pk and anti-drug antibody immunoassays using covalent binding plates - case studies

PK and Anti-Drug Antibody Immunoassays Using Covalent Binding

Plates - Case Studies

Types of ELISA

1. Noncompetitive binding assay or Sandwich method

1) Antigen measuring system [Plate wells coated with antibodies ; Enzyme labelled antibodies]

2) Antibody measuring system [Plate wells coated with antigens ; Enzyme labelled anti-antibodies]

2. Competitive binding assay [Plate wells coated with antibodies ; Enzyme labelled antigens]

Noncompetitive or Sandwich Assay

• Antibody measuring system

– Plate wells coated with suitable antigen– Add sample containing the antibody– Incubate: till antigen antibody reaction is complete– Wash remove unbound antibody– Add Anti-antibody labelled with Enzyme– Incubate till labelled anti-antibodies binds antigen-

antibody complex– Wash remove unbound labelled anti-antibody– Add substrate ; incubate – Enzyme + Substrate Product measure colour– Colour proportional to antibody in patient sample

Non-competitive or Sandwich Assay

• Antigen measuring system

– Plate wells coated with suitable antibody– Add sample containing the antigen– Incubate: till antigen antibody reaction is complete– Wash remove unbound antigen– Add Antibody labelled with Enzyme– Incubate till antigen binds labelled antibody– Wash remove unbound labelled antibody– Add substrate ; incubate – Enzyme + Substrate Product measure colour– Colour proportional to antigen in patient sample

Competitive Binding assay

• Plate wells coated with antibodies• Known quantities of patient sample containing antigen +

antigen labelled with enzyme• Incubate: till antigen antibody reaction is complete• Wash remove unbound antigens• Add substrate ; incubate • Enzyme + Substrate Product measure colour• Colour inversely related to antigen in patient sample

Tox Study Experimental Design

Study title: A 4-Week Intravenous Infusion and Subcutaneous Injection Toxicity Study with ABC-001 in Cynomolgus Monkeys Followed by an 8-Week Recovery

Objective: ABC-001 is a humanized IgG1 monoclonal antibody which binds human and cynomolgus monkey 001 cytokines. The objectives of this study are to determine the potential toxicity of ABC-001 when given by either 15-minute intravenous infusion or subcutaneous injection once weekly for 4 consecutive weeks to cynomolgus monkeys, to evaluate the potential reversibility of any adverse findings following an 8-week dose-free recovery period, and to provide additional nonclinical safety data to support the use of ABC-001 in humans. In addition, the toxicokinetic (TK) and immunogenic characteristics of ABC-001 will be determined.


Group No.

No. of Animals Test Material

ROA Dose

(mg/kg) Main Study Recovery M F M F

1 3 3 2 2 Control Article IV and SC * 0

2 3 3 - -

ABC-001 SC only

1 3 3 3 2 2 10 4 3 3 2 2 30 5 3 3 2 2 100 6 3 3 - -

IV only 1

7 3 3 2 2 30


Study Day Groups No(s).

Time points (relative to the End of IV Infusion, EOI or SC

injection) TK MAHADay 1 1-7 Predose X X Day 1 1, 6-7 5 min, 1 hr X - Day 2 1-7 24 hours post Day 1 dose X - Day 3 1-7 48 hours post Day 1 dose X - Day 4 1-7 72 hours post Day 1 dose X - Day 5 1-7 96 hours post Day 1 dose X - Day 6 1-7 120 hours post Day 1 dose X - Day 8 1-7 Predose X X

Day 12 1-7 96 hours post Day 8 dose X - Day 15 1-7 Predose X X Day 19 1-7 96 hours post Day 15 dose X - Day 22 1-7 Predose X X Day 22 1, 6-7 5 min, 1 hr X - Day 23 1-7 24 hours post Day 22 dose X - Day 24 1-7 48 hours post Day 22 dose X - Day 25 1-7 72 hours post Day 22 dose X - Day 26 1-7 96 hours post Day 22 dose X - Day 27 1-7 120 hours post Day 22 dose X - Day 29 1-7 168 hours* post Day 22 dose X X Day 43 1, 3-5, 7 NA X - Day 57 1, 3-5, 7 NA X X Day 71 1, 3-5, 7 NA X - Day 85 1, 3-5, 7 NA X X

Toxicokinetic (TK) and Monkey Anti-Human Antibody (MAHA) Sample Collection Schedule

Toxicokinetic (TK) Assessment

Capture protein

ABC-

Chemiluminescent Substrate

Read plate @ 570 nm

Monkey -Absorbed SheepAnti -human IgG (H+L) pAb HRP

Dry coated plate

-001

Chemiluminescent Substrate

Read plate @ 570 nm

Monkey Absorbed SheepAnti -human IgG (H+L) pAb -

Y

Y

Dry coated plate

Chemiluminescence-based Immunoassay Using Covalent Binding Plate to Quantify ABC-001 in Monkey Serum

Assay Materials and Procedure

Equipment

• SpectraMax® L Luminescence Microplate Reader• Software: SoftMax Pro Gxp (Version 5.4.4)• Micro-plate Incubator/Shaker, Awareness Technology, Inc. • BioTek Elx405 96-well plate washer (System No. 262)• Precision Pipettes to deliver 10, 100, and 1000 µL• Vortex mixer• Adhesive sealing films for microplates, Excel Scientific, Inc.• Absorbent materials for blotting the strips

Assay Materials and Procedure

Biological Matrix

• Blank monkey serum was purchased from Bioreclamation Inc. The serum lots used in the assay were Lot Nos. CYN105050 and CYN111113.

• The pooled serum was used to prepare the standards and QC samples, and for sample dilution. The serum was stored at -70 ºC.

Reagents and Materials

• Reference Standard:ABC-001• Immobilizer amino F96 Black Plate: NUNC, Catalog # 436008• StabilGuard from SurModics, Catalog # SG01-1000• Human 001 Protein Carrier-Free, eBioscience, Catalog # 34-8239-85• Sheep anti-human IgG (H+L) Monkey–Adsorbed–Peroxidase (1:50 dilution in

StabilZyme HRP Conjugate Stabilizer), The Binding Site Group Ltd.• Pooled monkey serum, Bioreclamation Inc.• SuperSignal West Pico Chemiluminescent Substrate kit, Thermo Scientific,

Catalog # 34080• 10X DPBS, Mediatech, Inc, Catalog # 20-031-CV• 1X DPBS, Mediatech, Inc, Catalog # 20-031-CM• Tween 20, Sigma, Catalog # P1379• Sodium Chloride, Sigma, Catalog # P9416• ProClin300, Supelco, Catalog # 48912-U• SuperBlock, ScyTek, Catalog # AAA999• Purified H2O

Plate Preparation

• Plates can be coated and used freshly or stored at 2-8°C in airtight bag with desiccant. Prepare the plate as following:– Dilute Protein-001 Carrier- Free stock in 1X DPBS; make the final

concentration 0.5 µg/mL.– Label Nunc immobilizer 96 well plates.– Add 100 µL per well of the diluted protein solution.– Seal the plates and incubate at room temperature for 2-5 hours.– Place plates in a refrigerator (2-8°C) and incubate overnight.– Wash the plate(s) 3 times using a microplate washer.– Add 200 µL per well of StabilGuard to all wells by reverse pipetting

using a multichannel pipette and incubate at RT with shaking for 60±10 min.

– Dump StabilGuard and blot dry. Coated plates are ready for use.– For plates to be used later days, aspirate plate and dry at room

temperature over night. Store dried plate at 2-8°C in airtight bag with desiccant with an expiration date of three month.

Standard and QC

Calibration Standard

ABC-001Concentration, ng/mL

12345678

100250500125025005000800010000

Working Standard Concentrations

Quality Control ABC-001Concentration, ng/mL

QC LowQC MidQC High

30015007500

QC/Validation Sample Concentrations

Assay Performance- Standard Curves

Run ID STD01 100.0

STD02 250.0 STD03 500.0 STD04 1250

STD05 2500 STD06 5000

STD07 8000

STD08 10000

1STD09 50000

ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL

1 98.11 263.5 501.2 1238 2357 5459 9031 8615 50756

2 100.4 249.8 487.7 1278 2524 4789 8443 9781 49957

3 99.03 258.3 494.3 1295 2308 5332 8670 9111 50417

4 93.19 275.7 514.1 1203 2459 5097 9450 8602 50918

5 101.0 239.5 521.7 1237 2469 5097 8136 9736 50140

6 101.6 238.2 515.3 1268 2449 4916 8792 9352 50169

n 6 6 6 6 6 6 6 6 6 Overall Mean

(ng/mL) 98.89 254.2 505.7 1253 2428 5115 8754 9200 50393

S.D. 3.072 14.54 13.36 33.45 79.67 249.8 457.9 520.7 377.4

%CV 3.1 5.7 2.6 2.7 3.3 4.9 5.2 5.7 0.7

%RE -1.1 1.7 1.1 0.2 -2.9 2.3 9.4 -8.0 0.8

Back Calculated Standard Concentrations and Curve Parameters for ABC-001 Assay in Monkey Serum (5-Parameter Logistic Fit with 1/Y Weighting)

Precision and Accuracy

Nominal Concentration LLOQ LQC MQC HQC ULOQ ng/mL 100.0 300.0 1500 7500 10000

Mean Observed Concentration* 98.35 293.5 1458 8160 10822

Intra-Batch Accuracy (%RE) -1.7 -2.2 -2.8 8.9 8.2 Inter-Batch Accuracy (%RE) -1.7 -2.2 -2.8 8.8 8.2 Intra-Batch Precision (%CV) 3.3 9.7 8.4 7.0 16.9 Inter-Batch Precision (%CV) 10.8 10.8 8.4 7.0 16.9

%Total Error 12.5 13.0 11.2 15.8 25.1 n 18 18 18 17 18

Number of Runs 6 6 6 6 6

%Total Error = ABS(%RE) + %CV of inter-Batch *Calculated via inter-batch statistics (ANOVA)

Overview of Precision and Accuracy for QC Samples

Assay Performance- Selectivity

Selectivity Evaluation

Sample Name MS01 MS02 MS03 MS04 MS05 MS06 MS07 MS08 MS09 MS10 PMS1 PMS2 PMS3

Sample Unspiked Observed Conc. (ng/mL) 0 0 0 0 0 0 0 0 0 0 0 0 0

Sample Spiked Conc. (ng/mL) 100 100 100 100 100 100 100 100 100 100 100 100 100

Sample Spiked Observed Conc. (ng/mL) 99.36 102.8 112.2 106.6 110.2 118.8 105.2 100.1 88.59 99.09 114 109.5 106.9

%CV 1.8 0.2 3.4 5.7 1.4 1.9 1.2 0.3 17.9 0.5 8.1 0.6 11.7

%RE -0.6 2.8 12.2 6.6 10.2 18.8 5.2 0.1 -11.4 -0.9 14 9.5 6.9

Matrix Interference

Matrix Interference for ABC-001 Assay in Monkey Serum

Dilution Integrity

Dilution Linearity of ABC-001 in Monkey Serum

Original Concentration = 1.5 mg/mL Sample Name DL1 DL2 DL3 DL4 DL5 Dilution Factor 15 200 500 1500 3000 Nominal Conc. (ng/mL) 100000 7500 3000 1000 500 Observed Conc.(ng/mL) OOR 7985 3109 964.8 457.0 %CV of raw data 1.7 2.0 1.9 6.6 1.2 %RE NA 6.5 3.6 -3.5 -8.6

Original Concentration = 0.5 mg/mL Sample Name DL1 DL2 DL3 DL4 DL5 Dilution Factor 5 66.7 166.7 500 1000 Nominal Conc. (ng/mL) 100000 7500 3000 1000 500.0 Observed Conc.(ng/mL) OOR 7832 3338 1141 511.8 %CV of raw data 1.1 0.3 2.0 0.0 0.3 %RE NA 4.4 11.3 14.1 2.4 Note: OOR = Out of quantitation range (Assay response greater than that of ULOQ) NA = Not applicable

Assay Performance - RobustnessTested at the lower limit at each incubation step

Sample Name Observed Concentration (ng/mL) %RE %CV

LQC (300.0 ng/mL) Replicate 1 301.8 0.6 3.4 Replicate 2 309.7 3.2 3.6 Replicate 3 255.6 -14.8 6.6 Replicate 4 289.5 -3.5 2.6 Replicate 5 276.9 -7.7 1.0 Replicate 6 269.3 -10.2 3.0

Mean 283.8 %RE of Mean -5.4% %CV 7.2% MQC (1500 ng/mL) Replicate 1 1684 12.2 0.1

Replicate 2 1521 1.4 2.1 Replicate 3 1470 -2.0 0.9 Replicate 4 1611 7.4 3.4 Replicate 5 1304 -13.1 2.5 Replicate 6 1551 3.4 1.6

Mean 1524 %RE of Mean 1.6% %CV 8.6% HQC (7500 ng/mL) Replicate 1 9228 23.0* 2.8

Replicate 2 7087 -5.5 2.3 Replicate 3 8670 15.6 1.8 Replicate 4 9339 24.5* 1.7 Replicate 5 7285 -2.9 4.5 Replicate 6 8738 16.5 1.6

Mean 8391 %RE of Mean 11.9% %CV 11.6% Note: * Exceeding acceptance criteria of ±20%.

RobustnessTested at the upper limit at each incubation step

Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Replicate 1 294.2 -1.9 6.0

Replicate 2 307.5 2.5 3.6 Replicate 3 248.4 -17.2 12.2 Replicate 4 295.3 -1.6 9.0 Replicate 5 283.9 -5.4 2.2 Replicate 6 250.2 -16.6 0.6



Mean 1512 %RE of Mean 0.8% %CV 7.5% HQC (7500 ng/mL) Replicate 1 9605 28.1* 4.2

Replicate 2 6804 -9.3 3.0 Replicate 3 8761 16.8 3.5 Replicate 4 9870 31.6* 1.3 Replicate 5 6995 -6.7 3.6 Replicate 6 8889 18.5 1.4

Mean 8487 %RE of Mean 13.2% %CV 15.3% Note: * Exceeding acceptance criteria of ±20%.

Coated Plate StabilityStability of Coated Plates at 5°C ± 3°C After 27 Days

Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Replicate 1 290.3 -3.2 5.5

Replicate 2 286.8 -4.4 4.7 Replicate 3 249.6 -16.8 8.3 Replicate 4 283.1 -5.6 2.0 Replicate 5 290.4 -3.2 2.2 Replicate 6 267.9 -10.7 4.2



Mean 1490 %RE of Mean -0.7% %CV 6.1% HQC (7500 ng/mL) Replicate 1 8383 11.8 3.4 Replicate 2 7398 -1.4 0.1

Replicate 3 7815 4.2 2.6 Replicate 4 8803 17.4 4.5 Replicate 5 7015 -6.5 0.0 Replicate 6 7972 6.3 2.1

Mean 7898 %RE of Mean 5.3% %CV 8.2%

Freeze -Thaw Stability (4 cycles)

Sample Name Observed Concentration (ng/mL) %RE %CV

LQC (300.0 ng/mL) 260.4 -13.2 4.4 235.6 -21.5* 13.9 265.8 -11.4 6.7 265.3 -11.6 3.4 Mean 256.8 %RE of Mean -14.4% %CV 5.6% HQC (7500 ng/mL) 9402 25.4* 0.4 7540 0.5 1.7 8169 8.9 3.0 8622 15.0 2.0 Mean 8433 %RE of Mean 12.4% %CV 9.3% Note: * Value exceeded acceptance criteria of within ±20%.

Bench Top Stability of 20 Hours

Sample Name

Observed Concentration (ng/mL) %RE %CV

LQC (300.0 ng/mL) 265.9 -11.4 4.5 240.8 -19.7 5.4 260.7 -13.1 4.7 271.3 -9.6 6.9 Mean 259.7 %RE of Mean -13.4% %CV 5.1%

HQC (7500 ng/mL) 8290 10.5 3.3 7191 -4.1 4.8 8346 11.3 4.3 8628 15.0 2.6 Mean 8114 %RE of Mean 8.2% %CV 7.8%

Long-term Storage Stability

Nominal ConcentrationLQC, 300 ng/mL HQC, 7500 ng/mL

6-Month Stability(188 Days)

304.897 8630.903301.833 8689.788285.895 8051.411

Mean ng/mL 297.542 8457.367%RE -0.8 12.8%CV 3.4 4.2n 3 3

12-Month Stability(364 Days)

276.973 9398.378312.490 8455.593296.506 7452.369

Mean ng/mL 295.323 8435.447%RE -1.6 12.5%CV 6 11.5n 3 3

Method transfer (P/A Summary)

Sample Batch/plate Rep 1 Rep 2 Rep 3

Intrabatch (within-run) Statistics Absolute

n Mean(ng/mL) Stdv. %CV %RE ∑%CV+

%RE

LLOQ 1/1 117 81 99 3 99.0 18.0 18.2 -1.0 19.2

100 1/2 122 85 112 3 106.3 19.1 18.0 6.3 24.3ng/mL 2/3 103 105 96 3 101.3 4.7 4.6 1.3 5.9

Interbatch (Between-run) Statistics: 9 102.2 13.7 13.4 2.2 15.6

LQC 1/1 337 257 289 3 294.3 40.3 13.7 -1.9 15.6

300 1/2 330 285 336 3 317.0 27.9 8.8 5.7 14.5ng/mL 2/3 297 314 291 3 300.7 11.9 4.0 0.2 4.2

Interbatch (Between-run) Statistics: 9 304.0 27.2 8.9 1.3 10.2MQC 1/1 1625 1389 1506 3 1506.7 118.0 7.8 0.4 8.21500 1/2 1569 1438 1582 3 1529.7 79.7 5.2 2.0 7.2ng/mL 2/3 1560 1665 1625 3 1616.7 53.0 3.3 7.8 11.1

Interbatch (Between-run) Statistics: 9 1551.0 91.1 5.9 3.4 9.3HQC 1/1 7503 6899 7437 3 7279.7 331.3 4.6 -2.9 7.57500 1/2 7963 7414 7824 3 7733.7 285.4 3.7 3.1 6.8ng/mL 2/3 7308 7950 7912 3 7723.3 360.2 4.7 3.0 7.7

Interbatch (Between-run) Statistics: 9 7578.9 361.4 4.8 1.1 5.9ULOQ 1/1 9489 9648 10026 3 9721.0 275.8 2.8 -2.8 5.610000 1/2 10592 11239 11739 3 11190.0 575.1 5.1 11.9 17.0ng/mL 2/3 9755 9940 9560 3 9751.7 190.0 1.9 -2.5 4.4

Interbatch (Between-run) Statistics: 9 10220.9 799.5 7.8 2.2 10.0

Standard and QC performance of TK Sample Analysis

Concentration [ng/mL]

100 250 500 1250 2500 5000 8000 10000

n 48 48 50 50 50 50 50 50Overall Mean 101.708 249.312 487.196 1252.268 2540.942 5164.236 8075.762 9341.605

S.D. 6.868 11.255 19.802 38.548 106.490 197.440 386.785 441.922%CV 6.8 4.5 4.1 3.1 4.2 3.8 4.8 4.7%RE 1.7 -0.3 -2.6 0.2 1.6 3.3 0.9 -6.6

QC Low (300 ng/mL)

Mid (1500 ng/mL)

High (7500 ng/mL)

Mean 309.028 1535.604 7742.039S.D. 24.371 115.454 679.087%CV 7.9 7.5 8.8%RE 3 2.4 3.2

n 100 99 100

Representative TK Graphs

Group 5 SC 100 mg/kg Group 7 IV 30 mg/kg

ISR evaluation

• An ISR evaluation was performed on the 112 serum samples listed in the ISR Study Plan.

• 107 of 112 samples (95.5%) evaluated met the acceptance criteria : at least 2/3 of all the analyzed ISR samples had no more than a ± 30% difference when compared to the original analysis results.

General industrial guideline: For studies up to 1000 samples reanalyze 10% of samples and for studies with over 1000 samples reanalyze 10% of the first 1000 samples and 5% of any remaining samples.

Immunogenicity Assessment

Basic package:• Anti-drug binding antibody assays (ADA)• Screening• Confirmation• Titration • Neutralizing antibodies (Nab)

Basic package +/- based on:• Risk assessment • Results from pre-clinical and early clinical studies• Regulatory input

ADA Assay Platforms

PlatformsELISA

Bridging DirectIndirect

RIABiacoreElectrochemiluminescence (ECL)

No “perfect” assay currently existsIn general, assay format for ADA

Protein therapeutics: direct formatmAbs: bridging format

Popular ADA assay formats for Mab Drugs

ECL / MSD Homogeneous Bridging ELISA

ADA Assay

KEY PARAMETERS FOR VALIDATION Screening cut point Specificity/confirmation cut point Sensitivity System suitability controls(QCs) acceptance

criteria Selectivity/Interference

Matrix components Drug tolerance

Precision Robustness Stability Ruggedness

ECL vs. H-ELISA

H-ELISA Advantages• Antibody-drug interactions take place in solution–High capacity surface contributes to high drug tolerance• Broad dynamic range and good sensitivity• Instrumentation and consumables are available from multiple sources• Limitations-More complicated detection with an additional wash step

ECL Advantages• Antibody-Drug interactions take place in solution• High capacity surface contributes to high drug tolerance• Broad dynamic range and good sensitivity•Limitations–Single vendor technology

Anti-ABC01 Antibody Assay (Homogeneous Bridging Elisa format)

Key Reagents and Materials

– PC: Rabbit anti-ABC-001 pAb– Drug: ABC-001– Biotinylated ABC-001 .– Digoxigenin-conjugated ABC-001– Anti-Digoxigenin-POD (anti-Dig POD), Fab Fragments– Immobolizer Clear Streptavidin coated plates: Nunc, Cat #

436014.– TMB: KPL– 1N Sulfuric Acid

ADA Assay SummaryScreening Assay Cut Point 0.159

Normalization Factor 1.44Confirmation Cut-Point 14.79%High Positive Control Intra-Assay %CV: ≤ 12.6% , Inter-Assay %CV: 13.8 %

Low Positive Control Intra-Assay %CV: ≤ 14.5% , Inter-Assay %CV: 17.4%

Negative Control Intra-Assay %CV: ≤ 16.5% , Inter-Assay %CV: 21.1% Hook Effect No Hook effect up to 100000 ng/mL

Drug Tolerance 100-200 µg/mL at 500 ng/mL of PCInterference by Hemolysis and

Lipemia No Interference observedImmuno-depleted Control (LPC) Average: 37.20% Immuno-depleted Control (HPC) Average: 92.88%

Relative Sensitivity 100 ng/mL

Method Selectivity 90% of the spiked and unspiked samples were within 75%-125% of the respective controls.

Robustness Plates with longest incubation times at each step meet acceptance criteria

Bench Top Stability Stable up to 24 hrsRefrigerated Stability Stable up to 3 daysFreeze Thaw Stability Stable up to 6 cycles

Drug Tolerance Using H-ELISA

ADA at 500ng/mL

Drug Concentration µg/mL

OD1 OD2 OD Mean CV

500 0.2202 0.2054 0.213 4.9%

200 0.2227 0.2144 0.219 2.7%

100 0.2382 0.2274 0.233 3.3%

50 0.236 0.2409 0.238 1.5%

20 0.2509 0.2454 0.248 1.6%

5 0.2406 0.2485 0.245 2.3%

1 0.2735 0.2606 0.267 3.4%

0 0.3703 0.3595 0.365 2.1%

Cut-point: 0.229

Drug Interference: (µg/mL) 100~200 mg/mL

ADA sample Analysis

Total samples tested for screening assay

350

Samples confirmed positive

60

Titers 2-32

Impact of ADA on PK profile

No

ADA sample Analysis (QC Performance)

Run #OD

HPC_1 HPC_2 LPC_1 LPC_2 NCMean %CV Mean %CV Mean %CV Mean %CV Mean %CV

Run 1 0.997 1.2 0.952 0.9 0.110 2.6 0.102 2.8 0.070 7.5

Run 2 1.312 6.5 1.247 3.4 0.123 4.0 0.111 1.9 0.075 10.3

Run 3 1.286 4.9 1.184 2.1 0.113 5.6 0.099 3.6 0.068 8.6

Run 4 1.299 7.5 1.307 4.1 0.121 11.7 0.102 3.5 0.073 9.5

Run 5 1.195 1.1 1.156 1.2 0.123 1.7 0.108 2.6 0.073 7.5

Run 6 1.174 0.7 1.118 0.4 0.120 1.2 0.109 0.6 0.072 8.1

Run 7 0.926 2.0 0.824 5.5 0.106 4.7 0.102 4.9 0.072 7.0

Run 8 0.942 2.1 0.878 0.2 0.104 2.7 0.103 4.8 0.071 4.4

Run 9 0.875 1.3 0.927 4.0 0.099 6.4 0.101 0.7 0.070 4.1

Overall Mean 1.089 0.109 0.072

Overall SD 0.172 0.008 0.002

Overall CV% 15.8 7.6 2.9

Summary and Conclusions

•The H-ELISA format is generic and can be easily applied to other immunogenicity assays

•Performance characteristics of the H-ELISA meets industry requirement for ADA assays

•Frontage remains proactive in exploring new technologies and searching for alternative solutions to analytical problems

Acknowledgement

Study Sponsor:

http://tevausa.com/Default.aspx

Shawn Li, M.D., Ph.D.

Director, Biologics Services Frontage Lab. Inc.

[email protected]

Exton, PA Headquarters

Thank You

pk and anti-drug antibody immunoassays using covalent binding plates - case studies

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