Qualifying SPR immunogenicity assays Dr. Christian Kühne

Transcription

1 Qualifying SPR immunogenicity assays Dr. Christian Kühne

2 Bioagilytix / IPM Biotech global CRO for large molecule bioanalysis supporting pre-clinical & clinical studies Biomarker, Pharmacokinetics, Immunogenicity 2

3 Bioagilytix / IPM Biotech Implemented SPR technology for: GLP and GCP-compliant Immunogenicity testing Drug candidate profiling GMP batch release testing 3

4 Bioagilytix / IPM Biotech Implemented SPR technology for: Immunogenicity testing 4

5 Bioagilytix / IPM Biotech Implemented SPR technology for: Immunogenicity testing ADA Screening binding of ADA s to immobilized drug 5

6 Bioagilytix / IPM Biotech Implemented SPR technology for: Immunogenicity testing ADA Screening binding of ADA s to immobilized drug ADA Confirmatory differentiation between specific and unspecific binding by adding excess drug in mobile phase 6

7 Bioagilytix / IPM Biotech Implemented SPR technology for: Immunogenicity testing ADA Screening binding of ADA s to immobilized drug ADA Confirmatory differentiation between specific and unspecific binding by adding excess drug in mobile phase ADA Isotyping determination of Ig subclass by adding secondary anti-ig antibody on top 7

8 Introduction Biologic produced with a His-tag for purification His-tag enzymatically removed after purification Immunogenic byproducts/residuals? His enzyme His 8

9 Introduction If ADA s are formed, authorities ask against which target. His His 9

10 Biacore T200 CM5 Chip: Blank His His Fc1 Fc2 Fc3 Fc4 10

11 Chip ph Scouting G-CSF His-G-CSF His RU 3000 Result Plot Best results G-CSF: ph 4.5 His-G-CSF: ph 4.5 His: ph 4.0 Relative Response - Stop mm Acetate 4 10 mm Acetate mm Acetate 5 10 mm Acetate File & Cycle 11

12 Ligand Immobilization 74 RU 1960 RU 1900 RU Blank His His Fc1 Fc2 Fc3 Fc4 12

13 Sensorgram & Report Points RU Sensorgram Example G-CSF Fc4-1 sample regeneration Response (0 = baseline) baseline stability Time s 13

14 Chip Regeneration Test Binding response 14

15 Chip Regeneration Test Baseline response 15

16 Chip Stability RU 220 Stability Relative Response - Stability late Cycle 16

17 Minimum Required Dilution (MRD) His 5% serum His-G-CSF 5% serum G-CSF 5% serum Positive Controls anti-gcsf and anti-his from 10 µg/ml to 1.25 µg/ml in 100 % serum 17

18 Minimum Required Dilution (MRD) His 10% serum His-G-CSF 10% serum G-CSF 10% serum Positive Controls anti-gcsf and anti-his from 10 µg/ml to 1.25 µg/ml in 100 % serum 18

19 Minimum Required Dilution (MRD) His 20% serum His-G-CSF 20% serum G-CSF 20% serum Positive Controls anti-gcsf and anti-his from 10 µg/ml to 1.25 µg/ml in 100 % serum 19

20 Cut Point G-CSF (FCP = NC.IS RU) RU 80 G-CSF individual negative patient sera Relative Response - stability Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF Cycle 20

21 Cut Point G-CSF (FCP = NC.IS RU) RU 80 G-CSF 60 Relative Response - stability Control sample Sample Cycle 21

22 Cut Point His-G-CSF (FCP = NC.IS RU) RU 140 His-G-CSF individual negative patient sera Relative Response - stability Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF Cycle 22

23 Cut Point His-G-CSF (FCP = NC.IS RU) RU 140 His-G-CSF 120 Relative Response - stability Control sample Sample Cycle 23

24 Cut Point His (FCP = NC.IS RU) RU 25 His individual negative patient sera Relative Response - stability Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF Cycle 24

25 Cut Point His (FCP = NC.IS RU) RU 25 His Relative Response - stability Control sample Sample Cycle 25

26 Sensitivity G-CSF G-CSF calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a His-G-CSF G-CSF n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to µg/ml in 100 % serum 26

27 Sensitivity His-G-CSF His - G-CSF calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a His-G-CSF G-CSF n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to µg/ml in 100 % serum 27

28 Sensitivity - His His calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a His-G-CSF G-CSF n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to µg/ml in 100 % serum 28

29 Reproducibility - intra assay G-CSF - 5 duplicates in one run - antibody mix injected - all CV s below 20% red line: mean NC + 2x SD -> acceptance criteria set 10 µg/ml PC mix to monitor chip stability 29

30 Reproducibility - inter assay G-CSF Positive Control 10 µg/ml - 3 independent runs (duplicates each) - merged in one evaluation file - antibody mix injected - all CV s below 20% High intra assay sample 5 µg/ml Low intra assay sample 1,25 µg/ml red line: mean NC + 2x SD Negative Control 0 µg/ml -> acceptance criteria set 10 µg/ml PC mix to monitor chip stability 30

31 Summary Summary Cut Point His His-G-CSF G-CSF CF 19.5 RU 1.6 RU 8.3 RU 31

32 Summary Summary Cut Point His His-G-CSF G-CSF CF 19.5 RU 1.6 RU 8.3 RU calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a His-G-CSF G-CSF n.d. 32

33 Summary Summary Cut Point His His-G-CSF G-CSF CF 19.5 RU 1.6 RU 8.3 RU calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a His-G-CSF G-CSF n.d. Reproducibility Intra CV (highest) His 5.2 % His-G-CSF 3.6 % G-CSF 2.8 % Reproducibility Inter CV (highest) His 4.8 % His-G-CSF 3.4 % G-CSF 3.6 % 33

34 Sample Analysis Sample analysis performed: comparison between pre-dose and post-dose samples post-dose had to be 3x higher than pre-dose to be considered drug induced / treatment emergent 34

35 Thank You Thank you for your attention 35