Molecular Interactions Research Group (MIRG)Group · PDF fileMolecular Interactions Research...

Molecular Interactions Research Group (MIRG)Group (MIRG)

Satya P. Yadav, Aaron P. Yamniuk, John Newitt Michael L Doyle Ed EisensteinNewitt, Michael L. Doyle, Ed Eisenstein, Thomas A. Neubert

Presented at:ABRF 2013 (RG9 i ) P l S i CAABRF 2013 (RG9 session), Palm Springs, CAMarch 4, 2013

Objective of the 2012 Benchmark Study

To determine the stoichiometry of protein-X binding to immobilized protein-Y

To measure the equilibrium dissociation constant (KD) value(s) for the interaction of protein-X to protein-Yprotein X to protein Y

Optional –What are the measured or estimated associationWhat are the measured or estimated associationrate (ka) and dissociation rate (kd) for protein-XBinding to protein-Y

Samples Provided to Participants

P t iQuantity

id dVolume

id Mw E280 concentration B ff

p p

Protein provided (mg)

provided (ml)

Mw (kDa)

E280 (M-1 cm-1)

concentration (mg/ml) Buffer

X 2 5 0 28 11 9 20970 9 050 mM NH4C2H3O2, 100 mM NaCl 0 1% EDTAX 2.5 0.28 11.9 20970 9.0 mM NaCl, 0.1% EDTA, 0.01% Na-azide, pH 8.0

Y 0.7 0.26 26.3 55350 2.6850 mM NH4C2H3O2, 100 mM NaCl, 0.1% EDTA, 0 01% N id H 8 00.01% Na-azide, pH 8.0

E i t l G idExperimental GuidanceRecommended Experimental Conditions to participants:Recommended Experimental Conditions to participants:

Experimental temperature = 25o C Running buffer should be pH (8.0) and ~100 mM NaCl.g p ( ) Immobilize protein “Y” using standard EDC/NHS chemistry (recommended buffer

for immobilization is Na-acetate pH 5.5) Protein-X dissociates from a protein-Y surface quite rapidly, and complete

dissociation is expected within a few minutes using the recommended running buffer conditions above (pH 8.0, ~100mM NaCl). Therefore, it should not be necessary to use a unique “regeneration solution” other than allowing sufficient dissociation time in the running buffer.

It was recommended that the participants test a large range of protein-X analytet ti i f t l t 300 M d t 10 9 M N t th t t i Xconcentrations ranging from at least 300 uM down to ~10-9 M. Note that protein-X

is delivered at a concentration of 9.0 mg/ml corresponding to a molar concentration of 760 uM.

Proteins were shipped on blue ice.

NOTE on interpretation and pcompilation of participant data

Data was received between Nov, 2012 – Feb, 2013 Report forms interpreted by MIRG, compiled and summarized

for ABRF2013 presentation no discussions with participants to clarify reported data

Post-ABRF2013 plans: Summarize Benchmark Study results for publication in JBT*discussions with participants for clarification of reported

data, and to obtain further information as necessary

Summary of DataID # Instrument

usedSensor

ChipImmob.Method

Y-ImmobilizedRU

Running Buffer

#6 Biacore 3000 CM5 EDC/NHS 57, 270, 942 1

#7 Biacore 3000 CM5 EDC/NHS >300 2

#19 Biacore 3000 CM5 EDC/NHS 425.5 HBS-EP

#3 T100 CM5 EDC/NHS 581, 983 HBS-P,

#11 T100 CM5 EDC/NHS Y-194; X-175 PBS

#14 T100 CM5 EDC/NHS 28,289,1071 3

#22 T100 CM5 EDC/NHS 100 700 2500 1xPBS-T, pH8#22 T100 CM5 EDC/NHS 100,700,2500 , p

#26 T100 CM5 EDC/NHS 402, 432 4

#12 T200 CM5 EDC/NHS 58, 113, 293 5

#17 T200 CM5 EDC/NHS 20 4#17 T200 CM5 EDC/NHS 20 4

#24 T200 CM5 EDC/NHS 100.6, 67 HBS-EP+

#4 Reichert DC7500 R13206061 EDC/NHS 250 uRIU 4

#9 Wyatt Calypso II N/A N/A N/A 4#9 Wyatt Calypso II N/A N/A N/A 4

Buffers used: 1. 50 mM Amm acetate +100 mM NaCl + 0.1% EDTA, pH 8.0: 2. 50 mM Tris-Hcl + 100 mM NaCl, pH 8.0 3. 50 mM Amm acetate +150 mM NaCl + 0.05% P-20, pH 8.0 ; 4. 50 mM Amm acetate + 100 mM NaCl +0.1% EDTA + 0.01% Sod Azide, pH 8.0; 5. HBS-EP + 1 mg/ml BSA , pH 8.0

Summary DatayID # Flow

rateAssociation

TimeDissociation

TimeRegen. Total Time

used#6 - 3000 25 ul 600 sec 300 sec 25mM NaOH 1h+7h+2h

#7 - 3000 25 ul 120 sec 600 sec None 0.5h+11h+1d

#19 - 3000 30 ul 60 sec 300 sec None 3h+1d+5d

#3 - T100 30 ul 60 sec 300 sec None 2h+6h+5h#3 T100 30 ul 60 sec 300 sec None 2h 6h 5h

#11 - T100 10 ul 180 sec 600 sec None 2h+2d+4-5h

#14 - T100 30 ul 120 sec 300 sec None 0.5h+8 h+5 d

#22 T100 30 l 180 300 N 20 +6h+1 5h#22 - T100 30 ul 180 sec 300 sec None 20m+6h+1.5h

#26 - T100 30 ul I run-700 secII run -150 sec

I run – 100 secII run – 300 sec

None 4h+12h+8h

#12 - T200 50 ul 300 sec 900 sec None 2h+1.5d+1.5h#12 T200 50 ul 300 sec 900 sec None 2h 1.5d 1.5h

#17 - T200 30 ul 30 sec 60 sec None 30m+2.5h+30m

#24 - T200 30 ul 20 sec 30 sec None 25m+40m+20m

#4 - Reichart 35 ul 240 sec 240 sec None 20m+5h+1h

#9 - Wyatt Calypso 1 ml 180 sec N/A 10m+3h+30min

Summary DatayID # Analyte Conc.

TestedSoftware Model

#6 3000 0 125 M 2 M Biae al ation Stead State V3 2#6-3000 0.125 uM – 2 uM Biaevaluation Steady State V3.2

#7-3000 0.156 uM – 300 uM Biaevaluation Steady State V4.01

#19-3000 0.01 uM – 160 uM Biaevaluation R programming Biaevaluation R programming

#3-T100 0.001 uM – 100 uM Biaevaluation+Excel Biaevaluation+Excel

#11-T100 0.5 uM – 300 uM Biaevaluation Steady State


#22-T100 0.0057 uM – 300 uM Biavaluation Global, Steady State(T100)#22 T100 0.0057 uM 300 uM Biavaluation Global, Steady State(T100)

#26-T200 0.001 uM – 500 uM Biaevaluation (T100 V2.0.3) T100 V2.0.3 Steady State


#17-T200 0.13 uM – 300 uM Biaevaluation T200 Heterogeneous

#24-T200 0.001 uM – 300 uM Biaevaluation T200, V1.0 Steady State

#4-Reichart 0.001 uM – 87 uM Scrubber-2 Clamp 99 v3.4 Scrubber-2 Clamp 99 v3.4

#9-Wyatt 0.3 uM - 30 uM Calypso 2.1.2 Calypso 2.1.2

ID # 6 – Biacore 3000N = 2Kd1 = 15-38 nMKd2 = 454-657 nM

60

70

80

RU

250

300

350

RU

6

8

10

12

RU

Y immobilized = 942 RUY immobilized = 270 RUY immobilized = 57 RU

Kd2 = 454 657 nM

-10

0

10

20

30

40

50

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

Tim e s

Res

p. D

iff.

-50

0

50

100

150

200

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

Tim e s

Res

p. D

iff.

-10

-8

-6

-4

-2

0

2

4

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

Res

p. D

iff.

sTim e

20

25

30

35

40

iff.

RU

X immobilized = 31 RU

60

80

100

120

140

160

p. D

iff.

RU


-5

0

5

10

15

100 200 300 400 500 600 700 800 900 1000 1100Tim e s

Res

p. D

-20

0

20

40

60

100 200 300 400 500 600 700 800 900 1000 1100Tim e s

Res


100

200

300

400

500

Res

p. D

iff.

RU


-100

0

100 200 300 400 500 600 700 800 900 1000 1100

Tim e s

ID # 7 Biacore 3000

600

700RU Protein X binding to protein Y Sigma plot fitting using one site saturation

N = 2

100

200

300

400

500

600

Res

p. D

iff.

N = 2Kd1 = 234 nMKd2 = 84 uM

-100

0

100

0 200 400 600 800 1000 1200

sTim e Sigma plot fitting using two site saturation

Kd [M ]Std. ErrorsKd [M ] Errors

Kd1 234 nM 7.74E-08Kd2 84 uM 4.06E-05

ID # 19 Biacore 3000ID # 19 Biacore 3000

N 2N = 2Kd1 = 42.5 uMKd2 = 19.2 mM

SPR analysis of sample X binding to sample Y. Conc of X from 10nM to 160 uM

Double reciprocal plot of 1/Req vs 1/[X]. Curved double reciprocal plot suggests at least two binding Conc of X from 10nM to 160 uM phenomena of X to immobilized Y: one high affinity and one low affinity.

Stoichiometry of protein-X binding to protein-Y 2:1Kd value(s) for protein X Kd1 4 25 10 5 M Kd2Kd value(s) for protein-X binding to protein-Y

Kd1: 4.25 x 10-5 M , Kd2: 1.92x10-2 M

association rate (ka) & dissociation rate (kd) for

Ka1: 7.76 x 106 1/MSKd1: 0.33 M-1

The concentrations of X used to fit the model were 40nM to 160 uM

d ssoc at o ate ( d) oprotein-X binding to protein-Y

Ka2: 5.41X103 1/MSkd2:≤0.10 M-1

ID # 3 Biacore T100

N = 1N 1Kd1 = 190 nM

Experimental data for the binding of protein-X to protein-Y

X concentration used 1 nM to 100 uMStoichiometry of protein-X binding to protein-Y? 1:1Binding affinity Kd value(s) for protein-X binding

to protein-Y?190 nM

(Av FC 2 & 4)Measured association rate (ka) & dissociation ka: 5 6E+06 (1/Ms)Measured association rate (ka) & dissociation

rate (kd) for protein-X binding to protein-Y?ka: 5.6E+06 (1/Ms)

kd: 1.1 (1/s)

ID# 11- T100 N = 2Kd1 = 26.6 uM

Heterogenous Ligand d l

KD = 2.66x10-5 (M) Kd2 = ?

model:ka1 = 9.520x106(1/Ms)kd1 = 2.866 (1/s)KD1 = 3.011x10-7 (M)ka2 = 2.942x105 ( 1/RUs)a2kd2 = 0.2037 (1/s)KD2 = 0.2037x10-7 (M)

Interaction of protein X with immobilized protein Y evaluated by Biacore T-100. Sensorgrams obtained from the injections of increasing concentrations of Protein X over Protein Y (0, 1, 2.5, 5, 10, 25, 50, 100, 200 and 300 μM) in running buffer (PBS). The10, 25, 50, 100, 200 and 300 μM) in running buffer (PBS). The kinetic assays were carried out at 25°C and a flow rate of 10 μl/min. Global curve fits are shown, using a heterogeneous binding.

ID # 14 Biacore T100

N = 2Kd1 = 156 nMKd2 = 75 uM


1:1 Global Fitting of Protein X at 1.7uM-0.006uM binding to 100RU immobilized Protein Y

Steady State Fitting of Protein X at 1.7 uM-0.006 uM binding to 100 RU immobilized Protein Y

N = 1

Steady State Fitting of Protein X at 4.7 uM-0.006 uM binding to 100 RU immobilized Protein Y

N 1Kd1 = ~400 nM

1:1 Global Fitting of Protein X at 4.7uM-0.006uM binding to 100RU immobilized Protein Y

Steady State Fitting of Protein X at 1 7 uM 0 006 uMSteady State Fitting of Protein X at 1.7 uM-0.006 uM binding to 100 RU immobilized Protein Y

Highest Concentration of Protein Rmax Chi²

1:1 Global Fitting of Protein X at 18.8 uM-0.006uM binding to 100RU immobilized Protein Y

X (uM) KD (M) (RU) (RU²)1.7 4.04E-07 31 1.094.7 4.15E-07 31 0.97

17.8 5.94E-07 35 2.85

ID # 22 continued (SEC-MALS)

/mol

)43.0x10

44.0x10

45.0x10 LS

Mol

ar M

ass

(g/

42.0x10

time (min)20.0 21.0 22.0 23.0

9000.041.0x10

SEC-MALS: 50uM Protein X (green trace), 50uM Protein Y (red trace), 50uM Protein Y + 12uM Protein X (blue trace), 50uM Protein Y + 100uM Protein X (pink trace)

Approximately 10% of Protein X by mass appeared to be dimeric at 50uM, but the combination of Protein X and Protein Y does not appear to produce a shift consistent with oligomerized Protein X binding to Protein Y

Poor resolution of peaks and possible dilution effect occurring on column also limit interpretation of the MALS results and do not provide significant insight into the behaviors seen in the Biacoreexperimentsexperiments

Did not see much evidence for complex formation by SEC-MALS, likely due to the very rapid dissociation of complex and separation of components on the SEC column

ID # 26 Biacore T1001.25

Kd1 Bmax1

Rm

ax 0.75

1.00Kd1 Bmax1223 + 46 nM 0.455 + 0.032

Kd2 Bmax257.6 + 16.4 uM 0.684 + 0.049

RU

/ R

0.25

0.50

[Prot X] mol/L1.0e-9 1.0e-8 1.0e-7 1.0e-6 1.0e-5 1.0e-4 1.0e-3

0.00

Figure(s) showing experimental data

N = 2Kd1 = 223 nMKd2 = 58 uM

Figure(s) showing experimental data for the binding of protein-X to protein-Y. Conc of X is from 1nM to

Steady-state binding data, fitto 2-site model, SigmaPlot 11.0

530 nM & 1uM to 500 uM

ID # 12 Biacore T200Immobilized Protein Y

Protein X injected from 6,000 nM-0.384 nM

Reference Subtracted Blank Subtracted

SensogramsSensograms Sensograms

N = 1Kd1 = 300 nM

Steady State Affinity Model

Kd1 300 nM


40

50

60RU

Our original experiments on a T200 showed very rapid binding kinetics Subsequent experiments

N = 3Kd1 = 1.3uMKd2 = 100uM

0

10

20

30

Res

pons

e

kinetics. Subsequent experiments indicated this T200 was not performing correctly (a failure in the high quality injection start). The chip was moved to our second

Kd2 = 100uM

-20

-10

-20 0 20 40 60 80 100Time s

T200 and now the binding kinetics where much slower, as shown above.

There was significant non-specific binding to the reference flow cellsbinding to the reference flow cells. We assume non-specific binding as we used the same running buffer the samples were prepared in to minimize bulk shift from buffer

- Likely issue with sample mishandling (freeze/thaw)

mismatches.Stoichiometry of protein-X binding to protein-Y? 3Binding affinity Kd value(s) for protein-X binding to protein-Y?

Kd1: 1.3 uMKd2: 100 uM

Measured ka & kd for protein-X binding to protein-Y? For High affinity site

ka1: 1.08 x103; kd1: 0.11ka2: 6.74 x103; kd2: 8.7x10-3

ID # 24 Biacore T200Figure(s) showing experimental data for the binding of protein-X to protein-Y:

490

492

494SensorgramRU

Steady State Affinity

N = 1Kd1 = 278 nM

476

478

480

482

484

486

488

0 20 40 60 80 100 120 140 160

Res

pons

e

sTim e

0

0.001

0.02

0.039

0.078

0.156

0.313

0.625

1.25

2.5

6

8

10

12

14RU

Res

pons

e

1:1 Binding model fit (black line) to concentrations; Residuals generally between -0.2 and 0.2

0

2

4

0 5e-7 1e-6 1.5e-6 2e-6 2.5e-6Concentration M

Flow Cell 2-1 4-3 4-3 4-3Conc of X 0 02 – 2 5 0 02 – 2 5 0 02 – 2 5 0 001 – 300

Removing injection and post-injection spikes did

Conc. of X 0.02 2.5 µM

0.02 2.5 µM

0.02 2.5 µM

0.001 300 µM

Buffer HBS-EP+ HBS-EP+ Acetate, pH 8.0

HBS-EP+

Rmax(affinity)

19.54 RU 12.54 RU 15.47 RU 28.07 RU

Kd (affinity) 2.415×10-7 2.782×10-7 4.017×10-7 7.235×10-6Removing injection and post injection spikes did not significantly change kinetic parameters.

ka (rate) 2.322×106 3.037×106 1.360×106 1.374×106

kd (rate) 0.5694 0.6115 0.3625 0.7223Kd (from

rates)2.452×10-7 2.013×10-7 2.665×10-7 5.257×10-7

ID# 4 Reichert DC7500

N = 2Kd1 = 142 nMKd1 = 142 nMKd2 = 71 uM

Low concentration High concentration

Stoichiometry of protein-X binding to protein-Y? 2Binding affinity Kd value(s) for protein-X binding to

protein-Y?Kd1: 42 nM

Kd2: 71 mMMeasured ka & kd for protein-X binding to protein-Y? ka: 1 7 E+06 (1/Ms)Measured ka & kd for protein X binding to protein Y?

For High affinity siteka: 1.7 E+06 (1/Ms)

kd: 0.054 (1/s)

ID # 9 (Wyatt Calypso)

N = 2N = 2Kd1 = 10 nMKd2 = 14 uM

Best fit of light scattering data during hetero-association gradients for low concentration experiment 1 (left) and high concentration experiment 2 (right). Blue circles indicate measured light scattering data. The red open circles indicate the best fit to the light scattering data, consisting of the sum of the contributions from the free monomer X (blue diamonds), free monomer Y (red squares), XY complex (green triangles), and X2Y complex (purple circles).

M l di t ib ti f i i l ti d i h t i ti di t f lMolar distribution of species in solution during hetero-association gradients for low concentration experiment 1 (left) and high concentration experiment 2 (right). In each case, the mole fraction of XY reaches a maximum at a 1:1 overall molar ratio of X:Y, and the mole fraction of X2Y reaches a maximum at a 2:1 overall molar ratio of X:Y.

Participant Data Summary: D t C i t t ith MIRG D t

ID # N Affinity KD Model Comments

Data Consistent with MIRG Data

(M)#7-3000 2 KD1: 234 nM;

KD2: 84 uMSteady State

V4.01Expected result based on MIRG data

#14 T100 2 K 1: 156±41 nM Biaevaluation Expected result based on MIRG data#14-T100 2 KD1: 156±41 nMKD2: 75±16 µM

Biaevaluation Expected result based on MIRG data

#26-T200 2 KD1: 223±46 nM;KD2: 58±16 µM

T100 V2.0.3

Expected result based on MIRG data

#4-Reichart 2 KD1: 42 nMKD2: 71 µm

Scrubber-2Clamp 99 v3.4

Indicated non-ideal binding requiring regeneration, but close to expected result

based on MIRG data

SPR (i bili d t i Y) d t bt i d b MIRGSPR (immobilized protein Y) data obtained by MIRG:Kd1 = 350 +/- 140 nMKd2 = 79 +/- 45 uM

Participant Data Summary: p yData Consistent with MIRG Data

ID # N Affinity KD(M)

Model Comments

#9-Wyatt 2 KD1: 10 nM Calypso 2.1.2 Only “in solution” participant data. y DKD2: 14 µM

y yAgrees well with MIRG data for in solution measurement (ITC, AUC)

In solution data obtained by MIRG:

ITC:Kd1 = 8 – 34 nM

AUC:Kd1 = 5 - 40 nM

y

Kd2 = 3 – 17 uM Kd2 = 16 – 46 uM

Participant Data Summary: Fitti t i t bi di d l


Model Comments

Fitting to an incorrect binding model

(M)#6-3000 1 KD 302-320 nM Steady State

V3.2Poor fit to model (incorrect model)

#3-T100 1 KD 190 nM Biaevaluation+Ex Only fit low concentration data in “linear#3 T100 1 KD 190 nM Biaevaluation+Excel

Only fit low concentration data in linear range of Scatchard plot” – incorrect model

#11-T100 2 KD 26.6uM Biaevaluation Multiple values reported using different (incorrect) models. Need to clarify with

participant.p p#22-T100 ? ~ KD 400 nM T100: Global

Steady StateRecognized poor fit to 1:1 model, but

incorrectly attributed to aggregation rather than 2nd site

#12-T200 1 K 300 nM Steady State Dilution series too broad (1:5) and excluded#12-T200 1 KD 300 nM Steady State Dilution series too broad (1:5) and excluded higher [analyte] data – incorrect model

#24-T200 1 KD 278 nM T200, V1.0Steady State

Incorrect model

- Incorrectly assumed deviation from 1:1 binding due to non-specific binding, aggregation, or other non-ideality, when it should have been attributed to a second binding event

Participant Data Summary: Oth i P ibl d t tiOther issues: Possibly data reporting

error, sample mishandling.


Model Comments(M)

#19-3000 2 KD1: 4.25 x 10-

5 MKD2: 1.92 x 10-

2 M

BiaevaluationR

programming

Error in units? Need to clarify with participant.

2 M#17-T200 3 KD1: 1.3 µM;

KD2: 100 µMT200

HeterogenousPossible issues with sample

handling (freeze/thaw).#15 No report. Instrument / experimental issuesp p

Take Home SummaryTake Home Summary15 samples prepared, 14 participant requests, 13 reports p p p , p p q , p

received Five instruments used: Biacore 3000, T100, T200, Reichart,

Wyatt CalypsoWyatt Calypso7/13 (54%) of participants reported correct stoichiometry, i.e.

two binding site for protein-X to protein-Y / ( )5/13 (38%) of participants reported similar Kd values to those

determined by MIRGMost common issue = incorrect assumption on binding model, p g ,

and fitting only part of the data

AcknowledgementsAcknowledgements

All the MIRG membersABRFParticipants who took time to participate in

the study!

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