Using Statistical Analyses to Demonstrate Equivalency ......15.98 16.71 17.45 18.55 XIC of +MRM (12 pairs): 497.280/469.100 Da ID: THRX-112341 from Sample 1 (Blank) of CB-0411-DV-PB-B02_Cassette

HT-Automation

Figure 1a: Chromatograms of known compounds demonstrate high quality peak shape in

gradient (left side) and T/E (right side) acquisition

Microsomal Stability using Venus on Vantage

(Hamilton)

Protein Binding by Rapid Equilibrium Dialysis (RED)

using Venus on Vantage

(Hamilton)

Descriptive Statistical Data Analysis Approach

• Graphical assessment of ratio vs. geometric mean

• Graphical assessment of degree of departure from

concordance

• Concordance correlation, with Pearson & Spearman

• Minimum Significant Ratio (MSR)

• Mean Ratio & 95% confidence interval

Using Statistical Analyses to Demonstrate Equivalency Between

Automated and Manual Processes to Deliver High Quality In Vitro ADME

Data Even FasterJakal Amin, David Plourde, Allison Lewia, Patty Walton, Françoise Powell, Gaurang Patel, Susan Dearborn and Adrian Sheldon

Charles River Laboratories – Worcester, MA

1 PURPOSE

The role of lab automation to enable drug discovery and compound screening

related to in-vitro ADME assays has been well established over the past 10-20

years. As bio-pharmaceutical companies continue to evolve from performing

ADME assays in-house to outsourcing, it has become necessary for contract

research organizations (CROs) to adopt this paradigm shift and invest in

automation technologies to enable and support large scale ADME analysis. Based

on client expectations, our objective was to provide high quality data within 5 days

for up to 500 compounds per week for three primary screening assays: Metabolic

Stability, Plasma Protein Binding using RED, and Permeability (Caco-2 and

MDCK-MDR1). To enable this, dual liquid-handlers (Hamilton Vantage®) were

established to perform multiple biological experiments with minimal human

intervention. The implementation of the high-throughput work-flow involved

deployment of key features such as cherry-picking and dilution of stock solutions

(10 mM compound in DMSO) from multiple barcoded source plates to intermediate

plates prior to the commencement of biological experiments. For the LC-MS/MS

analysis, Apricot Designs Dual Arm (ADDA) was selected as the primary

autosampler coupled with Sciex 5500 LC-MS/MS systems. ADDA provided

flexible high-throughput bioanalysis capability in 384-well plate format to perform

both trap/elute (T/E) methods (~10 seconds/cycle) along with gradient methods

when required. Gubbs® GMSU ADDA feature was used to provide high-

throughput post-acquisition data review, processing and reporting. Multiple

validation experiments were performed to demonstrate an agnostic statistical

equivalency of T/E vs. gradient-based analyses for diverse new chemical entities

(NCEs) and marketed compounds. Statistical correlation of data between different

platforms was based on the confidence and tolerance, concordance correlation

and Minimum Significant Ratio (MSR).

2 WORKFLOW: SOURCE TO

BIOANALYTICAL5 STATISTICAL ANALYSIS:

PERMEABILITY ASSAY

8 CONCLUSIONSWe have successfully validated In vitro ADME HT-platform utilizing latest automation by deploying Hamilton Vantage liquid-handler and Apricot ADDA-MS to meet the

TAT demands of bio-pharmaceutical drug discovery without compromising data quality. By deploying descriptive statistical approach (e.g. MSR, F-Test, etc.) and

best analytical practices for LC-MS/MS analysis, a very high concordance between T/E-MS/MS and UPLC-MS/MS was demonstrated for cell-based and whole

blood/plasma samples. The results of control compounds demonstrated excellent consistency. The HT- platform is currently has the capability and capacity to

support up to 500 NCEs per week for microsomal, PPB and permeability assays.

3 MATERIALS AND METHODS

MS: Sciex Triple Quad 5500

LC: Agilent 1260 Infinity Binary Pump

Autosampler: ADDA High-Speed Dual Arm Autosampling System (Apricot Designs)

Mobile Phases: A: 0.1% Formic Acid in H2O B: 0.1% Formic Acid in Acetonitrile

Flow Rate: 1200 µL/min (67:33 MS:Waste Split)

Injection Volume: 7 µL (T/E), 10 µL (Gradient)

Format: Trap and Elute Gradient

Columns: Sprite echelon C18, 4 µm, 20x2.1 mmGemini NX-C18, 110 Å Mercury MS, 3 µm, 10x2.1 mm

Gemini NX-C18, 110 Å Mercury MS, 3 µm, 20x2.1 mm

LC Timing:

Time (min) Stream A (%) B (%) Time (min) A (%) B (%)

0 1 99 1 0 99 1

0 2 10 90 0.2 99 1

0.1 1 10 1 0.5 10 90

0.1 2 99 90 0.7 10 90

Hardware: Hamilton Vantage

Software: Venus on Vantage

Run Control: Version 4.4.07740

Deck Features:

8-channel spanning head allows for easy conversion between plate sizes

96-channel fixed head allows for quick stamping/liquid transfers

6 temperature controlled positions allow for heating or cooling of reagents or plates

Flexible deck layout allows for various sizes of tips, troughs and plates

CO-RE Paddle Grippers allows for transport of consumables around the deck

Barcode reader allows for tracking of several plates on deck

Consumables:

Conductive tips allow for liquid level sensing

Nested Tip Racks (NTRs) allow for additional deck flexibility

Specifications for various labware are taught in the system allowing for precise liquid transfers while

avoiding sedimented proteins, etc.

4 RESULTS: CHROMATOGRAMS

Warfarin

Carbutamide

XIC of +MRM (12 pairs): 255.000/153.000 Da ID: Chrysin from Sample 1 (Blank) of CB-0411-DV-PB-B02_Cassette 1.wiff (Heated Nebulizer) Max. 1.2e6 cps.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.18e6

Intensi

ty, cps

1.13

13.60 21.301.50 7.006.63 25.7019.8312.13 21.677.372.60 16.173.70 15.80 16.53 27.5324.6010.67 22.7718.738.839.575.904.07

24.23 26.800.97 3.338.47

5.17 27.90

15.63

13.97

14.17

XIC of +MRM (12 pairs): 417.200/293.990 Da ID: THRX-544756 from Sample 1 (Blank) of CB-0411-DV-PB-B02_Cassette 1.wiff (Heated Neb... Max. 1.6e5 cps.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time, 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

Inten

sity, cp

s

27.7226.6125.52

20.027.18 20.3810.85 23.6813.057.55 13.426.82 28.0821.8511.22 21.489.75 22.225.72 24.42

11.95 23.327.92

5.35

13.786.45

8.28

16.7115.98 17.45 18.55

XIC of +MRM (12 pairs): 497.280/469.100 Da ID: THRX-112341 from Sample 1 (Blank) of CB-0411-DV-PB-B02_Cassette 1.wiff (Heated Neb... Max. 1.0e6 cps.

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.04e6

Inten

sity, cp

s

12.1227.15

27.8826.7825.6812.48

13.58

24.5828.6210.28 11.38

9.92

20.185.51 7.72 19.45 20.556.98

5.884.41 9.55 20.92 22.0124.2215.78

1.12 8.0818.35

1.48 16.15

4.053.31

Abbreviation Definition

N # of Data Points

MR Mean Ratio

MSR Minimum Significant Ratio [Acceptable Limit <3]

LsA Limits of Agreement [0.33 – 3.0]

SD Standard Deviation

RLs Ratio Limits [Statistical Limits of Mean Ratio]

MSD Minimum Significant Difference

LsAd Limits of Agreement for the Difference

Statistical Analysis of "Bland-Altman" Plot

N 44 Reproducibility Results

MR 1.14 MSR [Within Run] 1.94

RLs1.03

LSA0.59

1.26 2.22

95% CI [1.18, 1.23] SD 0.1018

Significant

Difference

Between Runs,

p= 0.0108




RLs0.95

LSA0.62

1.16 1.70

95% CI [1.06, 1.10] SD 0.0742

Significant

Difference

Between Runs,

p= 0.3472

6 STATISTICAL ANALYSIS:

METABOLIC CL

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0 5 10 20 40 60

Res

pons

e [P

eak

Are

a R

atio

]

Time (min)

Chlorpromazine

Gradient-MS/MS T/E-MS/MS

50.0 49.7 49.546.9

0

10

20

30

40

50

60

CLi

nt (

mL/

min

/kg)

CLint of Chlorpromazine in HLM

Gradient-MS/MS [All TimePoints] Gradient-MS/MS [T0,60]

T/E-MS/MS [All TimePoints] T/E-MS/MS [T0,60]

t-Test: Paired Two Sample for

MeansChlorpromazine


MeansHaloperidol


Means

7-

Ethoxycoumarin

Gradient T/E Gradient T/E Gradient T/E

Half-Life (min) 23.7 31.4 Half-Life (min) 76.8 78.5 Half-Life (min) 6.0 6.4

Mean 25.4 24.8 Mean 87.6 96.0 Mean 7.1 7.4

Variance 6.03 8.23 Variance 92.25 366.45 Variance 0.63 0.63

Observations 7 7 Observations 7 7 Observations 7 7

Pearson Correlation 0.856 Pearson Correlation 0.708 Pearson Correlation 0.524

Hypothesized Mean Difference 0 Hypothesized Mean Difference 0 Hypothesized Mean Difference 0

df 6 df 6 df 6

t Stat 0.972 t Stat -1.569 t Stat -1.301

P(T<=t) one-tail 0.184 P(T<=t) one-tail 0.084 P(T<=t) one-tail 0.121

t Critical one-tail 1.943 t Critical one-tail 1.943 t Critical one-tail 1.943

P(T<=t) two-tail 0.369 P(T<=t) two-tail 0.168 P(T<=t) two-tail 0.241

t Critical two-tail 2.447 t Critical two-tail 2.447 t Critical two-tail 2.447


MeansImipramine


MeansPropranolol


MeansQuinidine

Gradient T/E Gradient T/E Gradient T/E

Half-Life (min) 50.3 52.3 Half-Life (min) 41.7 53.6 Half-Life (min) 91.1 104.3

Mean 57.1 57.9 Mean 48.3 47.0 Mean 98.9 131.5

Variance 7.19 11.21 Variance 19.52 105.37 Variance 1655.10 8157.38

Observations 7 7 Observations 7 7 Observations 7 7

Pearson Correlation 0.011 Pearson Correlation 0.711 Pearson Correlation -0.589

Hypothesized Mean Difference 0 Hypothesized Mean Difference 0 Hypothesized Mean Difference 0

df 6 df 6 df 6

t Stat -0.522 t Stat 0.443 t Stat -0.727

P(T<=t) one-tail 0.310 P(T<=t) one-tail 0.336 P(T<=t) one-tail 0.247

t Critical one-tail 1.943 t Critical one-tail 1.943 t Critical one-tail 1.943

P(T<=t) two-tail 0.620 P(T<=t) two-tail 0.673 P(T<=t) two-tail 0.495

t Critical two-tail 2.447 t Critical two-tail 2.447 t Critical two-tail 2.447

Compound ID

Mean % Remaining at T60

minMean T1/2 (min) Mean CLint (mL/min/kg)

Automated Manual Automated Manual Automated Manual

7-EC 2.8 ± 2.5 1.8 ± 0.2 9.7 ± 1.4 9.1 ± 0.3 131 ± 15 137 ± 4

Testosterone 22.3 ± 6.3 24.0 ± 4.6 24.9 ± 2.8 29.0 ± 2.7 50.7 ± 5.5 43.3 ± 3.9

Imipramine 52.4 ± 4.2 57.8 ± 6.6 66.7 ± 10.9 79.0 ± 17.0 19.2 ± 3.0 16.4 ± 3.2

Terfenadine 1.5 ± 0.8 1.5 ± 1.1 11.3 ± 1.7 10.4 ± 1.8 113 ± 17 123 ± 20

Quinidine 62.9 ± 5.9 70.3 ± 5.7 103 ± 31 108 ± 25 13.3 ± 4.0 12.1 ± 3.1

7 STATISTICAL ANALYSIS:

PROTEIN BINDING (RED)

ASSAY




RLs0.88

LSA0.52

1 1.71

95% CI [0.931, 0.949] SD 0.0917

Significant

Difference

Between Runs,

p= 0.0471

90

91

92

93

94

95

96

97

98

99

100

0 20 40 60 80 100 120 140

% B

ou

nd

Datapoints

% Bound(n=134 from 4 Assays; Warfarin)

Mean: 99.1 ± 0.40

10

20

30

40

50

60

70

80

90

100

1 6 11 16 21 26 31

% B

ou

nd

Datapoints

% Bound(n=32 from 4 Assays; Propranolol)

Mean: 83.5 ± 3.2

Compound IDMean % Bound

Automated Manual

Caffeine 20.1 ± 10.9 21.8 ± 0.2

Chlorpromazine 97.3 ± 1.4 95.8 ± 5.1

Digoxin 47.7 ± 18.3 45.6 ± 12.2

Metoprolol 4.7 ± 4.3 5.8 ± 2.6

Propranolol 83.5 ± 3.2 83.8 ± 0.6

Quinidine 63.0 ± 8.0 61.5 ± 1.1

Verapamil 87.4 ± 0.8 87.7 ± 3.6

Warfarin 99.1 ± 0.4 99.1 ± 0.3

9 ACKNOWLEDGMENTSWe are very grateful to Drs. James Jersey and Viswanath Devanarayan for performing and/or providing guidance related to descriptive statistical analyses of HT-LC-

MS/MS data. We also wish to express sincere gratitude to rest of the CRL-Worcester In Vitro ADME team members for performing biological and bioanalytical

experiments.

NCE

NCE

IS

LC-MS/MS Acquisition and ProcessingLeadScapeTM

• LC-MS Acquisition

• DiscoveryQuantTM Optimization

• Peak Integration for T/E

GMSU

• Customized Scripts built to process T/E Data

• Peak Data Review

• Raw Data Summary

Figure 1b: Chromatograms of 2 NCEs and IS demonstrate the ability of dual arm T/E

acquisition to acquire 160 samples in under 30 min

ADDA LC-MS/MS Conditions Hamilton Conditions

Figure 2a: NCEs assayed in T/E mode and

Gradient Mode-MS/MS

Figure 2b: Control Compounds assayed in

T/E mode and Gradient Mode-MS/MS

Figure 3: Example Control, Chlorpromazine [N=8], in HLM Assayed in T/E mode and Gradient

Mode-MS/MS [Incubation Conc: 1 µM, Protein Conc: 0.5 mg/mL]

Figure 4: Statistical Analysis of Control Compounds [N=8] for HLM Assayed in T/E mode and

Gradient Mode-MS/MS [Incubation Conc: 1 µM, Protein Conc: 0.5 mg/mL]

Figure 5: Assay results comparing Hamilton vs. Manual for Metabolic Clearance Assay

Figure 6: Fraction Unbound summary of NCEs and Control Compounds [N=93] for PPB Assay

samples assayed in T/E mode and Gradient Mode-MS/MS

[Incubation Conc: >1 µM, PPB Values accepted: up to 99.5% bound]

Figure 7: Summary of results generated using HT-Automation compared to traditional manual

approach

Lessons Learned

Documents

Using Statistical Analyses to Demonstrate Equivalency ......15.98 16.71 17.45 18.55 XIC of +MRM (12 pairs): 497.280/469.100 Da ID: THRX-112341 from Sample 1 (Blank) of CB-0411-DV-PB-B02_Cassette