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Noncompetitive binding assay or Sandwich method

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1 PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies

2 Noncompetitive binding assay or Sandwich method
Types of ELISA Noncompetitive binding assay or Sandwich method Antigen measuring system [Plate wells coated with antibodies ; Enzyme labelled antibodies] Antibody measuring system [Plate wells coated with antigens ; Enzyme labelled anti-antibodies] Competitive binding assay [Plate wells coated with antibodies ; Enzyme labelled antigens]

3 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

4 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

5 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

6 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.

7 Tox Study Experimental Design
Group No. No. of Animals Test Material ROA Dose (mg/kg) Main Study Recovery M F 1 3 2 Control Article IV and SC * - ABC-001 SC only 10 4 30 5 100 6 IV only 7

8 Tox Study Experimental Design
Toxicokinetic (TK) and Monkey Anti-Human Antibody (MAHA) Sample Collection Schedule Study Day Groups No(s). Time points (relative to the End of IV Infusion, EOI or SC injection) TK MAHA Day 1 1-7 Predose X 1, 6-7 5 min, 1 hr - Day 2 24 hours post Day 1 dose Day 3 48 hours post Day 1 dose Day 4 72 hours post Day 1 dose Day 5 96 hours post Day 1 dose Day 6 120 hours post Day 1 dose Day 8 Day 12 96 hours post Day 8 dose Day 15 Day 19 96 hours post Day 15 dose Day 22 Day 23 24 hours post Day 22 dose Day 24 48 hours post Day 22 dose Day 25 72 hours post Day 22 dose Day 26 96 hours post Day 22 dose Day 27 120 hours post Day 22 dose Day 29 168 hours* post Day 22 dose Day 43 1, 3-5, 7 NA Day 57 Day 71 Day 85

9 Toxicokinetic (TK) Assessment
Chemiluminescence-based Immunoassay Using Covalent Binding Plate to Quantify ABC-001 in Monkey Serum Capture protein ABC - Chemiluminescent Substrate Read 570 nm Monkey Absorbed Sheep Anti human IgG (H+L) pAb HRP Dry coated plate -001 Y

10 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 Elx 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

11 Assay Materials and Procedure
Biological Matrix Blank monkey serum was purchased from Bioreclamation Inc. The serum lots used in the assay were Lot Nos. CYN and CYN The pooled serum was used to prepare the standards and QC samples, and for sample dilution. The serum was stored at -70 ºC.

12 Reagents and Materials
Reference Standard:ABC-001 Immobilizer amino F96 Black Plate: NUNC, Catalog # StabilGuard from SurModics, Catalog # SG Human 001 Protein Carrier-Free, eBioscience, Catalog # 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 # 10X DPBS, Mediatech, Inc, Catalog # CV 1X DPBS, Mediatech, Inc, Catalog # CM Tween 20, Sigma, Catalog # P1379 Sodium Chloride, Sigma, Catalog # P9416 ProClin300, Supelco, Catalog # U SuperBlock, ScyTek, Catalog # AAA999 Purified H2O

13 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.

14 Standard and QC Working Standard Concentrations
Calibration Standard ABC-001 Concentration, ng/mL 1 2 3 4 5 6 7 8 100 250 500 1250 2500 5000 8000 10000 QC/Validation Sample Concentrations Quality Control ABC-001 Concentration, ng/mL QC Low QC Mid QC High 300 1500 7500

15 Assay Performance- Standard Curves
Back Calculated Standard Concentrations and Curve Parameters for ABC-001 Assay in Monkey Serum (5-Parameter Logistic Fit with 1/Y Weighting) Run ID STD STD STD STD STD STD STD STD 1STD 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 8136 9736 50140 6 101.6 238.2 515.3 1268 2449 4916 8792 9352 50169 n 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 79.67 457.9 520.7 377.4 %CV 3.1 5.7 2.6 2.7 3.3 4.9 5.2 0.7 %RE -1.1 1.7 1.1 0.2 -2.9 2.3 9.4 -8.0 0.8

16 Precision and Accuracy
Overview of Precision and Accuracy for QC Samples 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) 8.8 Intra-Batch Precision (%CV) 3.3 9.7 8.4 7.0 16.9 Inter-Batch Precision (%CV) 10.8 %Total Error 12.5 13.0 11.2 15.8 25.1 n 18 17 Number of Runs 6 %Total Error = ABS(%RE) + %CV of inter-Batch *Calculated via inter-batch statistics (ANOVA)

17 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) Sample Spiked Conc. (ng/mL) 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

18 Matrix Interference Matrix Interference for ABC-001 Assay in Monkey Serum

19 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 1000 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 5 66.7 166.7 500.0 7832 3338 1141 511.8 1.1 0.3 0.0 4.4 11.3 14.1 2.4 Note: OOR = Out of quantitation range (Assay response greater than that of ULOQ) NA = Not applicable

20 Assay Performance - Robustness
Tested 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% 7.2% MQC (1500 ng/mL) 1684 12.2 0.1 1521 1.4 2.1 1470 -2.0 0.9 1611 7.4 1304 -13.1 2.5 1551 1.6 1524 1.6% 8.6% HQC (7500 ng/mL) 9228 23.0* 2.8 7087 -5.5 2.3 8670 15.6 1.8 9339 24.5* 1.7 7285 -2.9 4.5 8738 16.5 8391 11.9% 11.6% Note: * Exceeding acceptance criteria of ±20%.

21 Robustness Tested 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 279.9 %RE of Mean -6.7% 8.9% MQC (1500 ng/mL) 1594 6.3 2.3 1520 1.3 0.2 1424 -5.1 2.7 1662 10.8 1345 -10.3 1527 1.8 1512 0.8% 7.5% HQC (7500 ng/mL) 9605 28.1* 4.2 6804 -9.3 3.0 8761 16.8 3.5 9870 31.6* 6995 -6.7 8889 18.5 1.4 8487 13.2% 15.3% Note: * Exceeding acceptance criteria of ±20%.

22 Coated Plate Stability
Stability 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 2.2 Replicate 6 267.9 -10.7 4.2 Mean 278.0 %RE of Mean -7.3% 5.8% MQC (1500 ng/mL) 1583 2.1 1543 2.9 0.6 1414 -5.7 0.1 1550 3.4 0.8 1346 -10.2 1505 0.4 6.5 1490 -0.7% 6.1% HQC (7500 ng/mL) 8383 11.8 7398 -1.4 7815 2.6 8803 17.4 4.5 7015 -6.5 0.0 7972 6.3 7898 5.3% 8.2%

23 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% 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 8433 12.4% 9.3% Note: * Value exceeded acceptance criteria of within ±20%.

24 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% 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 8114 8.2% 7.8%

25 Long-term Storage Stability
Nominal Concentration LQC, 300 ng/mL HQC, 7500 ng/mL 6-Month Stability (188 Days) Mean ng/mL %RE -0.8 12.8 %CV 3.4 4.2 n 3 12-Month Stability (364 Days) -1.6 12.5 6 11.5

26 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+ LLOQ 1/1 117 81 99 3 99.0 18.0 18.2 -1.0 19.2 100 1/2 122 85 112 106.3 19.1 6.3 24.3 ng/mL 2/3 103 105 96 101.3 4.7 4.6 1.3 5.9 Interbatch (Between-run) Statistics: 9 102.2 13.7 13.4 2.2 15.6 LQC 337 257 289 294.3 40.3 -1.9 300 330 285 336 317.0 27.9 8.8 5.7 14.5 297 314 291 300.7 11.9 4.0 0.2 4.2 304.0 27.2 8.9 10.2 MQC 1625 1389 1506 1506.7 118.0 7.8 0.4 8.2 1500 1569 1438 1582 1529.7 79.7 5.2 2.0 7.2 1560 1665 1616.7 53.0 3.3 11.1 1551.0 91.1 3.4 9.3 HQC 7503 6899 7437 7279.7 331.3 -2.9 7.5 7500 7963 7414 7824 7733.7 285.4 3.7 3.1 6.8 7308 7950 7912 7723.3 360.2 3.0 7.7 7578.9 361.4 4.8 1.1 ULOQ 9489 9648 10026 9721.0 275.8 2.8 -2.8 5.6 10000 10592 11239 11739 575.1 5.1 17.0 9755 9940 9560 9751.7 190.0 1.9 -2.5 4.4 799.5 10.0

27 Standard and QC performance of TK Sample Analysis
Concentration [ng/mL] 100 250 500 1250 2500 5000 8000 10000 n 48 50 Overall Mean S.D. 6.868 11.255 19.802 38.548 %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 S.D. 24.371 %CV 7.9 7.5 8.8 %RE 3 2.4 3.2 n 100 99

28 Representative TK Graphs
Group 5 SC 100 mg/kg Group 7 IV 30 mg/kg

29 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.

30 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

31 ADA Assay Platforms Platforms ELISA Bridging Direct Indirect RIA
Biacore Electrochemiluminescence (ECL) No “perfect” assay currently exists In general, assay format for ADA Protein therapeutics: direct format mAbs: bridging format

32 Popular ADA assay formats for Mab Drugs
ECL / MSD Homogeneous Bridging ELISA

33 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

34 ECL vs. H-ELISA ECL Advantages H-ELISA 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 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

35 Anti-ABC01 Antibody Assay (Homogeneous Bridging Elisa format)

36 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 # TMB: KPL 1N Sulfuric Acid

37 ADA Assay Summary Screening Assay Cut Point 0.159 Normalization Factor
1.44 Confirmation Cut-Point 14.79% High Positive Control Intra-Assay %CV: ≤ 12.6% , Inter-Assay %CV: % 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 ng/mL Drug Tolerance µg/mL at 500 ng/mL of PC Interference by Hemolysis and Lipemia No Interference observed Immuno-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 hrs Refrigerated Stability Stable up to 3 days Freeze Thaw Stability Stable up to 6 cycles

38 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% 0.3703 0.3595 0.365 2.1% Cut-point: 0.229 Drug Interference: (µg/mL) 100~200 mg/mL

39 ADA sample Analysis Total samples tested for screening assay 350
Samples confirmed positive 60 Titers 2-32 Impact of ADA on PK profile No

40 ADA sample Analysis (QC Performance)
Run # OD HPC_1 HPC_2 LPC_1 LPC_2 NC 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 1.307 4.1 0.121 11.7 3.5 0.073 9.5 Run 5 1.195 1.1 1.156 1.7 0.108 Run 6 1.174 0.7 1.118 0.4 0.120 0.109 0.6 0.072 8.1 Run 7 0.926 2.0 0.824 5.5 0.106 4.7 7.0 Run 8 0.942 0.878 0.2 0.104 2.7 0.103 4.8 0.071 4.4 Run 9 0.875 1.3 0.927 6.4 0.101 Overall Mean 1.089 Overall SD 0.172 0.008 0.002 Overall CV% 15.8 7.6 2.9

41 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

42 Acknowledgement Study Sponsor:

43 Director, Biologics Services
Thank You Shawn Li, M.D., Ph.D. Director, Biologics Services Frontage Lab. Inc. Exton, PA Headquarters


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