bioanalytical method development for therapeutic peptides · ©2010 waters corporation | company...

©2010 Waters Corporation | COMPANY CONFIDENTIAL 1

Bioanalytical Method Development for

Therapeutic Peptides


Challenge: Quantify Therapeutic Peptides in Human Plasma

Desmopressin MW 1069


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


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


1.90e3Area

1.0029

1.0029

Red trace = blank human plasmaGreen trace= 5 pg/mL desmopressin


Goals

Provide tools and techniques to facilitate the

development of methods for accurate peptide

quantitation in biological matrices

—Mass spectrometry

—Chromatography

—Sample preparation


Goals





—Chromatography


Integrate the workflow and demonstrate

performance


Goals





—Chromatography


Integrate the workflow and demonstrate

performance

Understand parameters affecting successful

method development


Mass Spectrometry

Chromatography Sample

Preparation

Method Development

Bioanalytical Method Development for Peptides


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


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


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


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)


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


Mass Spectrometry


Preparation

Method Development

MS Method Development for Peptides

Goal: Develop a reliable, sensitive MRM method


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


Why Mass Spectrometry

Reproducibility

—Multi-analyte assays

Accuracy

One analytical technique for many diverse peptides

Common, comfortable technology

Follows familiar regulatory guidelines


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


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


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


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


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


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


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…..


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


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


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


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


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


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


Bivalirudin Fragment Matching with BioLynx

Singly charged fragments at m/z 650 and 1530:b and y ions, cleavage at proline residue


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


Mass Spectrometry


Preparation

Method Development

Chromatography

Goal: Develop fast, sensitive, and selective starting LC method


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


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


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


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


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


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


Mass Spectrometry


Preparation

Method Development

Sample Preparation

Goal: Develop simple and selectivesample preparation method


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


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.


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


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?


Solid-Phase Extraction: reversed-phase backbone


Mixed-mode Ion Exchange and Reversed-phase


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



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


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



Load Diluted Plasma

Wash:5% NH4OH

Elute 1:100% MeOH

Elute 2:2% HCOOH in 60:40 ACN:MeOH

Protocol 2



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


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


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


Path to Peptide SPE Screening Protocol

Oasis® WCXµElution

Oasis® MAXµElution


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





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


Oasis® PST SPE Protocol for Peptides





Load Diluted Plasma

Wash 1:5% NH4OH

Wash 2:20% ACN


Dilute:H2O

Protocol


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


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


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

?


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


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 *


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


Mass Spectrometry


Preparation

Method Development

Total Solution for Peptide Method Development


Bioanalytical Method Development for Peptide Therapeutics: The Tools

ACQUITY UPLC® Xevo™ TQ MS

Method Development Kits


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


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


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


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


PST Method Development Kits

UPLC® PST Therapeutic Peptide Method Development Kit: Part# 176001835

ACQUITY UPLC® BEH300 C18 1.7 µm column




HPLC PST Therapeutic Peptide Method Development Kit: Part# 176001836

XBridge BEH300 C18 3.5 µm column





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


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


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


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


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


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


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


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


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





Load Diluted Plasma

Wash 1:5% NH4OH

Wash 2:20% ACN


Dilute:H2O

Protocol

% S

PE R

eco

very

0

20

40

60

80

100

Oasis® MAX Oasis® WCX


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


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


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


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


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


1.90e3Area

1.0029

1.0029

Red trace = blank human plasmaGreen trace= 5 pg/mL desmopressin


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


3.64e3Area

0.7810


3.64e3Area

0.799


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


1.54e3Area

0.8519


1.54e3Area

0.8712


1.54e3Area

0.863

1 pg/mL

10 pg/mL

5 pg/mL

Blank plasma


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


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

bioanalytical method development for therapeutic peptides · ©2010 waters corporation | company...

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