Determine Kinetics Earlier in the Drug Discovery Process
App Note / Case Study
Published: October 25, 2023
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Determination of accurate binding kinetics and affinity along with other critical quality attributes (CQAs) plays an ever-increasingly important role in identifying and isolating therapeutic molecules.
During high-throughput screening (HTS), drug candidates are typically prepared as a single concentration and injected across the target. A major restriction of this approach is that no quantitative information about kinetic parameters can be determined and that any hits need subsequent characterization using a more laborious multi-concentration assay.
This app note highlights how reliable kinetics for high-affinity interactions can be obtained from single injections.
Download this app note to learn more about:
- Generating accurate binding kinetics from a single concentration gradient
- Reducing sample preparation with lower analyte concentrations
- A cost-effective method for the early drug discovery process
Application Note
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 predicated
on the identification of therapeutic targets. The drug development process is generally a long and costly process and only a very
small 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 important
role 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 lead
candidates to promote.
September 18, 2023
Keywords or phrases:
Octet®, OneStep®, SF3, Kinetics Determination
2
Modern label-free analytical techniques allow kinetics interactions to be monitored with high resolution in real-time, which when combined with high-throughput capabilities, can significantly reduce the time to the discovery, streamlining the selection of optimal drug candidates with the best chances for success downstream.
Typically, during high-throughput screens (HTS) drug candidates are prepared as a single concentration and injected across the target and as such only the binding levels are evaluated. As no reliable kinetic parameters can be determined from such a limited data set the HTS is reduced to ‘yes/no’ binding depending on whether the observed response falls within a pre-set value range and doesn’t indicate additional non-specific interactions with the 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 more laborious multi-concentration assay, which can often lead to 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 time frame 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 that provides kinetics and affinity across a range of concentration values in a fraction of the time and with substantial savings in sample and buffer usage compared to standard multi-cycle kinetics (MCK).
Here, we demonstrate that reliable kinetics and affinity for high-affinity interactions can be obtained from significantly less data than is commonly used and the number of measurements necessary for accurate kinetics and affinity determination can be reduced to a single measurement by using OneStep® injections, increasing sample throughput and saving sample material.
This removes the barrier for SPR as a tool for large screens and allows rapid progression for additional assay development of potential therapeutics.
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. Unless indicated, 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 purchased from Midwinter Solutions and anti-VEGF (bevacizumab biosimilar) was purchased from Absolute Antibody. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (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 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 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 M ethanolamine HCl pH 8.5 across flow cells 1, 2 and 3 using a flow rate of 10 μl/min for 7 minutes. Approximately 400 RU of 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 manual mode function of the Octet® SF3 bHER2 was injected at 10 μl/min across flow cell 1 until a capture level of ~150 RU was reached.
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-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 used for regeneration injections. The sample rack was sealed using
3
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 and
conditioned 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 of
3600 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 to
100% here and the same dissociation parameters used as
for multi-cycle kinetics. A buffer blank injection was
performed for each analyte concentration in order to
generate accurate double referenced data.
The trastuzumab-bHER2 complex was regenerated with a
single 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) and
constraining the lower concentrations (420 sec
dissociation) to the same value. All data was locally fitted to
a simple 1:1 interaction model.
VEGF 165 and VEGF 121a
Recombinant VEGF 165 and VEGF 121a were immobilized
on 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) across
flow cell 3 until a response of 35 RU was achieved for both
ligands. The surface was then deactivated by injecting 1 M
ethanolamine HCl pH 8.5 across flow cells 1, 2 and 3 using a
flow rate of 10 μl/min for 7 minutes.
Kinetics and affinity of the interaction between a
bevacizumab 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
used for regeneration 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 and
conditioned 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 of
3600 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 to
100% here and the same dissociation parameters used as
for multi-cycle kinetics. A buffer blank injection was
performed for each analyte concentration in order to
generate 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) and
constraining the lower concentrations (420 sec
dissociation) to the same value. All data was globally fitted
to a simple 1:1 interaction model.
4
Results and Discussion
The objective was to demonstrate that the kinetic and
affinity 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 in
determining the initial analyte concentration choice is
considered.
Kinetic analysis trastuzumab binding HER2
High affinity kinetic interactions are often poorly defined
due to the lack of curvature in the association phase and
may 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 are
shown in Table 1 and the associated sensorgram shown in
figure 1.
Table 1
ka (M–1s–1) kd (s–1) KD (M)
Trastuzumab 1.30*106 1.00*10-5 7.38*10-12
Figure 1
A single injection of each analyte concentration was then
injected 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 the
rationale of performing multiple injections here was to
determine the tolerance levels between standard multicycle
kinetics and OneStep® injections. Kinetics parameters
for local fits of each concentration are shown in Table 2 with
corresponding sensorgrams in figure 2a – d (note: the full
3600 sec dissociation phase is shown for 25 nM).
Table 2
Concentration
(nM)
ka
(M–1s–1)
kd
(s–1)
KD
(M)
Trastuzumab 25 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
Figure 2a. 25 nM
Figure 2b. 8.33 nM
Figure 2c. 2.78 nM
80
70
60
50
40
30
20
10
0
Time (sec)
Response (RU)
0 100 200 300 400 500
100
80
60
40
20
0
Time (sec)
Response (RU)
0 500 1000 1500 2000 2500 3000 3500
Time (sec)
70
60
50
40
30
20
10
0
Response (RU)
0 100 200 300 400 500
Time (s)
35
30
25
20
15
10
5
0
Response (RU)
0 100 200 300 400 500
Time (sec)
5
Figure 2d. 0.926 nM
It is important to define what an acceptable level of
precision is for defining kinetic parameters prior to
comparing data.
Four landmark studies have reported benchmarks for
variability in the measurements of acceptable association
and dissociation rate constants based on interactions
characterized by multiple independent laboratories
(Cannon 2004, Katsamba 2006, Papalia 2006, Navratilova
2007). The variability between 59 independent
measurements was approximately 20% for both the
association and dissociation rate constants. Therefore,
kinetic constants that deviate by less than 20% are
considered acceptable in this assessment of OneStep® vs
multi-cycle kinetics.
As shown in Table 3, all OneStep® injections of trastuzumab
show acceptable association kinetic values compared to
the association kinetic rate constant determined by multicycle
kinetics (Table 1). Therefore, even with high affinity
interactions a single OneStep® injection is sufficient to
determine accurate association kinetics compared to multicycle
kinetics.
Table 3
Concentration
(nM)
ka
(M–1s–1)
Acceptable
association (ka)
range (M–1s–1)
Trastuzumab 25 1.06*106
1.04*106 - 1.56*106
8.33 1.18*106
2.78 1.45*106
0.926 1.16*106
Kinetic analysis bevacizumab binding VEGF 165 and
VEGF 121a
Unlocking the potential of high throughput screening
involves generating accurate data quickly that allows you to
progress 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 injection
that accurately resolves association and dissociation
kinetics is highly desirable in terms of saving precious
sample, time and importantly, how many samples can be
assessed in a single run.
VEGF 165 and VEGF 121a were immobilized as described
in the methods section and its binding to a bevacizumab
biosimilar assessed using standard multi-cycle kinetics and
a 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 associated
sensorgram shown in figures 3a (VEGF 165) and
b (VEGF 121a).
Table 4
ka (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
Figure 3a
16
14
12
10
8
6
4
2
0
Response (RU)
Time (sec)
0 100 200 300 400 500
40
35
30
25
20
15
10
5
0
Response (RU)
Time (sec)
0 100 200 300 400 500 600
6
Figure 3b
A single injection of the top concentration assessed in
multi-cycle kinetics (100 nM) was then injected across the
same 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 5
ka (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
Figure 4b
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 of
accuracy for the interaction compared to multi-cycle
kinetics.
Table 6
ka (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
Previous attempts at determining the minimum data set
required for multi-cycle kinetics have shown that in a
confidently measurable region, significant curvature is
required (Onell and Andersson 2005) to determine
accurate kinetics, and even then, a screening process is
required 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 gradient
formation, 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 be
used 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 the
lack of necessity for serial dilutions, OneStep® injections
offer significant time savings. As a fair comparison, multicycle
kinetics assays here were performed using short and
long dissociation times which collect dissociation
information for the highest analyte concentration which is
then combined with short dissociation times for lower
concentrations. This method works because the reduction
in 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 is
concentration independent. This shortens the time
required 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 a
higher number of samples.
50
40
30
20
10
0
Response (RU)
Time (sec)
0 100 200 300 400 500 600
50
40
30
20
10
0
Response (RU)
Time (sec)
0 100 200 300 400 500 600 700
50
40
30
20
10
0
Response (RU)
Time (sec)
0 100 200 300 400 500 600 700 800
7
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 from
a single top concentration injection without the need to
determine an optimum concentration series of analyte, as
would be required for multi-cycle kinetics.
OneStep® injections offer additional benefits when
assessing 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 critical
to achieving stable baselines regardless of whether the
ligand is directly immobilized to the sensor chip surface or a
capture molecule is being used.
OneStep® injections remove the guess work from high
throughput 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 then
potentially extensive assay development. Full kinetic
information early on in the drug development process
allows rapid progress of potential therapeutics through the
development 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 and
subsequent calculation of kinetic constants for those
molecules deemed hits involves an unnecessarily large
amount of sample and lab time. OneStep® injections are
shown to generate accurate association kinetics for even
high affinity interactions and offer a rational and costeffective
method to determine kinetics much earlier in the
drug discovery process.
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 in
solution, which affects the accuracy of ka, and subsequently
the accuracy of KD. OneStep® injections reduce the risk of
this error due to the necessity of preparing only a single
analyte concentration with no requirement to prepare a
concentration series, meaning fewer errors in sample
preparation but also in that the decreased time to generate
accurate kinetics means that samples are less prone to
experience evaporation or any side-effects from sitting in a
sample plate. As shown here, lower analyte concentrations
for OneStep® are still capable of generating accurate
association kinetics and as such the advantage of these
lower 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 normally
required; 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 within
easy 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):208-
221
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
Specifications subject to change without notice.
Copyright Sartorius Lab Instruments GmbH & Co. KG.
Publication No.: Octet-SF3-High-Affinity-Interactions-Application-Note-en-L-RevB-4077
Status: 09 | 2023
Germany
Sartorius Lab Instruments GmbH & Co. KG
Otto-Brenner-Strasse 20
37079 Goettingen
Phone +49 551 308 0
USA
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
Phone +1 631 254 4249
Toll-free +1 800 635 2906
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