** C CORRESPONDING AUTHORORRESPONDING AUTHOR

Bioanalysis of Exenatide: Intact Versus Signature Peptide Approach Bioanalysis of Exenatide: Intact Versus Signature Peptide Approach to Reach Optimal Sensitivity in Large Molecule Quantification by LC-MSto Reach Optimal Sensitivity in Large Molecule Quantification by LC-MS

Jean-Nicholas Mess, Daniel Villeneuve and Fabio GarofoloJean-Nicholas Mess, Daniel Villeneuve and Fabio Garofolo**

Algorithme Pharma Inc., Laval (Montréal), QC, CanadaAlgorithme Pharma Inc., Laval (Montréal), QC, Canada

INTRODUCTIONINTRODUCTION

CONCLUSIONCONCLUSION

LC-MS is an emerging technique for the quantification of large molecules such as therapeutic peptides, proteins and biomarkers. However, due to the limited mass range of triple quadrupole mass spectrometers, these analytes are typically monitored as multicharged species. This dispersion of MS signal combined with poor fragmentation (CID) efficiency may affect the achievable LOQs. Another approach is to generate smaller signature peptides through tryptic digestion. These tryptic peptides will generally show less charge state distribution and clearer CID pattern than the intact analyte. However, the efficiency of the digestion process could be poor and compromise its sensitivity. In this study, both approaches will be compared in order to reach optimal sensitivity for the quantification of exenatide in human plasma.

METHODSMETHODSOVERVIEWOVERVIEW

• PurposePurpose– Direct comparison of two different quantification

approaches: intact versus signature peptide to reach optimal sensitivity for the quantification of exenatide in human plasma.

• MethodMethod– Sensitivity comparison was performed using

reference standard material.– Samples were extracted by SPE, digested with

trypsin and analysed by LC-MS/MS on an AB SCIEX QTRAP®5500 operated in ESI+

• ResultsResults– The detection of the signature peptide was 5 times

more sensitive than the intact exenatide.– The digestion efficiency was evaluated to be >80%– Using the digestion approach, an LLOQ of 10 pg/mL

was achieved with an assay showing good precision and accuracy.

SAMPLE EXTRACTIONSAMPLE EXTRACTION• Range of 10 – 2000 pg/mL in human plasma• 500 µL of sample extracted by solid phase extraction using Oasis ion exchange SPE.• Following elution and drying step, the samples were reconstituted with trypsin (Sigma T1426) and incubated for 3 hours at 37°C

CHROMATOGRAPHYCHROMATOGRAPHY• Agilent Technologies Series 1100 pumps and autosampler • Ascentis Express C18, 50x2.1mm, 2.7µm• Gradient elution of 0.1% HCOOH and ACN• 3.5 minutes run time

DETECTIONDETECTION• AB SCIEX QTRAP®5500 • MRM mode ESI(+)• The [M+5H]5+ (m/z 838.3 → 396.2) was monitored for exenatide and the [M+2H]2+ (m/z 475.1 → 688.4) was monitored for the signature peptide.

Exenatide amino acids sequence analysis showed 3 theoretical trypsin cleavage sites which would result in the generation of 4 tryptic peptides. However, experimental digestion data performed in solution showed the presence of only 3 tryptic peptides. One of the tryptic peptide (LFIEWLK) had good characteristic for quantification (retention on C18 column and MS sensitivity) (Table 1Table 1). This peptide mainly ionized to the +2 charge state (m/z 474.8) and clearly fragmented to one singly charged product ion (m/z 688.4).

Table 2: Summary of the Evaluations PerformedTable 2: Summary of the Evaluations Performed

Figure 4: Representative Chromatograms of Extracted Figure 4: Representative Chromatograms of Extracted and Digested Exenatide in Human Plasmaand Digested Exenatide in Human Plasma

RESULTSRESULTSA) No Trypsin AddedA) No Trypsin Added

B) Following Digestion (3 hrs at 37°C)B) Following Digestion (3 hrs at 37°C)

The extraction efficiency of exenatide (SPE process) was on average 60.9% and the digestion efficiency was 80.4%. No intact exenatide was found in the extracted samples following tryptic digestion (Figure 3Figure 3). Overall, the entire extraction efficiency process (SPE + digestion) was found to be 49.0%.

The lower limit of quantification (LLOQ) achievable using the tryptic digestion approach was 10 pg/mL (Figure 4Figure 4), which is significantly lower than the reachable LLOQ for the intact exenatide.

A) Extracted BlankA) Extracted Blank

B) Extracted LLOQ (10.0 pg/mL)B) Extracted LLOQ (10.0 pg/mL)

C) Extracted ULOQ (2000.0 pg/mL)C) Extracted ULOQ (2000.0 pg/mL)

• This study demonstrates that the tryptic digestion approach is suitable for the quantification of exenatide in human plasma by LC-MS/MS.

• The digestion step was efficient (within 3 hours) and reproducible.

• The tryptic peptide generated allowed to decrease the LLOQ of the exenatide assay.

• The LLOQ achieved by this LC-MS/MS assay is comparable, if not lower, to the commercial LBA assay kits.

• On-going work is currently being performed on the novel triple quadrupole QTRAP®6500 to decrease the LLOQ to the low pg/mL level.

Figure 1: Structure of Exenatide and Signature PeptideFigure 1: Structure of Exenatide and Signature Peptide

Peptide Charge State

Monoisotopic (m/z)

tR

(min)Intensity

(cps)HGEGTFTSDLSK +1 1278.5961 Outside calibration

+2 639.8017 0.63 8.84e5+3 426.8702 0.63 7.73e5

QMEEEAVR +1 991.4513Not Detected+2 496.2293

+3 331.1553LFIEWLK +1 948.5553 Not Detected

+2 474.7813 2.33 5.37e6+3 316.8566 2.33 5.74e4

NGGPSSGAPPPS-NH2 +1 1023.4854 Not Detected+2 512.2463 0.34 7.30e5+3 341.8333 0.34 1.56.e4

Table 1: Exenatide Tryptic Digestion Table 1: Exenatide Tryptic Digestion

When comparing the synthetic reference standard material for exenatide and its tryptic peptide prepared at an equimolar concentration (Figure 2Figure 2), the detection of exenatide tryptic peptide was 5 times more sensitive than the intact exenatide.

XIC of +MRM (10 pairs): 475.100/688.400 Da from Sample 11 (NE) of Digestion.wiff (Turbo Spray) Max. 3.8e4 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

3.8e4

In

te

ns

ity

, c

ps

1.73

Intact exenatideS/N = 84.2

Signature peptideS/N = 431.5

Inte

nsity

(cp

s)

Figure 2: Sensitivity comparison of Intact Exenatide and Figure 2: Sensitivity comparison of Intact Exenatide and Exenatide Signature Peptide Prepared at Equimolar Exenatide Signature Peptide Prepared at Equimolar Concentration (1 nM)Concentration (1 nM)

Figure 3: Tryptic Digestion Efficiency of Exenatide to its Figure 3: Tryptic Digestion Efficiency of Exenatide to its Signature Peptide in Human Plasma SPE ExtractsSignature Peptide in Human Plasma SPE Extracts

XIC of +MRM (10 pairs): 475.100/688.400 Da from Sample 40 (REF 25ug ACN) of Digestion.wiff (Turbo Spray) Max. 160.0 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, min

0

20

40

60

80

100

120

140

160

In

te

ns

ity

, c

ps

2.772.04

0.22 3.00

3.030.37 2.751.70 2.461.78

1.651.360.990.85 3.19

XIC of +MRM (10 pairs): 838.300/396.200 Da from Sample 40 (REF 25ug ACN) of Digestion.wiff (Turbo Spray) Max. 8160.0 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

In

te

ns

ity

, c

ps

2.01

XIC of +MRM (10 pairs): 475.100/688.400 Da from Sample 63 (REF 25ug MeOH) of Digestion.wiff (Turbo Spray) Max. 3.2e4 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e43.2e4

In

te

ns

ity

, c

ps

1.73

XIC of +MRM (10 pairs): 838.300/396.200 Da from Sample 63 (REF 25ug MeOH) of Digestion.wiff (Turbo Spray) Max. 400.0 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time, min

0

50

100

150

200

250

300

350

400

In

te

ns

ity

, c

ps

0.39

1.63

2.001.76

2.441.870.46

1.27 2.592.321.20 1.61

2.842.161.55

1.571.240.27 0.50 2.88

1.160.86 3.04

Signature PeptideSignature Peptide

ExenatideExenatide

Signature PeptideSignature Peptide

ExenatideExenatide

Inte

nsity

(cp

s)In

tens

ity (

cps)

Inte

nsity

(cp

s)In

tens

ity (

cps)

XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 10 (CR04-ZRA) of MSMS27.wiff (Turbo Spray), Smoothed Max. 3296.5 cps.

2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0Time, min

0

50

100

150

200

250

300

In

te

ns

ity

, c

ps

2.672.68

XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 11 (CR04-P1A) of MSMS27.wiff (Turbo Spray), Smoothed Max. 6100.0 cps.

2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0Time, min

0

50

100

150

200

250

300

350

In

te

ns

ity

, c

ps

2.68

XIC of +MRM (2 pairs): 474.800/688.400 Da from Sample 6 (CR04-P10A) of MSMS27.wiff (Turbo Spray) Max. 4.8e4 cps.

2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

4.8e4

In

te

ns

ity

, c

ps

2.45

The calibration curve was linear (weighted 1/x2) from 10.0 to 2000.0 pg/mL with a coefficient of correlation of 0.9951 (Figure 5Figure 5). Summary of the preliminary data obtained during method development is shown in Table 2Table 2.

Inte

nsity

(cp

s)In

tens

ity (

cps)

Inte

nsity

(cp

s)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0 1800.0 2000.0

Concentration (pg/mL)

Pea

k A

rea

Rat

io

Regression type: Linear 1/xRegression type: Linear 1/x22 Coefficient Correlation: r = 0.9951Coefficient Correlation: r = 0.9951

Calibrant Nominal Back-Calculated Deviation(pg/mL) (pg/mL) (%)

P1 10.0 10.7 6.6P2 20.0 17.7 -11.3P3 40.0 37.4 -6.5P4 100.0 105.2 5.2P5 300.0 301.8 0.6P6 500.0 537.9 7.6P7 800.0 776.1 -3.0P8 1400.0 1477.9 5.6P9 1700.0 1655.4 -2.6

P10 2000.0 1956.8 -2.2

Figure 5: Calibration Curve for ExenatideFigure 5: Calibration Curve for Exenatide

   

Evaluation Results   

Inter Batch Precision (%CV) and Accuracy (%Bias) 8.2% and 11.7% at LLOQ QC7.1% and 2.2% at Low QC3.7% and 1.7% at Mid QC4.6% and 3.0% at High QC

Extraction Process (SPE) Yield 64.7% at Low QC 57.1% at High QC

Digestion Process Yield 75.3% at Low QC 85.5% at High QC

Total Process Yield 48.7% at Low QC 48.8% at High QC

Matrix Effect Acceptable for 9/10 donors of human plasma including lipemic samples. Hemolysed samples under investigation.

Selectivity No significant interference at analyte and IS retention times for 10 donors including lipemic and hemolysed samples

Dilution Integrity up to 4 000 pg/mL, diluted 5-fold

Processed Stability 45.8 hrs at 4°C

Bench-Top Stability (K2EDTA + Inhibitor) 20.0 hrs on ice/water bath2.2 hrs at room temperature

Long-Term Storage Stability (K2EDTA + Inhibitor) On going

Freeze-Thaw Stability (K2EDTA + Inhibitor) 3 Freeze Thaw Cycles

Stability in Whole Blood (K2EDTA) 2.0 hr on ice/water

Stock Solution Stability 100 µg/mL (Days) 48 days at 4°C in aq.solution