Application Note

September 18, 2023
Keywords or phrases:
Octet®, OneStep®, SF3, Kinetics Determination

Kinetics Determination of High Affinity
Molecular Interactions Using OneStep®
Injections

Introduction
Drug development using protein-based therapeutics has become particularly important in medical research and is predicatedon the identification of therapeutic targets. The drug development process is generally a long and costly process and only a verysmall percentage of drug candidates under development make it through to approval by the regulatory bodies.
Determination of accurate kinetics and affinity along with other critical quality attributes play an ever-increasing importantrole in identifying and isolating therapeutic molecules to drug-target. It is important therefore, that any systems developed
for these purposes can match not only the high throughput needs of the user but also their sensitivity needs; allowing assays
to be performed earlier in the workflow with minimal amounts of precious samples. This faster time to results allows
assessment of accurate and precise data earlier in the workflow and as such quicker decisions can be made on which leadcandidates to promote.

Modern label-free analytical techniques allow kinetics
interactions to be monitored with high resolution in realtime, which when combined with high-throughput
capabilities, can significantly reduce the time to thediscovery, streamlining the selection of optimal drugcandidates with the best chances for success downstream.
Typically, during high-throughput screens (HTS) drugcandidates are prepared as a single concentration andinjected across the target and as such only the binding
levels are evaluated. As no reliable kinetic parameters canbe determined from such a limited data set the HTS is
reduced to ‘yes/no’ binding depending on whether theobserved response falls within a pre-set value range anddoesn’t indicate additional non-specific interactions withthe target or sensor chip surface. Therefore, a major
restriction of this method is that no quantitative information
about kinetic parameters can be determined and that any
hits need subsequent characterization using a morelaborious multi-concentration assay, which can often leadto a labor-intensive assay development period.
Due to these limitations, the use of surface plasmon
resonance (SPR) in HTS is often limited to hit validation as
screening multiple concentrations of the drug candidate is
not suitable for primary screening due to an extended timeframe for screening and preparing samples. Additionally,
there are other considerations, such as how many
regeneration cycles the drug target can tolerate and space
limitations as to how many samples can be accommodated
in the system.
Based on these restrictions there has always been a
tradeoff between throughput and precision but this can be
remedied using OneStep® injections which is a feature
unique to the Octet® SF3 SPR system. OneStep® injections
offer a unique single concentration injection profile thatprovides kinetics and affinity across a range ofconcentration values in a fraction of the time and with
substantial savings in sample and buffer usage compared tostandard multi-cycle kinetics (MCK).
Here, we demonstrate that reliable kinetics and affinity forhigh-affinity interactions can be obtained from significantly
less data than is commonly used and the number ofmeasurements necessary for accurate kinetics and affinity
determination can be reduced to a single measurement byusing OneStep® injections, increasing sample throughput
and saving sample material.
This removes the barrier for SPR as a tool for large screensand allows rapid progression for additional assay
development of potential therapeutics.

2

Methods
Instrument and Reagents
All assays were performed using an Octet® SF3 SPR system.
Hepes buffered saline with 0.05% Tween 20 (HBS-EP+), pH
7.4 was used as running buffer throughout. Unlessindicated, all assays were performed at 37 °C.
Recombinant biotinylated HER2, Vascular endothelial
growth factor (VEGF) 165 and 121a were purchased from
Sino Biological. Herceptin (Trastuzumab) was purchasedfrom Midwinter Solutions and anti-VEGF (bevacizumab
biosimilar) was purchased from Absolute Antibody.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) was purchased from Thermo Fisher
Scientific. All other reagents were purchased from Sigma
Aldrich and prepared in-house.

Kinetics and Affinity Determination
HER2
Recombinant streptavidin was immobilized on an Octet®
SPR CDL Sensor Chip using standard amine couplingchemistry. A 50:50 mixture of 0.4 M 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) and 0.1 M N-Hydroxysulfosuccinimide (NHS) was
injected across flow cells 1, 2 and 3 using a flow rate of 10µl/min for 7 minutes. Recombinant streptavidin (5 µg/mL in
sodium acetate pH 4.0) was then injected across flow cells
1, 2 and 3 using a flow rate of 10 µl/min for 7 minutes. The
surface was then deactivated by injecting 1 Methanolamine HCl pH 8.5 across flow cells 1, 2 and 3 using aflow rate of 10 µl/min for 7 minutes. Approximately 400 RUof recombinant streptavidin was immobilized on each flow
cell.
Next, biotinylated HER2 (bHER2) was prepared to a final
concentration of 1.25 µg/mL in HBS-EP+. Using the manualmode function of the Octet® SF3 bHER2 was injected at 10µl/min across flow cell 1 until a capture level of ~150 RU wasreached.
Kinetics and affinity of the interaction between
Trastuzumab and bHER2 was performed using standard
multi-cycle kinetics and OneStep® injections, which are
unique to the Octet® SF3.
Trastuzumab was prepared to a final top concentration of
25 nM in HBS-EP+ running buffer and a 6-foldconcentration series prepared using a 1:3 dilution into HBSEP+. Samples were placed into Octet® SPR 0.9 mL vials andplaced into a mixed format sample rack. 3% sucrose wasprepared using HBS-EP+ as the bulk reference standard for
OneStep® injections and 100 mM HCl was used forregeneration injections. The sample rack was sealed usingresealable septa and placed in the sample tray set to 15 °C.
The Octet® SF3 system was primed 3 times into HBS-EP+
running buffer and the sensor chip hydrated andconditioned using injections of HBS-EP+ and 100 mM HCl.
A short and long assay format was used (Katsamba 2006)
with a common association time of 180 sec at 50 µL/min
used for multi-cycle kinetics and a ‘long’ dissociation of3600 sec used for the highest concentration and a ‘short’dissociation of 420 sec used for all other concentrations.
Association parameters for OneStep® are fixed based upon
the volume of the injection loop used, which was set to100% here and the same dissociation parameters used as
for multi-cycle kinetics. A buffer blank injection was
performed for each analyte concentration in order togenerate accurate double referenced data.
The trastuzumab-bHER2 complex was regenerated with asingle injection of 100 mM HCl at 50 µL/min for 60 sec,
followed by a 180 sec stabilization period.
Data were analyzed using the standard short and long
method of determining the dissociation rate constant (kd)for the highest concentration (3600 sec dissociation) andconstraining the lower concentrations (420 sec
dissociation) to the same value. All data was locally fitted toa simple 1:1 interaction model.
VEGF 165 and VEGF 121a
Recombinant VEGF 165 and VEGF 121a were immobilizedon flow cells 1 and 3, respectively of an Octet® SPR CDL
Sensor Chip using standard amine coupling chemistry. A
50:50 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC) and 0.1 M
N-Hydroxysulfosuccinimide (NHS) was injected across flow
cells 1, 2 and 3 using a flow rate of 10 µl/min for 7 minutes.
Recombinant VEGF 165 (0.25 µg/mL in sodium acetate pH
5.0) was then injected across flow cell 1 and Recombinant
VEGF 121a (0.25 µg/mL in sodium acetate pH 5.0) acrossflow cell 3 until a response of 35 RU was achieved for both
ligands. The surface was then deactivated by injecting 1 Methanolamine HCl pH 8.5 across flow cells 1, 2 and 3 using aflow rate of 10 µl/min for 7 minutes.

used for regeneration injections. The sample rack wassealed using resealable septa and placed in the sample tray
set to 15 °C.
The Octet® SF3 system was primed 3 times into HBS-EP+
running buffer and the sensor chip hydrated andconditioned using injections of HBS-EP+ and 100 mM HCl.
A short and long assay format was used (Katsamba 2006)
with a common association time of 180 sec at 50 µL/min
used for multi-cycle kinetics and a ‘long’ dissociation of3600 sec used for the highest concentration and a ‘short’dissociation of 420 sec used for all other concentrations.
Association parameters for OneStep® are fixed based upon
the volume of the injection loop used, which was set to100% here and the same dissociation parameters used as
for multi-cycle kinetics. A buffer blank injection was
performed for each analyte concentration in order togenerate accurate double referenced data.
The bevacizumab VEGF complex was regenerated with two
injections of 100 mM HCl at 50 µL/min for 30 sec, followed
by a 180 sec stabilization period.
Data were analyzed using the standard short and long
method of determining the dissociation rate constant (kd)for the highest concentration (3600 sec dissociation) andconstraining the lower concentrations (420 sec
dissociation) to the same value. All data was globally fitted
to a simple 1:1 interaction model.

Kinetics and affinity of the interaction between abevacizumab biosimilar and VEGF 165 and VEGF 121a was
performed using standard multi-cycle kinetics and
OneStep® injections, which are unique to the Octet® SF3.
The bevacizumab biosimilar was prepared to a final top
concentration of 100 nM in HBS-EP+ running buffer and a
6-fold concentration series prepared using a 1:3 dilution
into HBS-EP+. Samples were placed into Octet® SPR
0.9 mL vials and placed into a mixed format sample rack. 3%
sucrose was prepared using HBS-EP+ as the bulk reference
standard for OneStep® injections and 100 mM HCl was
3Results and Discussion

Table 2Kinetic analysis trastuzumab binding HER2High affinity kinetic interactions are often poorly defineddue to the lack of curvature in the association phase andmay require in excess of an hour to approach equilibrium.
Therefore, the resolving power of a single analyte injection
must not be at the expense of resolution or throughput.
Initial analysis of the trastuzumab HER2 kinetics and affinity
determined by multi-cycle kinetics on the Octet® SF3 areshown in Table 1 and the associated sensorgram shown infigure 1.

Concentra- ka
tion (nM) (M–1s–1)
Trastuzumab 25

kd
(s–1)

KD
(M)

1.06*106 1.00*10-5 9.85*10-12

8.33

1.18*106 1.00*10-5 8.49*10-12

2.78

1.45*106 1.00*10-5 11.73*10-12

0.926

1.16*106 1.00*10-5 8.60*10-12

500

1000

1500

2000
Time (sec)

100

200

300
Time (sec)Figure 2a. 25 nM
100
80
Response (RU)

The objective was to demonstrate that the kinetic andaffinity constants determined using OneStep® injections
compared favorably to those derived from standard multicycle kinetic assays, which require multiple analyte
concentration injections. In addition, as the affinity of the
drug – therapeutic is often unknown, the flexibility indetermining the initial analyte concentration choice isconsidered.

60
40
20
0

Table 10

Trastuzumabka (M–1s–1) kd (s–1)

KD (M)

1.30*106

7.38*10-12

1.00*10-5

2500

3000

3500

Figure 2b. 8.33 nM
7060
Response (RU)

Response (RU)

80
70
60
50
40

50
40
30
20

30

10

20

0

10

0 		

0
0 		

100

200

300
Time (sec)

400

500

400

500

500

Figure 2c. 2.78 nM

Figure 13530
Response (RU)

A single injection of each analyte concentration was theninjected across the same surface using OneStep®
injections. 0.3 nM was rejected as the dissociation phase
was not well described by the observed kd at higher
concentrations in both multi-cycle kinetics and OneStep®
injection. It is important to note that multiple OneStep®
injections are not required in normal screening and therationale of performing multiple injections here was todetermine the tolerance levels between standard multicycle kinetics and OneStep® injections. Kinetics parameters
for local fits of each concentration are shown in Table 2 withcorresponding sensorgrams in figure 2a – d (note: the full
3600 sec dissociation phase is shown for 25 nM).
4

400

25
20
15
10
Time (s)

5
0
0 		

100

200

300
Time (sec)Figure 2d. 0.926 nM

14
12
10
8
6
4
2
0
0 		

100

200

300
Time (sec)

400

500

It is important to define what an acceptable level ofprecision is for defining kinetic parameters prior tocomparing data.
Four landmark studies have reported benchmarks forvariability in the measurements of acceptable associationand dissociation rate constants based on interactions
characterized by multiple independent laboratories(Cannon 2004, Katsamba 2006, Papalia 2006, Navratilova
2007). The variability between 59 independentmeasurements was approximately 20% for both theassociation and dissociation rate constants. Therefore,
kinetic constants that deviate by less than 20% areconsidered acceptable in this assessment of OneStep® vsmulti-cycle kinetics.
As shown in Table 3, all OneStep® injections of trastuzumab
show acceptable association kinetic values compared tothe association kinetic rate constant determined by multicycle kinetics (Table 1). Therefore, even with high affinity
interactions a single OneStep® injection is sufficient todetermine accurate association kinetics compared to multicycle kinetics.
Table 3

Concentra- ka
tion (nM)(M–1s–1)
Trastuzumab 25

Acceptableassociation (ka)range (M–1s–1)

1.18*102.78

1.45*1060.926

1.16*106

VEGF 165 and VEGF 121a were immobilized as describedin the methods section and its binding to a bevacizumab
biosimilar assessed using standard multi-cycle kinetics anda single top concentration OneStep® injection.
Initial analysis of the bevacizumab biosimilar VEGF 165 and
VEGF 121a kinetics and affinity determined by multi-cycle
kinetics are shown in Table 4 and the associatedsensorgram shown in figures 3a (VEGF 165) andb (VEGF 121a).
Table 4ka (M–1s–1)

kd (s–1)

KD (M)

VEGF 165

8.22*104

6.12*10-5

744.4*10-12

VEGF 121a

9.40*104

3.93*10-5

417.9*10-12

100

300
Time (sec)

500

40
35
30
25
20
15
10
5
0

1.06*106

8.33

Kinetic analysis bevacizumab binding VEGF 165 and
VEGF 121a
Unlocking the potential of high throughput screening
involves generating accurate data quickly that allows you toprogress potential candidates quickly through downstream
stages. As shown for trastuzumab binding to HER2,
OneStep® injections are able to produce accurate
association kinetics and a single concentration injectionthat accurately resolves association and dissociation
kinetics is highly desirable in terms of saving precious
sample, time and importantly, how many samples can beassessed in a single run.

Response (RU)

Response (RU)

16

0

6

1.04*106 - 1.56*106

200

400

600

Figure 3a5Applying the same accuracy measure for the association
kinetic parameters as discussed for trastuzumab binding to
HER2, the association kinetics determined from a single
OneStep® injection are within the acceptable range ofaccuracy for the interaction compared to multi-cycle
kinetics.

Figure 3b

Response (RU)

50
40
30
20

Table 610ka (M–1s–1)

Acceptable association (ka)range (M–1s–1)

VEGF 165

8.20*104

6.58*104 - 9.87*104

VEGF 121a

1.05*104

7.52*104 - 1.13*105

0
0

100

200

300
Time (sec)

400

500

600

A single injection of the top concentration assessed in
multi-cycle kinetics (100 nM) was then injected across thesame surfaces using OneStep® injections. Kinetics
parameters for local fits of each concentration are shown in
Table 5 and the corresponding sensorgrams in figure 4a(VEGF 165) and 4b (VEGF 121a).
Table 5ka (M–1s–1)

kd (s–1)

KD (M)

VEGF 165

8.20*104

6.12*10-5

746.2*10-12

VEGF 121a

1.05*104

3.93*10-5

371.14*10-12

Figure 4a

Response (RU)

50
40
30
20
10
0
0

100

0

100

200

300
400
Time (sec)

500

600

700

Figure 4b

Response (RU)

50
40
30
20
10
0

6

200

300

400
Time (sec)

500

600

700

800

Previous attempts at determining the minimum data set
required for multi-cycle kinetics have shown that in aconfidently measurable region, significant curvature isrequired (Onell and Andersson 2005) to determine
accurate kinetics, and even then, a screening process isrequired before hand to determine the necessary
concentrations. As shown here for trastuzumab binding to
HER2 and a bevacizumab biosimilar binding to VEGF 165
and VEGF 121a and also exemplified in (Quinn 2012),
OneStep® injections provide significant curvature during
the single injection thanks to the analyte gradientformation, which allows accurate association kinetics to be
determined for even high-affinity interactions. As shown
here, a single top/high concentration matching that used
for multi-cycle kinetics is sufficient to derive accurate
kinetics and affinity. Importantly, as shown for trastuzumab
binding to HER2, even if the affinity of the interaction is
unknown, a wide range of analyte concentrations can beused to determine association and dissociation kinetics,
these concentrations are typically linear in multi-cycle
kinetics and therefore, yield no usable kinetic information.
In addition to a significant sample saving of ~50% due to thelack of necessity for serial dilutions, OneStep® injections
offer significant time savings. As a fair comparison, multicycle kinetics assays here were performed using short andlong dissociation times which collect dissociation
information for the highest analyte concentration which isthen combined with short dissociation times for lower
concentrations. This method works because the reductionin the observed signal at the highest analyte concentration
is sufficient (>5%) to establish the dissociation rate constant(kd) for the entire assay as the dissociation phase isconcentration independent. This shortens the timerequired for a multi-cycle assay significantly. The total time
taken for the multi-cycle kinetics assay was approximately 4
hours and 30 min and 2 hours and 50 min for the OneStep®.
This represents a significant saving it time of over 90 mins
for a single analyte, which would only be compounded at ahigher number of samples.

As shown above, kinetics and affinity determined by
OneStep® injections compared favorably to those derived
from standard multi-cycle kinetic assays. OneStep®
injections allow flexibility in assay design as accurate
association and dissociation kinetics can be generated froma single top concentration injection without the need todetermine an optimum concentration series of analyte, as
would be required for multi-cycle kinetics.
OneStep injections offer additional benefits whenassessing binding interactions thanks to the necessity of
only a single injection. Surface regeneration is an integral
part in assessing high affinity interactions and unlike
standard multi-cycle kinetic assays, where several
concentrations require an equal number of regenerations,
OneStep® injections only require a single regeneration step,which over the course of a high throughput screen is criticalto achieving stable baselines regardless of whether theligand is directly immobilized to the sensor chip surface or acapture molecule is being used.
®

OneStep® injections remove the guess work from highthroughput screens and allows a full kinetics panel to be
determined in a fraction of the time compared to standard
techniques that require initial screening and thenpotentially extensive assay development. Full kinetic
information early on in the drug development process
allows rapid progress of potential therapeutics through thedevelopment pipeline and forms a solid ground for any
additional assay development.

Conclusions
In conclusion, this application note shows that current
common practices for high throughput screening using
SPR, which rely on a limited data set of ‘yes/no’ binding andsubsequent calculation of kinetic constants for thosemolecules deemed hits involves an unnecessarily large
amount of sample and lab time. OneStep® injections areshown to generate accurate association kinetics for evenhigh affinity interactions and offer a rational and costeffective method to determine kinetics much earlier in the
drug discovery process.

accurate kinetics means that samples are less prone toexperience evaporation or any side-effects from sitting in asample plate. As shown here, lower analyte concentrations
for OneStep® are still capable of generating accurate
association kinetics and as such the advantage of theselower concentrations is that adverse effects such as
aggregation or solubility issues are minimized.
Therefore, the barrier to the tradeoff between throughput
and precision has been removed by OneStep® injections
and reliable kinetic estimates can be obtained much earlier
in drug discovery with much less data than is normallyrequired; increasing sample throughput and saving sample
material, particularly for protein-based therapeutics.
Thanks to the flexible sample format of the Octet® SF3 up
to 768 unique analytes can be assessed in a single run.
Combined with the ability to measure dissociation rates for
up to 12 hours, full kinetics for even the highest affinity
interactions and high throughput screens are now withineasy reach for all SPR users.

References
1.

Cannon MJ et al. Anal. Biochem. 2004 1;330(1):98-113

2.

Karlsson R and Larsson A. Methods Mol Biol.
2004;248:389-415

3.

Katsamba P et al. Anal. Biochem. 2006 15;352(2):208221

4.

Navratilova I et al. Anal. Biochem. 2007 1;364(1):67-77

5.

Papalia GA et al. Anal. Biochem. 2006 1;359(1):94-105

6.

Onell A and Andersson K. J. Mol Recognit.
2005;18(4):307-17

7.

Quinn J. Anal Biochem. 2012 15

A neglected source of error in SPR assays is the nature of
the sample preparation (Karlsson and Larsson, 2004).
Pipetting errors and evaporation can lead to erroneous
estimates of the concentration of the binding partner insolution, which affects the accuracy of ka, and subsequently
the accuracy of KD. OneStep® injections reduce the risk ofthis error due to the necessity of preparing only a single
analyte concentration with no requirement to prepare aconcentration series, meaning fewer errors in sample
preparation but also in that the decreased time to generate
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Publication No.: Octet-SF3-High-Affinity-Interactions-Application-Note-en-L-RevB-4077
Status: 09 | 2023