Figure 2. Sample Processing Workflow Used for Bevacizumab Enrichment and Digestion.

Results

Selection and Optimization of Signature Peptides

MRM experiments of a Bevacizumab digest were performed on 5500 QTRAP to screen for the 21 candidate tryptic peptides generated:

The most sensitive precursor ion charge state was selected for each peptide.

The relative abundance of the MRM transitions was evaluated to identify the 2 best MRM transitions for each peptide.

The CE and DP values were optimized for each of the selected MRM transitions.

An overview of the strategy used for the selection and optimization of tryptic peptides is shown in Fig. 3.

Figure 3. Selection and optimization of signature peptides using Skyline. Skyline allowed to reduce the list of MRM transitions down to the 15 signature peptides and to select the optimal instrument settings for each of the 30 selected MRM transitions. The optimized DP and CE values were recorded and used in the final MRM method.

Final MRM Method Tested on Bevacizumab Digest (pure solution)

The final MRM method with optimized conditions was tested on a Bevacizumab tryptic digest. The chromatogram obtained is shown in Fig. 4.

Figure 4. Representative LC-MS/MS chromatogram of Bevacizumab digest using the optimized MRM method created from Skyline software. Bevacizumab was spiked at 10μg/mL in pure solution, processed and analyzed by LC-MS/MS. Note: no immunocapture enrichment was performed.

Introduction

LC-MS/MS bioanalytical methods using signature peptides as stoichiometric representations have emerged for quantification of monoclonal antibodies (mAbs) in biological matrices. While promising, the selection of sensitive and selective signature peptides remains a critical step in the development of a robust bioanalytical method to deliver high-quality data for mAb quantitation.

The aim of this study was to develop a LC-MS/MS method for the determination of Bevacizumab, a humanized anti-VEGF monoclonal antibody, in rat plasma. This approach combines specificity of immunocapture for sample preparation and selectivity of mass spectrometry for detection. This poster describes the strategy used to select and optimize MRM signature peptides, and to identify the most sensitive and selective signature peptides of a Bevacizumab digest in rat plasma using Skyline software.

Method

Analyte Bevacizumab (Avastin®, GENENTECH/ROCHE)

recombinant humanized monoclonal antibody

to VEGF

Matrix Rat plasma

Sample volume 100µL

Immunocapture method Streptavidin immobilized-tips (Thermo Scientific)

Biotin conjugated anti-mAb-Fc (Human)

antibody (CaptureSelectTM)

Enzymatic digestion Trypsin, MS Grade

LC system Waters Acquity UPLC I-Class System (SM-FTN)

LC Method UPLC reverse phase

Analytical column BEH C18, 2.1mm x 50mm, 1.7µm particles

Mobile phase A 0.1% formic acid in water

Mobile phase B 0.1% formic acid in acetonitrile

Flow rate, elution mode 300µL/min, linear gradient

MS system Sciex 5500QTRAP

Acquisition mode, polarity MRM, positive ESI

Injection volume 2µL

Selection and optimization of signature peptides suitable for quantification

Candidate tryptic peptides were selected according to defined criteria. Details on the method used to select and optimize signature peptides with Skyline are shown in Fig.1.

Figure 1. Method used for identification and selection of the best peptide MRM transitions and optimization of CE and DP parameters of selected MRM transitions. Unscheduled MRM methods (diagram A) were generated to identify peptide’s retention times and to select the best MRM transitions for each tryptic peptide. Scheduled MRM methods (diagram B) were used to optimize CE and DP parameters.

Sample Preparation and Tryptic Digestion Workflow

Bevacizumab was purified from rat plasma by immunocapture using streptavidin MSIA-D.A.R.T.’S previously activated with a biotin conjugated antibody specific to the Bevacizumab Fc region. The sample processing workflow is depicted in Fig. 2.

A Hybrid Immunocapture-LC-MS/MS Method for the Determination of Humanized Monoclonal Antibody in Rat PlasmaH. Kharbouche, S. Denham and P. StruweCelerion Switzerland AG, 8320 Fehraltorf, Switzerland

Poster presentation at EBF 2016

ConclusionsOur approach demonstrated its utility in identifying the most effective signature peptides for quantitation of Bevacizumab in rat plasma. The method offers the advantages to:

Quantify other mAbs in preclinical studies by monitoring the 3 signature peptides which were found to be conserved among different human mAbs.

Quantify Bevacizumab in clinical studies by monitoring the 2 signature peptides which were found to be unique.

This method will be tested, together with a heavy isotope labeled mAb internal standard, for bioanalytical assay performance.

Figure 6. Extraction chromatogram (XIC) of GPSVFPLAPSSK peptide (+2 charge state) from a digested immuno-enriched sample containing 10μg/mL Bevacizumab in pure solution (A) or in rat plasma (B).

Bevacizumab sequence

List of candidate peptides

ASelect best

MRM transitions

BOptimizeCE / DP

Peptide con�rmation in

�nal assay

2Run MRM

experiments

5Export method

1Create MRM

methods

UnscheduledMRM

A 2Run CE/DP optimization experiments

5Export method with optimized

1Create CE/DP

optimization MRM methods

B

3Load generated

MRM data

4Analyze results

Identify peptides & their respective RT

with selected MRM transitions

MRM3

4Analyze results

Identify best CE/DP parameters

with optimized CE/DP Load generated

MRM data

MRMScheduled

Light chain

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP

EDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS

TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Signature peptides being the most sensitive

Selected and optimized signature peptides

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

DTLMISR

ALPAPIEK

GPSVFPLAPSSK

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

Inte

nsity

, cps

FTFSLDTSK

NQVSLTCLVK

Position Selected peptide Sequence

MRM transition MW pI # of residues

Region in Bevacizumab

254 - 260 DTLMISR 418.2 (2+) 619.4 (y5+)

418.2 (2+) 506.3 (y4+)

834.99 7.4 7 Fc

332 - 339 ALPAPIEK 419.8 (2+) 654.4 (y6+)

419.8 (2+) 486.3 (y4+)

838.01 6.4 8 Fc

127 - 138 GPSVFPLAPSSK 593.8 (2+) 846.5 (y8+)

593.8 (2+) 699.4 (y7+)

1186.37 10.1 12 Fab

366 - 375 NQVSLTC[+57]LVK 581.3 (2+) 919.5 (y8+)

581.3 (2+) 820.5 (y7+)

1161.38 9.0 10 Fc

67 - 75 FTFSLDTSK 523.3 (2+) 797.4 (y7+)

523.3 (2+) 650.3 (y6+)

1045.16 6.4 9 Fab

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

Inte

nsity

, cps

4.21

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Inte

nsity

, cp

s

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

Inte

nsity

, cps

4.22

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4Ti i

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

Bevacizumab in rat plasma

Bevacizumab in pure solution

GPSVFPLAPSSK

GPSVFPLAPSSK

593.8 � 699.4

593.8 � 846.5

593.8 � 699.4

593.8 � 846.5

Table 1. Characteristics of the selected signature peptides. The three peptides DTLMISR, ALPAPIEK and NQVSLTC[+57]LVK are generated from the constant region (Fc) of the Bevacizumab and can thus be used as generic signature peptides since they are conserved among different human mAb (IgG) subclasses. The two other peptides GPSVFPLAPSSK and FTFSLDTSK are generated from the variable region (Fab) of Bevacizumab and can thus be used as unique signature peptides since they are only present in Bevacizumab.

The background noise levels were compared for the 5 selected signature peptides, in rat plasma and in pure solution for selectivity assessment. The immuno-enrichment procedure, combined to mass spectrometry, allowed to remove endogenous interferences from rat plasma, and resulted in good selectivity (Fig. 6).

Figure 5. Amino acid sequence information of the light and heavy chain of Bevacizumab. The blue-underlined sequences represent the 15 optimized signature peptides. The red-squared sequences represent the most sensitive signature peptides.

mAb DENATURATION (protein unfolding)

Neutralisation (Ammonium bicarbonate)Incubation: RapiGest surfactants

mAb REDUCTION (breaking disulfide bonds)

Incubation: Dithiothreitol (DTT)

mAb ALKYLATION (avoid non-specific S-S formation)

Incubation: Iodoacetamide (IAA)

mAb DIGESTION (peptide formation)

Incubation: Trypsin (enzyme:protein ratio 1:20)Quench with formic acid

mAb IMMUNO-CAPTURE (mAb enrichment)

DESALTING (salt removal)

Incubation: activated MSIA with rat plasma (100µL)Wash: 1xPBS / waterElution: 0.4% aqueous TFA

Oasis HLB µElution plate

~1.5hour at RT

10min at RT

30min at 56°C

30min at RT

3hours at 37°C

30min at RT

Streptavidin MSIA D.A.R.T.’S ACTIVATION

Incubation: Biotin-conjugated anti-mAb-Fc antibody with immobilized streptavidin

~30min at RT

En

ab

le d

ata

acq

uis

itio

n w

ithin

a d

ay (

pro

ced

ure

take

s <

7h

ou

rs)

LC-MS/MS analysis (2µL)

List of candidate peptides

Selected MRM transitionsCE / DP optimization

21 peptides / 42 precursor ions 416 transitions

15 peptides / 15 precursor ions 30 transitions

Selection criteria applied

Identi�ed MRM transitions

Experimentaldata

On-column tuningScheduled MRM methods

Candidate tryptic peptide selection criteria

Included 7 to 25 amino acid-containing peptides, multiple positively charged precursor ions (+2 and +3 charge states), b and y product ions (+1 and +2 charge states).

Product ions with m/z higher than the precursor ions.

Excluded Peptides containing residues susceptible to posttranslational or artefactual modifications.

Peptides containing sequence that commonly results in missed cleavage.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e66.7e6

Inte

nsity

, cp

s

3.03

VVSVLTVLHQDWLNGK

TTPPVLDSDGSFFLYSK

LSCAASGYTFTNYGMNWVR

FTFSLDTSK

GPSVFPLAPSSK

NQVSLTCLVK

STAYLQMNSLR

AEDTAVYYCAK

ALPAPIEK

STSGGTAALGCLVK

TPEVTCVVVDVSHEDPEVK

FNWYVDGVEVHNAK

DTLMISR

VYACEVTHQGLSSPVTK

DSTYSLSSTLTLSK

Identification of the Most Sensitive Signature Peptides in Rat Plasma

The final MRM method was tested on Bevacizumab in rat plasma. The 15 signature peptides were simultaneously monitored to identify peptides that produced the best sensitivity. The best sensitivity was achieved using the signature peptides shown in Fig. 5. The key characteristics of the selected signature peptides are shown in Table 1.