Precipitation and Acid (PandA) to Resolve the Drug and Target Interference Problems in Immunogenicity Assays

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1 Precipitation and Acid (PandA) to Resolve the Drug and Target Interference Problems in Immunogenicity Assays Jad Zoghbi Senior Scientist Boston BCB, Sanofi, Framingham, MA

2 Introduction Circulating therapeutic antibody drug and or drug target can complex with anti-drug antibodies (ADA) present in patient samples and interfere with the detection of these ADAs during immunogenicity assessment. Immunoassays using acid dissociation have been have been introduced several years ago and used to improve the assay drug tolerance. Traditional acid dissociation may not always be suitable as re-association of drug and ADA occurs upon ph neutralization. Other methods involving target competition or removal are also being evaluated. PandA: a new method with exceptional drug tolerance and also reduced our drug target interference 2

3 Interferences in Immunogenicity Assays Specific: Drug Endogenous target Pre-existing antibody to other similar drug products Non-specific: Rheumatoid factors Human serum proteins (IgM, IgG) Other disease specific interference factors 3

4 Options to deal with the interference Increasing the sample dilution May work but can also negatively impact assay sensitivity Extraction Efficiency should be examined Potential assay throughput impact Competition of interference Availability of antibodies needed May be costly Efficiency needs to be examined New technologies with great sensitivities Can potentially improve the tolerance May not be available at preferred external CROs Acid dissociation Acid may alter analyte (and/or binding reagent) structure Under neutralizing assay condition, matrix effect may reappear 4

5 Key concepts for our new method Use what we learned from past experiences Understand why some described methods do not work that great Use of interference to your advantage 5

6 PandA method publication 6

7 Drug B Specific Challenge Drug B is a full human IgG4 that neutralizes a soluble cytokine binding to its cell surface receptor in the target tissue for a fibrosis indication. The drug has a long half life and a higher dosing regimen that will require drug tolerance greater than 250 ug/ml. Bridging immunogenicity assay with acid dissociation cannot be used since the target for Drug B changes from a monomer to a dimer at low ph causing false positive results. The dimerization effect is seen in majority of normal serum samples and disease baseline samples in the MSD bridging assay with acid dissociation. 7

8 Drug Tolerance Assessment (Bridging Assay without Acid Dissociation) S /B 4 0 D ru g a t g /m L D ru g a t 5 0 g /m L 3 0 D ru g a t 1 0 g /m L N o D ru g R a b b it a n ti-d r u g B C o n c e n tra tio n (µ g /m l) For Drug B, all concentrations of rabbit anti-drug B antibody were inhibited by the presence of as low as 10 µg/ml Drug B. 8

9 Target Interference in Bridging Assay with Acid S/B S/B Thirty-two serum samples from healthy donors and sixteen Disease baseline serum samples were assayed in the bridging assay with and without acid treatment. Normal population samples Disease Population Baseline Samples Bridging no Acid Bridging with Acid 0.8 Bridging No Acid Bridging with Acid 9

10 Precipitation and Acid dissociation (PandA) Method 10

11 Optimization ideas Optimal drug concentration for complex formation PEG concentration: High concentration of PEG will precipitate smaller molecular weight components Sample dilution: Volumes of sample and drug mixture (1/2, 1/5, 1/10) Volume of PEG added (equal volume) Addition dilution with acid optimized to ensure lack of competition from non specific proteins on plate surface Overall sample screen dilutions used ranged from 1/10-1/50 with acceptable results. 11

12 Target Interference reduced with PandA method S /B D is e a s e P o p u la tio n B a s e lin e S a m p le s B rid g i n g N o A c i d P a n d A M e th o d 12

13 Drug Interference resolved with PandA Method S/B 20 Drug at 250 g/ml 15 Drug at 50 g/ml Drug at 10 g/ml No Drug Rabbit anti-drug B Concentration (µg/ml) For Drug B, all concentrations of rabbit anti-drug B antibody were not inhibited by the presence of up to 250 µg/ml Drug B. Based on the sensitivity of 62.5 ng/ml of rabbit anti-drug B antibody, the maximum drug tolerance will correspond to a 4000-fold excess Drug B. 13

14 PandA Assay Validation Conclusions Use of PandA resulted in a validated method capable of detecting 62.5 ng/ml of ADA in the presence of 250 μg/ml of soluble Drug B (corresponding to a 4000-fold drug tolerance). In contrast, the traditional bridging ADA assay had poor drug tolerance and could not be used in combination with acid dissociation for this therapeutic due to the drug target dimerization and activation at low and high ph. Method fully and successfully validated and method applied to clinical program 14

15 Drug C a similar challenge Drug C is a humanized IgG1 The drug has a high dosing regimen that will require drug tolerance greater than 250 μg/ml. Bridging immunogenicity assay with acid dissociation was not sufficient to improve the drug tolerance Awaiting drug clearance was not an option 15

16 S /B % R e c o v e r y Proof of principle 2- ECL Bridging Assay with Acid Dissociation 1 5 D ru g a t g /m L A D ru g a t 2 5 g /m L 1 0 D ru g a t 2.5 g /m L N o D ru g B D ru g a t g /m L D ru g a t 2 5 g /m L D ru g a t 2.5 g /m L N o D ru g R b t a n ti-d ru g C C o n c (µ g /m l) R b t a n ti-d ru g C C o n c (µ g /m l) The ECL bridging assay format resulted in an acceptable sensitivity in the absence of drug but inhibition was seen with as little as 2.5 μg/ml of drug despite acid treatment in the bridging immunoassay. The sensitivity of the assay changed from 46 ng/ml to 1400 ng/ml in the presence of 25 μg/ml and antibodies were completely undetectable in the presence of 250 μg/ml of Drug, levels expected in some clinical samples. 16

17 S /B % R e c o v e r y Proof of principle 2- PandA method 2 0 D ru g a t g /m L A D ru g a t 2 5 g /m L 1 5 D ru g a t 2.5 g /m L N o D ru g B D ru g a t g /m L D ru g a t 2 5 g /m L D ru g a t 2.5 g /m L N o D ru g R b t a n ti-d ru g C C o n c (µ g /m l) R b t a n ti-d ru g C C o n c (µ g /m l) In the PandA method, antibody detection was maintained with complete recovery even in the presence of 250 μg/ml of Drug. In most instances, the percent recoveries were between % regardless of the drug amount present. The assay detection sensitivity was maintained around 75 ng/ml despite drug present at 250 µg/ml. 17

18 Final Conclusions The results demonstrate that the use of the newly developed and validated PandA method has successfully eliminated the drug interference seen in the previous assay and resolves the target interference problem that would be observed in the bridging assay with acid dissociation. The PandA method was applied to other programs with observed drug tolerance in excess of 2 mg/ml without any effect on the assay sensitivity. The PandA method is a potential solution to deal with interferences and has a broader potential for applicability (PK, NAb, etc.) 18

19 Acknowledgment Ryan Grabert Mary Brock Allan Mcphee Yuanxin Xu Valerie Theobald Susan Richards Lakshmi Amaravadi Alison Schroeer (Biomedical Media Services) 19

20 Thank you for your attention! 20