Best Practice Guide

April 14, 2022
Keywords or phrases:
Active Surface, Reference Surface, Surface Capacity,
Mass Transport Limitation, Kinetic Limitation

Best Practice Guide:
Minimize Artifacts
Stuart Knowling, PhD, Senior Scientist
Sartorius Corporation, 47661 Fremont Blvd, Fremont, CA 94538 USA
Correspondence
Email: octet@sartorius.com

Introduction
Surface plasmon resonance (SPR) is an established technique for characterizing the mechanism and rate constants
associated with molecular interactions. To determine meaningful kinetic rates and affinity constants and characterize
the mechanism associated with molecular interactions, high-quality SPR data must be generated.
It has long been known that many of the artifacts associated with binding data and the inability to fit data to a simple
model can be minimized or eliminated by the use of careful assay design, which allows the user to collect data under
optimum conditions and process the data with reference surfaces.
Although the application of SPR is often described as an art, adhering to some basic “good practices” along withhands-on experience is fundamental to producing high-quality SPR data that can be readily interpreted to deduce
meaningful kinetic rate and affinity constants.

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Whether using low-throughput SPR as a secondary
characterization tool to study binding interactions, or
high-throughput SPR as a preliminary screening tool tosurvey hundreds of binding interactions in a single assay,
there are some established best practices that aid in
improving the quality of biosensor data and minimizing
artifacts. When the goal is determining the kinetic rateand affinity constants of a biomolecular binding
interaction, a checklist of a few key points should be
followed to minimize assay artifacts. Here, we provide
a synopsis of those recommendations.

One of the most critical parameters in assay design canbe the inclusion of a suitable reference surface in addition
to the active surface where binding measurements will beperformed. It has been shown that a suitable reference
surface can help correct for multiple artifacts such as
refractive index changes, non-specific binding, assay drift
and injection noise.
Where possible, the reference surface should containthe same immobilization conditions as the active surface.
As shown in Figure 1A, the active and reference surface
both have the capture molecule immobilized on theirsurfaces, providing a more relevant reference surface fordata analysis than Figure 1B, where the reference surface
is left either unmodified or activated | deactivated.

B.

Active surface

Active surface

Reference surface

Reference surface

Note. Correct reference surfaces have a large impact on data quality. The
capture molecule (teal) should ideally be present on both the active andreference surface to ensure the best data quality possible.

Where a capture assay is not possible, a non-interacting
protein, such as a non-binding variant, should be
immobilized on the reference surface to the same
response level as the active surface. The Octet® SF3
allows the user to specify which channel acts as thereference surface and due to the design of the flow cell,a single channel can act as a parallel reference surface
for two active surfaces, which aids in generating highquality data through double reference subtraction.
2In addition to correct assay orientation (see best practiceguide Assay Orientation), low surface capacities minimize
artifacts associated with mass transport, steric hindrance,
crowding and aggregation, and subsequently simplify data
analysis. However, determining how low is “low enough” isan empirical exercise, although for kinetic assays, an Rmax
of ~50 RU will generally yield excellent results.

Figure 2A.

Reference Surface

Figure 1A.

Work at an Appropriate
Surface Capacity

Ligands

B.

LigandNote. (A) When using a 3D-hydrogel, the potential to crosswalk toanother ligand exists if the surface density is too high and will result in
an incorrect rate constant being determined during the dissociation
phase. (B) Use of a planar sensor chip reduces the surface density andcan minimize | remove the avidity effects observed with a 3D-hydrogel
sensor chip.

In addition to exploring a range of surface capacities, theuse of planar chips such as the Octet® SPR COOH1
Sensor Chip (Cat. No. 19-0053) instead of the commonlyused 3D-hydrogels such as the Octet® SPR CDH SensorChip (Cat. No. 19-0128) enables the more rigid tetheringof ligand molecules, minimizing ligand crosstalk that may
persist even in sparsely coated 3D-hydrogels (Figure 2).
While low surface capacities are generally advisable,binding responses should be sufficiently above theinstrument noise to be meaningful and not “lost within
the noise.”Mass transport limitation

Mass Transport and Flow Rates

Figure 4
Decreased flow rate
Increased surface capacity

For binding to occur in SPR assays the analyte must betransported laterally (diffusion) in the flow cell from the bulk
solution to the surface before any interaction with a ligandcan take place (Figure 3). This is referred to as mass tran­
sport and is a critical component of SPR as a technique.

Mass transport limitation

As discussed above, low surface capacities minimize
multiple artifacts associated with SPR but also play a keyrole in minimizing the risk of mass transport limitation.
Mass transport limitation (MTL) occurs when the analytecannot diffuse to the sensor chip surface rapidly enough,and the measurement becomes diffusion limited rather
than kinetics limited, where the analyte can diffuse to thesensor chip surface rapidly enough and there is an abun­dance of analyte for accurate kinetics determination.
It is important to not only consider the surface capacity
during assay development but also the analyte flow rate
so as to ensure that MTL is minimized.

Kinetic limitation

Note. Low flow rates and high immobilization levels can drive assays
towards mass transport limitation and lead to incorrect kinetics andaffinity.

As discussed above, use of a planar sensor chip reduces
the surface density and can minimize | remove the avidity
effects observed with a 3D-hydrogel sensor chip. This isdue to a reduced immobilization level (and subsequent
decrease in Rmax) but it’s clear that flow rate also plays
an important role in preventing MTL and allowing
accurate kinetic data (Figure 5) to be derived.
In general, for biologics assays, a flow rate of 50 µL/minshould be used (with 30 µL/min being the lowest) althoughhigher flow rates can be tested if required. Higher flow rates
should generally be used for small molecule assays.

Figure 3Analytes

Kinetic limitation

FlowFigure 5
Increased flow rate
Decreased surface capacity

AnalyteDiﬀusion

Mass transport limitation

Note. Measurement of binding interactions requires diffusion of theanalyte from the bulk flow to the sensor chip surface before binding
can be observed.

When designing an assay, it is important to consider thatat low flow rates and high surface capacity there is anincreased risk of experiencing MTL (Figure 4). Although
this assay setup can have a role in determining the active
concentrations of the analyte, it is not suitable for deter­mining accurate kinetics as with high immobilizationlevels transport can be limiting and the analyte concen­
tration at the surface varies with the flow rate. This means
the analyte concentration is different at the sensor chipsurface than the analyte concentration in the bulk of thesample being used in the assay. Since observed binding
rates are proportional to analyte surface concentration,
this reduction in surface con­centration will result in slower
than expected observed binding rates.

Kinetic limitation

Note. Decreasing the surface capacity and increasing the flow rate
moves the assay away from MTL and towards measuring accurate
kinetics.
Decreased flow rate
Increased surface capacity

Conclusion
In summary, in kinetic limitation, the transport is efficientandanalyte
concentration at the surface isKinetic
identical
Massthetransport
limitation
limitation
to the analyte concentration in the bulk of the sample
being used in the assay. As observed binding rates areproportional to analyte surface concentration, kinetic
limitation means that accurate binding rates will beobserved for the interaction being assessed.

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Status 03 | 2022

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