two dimensional lc-srm assay for a therapeutic ...©2012 waters corporation 1 two dimensional lc-srm...

©2012 Waters Corporation 1

Two Dimensional LC-SRM Assay for a Therapeutic Monoclonal

Antibody and potential for Accurate Mass Quantitation

Dr Diego Rodriguez CabaleiroWaters Europe

Catalin E. Doneanu, PhD.


PBA general strategies PBA general strategies ––advantages and drawbacksadvantages and drawbacks

Involving protein isolation/purification

Pros Cons

-significantly less complex matrix -requires protein IS (expensive)-more sensitive -affinity chromatography is protein specific

-what format to choose (MB, tips, cartridges)-more elaborate workflows

No protein fractionation

Pros Cons

-affordable 13C15N-isotopically labeled -very complex serum digest matrixpeptide IS -variability of protein vs peptide IS digestion


ESIESI--MS spectrum of MS spectrum of VicamVicam G4 G4 mAbmAb

Sample amount: 1 µg mAb


Peptide Based Quantification

PEPTIDE

Mod

Mod

Mod

Sensitivity requirements:

mAbs: 1 nM or ~ 150 ng/mL or ~ 10 ppm (w/w) mAb in serum

Recombinant proteins: 10-100 ppm in serum

Unique peptide !


Dynamic range issuesDynamic range issues

DR of chromatographic separation

DR of trypsin digestion

DR of electrospray ionization

mass spectrometer DR


Complexity of serum proteomeComplexity of serum proteome


Peptide interferencesPeptide interferences

Unpredictable Interferences !

Possible solutions:

1D/2D chromatography

ion mobility

more sample prep

combination of the above

Predictable Interferences

(use software tools to avoid them)


Example of MRM interferenceExample of MRM interference

8.55

T43


General Workflow for Protein General Workflow for Protein BioanalysisBioanalysis

1. Identify unique tryptic peptides from the therapeutic protein and validate their presence in a protein digest by monitoring the corresponding MRM transitions (3-5 transitions per peptide).

2. Optimize the MRM transitions in terms of cone voltage, collision energy and dwell time.

3. Assess the linearity, detection limit and peak area RSD for each proteotypic peptide in the absence of the sample matrix (serum digest).

4. Assess the linearity, detection limit, peak area RSD and MRM interference for each proteotypic peptide in the sample matrix.

5. Evaluate the MRM interference phenomenon for several serum matrices (e.g. human, rat, monkey, rabbit sera). Also evaluate the MRM interference for the corresponding IS peptides (13C, 15N-isotopically labeled peptides) and select the IS peptides based on this evaluation.

6. Design the final MRM assay for the therapeutic protein based on steps 1-5 and obtain the IS peptides.

7. Optimize the trypsin digestion protocol for the digestion of the therapeutic protein in serum using 1-3 extended IS peptides (containing 1 missed cleavage).

8. Test the MRM assay for different protein concentrations spiked in the serum sample (QC samples).


PBA sample preparation workflow for PBA sample preparation workflow for trastuzumabtrastuzumab ((herceptinherceptin))

Spiking the 13C15N-isotopically labeled extended peptides)

Protein denaturation, disulfide bond reduction

Cys alkylation

SPE clean-up

RG precipitation, centrifugation

LC-MRM

0.05 % RG, 10 min @ 80 oC 20 mM DTT, 60 min @ 60 oC

10 mM IAM, 45 min @ RT

Protein : porcine trypsin = 25:1 (w/w), 16 h, @ 37 oC

Add TFA, 30 min @ 37 oC Spin at 10,000 rpm for 10 min

Oasis MCX µElution plate

2.1x 150 mm BEH column (P/N 3687)

300 µL/min, 10 min gradientXevo TQ-S

GRFTISADTSKDTYIHWVRQA

Herceptin is spiked into human serum

Trypsin digestion


MRM quantification of MRM quantification of trastuzumabtrastuzumab in human in human serum using serum using isotopicallyisotopically labeled peptideslabeled peptides

Serum digest blank

1 nM trastuzumabin human serum digest

5 nM trastuzumabin human serum digest

10 nM trastuzumab in human serum digest

Serum digest blank

1 nM 13C15N-labeled peptide in human serum digest



MRM quantification of trastuzumab in human serum on a Trizaic Xevo TQ-S platform. MRMchromatograms for the spiked trastuzumab (1,5,10 nM) and the corresponding 13C15N-isotopically labeledpeptide spiked at the same concentration in human serum.

Native peptide 13C15N-labeled peptide

ASMS 2011 poster


TrypsinTrypsin digestion optimization stepsdigestion optimization steps

Optimization of protein denaturation with RapiGest

Finding the appropriate protein : trypsin digestion ratio

Testing digestion reproducibility

Evaluation of different trypsin sources (vendors)

Digestion time optimization

Optimization of protein reduction/alkylation


Optimization of protein : Optimization of protein : trypsintrypsin ratioratio

20

30

40

50

60

70

80

90

100

10 20 30 50 100

Protein to trypsin ratio

Digestion Efficiencynative peptide13C15N peptide

Digestions were performed using Sigma T-6567 trypsin


Digestion Time OptimizationDigestion Time Optimization

native peptide13C15N peptide


0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

I II III IV V

Digestion replicates (20:1 protein to trypsin ratio)

Average Peak Area 12C14N

13C15N2.2

2.42.4 2.5

2.2

RSD for 12C/13C : 5.7% (n=5)

Reproducibility of Reproducibility of trypsintrypsin digestiondigestion

5 nM Herceptin and 5 nM 13C15N-peptides were spiked in human serum and digested with Sigma T-6567


Evaluation of Evaluation of trypsintrypsin vendorsvendors

PT-42316 ($150/mg)

GOLD ($1030/mg)

TRL3 ($0.3/mg)

T-0303 ($9.2/mg)

T-6567 ($473/mg)

T-1426 ($0.5/mg)

TRL ($0.2/mg)

TRTPCK ($0.7/mg)

V511 ($820/mg)

2.08 2.03

1.36

2.431.89

1.91

1.05

1.86

1.92

SIGMA WORTHINGTON PROMEGA NOVATEINBIO


2D2D--LC setup: 1LC setup: 1stst dimension separation (pH=10) dimension separation (pH=10)

XBridge C18 1.0 x 50 mm, 2.5 µm

BEH300 C18 2.1 x 50 mm, 1.7 µm

186003685 TQ-S MS300 µL/min

100 µL/min

High pH separation (pH 10)A: 20 mM ammonium formateB: 20 mM ammonium formate in 90% ACN

Low pH separation (pH 2.5)A: 0.1 % FAB: 0.1% FA in ACN

186003118


2D2D--LC setup: isolation of LC setup: isolation of analyteanalyte fraction fraction

TQ-S MS


2D2D--LC setup: 2LC setup: 2ndnd dimension separation (pH=2) dimension separation (pH=2)

TQ-S MS


Linearity of the 2DLCLinearity of the 2DLC--MRM assayMRM assay<2% RSD, n=4<2% RSD, n=4

0.1 nM

1 nM

50 nM

5 nM


RADAR monitoring of the SPE cleanRADAR monitoring of the SPE clean--up sampleup sample1DLC chromatographic separation1DLC chromatographic separation

Blank (Solvent A injection)


RADAR monitoring of the SPE cleanRADAR monitoring of the SPE clean--up sampleup sample2DLC chromatographic separation2DLC chromatographic separation

Blank (Solvent A injection)


Reproducibility of the 2DLCReproducibility of the 2DLC--MRM assay: MRM assay: 5 5 nMnM herceptinherceptin digest in 20 digest in 20 mMmM AmmAmm formateformate

12C14N 13C15N

RSD: 1.2% RSD: 1.1%


Reproducibility of the 2DReproducibility of the 2D--LC/MRM assay: LC/MRM assay: 5 5 nMnM herceptinherceptin digest in SPEdigest in SPE--cleaned serumcleaned serum

12C14N 13C15N

RSD: 4.1% RSD: 2.2%


0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

1 2 3

Average Peak Area

12C14N

13C15N

1.08

1.17

Neat solution

1LC-MRM 2DLC-MRM

1.14

5 nM Herceptin digest spiked in SPE cleaned serum digest

Evaluation of Matrix InterferencesEvaluation of Matrix Interferences

5 nM Herceptin digest

in 20 mM ammonium formate


LC/MS/MS Nominal and Accurate LC/MS/MS Nominal and Accurate MassMass

Robert S Plumb*1,Gordon Fujimoto2, JoanneMather3, Warren B PottsIII3, Paul D Rainville3,4,Nicholas J Ellor2,Christopher Evans5,Jonathan R Kehler5& Matthew E Szapacs51Department of Surgery & Cancer,Imperial College, South Kensington,London, SW7 2AZ, UK2Waters Corporation, 100Cummings Centre, Beverly, MA,USA3Waters Corporation, 34 Maple St,Milford, MA, USA4Micro Separations Group, King’sCollege London, 100 Stamford St,London, UK5DMPK Department,GlaxoSmithKline, Upper Merion,PA, USA


MethodsMethods

50 µL sample + IS + 100 nM NaHCO3 Lys C

Incubate Samples overnight

Addition of 4 % formic acid

Purification 10 mg/well SCX SPE

N2 evaporation, reconstitution, injection onto LC/MS system

Chromatography:10 µLMP A 0.1% formic acid inH2OMP B MeCNPST 2.1 x 50 1.7 µm 300A C1840 ºCGradient elution0.7 mL/min

Mass Spectrometry:HRMS Waters Synapt G2-Sm/z= 510.5, 616.3821Nominal MS Waters Xevo TQ-S510.5>616.4


Effect of Mass Extraction Window Effect of Mass Extraction Window on Signal on Signal to to NoiseNoise

Nominal Mass

100 mDa

40 mDa

20 mDa


Blank Plasma and 10ng/mL StandardBlank Plasma and 10ng/mL Standard40mDa40mDa Extract Extract Mass Window Mass Window 503.5 503.5 616.3821616.3821

Blank

10ng/mL Standard


Blank Plasma and 10ng/mL StandardBlank Plasma and 10ng/mL Standard20mDa20mDa Extract Extract Mass Window Mass Window 503.5 503.5 616.3821616.3821

Blank

10ng/mL Standard


Calibration Calibration Line in Line in Plasma Plasma 20mDa Extract Mass 20mDa Extract Mass Window Window 503.5 503.5 616.3821616.3821


Extracted Standard and Plasma Blank Extracted Standard and Plasma Blank XevoXevo TQTQ--SS

1ng/mL Standard

Plasma Blank


ConclusionsConclusions

Developed a general PBA workflow for quantification of herceptin in serum without pre-fractionation

Optimized the critical parameters for trypsin digestion

2DLC-MRM can significantly reduce the matrix interference in PBA

The 2DLC-MRM assay provides a 3 fold increase in sensitivity over 1DLC-MRM

HRMS was successful in the quantification of a protein therapeutic and offers and alternative or complementary option

two dimensional lc-srm assay for a therapeutic ...©2012 waters corporation 1 two dimensional lc-srm...

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