Octet SF3 SPR®

Powered and Prepared

Simplifying ProgressPowered and Prepared
for Whatever Challenge You Take On
To keep pace with the current global demand for antibody
fragment characterization, vaccine research and wholecell biologics, continuous technological advances are
crucial in helping turn initial research hypotheses into
actionable results. This is especially true within the field oflabel-free protein analysis.
With exceptional sensitivity for both small and large
molecules, low baseline noise and drift, large injection

2volumes and the novel OneStep® and NeXtStep™ GradientInjection Technologies, the new Octet® SF3 allows users
to generate high-quality kinetics and affinity data in a
fraction of the time compared to standard multi-cyclekinetics. Combined with software designed by users forusers, the Octet® SF3 offers a robust, high-throughput, lowmaintenance SPR solution for the rapid characterization ofa wide variety of biomolecular interactions.

Robust and LowMaintenance SPR
State-of-the-art fluidics
and optimized electronic
designs significantly reduce
the potential for instrument
failure and blockages,
minimizing instrument
downtime and resulting in
the generation of reliable,
high-quality data.

High-Throughput SPR
Generate complete binding
kinetics and affinity for up to768 samples in a single
unattended assay. The
combination of OneStep®
Injection Technology, morethan 72 hours unattended
run time and a uniquesample layout allows forhigh-throughput acquisition
and analysis of hundreds ofsamples in a single run.

OneStep® Injection
Technology
Streamline assay
development andperformance by eliminating
the need to prepare
multiple dilution series byusing OneStep® Injection
Technology. Simply preparea single analyte solution tocreate a comprehensiveconcentration gradient for
kinetics and affinity analysis.

Competition Assays
Determine an analyte’s fullkinetics and affinity in thepresence of multiplecompetitors from a single
analyte concentration using
NeXtStep™ Injection
Technology.

3The Octet® SF3: A Unique Blendof Innovation and Design
A unique combination of technological innovation, complemented by a robust engineering design, helps to makethe Octet® SF3 the system of choice for both frequent andinfrequent SPR users, offering low system maintenance,high sample throughput and a highly accurate characterization of both small and large molecules.

-Key advantages include:
Determine accurate kinetics and affinity using the unique
OneStep® Gradient Injection which eliminates the need--

for multiple analyte concentrations, reduces surface
regeneration injections and significantly reduces assay
development time.
High sensitivity sample measurement for both small andlarge molecule assays.
High precision over a broad range of kinetic rate constants
allows the measurement of even the highest affinity
interactions with confidence.
NeXtStep™ Gradient Injections for highly accuratecompetition assays.

5361424

1

Increased Buffer Volume
The buffer tray can hold up to three one-liter bottles, comprisedof one water and two buffer lines. Both buffer lines function
independently, allowing two running buffers to be used during anassay.

2

Optimized Fluidics
All aspects of the Octet® SF3 fluidic system have been designed
and engineered to minimize any potential sources of blockage,
resulting in a strong, robust, low maintenance system. Systemdesorb, clean, and decontaminate protocols ensure maximum
system up-time.

3Dedicated Water LineThe Octet® SF3 contains a dedicated water line to minimize therisk of buffer precipitation and blockage formation.

4Thermodynamic and Physiological Measurements
The Octet® SF3 allows you to study interactions across a wide
range of temperatures to rapidly assess binding kinetics andaffinity of therapeutics at physiological temperatures. An inline buffer degasser prevents air bubble formation and whencombined with a large syringe volume (500 µl), allows theaccurate calculation of dissociation rate constants.

5Sample Recovery
Recovering precious samples for further analysis can rapidlydecrease workflow times. The Octet® SF3 allows you to recoverbound analyte using a predefined Sample Recovery Injection,
which means you can assess the same analyte using alternative
analytical techniques.

6High-Throughput Sample Acquisition
Combining OneStep® and high-throughput modes allows 768
unique samples to be analyzed in less than 24 hours. With morethan 72 hours of uninterrupted run time, this makes the Octet®
SF3 suitable for even the most high-throughput library screens.

5

OneStep® Gradient Injections
Based on the Taylor dispersion theory, OneStep® GradientInjections diffuse a single concentration of analyte into amoving stream of buffer to create an analyte concentration
gradient of at least 3 orders of magnitude, allowing an
accurate measurement of molecule kinetics and affinity froma single analyte concentration.
While standard Multi-Cycle Kinetics (MCK) can also be
performed on the Octet® SF3, OneStep® Injections eliminate
the need to prepare a dilution series of each analyte.

A.

tstartDiffusion

Flow--OneStep® Gradient Injection advantages include:
Accuracy: negligible rate constant differences with multicycle kinetics.
Speed: significantly less time required for sensor chip
regeneration, sample preparation, acquisition, andanalysis to obtain kinetics and affinity.
Throughput: unlock the full potential of each sample plate
as only a single analyte concentration is required to assesseach interaction.

1B.

t2

C1/C0

t1

t3
0tstoptstart

t1

t2

t3

τ

tstop

Figure 1Note. (A) OneStep® gradient formation in the injection line, with (B) the corresponding analyte concentration measured within the flow cell. Blue color
indicates the running buffer and pink color indicates the analyte. The gradient formation and its relationship to analyte concentration at the flow cell is
illustrated using five simulated snapshots (t start – t stop) of the injection line at different times, and shows that a single injection can be used to assessa full analyte concentration series.

6A.

60

Ch 3Figure 20Note. Trastuzamab Fab antibody (analyte)
against HER2 (ligand). Results weregenerated using both (A) multi-cycle kinetics(short-long, full dissociation not shown), and(B-D) OneStep® Injections on the Octet® SF3.
Serial multi-cycle kinetic dilutions for theTrastuzamab Fab antibody were 25 nM, 8.3nM, 2.78 nM, 0.926 nM and 0.309 nM.

Response(RU)50403020100B.

100

100200300
Time (seconds)

400

500

Following the preparation, acquisition andglobal analysis of the 5 different Trastuzamab
dilutions using standard multi-cycle kinetics,
the global affinity constant (KD) wascalculated at 7.38 pM.

Ch 1

Response(RU)806040200C.
Response(RU)

600100200300
Time (seconds)

400500Ch 350403020100Response(RU)

D.

1816141210864200100

200
300
Time (seconds)

400

500

Using OneStep® Gradient Injections, nosignificant differences in KD values were foundat any of the concentrations prepared for themulti-cycle kinetic assays. For example, KDvalues of 9.85 pM (figure 2B), 8.49 pM (figure2C) and 8.60 pM (figure 2D) could beachieved using Trastuzamab concentrationsof 25 nM, 8.3 nM and 0.926 nM, respectively.
Conclusion
The use of a single analyte concentration
using OneStep® Gradient Injections resulted
in similar results compared to those generatedusing a serial dilution multi-cycle kinetic assay.
Significant time, cost and labor savings,
however, can be achieved for highthroughput hit identification screens, as wellas in everyday instrument use.

Ch 10100

200300Time (seconds)

4005007

NeXtStep™ Gradient Injections
Competition assays are very useful in drug discovery,
yielding the ability to find active site binders by competingfragment hits with a control molecule. NeXtStep™ Injection
Technology has the unique ability to determine thebehavior of an analyte in the presence of a competitor
molecule from a single injection. Since the competitor does
not need to be present in the running buffer, NeXtStep™
allows you to assess multiple analytes and competitors ina single assay. Full kinetic profiles, affinity and site-specificcompetition can be clearly identified as a modulation ofbinding in the presence of the competitor molecule.

NeXtStep™ Gradient Injections comprise of solutions A(analyte and competitor molecule) and B (competitormolecule only), with the competitor molecule
concentration kept constant throughout. Both solutions
are sequentially diffused into the sample holding line,
where upon reaching the flow cell, the order of thesolutions is inversed. The first part of the injection
consists almost entirely of solution B (Fig. 3). As theNeXtStep™ Injection progresses and the concentrationof the competitor molecule remains constant, the analyte
concentration increases in a gradient fashion (Fig. 4).

1.0

1.0

Solution A

0.4

Solution B

Analyte Fraction

Analyte Fraction

0.60.20Competitor

0.8

0.8

0.8
0.6

Analyte0.4
0.204060Time (seconds)

80

0050

150
Time (seconds)

250

Figure 3

Figure 4Note. Time course charting the fraction of each solution as it passes
through the flow cell. At the beginning of the injection, the solution
almost entirely consists of solution B (the competitor molecule),
however as the injection progresses, it is gradually replaced by solution
A (the analyte + competitor molecule), until the final stages of theinjection, which consists primarily of solution A.

Note. The concentration of the competitor molecule remains constantthroughout the assay. However, the concentration of the analyte slowly
increases as solution A becomes more prominent during the course ofthe injection.

84030Benzenesulfonamide

201000200
100
Time (seconds)

C.

50
Furosemide

40

Response (RU)

B.

50

Response (RU)

Response (RU)

A.

30

201003000100
200Time (seconds)

300

Benzenesulfonamideand Furosemide

50
Furosemide

4030

Benzenesulfonamide

20
1000100
200
Time (seconds)

300

Figure 5Note. NeXtStep™ Gradient Injections wereused to identify any competitive effects in thebinding of Furosemide and Benzenesulfonamide to biotinylated Carbonic Anhydrase II.
An Octet® SPR SADH Sensor Chip was prepared with biotinylated Carbonic Anhydrase II
as the ligand. Successive NeXtStep™ Gradient
Injections of (A) Benzenesulfonamide (blue),and (B) Furosemide (green) across the same
sensor chip surface with HBS-EP+ in place of therespective competitor molecule were performed.
(C) A competitive NeXtStep™ Gradient
Injection using single concentrations ofBenzenesulfonamide and Furosemide (as thecompetitor molecule) was then performed(brown line) and compared to the individual
uninhibited binding profiles of Furosemide(green) and Benzenesulfonamide (blue).
Comparison of the Furosemide (green),
Benzenesulfonamide (blue), Furosemide andBenzenesulfonamide (brown) sensorgrams
indicate the absence of any additive response,with the higher affinity interaction of
Furosemide fully inhibiting the binding profileof the weaker affinity Benzenesulfonamide. It istherefore likely that the two molecules share acommon binding pocket, since an additive
response would have been expected had eachmolecule been able to bind to the CarbonicAnhydrase II ligand individually.

Figure 6
Ch 1

Note. NeXtStep™ Gradient Injection assays can be run as a directcompetition assay between an analyte and a competitive molecule toassess any impact on binding.

Furosemide
Furosemideand HBS-EP+Response (RU)

15

As shown in Figure 6, Furosemide was injected with HBS-EP+ (green) to
determine the kinetic response in the absence of a competitive
molecule. A NeXtStep™ Gradient Injection was then performed with
Methanesulfonamide as the competitor molecule, with a clear shift inFurosemide binding (blue).

1050Methanesulfonamide

050100150200Time (seconds)

250300350

A key feature of NeXtStep™ Gradient Technology is that multiple competitive molecules may be injected directly from the assay plate to identify any
potential interaction with your target analyte, allowing a large number ofcompetition assays to be rapidly performed within a single, unattended run.

9Software SpecificallyDesigned for
Low Instrument
Maintenance andEase of Use
The high-quality data recorded on the Octet® SF3 system
is easily analyzed using Octet® SPR Analysis software.

Figure 7Note. Octet® SPR Discovery Software: Instrument tubing, probing, flow
cell and sensor surface clean menu to maximize instrument run-time
despite requiring low user maintenance.

Assay Set-Up
With its intuitive drag and drop layout, assay design has
never been easier. The Octet® SPR Discovery software
allows you to rapidly define sample parameters, createcommon or independent sample flow rates, add
injection volumes and report points. Generating data
has never been easier.
The method setup page allows the user to selectfrom numerous pre-written methods that help guidesetup and provides the flexibility to write, modify, andview bespoke methods.
Rapid Data Analysis
Data analysis of SPR-based fragment screens can becumbersome and require days of an analyst’s time for asingle plate screen. Octet® SPR Analysis software uses
a unified approach for the selection of hits from nonhits and allows normalizing of screening data acrossdifferent days and instruments, so an entire screen
campaign can be compared rapidly. This integration
results in a major reduction in post-processing time —from hour or days, to seconds or minutes.

Figure 8Note. Octet® SPR Discovery Software: Pre-defined method protocols
for all potential assays to aid in the easy generation of high quality data.

The software provides models incorporating kinetics,
affinity, mass transport corrections, and multi-sitebinding as required to fit the interaction. Primary
screening data are ready for KD analysis without having
to perform a laborious, time consuming, and potentiallyerror-prone secondary screening.
Maintenance
Maintenance of the Octet® SF3 requires minimal userinteraction, with less time spent on maintaining yoursystem and more time focussed on generating data.

Figure 9Note. Octet® SPR Discovery Software: Intuitive layout of the assay method
set-up page includes variables such as analyte identity, concentration,
injection type and common association and dissociation times.

10Octet® SF3 Ordering
Octet® SF3Product Number

Octet SF3 SPR System

Octet-SF3Octet SF3 SPR System Installation

84IAFF3®

®

Octet® SF3 Specifications
Baseline Properties
Refractive Index Range

1.33 — 1.40

Baseline Noise

Typically < 0.025 RU (RMS)

Baseline Drift

Typically < 0.3 RU/min

Molecular Weight Cut-Off

No lower limit for organic molecules

Variable Data Rate

1, 2, 5, 10, 20 and 40 Hz

Affinity RangefM to mM

Working Ranges
Association rate constant (ka)

102 — 109 M -1 s-1

Dissociation rate constant (kd)

10 -6 — 2.5 s-1

Global affinity constant (KD)

10 -3 — 10 -12 M

Concentration

Sample concentration >1 pM

Sample Acquisition
Unattended Run Time>72 hours with no built in run-time limit

Sample Capacity

Any 2 sample racks plus 2 reagent racks

Sample Rack Options

96 vial, deep well and PCR formats, 384-well microplates, custom high volume

System and Sample Temperature Control

4 to 40°C (max 15° below ambient)

Buffer Line Selection

Automatic switching between 3 independent lines

Inline Buffer Degasser

Yes

Flow Cell and Injections
Number of Flow Channels

3

Flow Path1, 1–2, 1–2–3, 3, 3–2, 3–2–1

Flow Channel Volume<90 nL

Channel-Channel Dead Volume<20 nL

Injection Volume

2-700 µL

Flow Rate

0.1–200 μL/min

Injection Rise and Fall Time< 0.75 second @ 25 μL/min

Gradient Injections

OneStep® (including in High-Throughput Mode), NeXtStep™

Simultaneous Injections

Yes

SoftwareOctet® SPR Analysis Hit Selection

Yes

Dimensions
Width x Height x Depth

24" x 24" x 20" (61 cm x 61 cm x 51 cm)

Weight66 kg11

North America
Sartorius Corporation
565 Johnson Avenue
Bohemia, NY 11716
USA
Phone +1 888 OCTET 75
or +1 650 322 1360
Email: orders.US07@sartorius.com

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Otto-Brenner-Strasse 2037079 Goettingen
Germany
Phone +49 551 308 0
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Asia Pacific
Sartorius Japan K.K.
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Shinagawa-Ku
Tokyo 140-0001
JapanPhone +81 3 6478 5202
Email: orders.US07@sartorius.com

Find out more: www.sartorius.com/octet-support

For questions, email: octet@sartorius.com

Specifications subject to change without notice. © 2022. All rights reserved. All names of Sartorius products are registered trademarks and the property of Sartorius AG and/or one of its affiliated companies.
Octet-SF3-Instrument-Brochure-en-L-Sartorius Status: 06 | 2022