peptide Method Development Kit and ACQUITY UPLC
with Xevo TQ MS systems.
P ST T hera p eut ic P e pt ide Met hod Dev elo pment Kit
T he PST T herapeutic Peptide Method Development Kit has been developed to
simplify the process of sample preparation and LC method development for
the analysis of therapeutic peptides in plasma. T he kit contains an Oasis PST
µElution Method Development Plate, a PST 300Å C18 reversed-phase column
and the detailed screening protocol which was used to generate the data shown
in this publication.
In addition, a comprehensive method development training seminar has been
created which describes all aspects of the method development process from the
MS conditions to the final validation of a method for the extraction of the
therapeutic peptide Desmopressin from human plasma.
For more information, visit www.waters.com/pepkit,
or contact your local Waters sales office.
Ordering Informat ion
Description
UPLC PST T herapeutic Peptide Method Development Kit
Part No.
Qty/Box
176001835
Includes:
Oasis PST µElution Method Development Plate
186004713
1
ACQUITY UPLC BEH 300 C18 2.1 x 50 mm, 1.7 µm
186003685
1
96-well 1 mL Collection Plate and Cap Mat
600001043
3
HPLC PST T herapeutic Peptide Method Development Kit
176001836
Includes:
Oasis PST µElution Method Development Plate
186004713
1
XBridge™ BEH 300 C18 2.1 x 50 mm, 3.5 µm
186003607
1
96-well 1 mL Collection Plate and Cap Mat
600001043
3
Oasis MAX 96-well µElution Plate
186001829
1
Oasis WCX 96-well µElution Plate
186002499
1
96-well 1 mL Collection Plate
186002481
50
Cap Mats for 1 mL Collection Plate
186002483
50
Disposable Reservoir Tray
WAT058942
25
Extraction Manifold for 96-well Plates
186001831
1
Vacuum Box Gasket Kit (includes foam top gaskets and orange O-rings)
186003522
2
SPE Vacuum Pump 115 V, 60 Hz
725000417
1
SPE Vacuum Pump 240 V, 50 Hz
725000418
1
Available Waters Products Not Included in Kit:
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ACQUITY UPLC, Xevo, UPLC and XBridge are trademarks of Waters Corporation. All other
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720003055EN June 2009 SC-FP