Qualifying SPR immunogenicity assayslanding1.gehealthcare.com/rs/005-SHS-767/images/Kuehne SPR...
Transcript of Qualifying SPR immunogenicity assayslanding1.gehealthcare.com/rs/005-SHS-767/images/Kuehne SPR...
Qualifying SPR immunogenicity assaysDr. Christian Kühne
© 2017 BioAgilytix. 2
Bioagilytix / IPM Biotech
• global CRO for large molecule bioanalysis• supporting pre-clinical & clinical studies• Biomarker, Pharmacokinetics, Immunogenicity
© 2017 BioAgilytix. 3
Bioagilytix / IPM Biotech
Implemented SPR technology for:
• GLP and GCP-compliant Immunogenicity testing• Drug candidate profiling• GMP batch release testing
© 2017 BioAgilytix. 4
Bioagilytix / IPM Biotech
Implemented SPR technology for:
• Immunogenicity testing
© 2017 BioAgilytix. 5
Bioagilytix / IPM Biotech
Implemented SPR technology for:
• Immunogenicity testing
ADA Screening
binding of ADA´s toimmobilized drug
© 2017 BioAgilytix. 6
Bioagilytix / IPM Biotech
Implemented SPR technology for:
• Immunogenicity testing
ADA Confirmatory
differentiation betweenspecific and unspecificbinding by adding excessdrug in mobile phase
ADA Screening
binding of ADA´s toimmobilized drug
© 2017 BioAgilytix. 7
Bioagilytix / IPM Biotech
Implemented SPR technology for:
• Immunogenicity testing
ADA Isotyping
determination of Igsubclass by addingsecondary anti-Igantibody on top
ADA Confirmatory
differentiation betweenspecific and unspecificbinding by adding excessdrug in mobile phase
ADA Screening
binding of ADA´s toimmobilized drug
© 2017 BioAgilytix. 8
Introduction
• Biologic produced with a His-tag for purification• His-tag enzymatically removed after purification
• Immunogenic byproducts/residuals ?
His Hisenzyme
© 2017 BioAgilytix. 9
Introduction
• If ADA´s are formed, authorities ask against which target.
HisHis
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Qualification
• Biacore T200• CM5 Chip:
Fc1 Fc2 Fc3 Fc4
Blank HisHis
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Qualification
• Chip pH Scouting
-500
0
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0 2 4 6 8 10 12 14
Result Plot
Rel
ativ
e R
espo
nse
- Sto
p
RU
File & Cycle
10 mM Acetate 4
10 mM Acetate 4.5
10 mM Acetate 5
10 mM Acetate 5.5
G-CSF His-G-CSF His
Best results
G-CSF: pH 4.5His-G-CSF: pH 4.5His: pH 4.0
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Qualification
• Ligand Immobilization
Fc1 Fc2 Fc3 Fc4
Blank HisHis
74 RU 1960 RU 1900 RU
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Qualification
• Sensorgram & Report Points
-1000
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400
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0 100 200 300 400 500 600 700
Sensorgram Example G-CSF Fc4-1RU
Res
pons
e (0
= b
asel
ine)
sTime
base
line
stab
ility
sample regeneration
© 2017 BioAgilytix. 14
Qualification
• Chip Regeneration Test – Binding response
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Qualification
• Chip Regeneration Test – Baseline response
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Qualification
• Chip Stability
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0 5 10 15 20 25 30
Stability
Rel
ativ
e R
espo
nse
- Sta
bilit
y la
te
RU
Cycle
1
2
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Qualification
• 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
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Qualification
• 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
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Qualification
• 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
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Qualification
• Cut Point – G-CSF (FCP = NC.IS + 8.3 RU)
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G-CSF
Rel
ativ
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espo
nse
- sta
bilit
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RU
Cycle
Control sample
Sample
30 individual negative patient sera
outlier removal by box plot analysis
Mean of NC (in-study) + CF
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Qualification
• Cut Point – G-CSF (FCP = NC.IS + 8.3 RU)
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0 10 20 30 40 50 60 70 80 90
G-CSF
Rel
ativ
e R
espo
nse
- sta
bilit
y
RU
Cycle
Control sample
Sample
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Qualification
• Cut Point – His-G-CSF (FCP = NC.IS + 1.6 RU)
-20
0
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40
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0 10 20 30 40 50 60 70 80 90
His-G-CSF
Rel
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espo
nse
- sta
bilit
y
RU
Cycle
Control sample
Sample
30 individual negative patient sera
outlier removal by box plot analysis
Mean of NC (in-study) + CF
© 2017 BioAgilytix. 23
Qualification
• Cut Point – His-G-CSF (FCP = NC.IS + 1.6 RU)
-20
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His-G-CSF
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espo
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- sta
bilit
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RU
Cycle
Control sample
Sample
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Qualification
• Cut Point – His (FCP = NC.IS + 19.5 RU)
30 individual negative patient sera
outlier removal by box plot analysis
Mean of NC (in-study) + CF
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His
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bilit
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Control sample
Sample
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Qualification
• Cut Point – His (FCP = NC.IS + 19.5 RU)
-25
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His
Rel
ativ
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espo
nse
- sta
bilit
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RU
Cycle
Control sample
Sample
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Qualification
• Sensitivity – G-CSF
Positive Controls anti-GCSF and anti-His from 10 µg/ml to 0.3125 µg/ml in 100 % serum
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.
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Qualification
• Sensitivity – His-G-CSF
Positive Controls anti-GCSF and anti-His from 10 µg/ml to 0.3125 µg/ml in 100 % serum
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.
His - G-CSF
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Qualification
• Sensitivity - His
Positive Controls anti-GCSF and anti-His from 10 µg/ml to 0.3125 µg/ml in 100 % serum
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.
His
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Qualification
• Reproducibility - intra assayG-CSF - 5 duplicates in one run
- antibody mix injected- all CV´s below 20%
red line: mean NC + 2x SD-> acceptance criteria set10 µg/ml PC mix to monitor chip stability
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Qualification
• Reproducibility - inter assayG-CSF
Positive Control10 µg/ml
Negative Control0 µg/ml
High intra assay sample5 µg/ml
Low intra assay sample1,25 µg/ml red line: mean NC + 2x SD
-> acceptance criteria set10 µg/ml PC mix to monitor chip stability
- 3 independent runs (duplicates each)- merged in one evaluation file- antibody mix injected- all CV´s below 20%
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Summary
• Summary
His 19.5 RUHis-G-CSF 1.6 RUG-CSF 8.3 RU
Cut Point CF
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Summary
• Summary
His 19.5 RUHis-G-CSF 1.6 RUG-CSF 8.3 RU
Cut Point CFcalculated 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.
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Summary
• Summary
His 19.5 RUHis-G-CSF 1.6 RUG-CSF 8.3 RU
Cut Point CFcalculated 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.
His 5.2 %His-G-CSF 3.6 %G-CSF 2.8 %
Reproducibility Intra
CV (highest)
His 4.8 %His-G-CSF 3.4 %G-CSF 3.6 %
Reproducibility Inter
CV (highest)
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Sample Analysis
• Sample analysis performed:
comparison between pre-dose and post-dose samplespost-dose had to be 3x higher than pre-dose to be considereddrug induced / treatment emergent
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Thank you for your attention
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