©2010 Waters Corporation | COMPANY CONFIDENTIAL 1
Bioanalytical Method Development for
Therapeutic Peptides
©2010 Waters Corporation | COMPANY CONFIDENTIAL 2
Challenge: Quantify Therapeutic Peptides in Human Plasma
Desmopressin MW 1069
©2010 Waters Corporation | COMPANY CONFIDENTIAL 3
Challenge: Quantify Therapeutic Peptides in Human Plasma
5 pg/mL desmopressin
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
1.0029
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
1.0029
1.0029
Red trace = blank human plasmaGreen trace= 5 pg/mL desmopressin
©2010 Waters Corporation | COMPANY CONFIDENTIAL 4
Goals
Provide tools and techniques to facilitate the
development of methods for accurate peptide
quantitation in biological matrices
—Mass spectrometry
—Chromatography
—Sample preparation
©2010 Waters Corporation | COMPANY CONFIDENTIAL 5
Goals
Provide tools and techniques to facilitate the
development of methods for accurate peptide
quantitation in biological matrices
—Mass spectrometry
—Chromatography
—Sample preparation
Integrate the workflow and demonstrate
performance
©2010 Waters Corporation | COMPANY CONFIDENTIAL 6
Goals
Provide tools and techniques to facilitate the
development of methods for accurate peptide
quantitation in biological matrices
—Mass spectrometry
—Chromatography
—Sample preparation
Integrate the workflow and demonstrate
performance
Understand parameters affecting successful
method development
©2010 Waters Corporation | COMPANY CONFIDENTIAL 7
Mass Spectrometry
Chromatography Sample
Preparation
Method Development
Bioanalytical Method Development for Peptides
©2010 Waters Corporation | COMPANY CONFIDENTIAL 8
Introduction
Why quantitative analysis for peptides?
1. Drug discovery/development activities need to be performed
o PK/PD, metabolic fate, bioequivalence, drug monitoring
2. Peptides as biomarkers
o Examples: Angiotensin II* used to monitor cardiovascular health
and natriuretic peptides* are biomarkers for cardiovascular
disease
3. Key peptides can be used to quantitate protein drugs and
biomarkers in complex matrices, after digestion of the sample
*included in this work
©2010 Waters Corporation | COMPANY CONFIDENTIAL 9
Today’s Focus
Why quantitative analysis for peptides?
1. Drug discovery/development activities need to be performed
o PK/PD, metabolic fate, bioequivalence, drug monitoring
2. Peptides as biomarkers
o Examples: Angiotensin II* used to monitor cardiovascular health
and natriuretic peptides* are biomarkers for cardiovascular
disease
3. Key peptides can be used to quantitate protein drugs and
biomarkers also, after digestion of the sample
*included in this work
©2010 Waters Corporation | COMPANY CONFIDENTIAL 10
Move Towards LC/MS/MS for Peptide Bioanalysis
Benefits of LC/MS/MS for peptides
—Broad dynamic range
—Accurate
—Universal
—Faster method development
—Excellent utilization of current instrumentation
LC/MS/MS assays that follow guidelines used for small
molecules would require
—Meeting matrix effects and ISR acceptance criteria
—Selective sample preparation
—High resolution chromatographic methods
—MSMS specificity
©2010 Waters Corporation | COMPANY CONFIDENTIAL 11
Addressing Peptide Diversity
Twelve therapeutic peptides identified:
Customer requests (9)
Biomarkers (2)
Generic drugs (1)
Broad range of chemical properties
—Mass range from 1019 to 4492
—Acidic (pI 3.9) to basic (pI 12)
—Very polar (HPLC index 7.6) to
very hydrophobic (HPLC index 156)
©2010 Waters Corporation | COMPANY CONFIDENTIAL 12
Chemical Properties of Therapeutic Peptides
Peptide MW pI # of Residues HPLC Index*
Octreotide 1019 9.3 8 40.8
Angiotensin II 1046 7.35 8 38.3
Desmopressin 1069 8.6 9 16.8
Vasopressin 1084 9.1 9 7.6
Goserelin 1270 7.3 10 31.7
Angiotensin I 1296 7.51 10 56.2
Somatostatin 1638 10.4 14 52.6
Neurotensin 1673 8.93 13 44.4
Bivalirudin 2180 3.87 20 46.2
BNP 3464 12 32 15.9
Teriparatide 4118 9.1 34 90.4
Enfuvirtide 4492 4.06 36 155.9
*higher number = more hydrophobic
©2010 Waters Corporation | COMPANY CONFIDENTIAL 13
Mass Spectrometry
Chromatography Sample
Preparation
Method Development
MS Method Development for Peptides
Goal: Develop a reliable, sensitive MRM method
©2010 Waters Corporation | COMPANY CONFIDENTIAL 14
Why Mass Spectrometry
Specificity
—Complex biological matrices
Sensitivity
—Multiply charged species, requires high sensitivity
—Accurate pK profiles
Rapid Method Development
Dynamic range
—3-4 orders magnitude
Dru
g C
on
c
Time
IV Dose
©2010 Waters Corporation | COMPANY CONFIDENTIAL 15
Why Mass Spectrometry
Reproducibility
—Multi-analyte assays
Accuracy
One analytical technique for many diverse peptides
Common, comfortable technology
Follows familiar regulatory guidelines
©2010 Waters Corporation | COMPANY CONFIDENTIAL 16
Multiply Charged Precursors
514515516517518519520521522523524525526527528529530531532533534m/z0
100
%
524.4
524.8
1038 1040 1042 1044 1046 1048 1050 1052 1054 1056 m/z0
100
%
1047.1
1048.2
1049.2
100 200 300 400 500 600 700 800 900 10001100120013001400150016001700180019002000m/z0
100
%
524.4
102.6371.6263.6
1047.1
Common to see 2+, 3+, 4+, 5+ depending on size of peptide
— Occasionally 1+ for smaller peptides
— Small molecules typically only 1+
Multiple charging: detection of
large peptides possible in lower
m/z ranges
Singly charged
Doubly charged
Example PeptideMW 1046
©2010 Waters Corporation | COMPANY CONFIDENTIAL 17
Singly Charged vs. Multiply Charged
m/z274 276 278 280 282 284 286 288 290 292 294 296 298 300 302 304 306
%
0
100 281
282
Small moleculeImipramine MW 280
Singly chargedObserved mass ESI+ = MW + 1 = 281
Calculation of expected m/z values for possible peptide
precursor ions
— Example (ESI pos): M= molecular weight
singly charged= (M+1)/1
doubly charged= (M+2)/2
triply charged= (M+3)/3
©2010 Waters Corporation | COMPANY CONFIDENTIAL 18
Calculation of Precursor Ions:Singly Charged vs. Multiply Charged
m/z274 276 278 280 282 284 286 288 290 292 294 296 298 300 302 304 306
%
0
100 281
282
m/z1086 1088 1090 1092 1094 1096 1098 1100
%
0
100 1090.85
1090.33
1091.36
1091.87
1092.38
1092.83
PeptideBivalirudin MW 2180
Doubly chargedObserved mass ESI+ = (MW + 2)/2 = 1091
* Resolution increased to show multiply charged precursor
Small moleculeImipramine MW 280
Singly chargedObserved mass ESI+ = MW + 1 = 281
Multiple charging: detection of large peptides
possible in lower m/z ranges
©2010 Waters Corporation | COMPANY CONFIDENTIAL 19
Process:Precursor Identification
Calculate m/z values for potential precursors
Infuse peptide standard into MS source1-10 µg/mL in 50/50 ACN/H2O
May be teed into LC flow
MS scan: identify the m/z of precursor ions presentLook for the calculated multiply charged precursor ion(s)
Optimize source parameters for maximum precursorsignal without in-source fragmentation
Note: Choice of mobile phase modifier and %, pH, LC flow rate, and specific amino acids
composition can impact formation of specific charge states and sensitivity 1,2
©2010 Waters Corporation | COMPANY CONFIDENTIAL 20
m/z400 600 800 1000 1200 1400 1600 1800
%
0
100
Scan ES+ 9.91e7
m/z1494 1495 1496 1497 1498 1499 1500 1501 1502 1503
%
0
100ENFUVIRTIDE Scan ES+
1498.32
1498.01
1498.64
1498.89
1499.34
Example: Precursor Identification
Enfuvirtide
MW 4492
Calculate possibilities— 3+ at m/z 1498
— 4+ at m/z 1124
Perform MS scan
3+ most intense— Requires at least 1500 amu
on first quad1498
©2010 Waters Corporation | COMPANY CONFIDENTIAL 21
MS Method Development: Fragmentation of Peptides
Each shape isan amino acid
Relative to small molecules, peptides may form many, less
intense fragments
— Can cleave at amide bond between amino acids
— MS sensitivity may be lower than for small molecules
Peptide
Possible Fragments
©2010 Waters Corporation | COMPANY CONFIDENTIAL 22
Fragmentation of Peptides
Goal: Identify stable, intense fragment
Fragments may appear at higher m/z values
than precursors
—Scan a wider m/z range for fragment identification
Tune collision energy and collision gas flow
Let’s look at an example…..
©2010 Waters Corporation | COMPANY CONFIDENTIAL 23
m/z400 600 800 1000 1200 1400 1600
%
0
100
Example: Peptide Fragmentation
Bivalirudin (MW 2180)
—MS scan shows 2+ precursor present at m/z 1091
—Perform MSMS of m/z 1091 from 100 to 1900
650
10911530
Doubly charged precursor
©2010 Waters Corporation | COMPANY CONFIDENTIAL 24
m/z400 600 800 1000 1200 1400 1600
%
0
100
Example: Higher Fragment m/z
Bivalirudin
— Major singly charged fragments at m/z 650 and m/z 1530
— Precursor appears at lower m/z even though MW is higher
— Higher m/z fragments require adequate mass range on 2cnd quadrupole
Singly charged fragments
Result:2 MRM Transitions identified
1091->6501091-> 1530
Note: If need be, area counts from several MRM transitions can be summed to increase sensitivity
650
10911530
©2010 Waters Corporation | COMPANY CONFIDENTIAL 25
Intellistart allows for automatic MSMS method
development
— Tunes and optimizes precursor
— Performs MSMS and tunes and optimizes fragments
— Generates report listing fragments and MSMS
conditions
— Automatically generates and populate MS method file
Narrowing down of fragment choices ideally done by
comparing Intellistart results to predictive software
(i.e. BioLynx) to aid in final fragment choice
— Fragments which are inherently selective for peptide
are ideal
o b or y ions for example
Intellistart: for automated fragment choice and optimization
©2010 Waters Corporation | COMPANY CONFIDENTIAL 26
MassLynx Molecular Weight Calculator: Bivalirudin
Used to calculate potential precursor m/z values for use in Intellistart
Infuse with on-board fluidics to determine which precursors are present
Run Intellistart
©2010 Waters Corporation | COMPANY CONFIDENTIAL 27
Input the
compound name
Calculate
potential
precursor
masses
Select Multiply
Charged Parents
Input the
multiply charged
precursor
mass(es) from
MW calculator
Select advanced
mode
Limit low mass
fragments
Click Start
Intellistart Set Up for Peptides
©2010 Waters Corporation | COMPANY CONFIDENTIAL 28
Intellistart Report for Bivalirudin
Abbreviated sample report for Bivalirudin showing optimization of collision energy for 1091-> 1531 and MSMS spectrum
List of fragments and conditions for multiple precursors, found by Intellistart
Report showing recommended MRM
transition and conditions
All parameters are automatically
populated into the an MS method and
saved
Automatic generation of a report showing
how parameters were selected
©2010 Waters Corporation | COMPANY CONFIDENTIAL 29
Bivalirudin Fragment Matching with BioLynx
Singly charged fragments at m/z 650 and 1530:b and y ions, cleavage at proline residue
©2010 Waters Corporation | COMPANY CONFIDENTIAL 30
MS Characteristics for Peptide Bioanalysis
Mass range—Need 1500-2000 amu both quadrupoles
o Ability to select higher m/z precursors for fragmentation
• 3+ precursor ions of larger peptides
o Ability to select higher m/z fragments
Fast scanning—Obtain sufficient data points across narrow UPLC peaks
o Typically 12-20
—Accurate and reproducible quantitation
Reproducible quantitation—Low RSD’s even when multiple MRMs monitored
simultaneously
o Multi-analyte assays
—Ensures accurate and precise quantitation to meet regulatory requirements
©2010 Waters Corporation | COMPANY CONFIDENTIAL 31
Mass Spectrometry
Chromatography Sample
Preparation
Method Development
Chromatography
Goal: Develop fast, sensitive, and selective starting LC method
©2010 Waters Corporation | COMPANY CONFIDENTIAL 32
Mass spectrometry compatible solvents and
modifiers
Acceptable peak shapes even for large (~5000 MW)
peptides
Short run times
Resolution from endogenous compounds
Maximize sensitivity
Ensure analyte solubility at all concentrations
— Injection solvent and needle washes contain 5-10% organic and
0.025-1% acid
Key Considerations for LC Screening Protocol
LC method chosen gives excellentperformance for diverse peptides
©2010 Waters Corporation | COMPANY CONFIDENTIAL 33
UPLC® Technology for Peptides:Chromatographic Screening Protocol
ACQUITY UPLC® BEH300 C18 2.1 X 50 mm, 1.7 µm
Peptide Separation Technology (PST) Column
—Columns are QC tested with peptide standards
—300Å PST column gave overall best performance (peak
shape) for diverse peptides
—2.1 X 50 mm provides adequate throughput
Generic gradients
o Mobile phase A = 0.1% formic acid
o Mobile phase B = acetonitrile
o Flow rate = 0.4 mL/min
o 15% B to 75% B over 2 minutes
• Start at 5% B for polar peptides
o Total cycle time 3.5 minutes
Note: formic acid used in mobile phase to avoid MS suppression associated with TFA
©2010 Waters Corporation | COMPANY CONFIDENTIAL 34
Single Screening Method: Diverse Peptides
Analyte MW
1. Vasopressin 1084
2. Angiotensin II 1046
3. Desmopressin 1069
4. Bivalirudin 2180
5. Enfuvirtide 4492
Broad molecular weight range2 very similar peptides; differ only by amino group
©2010 Waters Corporation | COMPANY CONFIDENTIAL 35
Single Screening Method: Diverse Peptides
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
MRM of 5 Channels ES+ 1 324
5
Peak widths 2-3 seconds wide at baseAdequate MS data pointsShort run times (3.5 min cycle time)
Analyte MW
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
Resolution between similar peptides
©2010 Waters Corporation | COMPANY CONFIDENTIAL 36
Importance of Particle Size for Sensitivity:HPLC vs. UPLC®
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
S/N:RMS=10725.03
S/N:RMS=18712.60
S:N 1.8X HPLC
Desmopressin standard 50 ng/mL
3.5 µm HPLC Column
1.7 µm UPLC Column
UPLC® significantly increased analyte S:N over HPLC,improving overall assay sensitivity
S:N Ratio
UPLC vs
HPLC
Enfuvirtide 1.5X
Desmopressin 1.8X
Bivalirudin 2.5X
Vasopressin 5.8X
Angiotensin II 6.0X
Note: Both columns tested on a UPLC system
©2010 Waters Corporation | COMPANY CONFIDENTIAL 37
Summary of Chromatography
For the diverse set of peptides tested, the ACQUITY
UPLC® BEH300 C18 1.7 µm columns provide
excellent peak shape
—Peak widths 2-3 seconds wide
Run times are 3 to 3.5 minutes
—Excellent throughput for bioanalytical samples
ACQUITY UPLC® PST columns provide the
sensitivity, resolution, and speed necessary when
run on a UPLC instrument
©2010 Waters Corporation | COMPANY CONFIDENTIAL 38
Mass Spectrometry
Chromatography Sample
Preparation
Method Development
Sample Preparation
Goal: Develop simple and selectivesample preparation method
©2010 Waters Corporation | COMPANY CONFIDENTIAL 39
Sample Preparation Requirements
Provides maximum analyte recovery
Minimizes matrix effects
Provides significant increase in sample
concentration to meet detection limits
Reproducible
Straightforward method development
Selectively separates peptides from matrix
components
Fast
©2010 Waters Corporation | COMPANY CONFIDENTIAL 40
Choice of Sample Preparation Technique
200 literature articles for peptide quantitation
surveyed 2
Most common sample prep techniques identified
—Reversed-phase SPE (RP SPE) and protein precipitation
(PPT) were very common
—Liquid-liquid-extraction (LLE) used in a few cases
Experiments in our labs
—2 peptides spiked into human plasma at 100 ng/mL
—RP SPE, PPT, LLE
—Criteria: high analyte recovery (>80%)
low matrix effects (<15%)
2 Van den Broek, I., Sparidans, R., Schellens, J., and Beijnen, J. J. Chromatogr. B, 2008, 872, 1-22.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 41
Performance of Current Sample Preparation Techniques
% Analyte Recovery
Bivalirudin (acidic) Desmopressin (basic)
* < 1% recovery for LLE
0
10
20
30
40
50
60
70
80
90
100
Reversed-phaseSPE
PPT LLE
*
Moderate to poor peptide recovery
©2010 Waters Corporation | COMPANY CONFIDENTIAL 42
Current Sample Preparation Techniques
% Analyte Recovery
Bivalirudin Desmopressin
* < 1% recovery for LLE
% Matrix Effects
0
10
20
30
40
50
60
70
80
90
100
Reversed-phaseSPE
PPT LLE-50
-40
-30
-20
-10
0
10
20
30
40
50
*
Reversed-phaseSPE PPT LLE
Moderate to poor recovery andSignificant suppression due to matrix effects
Can we apply what we know about selectiveextraction of small molecules to peptides?
©2010 Waters Corporation | COMPANY CONFIDENTIAL 45
Original Oasis® 2x4 Methodfor Small Molecules
Neutrals
For Strong AcidspKa <1.0
Use Oasis® WAX
For Strong BasespKa >10
Use Oasis® WCX
For AcidspKa 2-8
Use Oasis® MAX
Dilute plasma with 4% H3PO4
Condition/Equilibrate
Load Diluted Plasma
Wash:5% NH4OH
Elute 1:100% MeOH
Elute 2:2% HCOOH in 60:40 ACN:MeOH
Protocol 2
Dilute plasma with 4% H3PO4
Condition/Equilibrate
Load Diluted Plasma
Wash:2% HCOOH
Elute 1:100% MeOH
Elute 2:5% NH4OH in 60:40 ACN:MeOH
Protocol 1
Bases Strong Acids Strong Bases Acids
2 Elution steps:Elute 1 = reversed-phaseElute 2 = ion exchange
For Bases:pKa 2-10
Use Oasis® MCX
©2010 Waters Corporation | COMPANY CONFIDENTIAL 46
Original Oasis® 2x4 Methodfor Small Molecules
Neutrals
For Bases:pKa 2-10
Use Oasis® MCX
For Strong AcidspKa <1.0
Use Oasis® WAX
For Strong BasespKa >10
Use Oasis® WCX
For AcidspKa 2-8
Use Oasis® MAX
Dilute plasma with 4% H3PO4
Condition/Equilibrate
Load Diluted Plasma
Wash:5% NH4OH
Elute 1:100% MeOH
Elute 2:2% HCOOH in 60:40 ACN:MeOH
Protocol 2
Dilute plasma with 4% H3PO4
Condition/Equilibrate
Load Diluted Plasma
Wash:2% HCOOH
Elute 1:100% MeOH
Elute 2:5% NH4OH in 60:40 ACN:MeOH
Protocol 1
Bases Strong Acids Strong Bases Acids
What happens if we apply this approachto the extraction of peptides?
Protocol 1
For Bases:pKa 2-10
Use Oasis® MCX
©2010 Waters Corporation | COMPANY CONFIDENTIAL 47
Peptide Recovery Using Small Molecule SPE Screening Protocol
Recovery not sufficient to meet sensitivity requirements
Recovery split between reversed-phase and ion-exchange
elutions
Further modifications necessary
% SPE Recovery from Human Plasma
Desmopressin Bivalirudin
0
10
20
30
40
50
60
70
80
90
100
Oasis®
MCXOasis®
WAXOasis®
MAXOasis®
WCX
Elute 2
Elute 1
0
10
20
30
40
50
60
70
80
90
100
Oasis®
MCXOasis®
WAXOasis®
MAXOasis®
WCX
Elute 2
Elute 1
©2010 Waters Corporation | COMPANY CONFIDENTIAL 48
Peptide Recovery Using Small Molecule SPE Screening Protocol
Oasis® MAX and Oasis® WCX showed the most promise
1 protocol for both
Final eluate is compatible with mobile phase
% SPE Recovery from Human Plasma
Desmopressin Bivalirudin
0
10
20
30
40
50
60
70
80
90
100
Oasis®
MCXOasis®
WAXOasis®
MAXOasis®
WCX
Elute 2
Elute 1
0
10
20
30
40
50
60
70
80
90
100
Oasis®
MCXOasis®
WAXOasis®
MAXOasis®
WCX
Elute 2
Elute 1
©2010 Waters Corporation | COMPANY CONFIDENTIAL 49
Path to Peptide SPE Screening Protocol
Oasis® WCXµElution
Oasis® MAXµElution
Dilute plasma with 4% H3PO4
Condition MeOH/Equilibrate H2O
Load Diluted Plasma
Wash 1:5% NH4OH
Wash 2:100% MeOH
Elution:2% FA in 60/40 ACN/MeOH
Dilute:0.1 % TFA
Protocol
Oasis® WCXµElution
Oasis® MAXµElution
Dilute plasma with 4% H3PO4
Condition MeOH/Equilibrate H2O
Load Diluted Plasma
Wash 1:5% NH4OH
Wash 2:20% ACN
Elution:1% TFA in 75/25 ACN/H2O
Dilute:H2O
Protocol
Original Protocol Optimized Protocol
©2010 Waters Corporation | COMPANY CONFIDENTIAL 50
Oasis® PST SPE Protocol for Peptides
Oasis® WCXµElution
Oasis® MAXµElution
Dilute plasma with 4% H3PO4
Condition MeOH/Equilibrate H2O
Load Diluted Plasma
Wash 1:5% NH4OH
Wash 2:20% ACN
Elution:1% TFA in 75/25 ACN/H2O
Dilute:H2O
Protocol
©2010 Waters Corporation | COMPANY CONFIDENTIAL 51
SPE Recoveries Using Peptide Screening Protocol
Great results for diverse peptides:Screening protocol results in method for 75% of peptides!
% S
PE R
ecovery
0
20
40
60
80
100
120
Oasis MAX
Oasis WCX
©2010 Waters Corporation | COMPANY CONFIDENTIAL 52
Final SPE Results after BNP, Enfuvirtide and Somatostatin Methods Optimized
% S
PE R
ecovery
Minor, compound specific, modifications for 3 peptidesresult in excellent recovery for all peptides
0
20
40
60
80
100
120
Screening Protocol
Modified Protocol
©2010 Waters Corporation | COMPANY CONFIDENTIAL 53
Challenges in Peptide Extraction Development
Sample concentration required to meet detection limits
Evaporation of eluates may decrease peptide recovery due to
adsorption
Must meet throughput needs for bioanalysis
?
©2010 Waters Corporation | COMPANY CONFIDENTIAL 54
SPE FormatOasis® µElution Plates: Enabling Technology for Peptide Extraction
Oasis® µElution plate technology
Up to 15X concentration without evaporation
— Concentration often necessary to reach LOD’s with peptides
Minimizes analyte loss
— Thermally unstable peptides
— Peptides sticking to walls of collection plates
Speed: 96-well plate in <30 min,
<20 seconds/sample
©2010 Waters Corporation | COMPANY CONFIDENTIAL 55
Final SPE Summary
*= data being generated
Maximum recovery = enhanced sensitivityMinimum matrix effects = selectivity and sensitivity
Peptide pI MW
% SPE
Recovery
% Matrix
Effects
Octreotide 9.3 1019 88 <10%
Angiotensin II 7.35 1046 82 8%
Desmopressin 8.6 1069 104 <11%
Vasopressin 9.1 1084 100 -3%
Goserelin 7.3 1270 100 -2%
Angiotensin I 7.51 1296 109 *
Somatostatin 10.4 1638 94 *
Neurotensin 8.93 1673 114 6%
Bivalirudin 3.87 2180 100 10%
BNP 12 3464 84 *
Teriparatide 9.1 4118 97 9%
Enfuvirtide 4.06 4492 102 *
©2010 Waters Corporation | COMPANY CONFIDENTIAL 56
Summary of Sample Preparation
An efficient SPE screening strategy, based on 1 protocol and 2 Oasis® mixed-mode sorbents, simplifies method development for extraction of peptides from human plasma
Mixed-mode SPE provides selective clean-up of peptides from human plasma
— Matrix effects are <11% for those peptides tested
Oasis® µElution format SPE provides significant benefits for peptide extraction
— No evaporation step
— Up to 15X concentration, without evaporation
— 96 samples processed in <30 minutes, < 20 seconds per sample
©2010 Waters Corporation | COMPANY CONFIDENTIAL 57
Mass Spectrometry
Chromatography Sample
Preparation
Method Development
Total Solution for Peptide Method Development
©2010 Waters Corporation | COMPANY CONFIDENTIAL 58
Bioanalytical Method Development for Peptide Therapeutics: The Tools
ACQUITY UPLC® Xevo™ TQ MS
Method Development Kits
©2010 Waters Corporation | COMPANY CONFIDENTIAL 59
Xevo™ TQ MS for Peptide Bioanalysis
Highest Sensitivity
Adequate Mass Range
—2000 amu quadrupoles
—Enables selection of higher m/z precursors and fragments
Fast scanning
—10,000 amu/sec
—Maintain sensitivity even with minimum dwell times
—Obtain sufficient (12-20) data points across UPLC peaks
Novel Collision Cell Design
—ScanWave technology
o Enhanced MS sensitivity
©2010 Waters Corporation | COMPANY CONFIDENTIAL 60
Additional Xevo™ TQ MS Characteristics for Peptide Bioanalysis
Dual scan MRM to monitor background interferences during method development
Product Ion Confirmation (PIC) scan to confirm identity of peptide—Other very similar interferences
may be present, remember these are biological entities
Dual scan MRM to look for conformational or other chemical changes occurring during processing
©2010 Waters Corporation | COMPANY CONFIDENTIAL 61
Reducing Assay Variability
UPLC Xevo™ TQ MS/MS of a mixture of 6 peptides
— Cycle time 3.5 min
Reproducibility of MS response calculated when 6
peptide MRMs monitored simultaneously
— Dwell time = 0.03 seconds
RSD’s of area counts calculated, n= 10
Peptide
% RSD of Area
Counts
Points Across
MS Peak
Desmopressin 3.1 13
Vasopressin 3.1 14
Neurotensin 3.0 12
Angiotensin I 5.3 12
Angiotensin II 3.6 12
Bivalirudin 3.0 12
©2010 Waters Corporation | COMPANY CONFIDENTIAL 62
PST Method Development Kits
UPLC® PST Therapeutic Peptide Method Development Kit: Part# 176001835
ACQUITY UPLC® BEH300 C18 1.7 µm column
Oasis® µElution PST Method Development Plate
96-well 1 mL Collection Plate and Cap Mat
Detailed LC/SPE Screening Protocol
©2010 Waters Corporation | COMPANY CONFIDENTIAL 63
PST Method Development Kits
UPLC® PST Therapeutic Peptide Method Development Kit: Part# 176001835
ACQUITY UPLC® BEH300 C18 1.7 µm column
Oasis® µElution PST Method Development Plate
96-well 1 mL Collection Plate and Cap Mat
Detailed LC/SPE Screening Protocol
HPLC PST Therapeutic Peptide Method Development Kit: Part# 176001836
XBridge BEH300 C18 3.5 µm column
Oasis® µElution PST Method Development Plate
96-well 1 mL Collection Plate and Cap Mat
Detailed LC/SPE Screening Protocol
©2010 Waters Corporation | COMPANY CONFIDENTIAL 64
Total Solution:Bioanalysis of Desmopressin
Desmopressin is a synthetic peptide
— Modified version of human arginine vasopressin
or anti-diuretic hormone
Used to treat bed-wetting and diabetes
Advantages over recombinant vasopressin
— Degrades more slowly, enabling less frequent
dosing
— Does not raise blood pressure as seen with the
unmodified peptide
Typical “small molecule” acceptance criteria
applied
Limit of Quantitation (LLOQ) required is 20 pg/mL (21 fmol/mL)
—Sample pre-concentration
—Selective sample preparation
—High sensitivity LC and MS instrumentation and methods
©2010 Waters Corporation | COMPANY CONFIDENTIAL 65
MS Method Development for Desmopressin
Desmopressin acetate has a molecular weight of 1069
Doubly and triply charged precursor masses calculated
—Doubly: (1069 + 2)/2 = m/z 535.5
—Triply: (1069 + 3)/3 = 357.3
Infusion with on-board fluidics
—Only doubly charged precursor observed, m/z 535.5
Intellistart used for precursor and fragment
optimization
©2010 Waters Corporation | COMPANY CONFIDENTIAL 66
m/z300 400 500 600 700 800 900 1000 1100
%
0
100
Scan ES+ 2.77e6
535.31
375.31
329.01
441.28
1123.48
909.77 1069.10
MS spectrum of Desmopressin
m/z528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543
%
0
100
535.31
528.93
535.83
536.35
536.80
MS scan performed and desmopressin doublycharged precursor observed at m/z 535 as predicted
©2010 Waters Corporation | COMPANY CONFIDENTIAL 67
Intellistart Process
Intellistart Process for Peptides
Input the compound name
Calculate potential precursor masses
Input the multiply charged precursor mass(es)
Select advanced mode
Limit low mass fragments
Click Start
©2010 Waters Corporation | COMPANY CONFIDENTIAL 68
Intellistart Results
Report showing
recommended MRM
transition and conditions
All parameters are
automatically populated
into the an MS method
and saved
Automatic generation of a report showing how parameters were selected
Abbreviated sample report for desmopressin showing optimization of collision energy for 535-> 328 and MSMS spectrum
List of fragments and conditions found by Intellistart
©2010 Waters Corporation | COMPANY CONFIDENTIAL 69
MSMS of Desmopressin
MSMS of doubly charged parent->
Singly charged major fragmentobserved at m/z 328
Collision energy and flow optimized for generation of
fragments
—Desmopressin fragments at the proline amino acid residue
—Prolines are common cleavage points
m/z200 300 400 500 600 700 800 900 1000 1100
%
0
100
Daughters of 535ES+ 5.11e4
328.02
©2010 Waters Corporation | COMPANY CONFIDENTIAL 70
m/z200 300 400 500 600 700 800 900 1000 1100
%
0
100
Daughters of 535ES+ 5.11e4
328.02
Matching MSMS Spectrum:BioLynx Application Manager
MSMS spectrum can be matchedto predicted MS fragments
If peptide sequence is known, the BioLynx application
manager within MassLynx can be used to predict peptide pI,
HPLC index and MS fragments
©2010 Waters Corporation | COMPANY CONFIDENTIAL 71
Predicting pI and HPLC Index: BioLynx Application Manager
If peptide sequence is known, the BioLynx application
manager within MassLynx can be used to predict peptide pI,
HPLC index and MS fragments
©2010 Waters Corporation | COMPANY CONFIDENTIAL 72
SPE Method Development:PST Screening Protocol
SPE screening method from kit used
to extract desmopressin from 300
µL human plasma
Analyte recovery best on Oasis®
WCX
Matrix effects <10% on Oasis® WCX
Oasis® WCXµElution
Oasis® MAXµElution
Dilute plasma with 4% H3PO4
Condition MeOH/Equilibrate H2O
Load Diluted Plasma
Wash 1:5% NH4OH
Wash 2:20% ACN
Elution:1% TFA in 75/25 ACN/H2O
Dilute:H2O
Protocol
% S
PE R
eco
very
0
20
40
60
80
100
Oasis® MAX Oasis® WCX
©2010 Waters Corporation | COMPANY CONFIDENTIAL 73
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
-1
99
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
-1
99
MRM of 1 Channel ES+
1.08e3
2.50
MRM of 1 Channel ES+
1.08e31.09
2.42
Desmopressin Extracted from 300 µL Human Plasma: LLOQ
LLOQ
Required LLOQ met with no changes to basic protocol in kit
Blank humanPlasma
No interferences
20 pg/mL(21 fmol/mL)Desmopressin Time
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40%
-1
99x101.09
2.42
BackgroundMagnified 10X
©2010 Waters Corporation | COMPANY CONFIDENTIAL 74
Desmopressin: Method Linearity
Internal Standard= Octreotide spiked at 1 ng/mLr2 = 0.997, linear fit, 1/x weighting
Linear over wide dynamic rangeAccuracy and precision meet validation acceptance criteria
Standard
Conc.
ng/mL Area IS Area Conc. %Dev
0.02 15 3341 0.02 11.1
0.05 30 3159 0.05 7.5
0.1 42 2451 0.10 0.1
1 425 2654 0.98 -2.4
2 1009 3282 1.88 -6.2
10 4354 3089 8.62 -13.8
20 9168 2769 20.25 1.2
50 25101 2994 51.28 2.6
©2010 Waters Corporation | COMPANY CONFIDENTIAL 75
After Desmopressin Method Optimization
5 pg/mL desmopressin
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
1.0029
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
5 pg/mL
Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20
%
0
100
MRM of 2 Channels ES+ 535.5 > 328.2 (desmopressin)
1.90e3Area
1.0029
1.0029
Red trace = blank human plasmaGreen trace= 5 pg/mL desmopressin
©2010 Waters Corporation | COMPANY CONFIDENTIAL 76
Angiotensin II: Oasis® WCX Basic Starting LC and SPE Protocols
350 µL human plasma
1 pg/mL
5 pg/mL
Blank plasma
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
-2
98
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
-2
98
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
-2
98
MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)
3.64e3Area
0.7980
MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)
3.64e3Area
0.7810
MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)
3.64e3Area
0.799
©2010 Waters Corporation | COMPANY CONFIDENTIAL 77
Angiotensin I: Oasis® MAX Basic Starting LC and SPE Protocols
350 µL human plasma
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
4
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
4
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
4
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
4
MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)
1.54e3Area
0.8626
MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)
1.54e3Area
0.8519
MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)
1.54e3Area
0.8712
MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)
1.54e3Area
0.863
1 pg/mL
10 pg/mL
5 pg/mL
Blank plasma
©2010 Waters Corporation | COMPANY CONFIDENTIAL 78
Conclusions
Waters Xevo™ TQ MS triple quadrupole mass spectrometer
can be successfully used to reliably and reproducibly
quantitate peptides in human plasma
ACQUITY UPLC BEH300 PST 1.7 µm columns provide
excellent peak shape, sensitivity and selectivity for peptides
A simple, straightforward Oasis® µElution SPE method
development strategy for extraction of peptides from human
plasma has been developed
The combination of Oasis® µElution SPE, ACQUITY UPLC®, and Xevo TQ MS provide the sensitivity,
speed, reliability and selectivity required for bioanalysis of peptide therapeutics and peptide
biomarkers
©2010 Waters Corporation | COMPANY CONFIDENTIAL 79
Acknowledgements
Joe Arsenault
Geneen Baynham
Patrick Boyce
Erin Chambers
Diane Diehl
Beth Gillece-Castro
Gislinde Gundel
Yasser Ismail
Joanne Mather
Damian Morrison
Anne-Marie Orkild
Rob Plumb
Paul Rainville
Shunya Sasaki
Tom Wheat
Jessalynn Wheaton
Hannah White