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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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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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)


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)


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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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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Tandem blocking (with concentration series)= no ternary complex

Buffer

100 nM Molecule B + 1 nM Molecule C

Buffer + 1 nM Molecule C


Buffer + 10 nM Molecule C


Buffer + 10 nM Molecule CBuffer

Protein B

Protein C

100 nM Molecule B + Buffer

No ternary complex

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Buffer

100 nM Molecule C + 1 nM Molecule B

Buffer + 1 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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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


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)


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!!!

MIRG2010 Benchmark Study: Molecular Interactions in a ......Assoc. Time 30-120s 60-180s 180s 60s...

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