Bioanalytical Method Development Strategy for Therapeutic Peptides

Oasis® SPE sorbents are unique in their combination of purity, reproducibility, stability, and retention characteristics. The 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.

The 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.

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. The 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.

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.

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. The 2000 amu range of the first resolving quadrupole represents a critical element for the generic applicability of this approach.

samPle P reParat Ion

format

column

lc

mass sPect romet ry

The 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

The Tools

The 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.

A successful generic bioanalytical assay needs to be:

■ Applicable to a diverse range of therapeutic peptides

■ Selective and free from significant matrix interferences

■ Linear over a wide dynamic range

■ Capable of supporting high throughput assays.

Creating a Universal Approach to a Complex Problem

NH

O HN

O O

NH2

HN O

HN

O

OH

NH

SS

O

N

NH O

HNNH2

HN

HNH2N

O

O O

NH2

O

SCH2CH2C-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2•CH3COOH•3H2

O

0

desmoP ressIn ac etat e

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.

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

%

0

0

%

100MRM of 1 Channel ES+

1.28e3

MRM of 1 Channel ES+ 1.28e3

1.09

Blank Plasma

20 pg/mL (21.4 fmol/mL)Desmopressin in human plasma104% SPE RecoveryMatrix Effects < 5%

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 recursor

Therapeutic 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.

Enfuvirtide, mw 4492, has possible precursor m/z values of 1498 for 3+ charge state and 1124 for 4+ charge state. Under the analytical conditions used, the 3+ was the most intense charge state present, very little of the 4+ was observed.

m/z1494 1495 1496 1497 1498 1499 1500 1501 1502 1503

%

0

100Scan ES+

1498.32

1498.011498.64

1498.89

1499.34

m/z

400 600 800 1000 1200 1400 1600 1800

%

0

100Scan ES+

9.91e7

m/z1494 1495 1496 1497 1498 1499 1500 1501 1502 1503

%

0

100Scan ES+

1498.32

1498.011498.64

1498.89

1499.34

m/z

400 600 800 1000 1200 1400 1600 1800

%

0

100Scan ES+

9.91e7

Enfuvirtidemw 4492

oPt ImIzIng for f ragmentat Ion

The ultimate sensitivity of the triple quadrupole MS detection method depends upon the ability to optimize for, and detect the most intense fragment(s). The 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.

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.

Note: If need be, area counts from several MRM transitions can be summed to increase sensitivity.

m/z400 600 800 1000 1200 1400 1600

%

0

100

Doubly charged precursor

Singly charged fragments

MRM Transitions identified:

1091 > 650

1091 > 1530

650

10911530

chromatograPhIc Performanc e

A key parameter in the success of a generic method development approach is the correct choice of LC instrumentation and associated column chemistry. The 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. The 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.

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

%

100

0

MRM of 5 Channels ES+

1 324

5Analyte 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

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.

Bivalirudinmw 2180

Sample Preparation Challenge

The 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. The 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.

select Iv e samPle ext ract 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.

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 Therapeudic Peptide Method Development Kit and ACQUITY UPLC with Xevo TQ MS systems.

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

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

0

20

40

60

80

100

120

Oasis MAX

Oasis WCX% R

ecov

ery

Desmop

ressin

Vaso

pressi

n

Octreo

tide

Bivali

rudin

BNP

Gosere

lin

Enfuv

irtide

Angiot

ensin

I

Angiot

ensin

II

Neurot

ensin

Somato

statin

Terip

aratid

e

Pst t heraPeut Ic PePt Ide met hod dev eloPment K It

The PST Therapeutic 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. The 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 Part No. Qty/Box

UPLC PST Therapeutic Peptide Method Development Kit 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 Therapeutic 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

Available Waters Products Not Included in Kit:

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

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720003055EN June 2009 SC-FP

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