a hybrid immunocapture-lc-ms/ms method for the … · 2018-10-26 · included 7 to 25 amino...
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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.
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