mirg2010 benchmark study: molecular interactions in a ......assoc. time 30-120s 60-180s 180s 60s...
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MIRG2010 Benchmark Study: Molecular Interactions in a Three Component System
Simon Bergqvist, PfizerMike Doyle (Chair), Bristol-Myers SquibbEd Eisenstein, University Maryland Biotechnology InstituteMatthew Robinson, Fox ChaseSatya Yadav, Cleveland ClinicAaron Yamniuk (incoming Chair), Bristol-Myers SquibbTony Yeung (EB liaison), Fox Chase
March 20-23, 2010ABRF2010
CollaboratorsSuzanne Edavettal, Bristol-Myers Squibb (PERG)James Bryson, Bristol-Myers Squibb (PERG)
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Outline
Overview (purpose, timeline) Description of the protein system Participant data: Summary of participant technical data Snapshot of different experimental
designs and results Unexpected activity data Summary of non-biosensor data Conclusions
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Purpose of the MIRG 2010 study
to test capabilities for different laboratories and different biosensor instruments to characterize protein binding competition and ternary interactions for three component protein interaction systems
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X
? ?
Ternary complex? Competitive binding?
protein “A”
protein “B” protein “C”
Objective
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MIRG2010 Study Timeline
APR MAY JUN JUL AUG SEPT OCT NOV DEC JAN FEB MAR
Identify protein system
Proposal approved by ABRF (June 18)
Protein Expression, Purification
ProteinCharacterization
Advertise, Ship samples, Compile data
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Material / Information ProvidedMaterial 100ug Protein “A” (1mg/ml) = 12.1 KDa protein 100ug Protein “B” (1mg/ml) = 12.2 KDa protein 100ug Protein “C” (0.5mg/ml) = 26.3 KDa protein
Instructions Immobilize protein “A” and determine if B and C compete
for A or if a ternary complex can be formed
Guidance Provided general recommendations on experimental
buffers, temperature, immobilization chemistry, regeneration, and ballpark kinetic values
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protein “A” (Barstar)(12.1 KDa)
protein “B”(Barnase)(12.2 KDa) protein “C”
(Binase)(26.3 KDa)
Actual proteins used in the study
-Barnase and Binase domains bind to the same site on barstar so a ternary complex should not form
2 mutant barnasesconnected by linker
KD ~10-12 M
KD ~10-9 M
KD ~10-5 M
PDB: 2ZA4
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Samples prepared (30), sent (20), data (12)
Summary of Participation in MIRG 2010 Study
N/ABiotinylatedprotein “A” and SA-captured
Amine(EDC/NHS)
Amine(EDC/NHS)
Amine(EDC/NHS)
Immobilization
N/ASuper Streptavidin
COOH1CM5CM5Chip11145#Participants
CovalXHM2
ForteBio Octet Red 384
IcxNomadics -
SensiQPioneer
BiacoreT100
Biacore2000/3000
Instrument
HM-MALDI(Chemical X-linking + High Mass MALDI-Mass Spec)
BLI(BioLayer
Interferometry)
SPR(Surface Plasmon
Resonance)
SPR(Surface Plasmon
Resonance)
SPR(Surface Plasmon
Resonance)
Technique
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Summary of Biosensor data
Super Streptavidin
COOH1CM5CM5Chip
Biotin'd protein A and captured
Amine(EDC/NHS)
Amine(EDC/NHS)
Amine(EDC/NHS)
Immobilization
1.5h5h9h,20h,48h(X2)4h(x3), 6h, 24hTime spent
Fortebio Data Analysis 6.2
Qdat (ICxNomadics Inc.
& BioLogic)
Biaevaluation(1), T100
evaluation (3)
Biaevaluation(3), Scrubber2
(1), both (1)
Analysis Software
1200rpm25-65ul/min10-30ul/min20-50ul/minFlow rate120s300-600s180-600s120-300sDissoc. Time60s180s60-180s30-120sAssoc. Time
304nM (B), 328nM (C),
100nM10-200nM80-500nMAnalyte Conc's0.60-1.1 nm250 RU100-700 RU60-1000 RUImmob Level
1145#Participants
ForteBio Octet Red 384
Icx Nomadics -SensiQ Pioneer
Biacore T100Biacore2000/3000
InstrumentBLISPRSPRSPRTechnique
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Simple Example of Biosensor Data
-10
0
10
20
30
40
50
60
-50 0 50 100 150 200 250 300 350 400
Res
pons
e (0
= S
ampl
e 1
star
t)
sTime (0 = Sample 1 start)
(Participant #22 data)
Immobilizeligand
association
association
dissociation
dissociation
regeneration
regeneration
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Summary of Biosensor Data
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-20
0
20
40
60
80
100
120
140
160
0 100 200 300 400 500 600 700 800
Res
pons
e (0
= d
ual_
base
line)
sTime
Inj.#1 Inj.#2 dissociation
C then CC then (C+B premix)B then BB then (B+C premix)(100nM each)
- B then B+C = B has higher affinity than C and very slow off-rate, so B is not replaced by C (B remains bound)
- C then B+C = as C dissociates it is replaced by higher affinity B leading to a decrease in mass on the surface (lower RU signal)
Example MIRG Data (Biacore T100)
“tandem blocking (combo)” method
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Participant #34 Data (Biacore 3000)
tandem blocking experiment = no ternary complex
No ternary complex
300 nM each
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Participant #35 Data (Biacore 3000)
tandem blocking experiment = no ternary complex
No ternary complex
500 nM each
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Participant #22 Data (Biacore T100)
Premix experiment = no ternary complex
No ternary complex
C
B+C
B
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Participant #20 Data (Biacore 3000)
B
C
BC
B+C
C+B
tandem blocking (combo) experiment = no ternary complex premix B+C (red curve above) = no ternary complex
No ternary complex
82 nM “B”494 nM “C”
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Reference subtracted duplicate assaysData shown from sensors with 1.2 nm of “A” immobilized
A + B AB ABCX+CA + C AC ABCX+BData shows that:
“C” “B” “B” “C”
* * * * * ****R
Legend
Step information
* PBS
R Regeneration (3X 5 seconds 10mM glycine pH 2)
“B” Protein “B” at 328 nM
“C” Protein “C” at 304 nM
Legend
Step information
* PBS
R Regeneration (3X 5 seconds 10mM glycine pH 2)
“B” Protein “B” at 328 nM
“C” Protein “C” at 304 nM
Participant #33 Data (Fortebio Octet Red 384)
tandem blocking experiment = no ternary complex
No ternary complex
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1 2 12
Legend
Step information
1 Teal and blue = Protein “B” at 328 nMYellow and pink = Protein “C” at 304 nM
2 All sensors = solution mixture of “B” (328 nM) + ”C”(304 nM)
Raw data for interaction analysis follow-up experiment
AB +(B+C) ABCXA + (B+C) ABCX
AC +(B+C) ABCX
Participant #33 Data (Fortebio Octet Red 384)
tandem blocking (combo)experiments = no ternary complex
No ternary complex
B (B+C)
C (B+C)
(B+C)
(B+C)
B
C
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100nM B
500nM C
100nM C
500nm B
Premix 100nM (B + C)
Participant #39 Data (Biacore 2000)
No ternary complex
No ternary complex
Fast dissociation of C (can’t determine if ternary complex)
tandem blocking and premix experiments = no ternary complex
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Participant #36 Data (Biacore T100)
Tandem blocking and premix = no ternary complex
-20
-10
0
10
20
30
40
50
0 200 400 600 800 1000 1200
Res
pons
e (0
= b
asel
ine)
sTim e
1st injection/association 2nd
injection/association
Delay between injections
-20
-10
0
10
20
30
40
50
0 200 400 600 800 1000 1200
Res
pons
e (0
= b
asel
ine)
sTim e
1st injection/association 2nd
injection/association
Delay between injections
-15
-10
-5
0
5
10
15
20
0 100 200 300 400 500 600 700 800 900
Res
pons
e (0
= b
asel
ine)
sTim e
Association Dissociation
-15
-10
-5
0
5
10
15
20
0 100 200 300 400 500 600 700 800 900
Res
pons
e (0
= b
asel
ine)
sTim e
Association Dissociation
B then CC then B
B aloneB+C (pre-mix)
No ternary complex
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Participant #37 Data (Biacore T100)
Tandem blocking (with concentration series)= no ternary complex
Buffer
100 nM Molecule B + 1 nM Molecule C
Buffer + 1 nM Molecule C
100 nM Molecule B + 10 nM Molecule C
Buffer + 10 nM Molecule C
100 nM Molecule B + 100 nM Molecule C
Buffer + 10 nM Molecule CBuffer
Protein B
Protein C
100 nM Molecule B + Buffer
No ternary complex
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Participant #37 Data (Biacore T100)
Buffer
100 nM Molecule C + 1 nM Molecule B
Buffer + 1 nM Molecule B
100 nM Molecule C + 10 nM Molecule B
Buffer + 10 nM Molecule B
100 nM Molecule C + 100 nM Molecule B
Buffer + 10 nM Molecule B
100 nM Molecule C + Buffer
Tandem blocking (with concentration series)= no ternary complex
No ternary complex
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Tandem blocking (including “A”) Also performed various other single injection combinations
(ABC), (CAB), (ACB), (BAC)… (not shown here)
Participant #29 Data (SensiQ Pioneer)
cA AA
A
B c B B
A AB
ABccc
A
B C A C B A
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Tandem blocking (combo) “We propose a mechanism where
A has two binding sites. Both C and B bind both sites on A where B binds both with relatively high affinity and C binds one site with high affinity and the other with weaker affinity.”
Participant #29 Data (SensiQ Pioneer)
100nM ‘B’15-1000nM ‘C’
100nm ‘C’15-1000nM ‘B’
A
c B
1st 2ndA
Bc
Yes ternary complex
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Participant #32 Data (Biacore T100)
300
320
340
360
380
400
420
0 500 1000 1500 2000
Res
pons
e
Time
200
250
300
350
400
450
200 400 600 800 1000 1200 1400 1600
Res
pons
e
Time
100
150
200
250
300
350
400
450
0 200 400 600 800 1000 1200
Res
pons
e
Time
B BBR RR
C CCR RR
B+C B+CB+CR RR
90 RU 84 RU 84 RU
121 RU121 RU129 RU
104 RU104 RU107 RU
Test 50nM “B”
Established reproducibility Premix experiment = no ternary complex on surface
Test 50nM “C”
Test 50nM “B+C”
(each)No ternary complex
on surface
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150
200
250
300
350
400
450
0 500 1000 1500 2000
Res
pons
e
Time
B then C(50 nM each)
C then B(50 nM Each)
100
200
300
400
500
0 200 400 600 800 1000 1200 1400
Res
pons
e
Time
B BBR RRC CC
B BBR RRC CC
90 RU 90 RU 90 RU
126 RU 126 RU 126 RU
Participant #32 Data (Biacore T100)
tandem blocking = no ternary complex on surface
No ternary complex on surface
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150
200
250
300
350
400
450
500
550
600
650
0 500 1000 1500 2000 2500 3000 3500 4
Res
pons
e
Time
B A+B+CA+B
R RR
C A+C
R RR RR
A+B+C A+B A+C
100nM each protein
Reproducibility
Premix (analyte + ligand in solution) Ternary complex can form in solution (assumption that amine
coupling compromises activity of A, and that concentrations of A, B and C are concentration of active material)
Participant #32 Data (Biacore T100)
Evidence for ternary complex in solution?
Yes ternary complex in solution
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Summary of Experimental Designs (Blocking experiments) Tandem blocking Individual = B C, and C B (including + A) Individual (with conc. series of 2nd injection) Combination = B (B+C), and C (B+C)
Premix Analytes only = B alone, C alone, then (B+C) Analytes + Ligand = All combinations of A / B / C
alone and premixed Other: Classical sandwich assay not attempted
since instructions specified only using “A” on surface
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Stability of MIRG protein samples
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Unexpected loss of binding activity
Most participants observed a significant loss of protein “A” binding activity (stated by participants #20,#32,#36)
Unexpected considering “high stability” reported in literature, and previously observed in house
Reason for loss of activity is currently under investigation
0%
10%
20%
30%
40%
50%
60%
70%
80%
MIRG 20 22 25 29 32 33 34 35 36 37 39participant
% T
heor
etic
al R
max
% Rmax (B)% Rmax (C)
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Summary of Kinetic Data
(kinetic characterization was specified as optional)
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Summary of Reported Kinetic Values
10-2<0.1 nM10-437
Did not fit to 1:1 model and did not reach steady state so could not determine
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Not determinedNot determined20,25, 36,39
0.34 nM0.97 nM33B
1.99 nM1.43E-027.16E+0614.6 pM2.33E-041.57E+07351.17 nM1.22E-021.04E+0762.9 pM6.97E-041.11E+07340.11 nM4.60E-034.13E+065.99 nM5.36E-028.94E+0632A
6.079 pM9.34E-041.54E+088.231 pM6.00E-057.29E+0629KDkd (s-1)ka (M-1s-1)KDkd (s-1)ka (M-1s-1)ID#
Protein C binding Protein AProtein B binding protein A
A - kd was calculated with a separate 3600s dissociation timeB – KD calculated by steady state analysis. Dissociation slow enough that longer assay would need to be run to measure kd
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Examples of Participant Kinetic Data
-20
0
20
40
60
80
-100 0 100 200 300 400
RUR
espo
nse
Time s
-200
20406080
100120140
-100 0 100 200 300 400
RU
Res
pons
e
Time s
-5
0
5
10
15
20
0 50 100 150 200 250 300 350 400
Time s
Res
p. D
iff.
RU
-10
-5
0
5
10
15
20
25
30
0 50 100 150 200 250 300 350 400
Time s
Res
p. D
iff.
RU
Par
ticip
ant 3
5P
artic
ipan
t 29
Par
ticip
ant 3
2
KD = 5.99 nM
KD = 0.11 nM
KD = 14.6 pMKD = 1.99 nM
KD = 8.231 pMKD = 6.079 pM
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Binding of “B” to “A” Binding of “C” to “A”• Duplicate binding shown• In order to measure the dissociation, a longer assay is needed• The affinity based on the extrapolated Req is shown in the lower graphs.
20.5 41 82
163.9 327.9 20.5
41 82 163.9
327.9
0.00
0.06
0.12
0.18
nm
0 40 80 120 160 200 240
Time (sec)
Sample ID: sample B - by Conc. (nM)
19 38 76.1
152.1 304.2 19
38 76.1 152.1
304.2
0.00
0.08
0.16
0.24
nm
0 40 80 120 160 200 240
Time (sec)
Sample ID: Sample C - by Conc. (nM)
Participant #33
Example of Participant Kinetic Data
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Non-Biosensor dataChemical cross-linking followed
by high mass MALDI Mass Spectrometry
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Participant #31 Data (X-link + HM-MALDI)
A, B, or C alone = no multimers (not shown here) Mix A+B = A•B only (24.4 KDa) (see above) Mix A+C = A•C only (38.5 KDa) (see above) Mix B+C = no complex (not shown here)
2.6μM “A”, 2.5μM “B” 2.6μM “A”, 2.4μM “C”
A = 12.1 KDaB = 12.2 KDaC = 26.3 KDa
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Participant #31 Data (X-link + HM-MALDI)
A+B+C = A•B (24.5KDa), A•C (38.6KDa), and “A•B•C” (50.8KDa) “A•B•C” is most likely A•C•A
(*later supported by A+C experiment)
2.6μM “A”, 2.5μM “B”, 2.4μM “C”
A = 12.1 KDaB = 12.2 KDaC = 26.3 KDa
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Summary 8/11 Biosensor participants = no ternary complex 1/11 Biosensor participant = yes ternary complex on
surface (complex mechanism) 1/11 Biosensor participant = yes ternary complex in
solution, but not with molecule A on sensor surface 1/11 Biosensor participant = samples degraded due to
shipping problem 1/1 non-biosensor participant (HM-MALDI) identified all
possible complexes (A-B, A-C) including low affinity A-C-A complex not studied in the nanomolarconcentration range by biosensor technology
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Acknowledgements
MIRG Members Simon Bergqvist, Pfizer Mike Doyle (Chair), Bristol-
Myers Squibb Ed Eisenstein, University
Maryland Biotechnology Institute
Matthew Robinson, Fox Chase
Satya Yadav, Cleveland Clinic Aaron Yamniuk (incoming
Chair), Bristol-Myers Squibb Tony Yeung (EB liaison), Fox
Chase
Collaborators Suzanne Edavettal, Bristol-
Myers Squibb (PERG) James Bryson, Bristol-Myers
Squibb (PERG)
THANK YOU ALL PARTICIPANTS!!!