Embed Size (px)
Citation preview
1 2 3 4 5 6 7 8 9 1 0
0 .1
1
1 0G yro lab
TM - L o P C le v e l
D is e a s e -S ta te In d iv id u a l s e ra
Re
sp
on
se
S c re e n in g C o n firm a to ry
1 2 3 4 5 6 7 8 9 1 0
1
1 0
1 0 0
1 0 0 0
M S D
- L o P C le v e l
D is e a s e -S ta te In d iv id u a l s e ra
Re
sp
on
se
S cre e n in g C o n firm a to ry
Streptavidin
Bead
Biotinylated
Drug
Bridging ADA
(reference material)
Alexa Fluor®
Conjugated Drug
Mixing
Chamber
Streptavidin Affinity
Capture Column
Volume Definition
Chambers
Evaluation of GyrolabTM and Meso-Scale Discovery® platforms in
the development of a clinical immunogenicity assay Justine Collet, Anaïs Moreau*, Maureen Deehan, Walter Ferlin and Robert Nelson
Technologies used
Purpose
Evaluation of the impact of disease matrix
Conclusion
November, 2014 EBF Young Scientist Symposium www.novimmune.com
Determination of confirmatory solution
Hook effect and titer assessment
Immunogenicity principle and assay
Labeled Drug
Potential ADA
Labeled Drug
Bridging format assay Immunogenicity principle
Drug interference concept
GyrolabTM
GyrolabTM is a fully automated nano-liter scale platform. The system transfers samples
and reagents from microplates to each microstructure within a GyrolabTM CD. Results
are obtained in less than an hour using high sensitivity, laser-induced fluorescence.
MSD®
MSD® is a plate reader based on electrochemiluminescence (ECL) detection
technology using Sulfo-Tag labels. Multiple analytes can be measured in the same well.
Five individual human sera and a pooled human serum were spiked at the HiPC level
(20 000 ng/mL). Samples were screened and tested with a confirmatory solution
containing free drug at 500 µg/mL or 1000 µg/mL for GyrolabTM (Figure A); at 200 µg/mL
or 500 µg/mL for MSD ® (Figure B).
Confirmatory solution reduced signal to below the cut-point.
Confirmatory solutions were selected at 500 µg/mL for GyrolabTM and
at 200 µg/mL for MSD®, respectively.
Ten healthy and ten disease-state individual human sera were tested on GyrolabTM and
MSD®, blank in screening assay (Figures A and B) and spiked at the LoPC level (15 ng/mL) in screening and confirmatory assays (Figures C and D).
The disease-state matrix had no impact on either assay.
The development of an immunogenicity assay is a critical step in the safety
assessment process for a new biotherapeutic drug. Key parameters which have to be
considered include assay sensitivity, drug tolerance, and selectivity.
Drug tolerance, the binding of the free drug to the anti-drug antibodies (ADA), is
evaluated in order to limit the risk of false-negative results. Selectivity, which evaluates
the robustness of the assay in the presence of normal or disease matrix, is critical to
ensure consistency when assessing different samples within a clinical study.
In order to obtain the most suitable assay, a homogenous bridging ADA assay was
developed in parallel on both Gyrolab™ and Meso-Scale Discovery® (MSD®) platforms.
NovImmune SA, 14 Chemin des Aulx, 1228 Plan-Les-Ouates, Switzerland
Contact : [email protected]
* Presenting author
Acid dissociation optimization and drug tolerance
Concentration range of ADA positive control (rabbit polyclonal Ab) were prepared in
blank matrix or in matrix containing 50 or 200 µg/mL of free drug. Acid treatment with
0.5M Glycine pH 2.6 (Figure A) or pH 2.2 (Figure B) were applied to these samples.
The acid buffer 0.5M Glycine at pH 2.2 was selected.
Loss of sensitivity with increasing free drug concentration.
Free drug tolerance is pH dependent.
Two Hook Effect samples (HE-1 and HE-2) at a concentration of 100 000 ng/mL and
50 000 ng/mL, respectively, and the HiPC (20 000 ng/mL) were tested neat and diluted
by serial dilution on GyrolabTM (Figure A) and MSD ® (Figure B).
Dilution profile is linear.
No hook effect observed.
All of the samples at 5 ng/mL were below the cut-point.
More variability observed on GyrolabTM platform.
Similar results between both technologies except for two individual sera.
Both Gyrolab™ and MSD® platforms demonstrated excellent performance in assay
parameters such as sensitivity, drug tolerance, titer and selectivity.
Both assays were suitable for validation and clinical sample testing.
Automation of the Gyrolab™ platform resulted in less sample handling procedures
and a shorter assay time, but a limited number of samples per assay CD. In addition,
some variability was observed for the low response level.
The MSD® platform was more labour intensive, but showed lower variability and a
higher throughput per assay plate.
A B
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
In d iv id u a l s e ra
Re
sp
on
se
H iP C s c re e n in g
H iP C + C o n firm a to ry a t 2 0 0 g /m L
H iP C + C o n firm a to ry a t 5 0 0 g /m L
M S D
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
Ind
ivid
ual 1
Ind
ivid
ual 2
Ind
ivid
ual 3
Ind
ivid
ual 4
Ind
ivid
ual 5
Po
ole
d s
eru
m
0 .1
1
1 0
1 0 0
1 0 0 0
In d iv id u a l s e ra
Re
sp
on
se
H iP C s c re e n in g
H iP C + C o n firm a to ry a t 1 0 0 0 g /m L
H iP C + C o n firm a to ry a t 5 0 0 g /m L
G yro labTM
A B
1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0
1
1 0
1 0 0
1 0 0 0
M S D
- B la n k le v e l
S c re e n in g a s s a y
Re
sp
on
se
H e a lth y D is e a s e -S ta te
In d iv id u a l s e ra
A B
C D
0.0
001
0.0
01
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
A D A p o s it iv e c o n tr o l c o n c e n tra t io n
in n e a t s e ru m (n g /m L )
Sig
na
l /
No
ise
2
p H 2 .6
250
A B
1 0 0 0 0 0 5 0 0 0 0 2 0 0 0 0 2 0 0 0 2 0 0 4 0 2 0 5 2 .5
0 .1
1
1 0
1 0 0
1 0 0 0
A D A p o s itiv e c o n tro l c o n c e n tra tio n in n e a t s e ru m (n g /m L )
Re
sp
on
se
H E -1
H E -2
H iP C
G yro labTM
1 0 0 0 0 0 5 0 0 0 0 2 0 0 0 0 2 0 0 0 2 0 0 4 0 2 0 5 2 .5
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
1 0 0 0 0 0 0
A D A p o s itiv e c o n tro l c o n c e n tra tio n in n e a t s e ru m (n g /m L )
Re
sp
on
se
H E -1
H E -2
H iP C
M S D
0.0
001
0.0
01
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
A D A p o s itiv e c o n tro l c o n c e n tra tio n
in n e a t s e ru m (n g /m L )
Sig
na
l /
No
ise
A D A + 2 0 0 g /m L d ru g
A D A + 5 0 g /m L d ru g
A D A + 0 g /m L d ru g
A D A + 2 0 0 g /m L d ru g
p H 2 .2
M S D
G yro labTM
2
250
A D A + 0 g /m L d ru g
A D A + 5 0 g /m L d ru g
1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0
0 .1
1
1 0
G yro labTM
- B la n k le v e l
S c re e n in g a s s a y
Re
sp
on
se
In d iv id u a l s e ra
H e a lth y D is e a s e -S ta te
* *
* C o n firm e d a s fa ls e -p o s it iv e
Drug
Is there an anti-drug antibody (ADA) response?
pH 2.2
Sample containingADA:Drug complexes No signal
False-negative results
