Qualifying SPR immunogenicity assays Dr. Christian Kühne

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

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

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

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

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

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

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

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

Chip ph Scouting G-CSF His-G-CSF His RU 3000 Result Plot Best results 2500 2000 G-CSF: ph 4.5 His-G-CSF: ph 4.5 His: ph 4.0 Relative Response - Stop 1500 1000 10 mm Acetate 4 10 mm Acetate 4.5 10 mm Acetate 5 10 mm Acetate 5.5 500 0-500 0 2 4 6 8 10 12 14 File & Cycle 11

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

Sensorgram & Report Points RU 1000 800 Sensorgram Example G-CSF Fc4-1 sample regeneration 600 400 Response (0 = baseline) 200 0-200 -400 baseline stability -600-800 -1000 0 100 200 300 400 500 600 700 Time s 13

Chip Regeneration Test Binding response 14

Chip Regeneration Test Baseline response 15

Chip Stability RU 220 Stability 200 180 Relative Response - Stability late 160 140 120 100 80 1 2 3 4 60 40 0 5 10 15 20 25 30 Cycle 16

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

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

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

Cut Point G-CSF (FCP = NC.IS + 8.3 RU) RU 80 G-CSF 60 30 individual negative patient sera Relative Response - stability 40 20 0-20 Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF -40 0 10 20 30 40 50 60 70 80 90 Cycle 20

Cut Point G-CSF (FCP = NC.IS + 8.3 RU) RU 80 G-CSF 60 Relative Response - stability 40 20 0 Control sample Sample -20-40 0 10 20 30 40 50 60 70 80 90 Cycle 21

Cut Point His-G-CSF (FCP = NC.IS + 1.6 RU) RU 140 His-G-CSF 120 100 30 individual negative patient sera Relative Response - stability 80 60 40 20 0 Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF -20 0 10 20 30 40 50 60 70 80 90 Cycle 22

Cut Point His-G-CSF (FCP = NC.IS + 1.6 RU) RU 140 His-G-CSF 120 Relative Response - stability 100 80 60 40 20 Control sample Sample 0-20 0 10 20 30 40 50 60 70 80 90 Cycle 23

Cut Point His (FCP = NC.IS + 19.5 RU) RU 25 His 20 15 30 individual negative patient sera Relative Response - stability 10 5 0-5 -10-15 Control sample Sample outlier removal by box plot analysis Mean of NC (in-study) + CF -20-25 0 10 20 30 40 50 60 70 80 90 Cycle 24

Cut Point His (FCP = NC.IS + 19.5 RU) RU 25 His 20 15 Relative Response - stability 10 5 0-5 -10-15 Control sample Sample -20-25 0 10 20 30 40 50 60 70 80 90 Cycle 25

Sensitivity G-CSF G-CSF calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a. 8.301 His-G-CSF 0.120 2.198 G-CSF 0.937 n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to 0.3125 µg/ml in 100 % serum 26

Sensitivity His-G-CSF His - G-CSF calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a. 8.301 His-G-CSF 0.120 2.198 G-CSF 0.937 n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to 0.3125 µg/ml in 100 % serum 27

Sensitivity - His His calculated assay sensitivity [µg/ml] Ligand Analyte anti-gcsf anti-his His n.a. 8.301 His-G-CSF 0.120 2.198 G-CSF 0.937 n.d. Positive Controls anti-gcsf and anti-his from 10 µg/ml to 0.3125 µg/ml in 100 % serum 28

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

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

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

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. 8.301 His-G-CSF 0.120 2.198 G-CSF 0.937 n.d. 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. 8.301 His-G-CSF 0.120 2.198 G-CSF 0.937 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

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

Thank You Thank you for your attention 35