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2018
st

How to Buy Biophysical

Characterization Technology EDITION

A guide to help you choose between

SPR, BLI, ITC and MST technologies
2018 Biophysical Characterization Technology

How to Buy Biophysical

Characterization Technology
This guide aims to compare the
four most common techniques
used to quantify and characterize
Contents
biophysical interactions — surface
plasmon resonance (SPR), bio-layer 1. Introduction to Biomolecular
interferometry (BLI) isothermal, Interaction Techniques
titration calorimetry (ITC), and 2. Top Tips from the Experts for
microscale thermophoresis (MST) Choosing Which Technology to Buy
— with a view to providing all the
3. A Comparison of the Top 4 Biophysical
information and insight you’ll need to
Characterization Techniques
make the best purchasing decisions
for your laboratory. 4. Comparison of Biomolecular
Interaction Analysis
5. Summary & Acknowledgements
6. Editor’s Picks

Octet
Pall Fortebio

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2018 Biophysical Characterization Technology

1. Introduction to Biomolecular
Interaction Techniques
Binding data is critical for publications as
it enables the in-depth characterization
of biological interactions. Binding data
can be determined by a range of different
techniques, providing information on kinetics,
binding affinity and/or thermodynamics.
Binding affinity — the strength of interaction
between two biomolecules — is the most
widely available piece of interaction data,
however it does not tell the full story. Kinetics
— the speed at which an interaction happens
— as well as specificity are also important
for generating a better understanding of the
protein or biological system being studied.
In particular, a broad range of binding data is
required for applications such as disease and
drug discovery.
Currently, the four most common
biophysical interaction techniques are
surface plasmon resonance (SPR), bio-
layer interferometry (BLI), isothermal
titration calorimetry (ITC) and microscale
thermophoresis (MST). Each technique has its
assets and drawbacks, which are discussed in
this guide, along with expert insight on some
of the available technologies.

Expert Tip: Is there ever an instance when a

scientist would use a combination of the four
techniques – SPR, ITC, MST and BLI?
“ITC, SPR, MST and BLI can be considered orthogonal assays: they all can give a KD
value, by different assays. For complex binding interactions, all 4 techniques may
be needed to provide a more complete understanding of the interaction, including
binding affinity, stoichiometry, binding kinetics, and binding thermodynamics.”
Verna Fresca, Malvern Panalytical

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2018 Biophysical Characterization Technology

2. Top Tips from the Experts for

Choosing Which Technology to Buy

“
We asked a range of experts to share their top
tips and considerations when choosing which
technology to buy:
does it meet your requirements?
v. H
 ow long does it take to become
comfortable with the technique?

Understand your research Evaluate vendors

needs Most of the research is is
Measuring binding affinity
can be useful for a variety
Do your research done online: you can watch
videos, download brochures,
of applications such as:
characterizing receptor
thoroughly, by and read independent
peer-peer reviews on
binding properties, measuring SelectScience. When it
interactions with antibodies, reading reviews, comes time to contact
investigating enzyme the manufacturer, don’t
inhibition, optimizing leads,
and many more.

Evaluate the challenges you

are facing
watching videos
and contacting
manufacturers.
“ forget to ask questions that
allow you to gauge their
reputation and credibility
in the industry, such as:
Can you trial the product
Explore the challenges posed on-site? How long has the
by the current techniques company been around?
in your lab; have they arisen How many customers does
since you have added in new projects? it have? Is it global? Am I served by a local
representative? What kind of support does the
Explore available solutions based on your company offer, and are customers satisfied
needs and challenges with the support they receive?
Some things to consider include:
i. Are the instrument and software user-friendly? Make an informed decision
ii. What reagents and consumables are needed? If you follow the steps above, then you are
iii. What upgrade pathways are available? ready to select the technology that best fits
iv. What is the technique throughput, and your needs.

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2018 Biophysical Characterization Technology
3. A Comparison of the Top 4 Biophysical
Characterization Techniques
3.1 Surface Plasmon Resonance
Surface plasmon resonance (SPR) can be
“
used as a real-time, label-free detection
tool whenever you want to detect, measure
or quantify the ability of a molecular entity
(be it protein, antibody,
small molecule, vaccine)
to bind to something else
(target, native ligand). More SPR is a reference
specifically this could be:
method for the
• When a researcher needs
a deeper understanding FDA, ICH and
of biological processes
and the dynamic of
molecular interactions
by characterizing the
interactions in terms of
kinetics, affinity, transition
state thermodynamics, and
EMA, and as such
can be used in
biotherapeutics.
yes/no binding data to prove
hypotheses, in order to
understand the mechanism of action and link
structure to function.
• Screening applications — to rapidly
profile large libraries of chemical matter
(either antibodies or low molecular weight
compounds), differentiate binders from non-
binders and critically prioritize binders by the
most desirably characteristics.
• To drive decision making either in medicinal
chemistry programs during hit-to-lead
development in drug discovery or part of
the affinity maturation
process during antibody
development.
• Comparability, quality
control and release testing
of antibodies and protein
drugs.
It is also important to
note that SPR is a reference
“ method for the U.S. Food
and Drug Administration
(FDA), International Council
for Harmonisation of
Technical Requirements
for Registration of
Pharmaceuticals for Human
Use (ICH) and European
Medicines Agency (EMA), and as such can be
used in biotherapeutics, drug development
and characterization1.
Technique Overview:
SPR occurs when a beam of polarized light
hits an electrically conducting surface at the
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2018 Biophysical Characterization Technology
© Pall ForteBio

Figure 1: SPR is the resonant oscillation of conduction electrons at the

interface between negative and positive permittivity material stimulated Figure 3: Video highlighting one-step SPR measurements with the ForteBio
by incident light Pioneer System

interface between two media, generating have a range of improvements that can make
electron charge density waves, called your experimentation easier. For example, the
plasmons, which thus reduce the intensity of Pioneer Platform uses a OneStep gradient
the reflected light. injection method. This method, which is
The sensor typically comprises a glass available exclusively on these systems,
substrate and thin gold coating; most enables reliable kinetics determination
commonly the protein of interest is in a single injection instead of running a
immobilized on a prepared gold surface, full dilution series. This greatly increases
and then during experimentation a sample throughput, reduces sample consumption,
containing a potential interacting partner in and makes set-up faster and simpler
solution is injected onto it. than with traditional SPR. For screening
During experimentation, light passes applications, OneStep injections eliminate the
through the substrate and is reflected off the need for secondary screen, since a full kinetic
gold coating (Figure 1). The angle of minimum profile can be obtained for every sample in
intensity of reflected light is detected, and a primary screen with a single injection per
this changes as molecules bind and dissociate sample. Only one assay is needed for hit
– forming an interaction profile which is identification, ranking, and characterization
recorded on the sensorgram (Figure 2). (KD). For full details on the OneStep method,
Some of the SPR systems available today watch this video (Figure 3).
© GE Healthcare

Figure 2: The sensorgram shows the absolute response (RU) over time, this example highlights an ideal sensorgram for SPR

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2018 Biophysical Characterization Technology

3.2 Bio-Layer Interferometry (BLI):

Bio-layer interferometry (BLI) is a label-

free technology for measuring biomolecular
interactions. As the analysis platform is plate
based the sample analysis is non-destructive.
Like SPR BLI is a reference method for the
© Puwadol Jaturawutthichai 123rf.com

FDA/ICH/EMA, and as such can be used in

Figure 4: A brain affected by ischemic stroke. Identification of novel
therapeutics could help reduce and improve recovery from damage
caused during ischemic events
Find out how scientists have been using the
technology in this article (Figure 4), where
Dr. Anders Bach explains how scientists at
the University of Copenhagen are searching
for a new class of stroke therapeutic using
high-throughput fragment analysis, using the
Pioneer FE system from ForteBio. Plus, for full
insights into the broad range of applications
for the Pioneer Platform, download this
publication spotlight.
The Biacore systems from GE Healthcare
also boast real-time analysis, enabling the
scientist to view the entire binding event
as it happens, removing the limitations
surrounding end-point/affinity reading. The
Biacore systems have also been widely cited
in a broad range of papers and applications,
from viruses to ions – allowing the research
drug development and characterization. BLI
is a fluidics-free system which along with
the fact that the BLI technology is minimally
influenced by molecules in solution, means
that a wide range of sample types including
lysates, cell culture supernatants, and phage
preparations can be used with minimal
sample preparation – without the concerns
of system clogging. In addition, ease of use,
low maintenance and high data precision
speeds time to results throughout the drug
development process.
BLI is an optical analytical technique that
analyzes the interference pattern of white
light reflected from two surfaces: a layer
of immobilized protein on the biosensor
tip, and an internal reference layer (Figure
5). Any change in the number of molecules
bound to the biosensor tip causes a shift
in the interference pattern that can be
measured in real time (Figures 5 and 6). The
biosensors for BLI are disposable, allowing for
© Pall ForteBio

scientist to assess the use of the tool for their

own work.
The ability to carry out analytics,
interpretation and subsequent data Figure 5: BLI is an optical analytical technique, which measures
the interference pattern of white light reflected on two surfaces
comparison are key considerations when
choosing the technology system. The new
software platform, Biacore Insight Evaluations
Software — a workflow and application-
specific evaluation software tailored for a
broad range of Biacore systems — not only
supports fast evaluation of large data sets,
© Pall ForteBio

but also offers an easier way to export and

share results: transfer large data sets or
editable figures. The presentation generator
supports a more streamlined process to share
Figure 6: By changing the number of molecules bound to the biosensor tip,
and compare results. the interference pattern can shift which can be measured in real time

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2018 Biophysical Characterization Technology
experimental flexibility and minimal upkeep –
the Dip and Read Sensors from Pall ForteBio
simplify quantitation and kinetics assays by
eliminating throughput limitations caused by
microfluidic flow, surface regeneration, and
sample purification.
The binding between a ligand immobilized
on the biosensor tip surface and an analyte
in solution produces an increase in optical
thickness at the biosensor tip, which results
in a wavelength shift, Δ (Figure 7), which is a
direct measure of the change in thickness of
the biological layer. Interactions are measured
in real time, providing the ability to monitor
binding specificity, rates of association and
dissociation, or concentration, with precision
and accuracy.
© Pall ForteBio
Figure 7: Wavelength shift (Δ ) is a measure of the change in thickness of
the biological layer and is measured in real time
Only molecules binding to or dissociating
from the biosensor can shift the interference
pattern and generate a response profile on
the BLI system. Unbound molecules, changes
in the refractive index of the surrounding
medium, or changes in flow rate do not
affect the interference pattern. This is a
unique characteristic of BLI and extends its
capability to perform in crude samples used
in applications for protein:protein binding,
quantitation, affinity, and kinetics.
The Octet® System has become the
technique of choice for many scientists,
Key Benefits of BLI
• Label-free detection
• Real-time results
• Simple and fast
• Fluidics-free
• Improves efficiency
• Crude sample compatibility
including Professor Harald Kolmar, Technical
University Darmstadt. He is using the Octet®
RED96 in his research on shark antibodies,
which is explored in detailed in this article.
Plus, in this white paper, discover how the
Octet RED96e system offers the versatility
needed to accelerate and de-risk binding
kinetics and affinity analysis in drug discovery
and development.
Choosing between BLI and SPR:
BLI and SPR are both real-time, label-
free techniques that use light to measure
biomolecular interactions, such as binding
kinetics and concentrations, and BLI has been
demonstrated to produce comparable data
to SPR. One important note is that BLI does
not rely on microfluidics, enabling much more
multi-parallel measurements than SPR – up to
96 samples can be measured simultaneously.
One example of a BLI technology is
ForteBio’s Octet platform, which has been
commended for its ease of use. Octet
systems are fluidics-free, allowing them
to be compatible with a diverse range of
sample types, and are more tolerant to
organic solvents, which are used with some
low affinity small molecules where solubility
might be a problem for SPR. Octet biosensors
can be regenerated and reused, leading to
reduced assay costs, and the methods are
easily transferable to manufacturing and QC
environments due to the instrument’s ease of
use and an optional GxP package.
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2018 Biophysical Characterization Technology

3.3 Isothermal Titration Calorimetry (ITC) complete understanding of structural biology.

This can lead to further understanding of
Isothermal titration calorimetry (ITC) can disease pathways. ITC can also be used to
be used to study the binding interactions study enzyme kinetics and enzyme inhibition.
between any two molecules. ITC measures In this white paper, find out how the MicroCal
the heat changes associated with binding system from Malvern Panalytical was used to
events, determining the binding affinity of any characterize epigenetic protein interaction in
biomolecular interaction. proteins, nucleic acids and lipids.
Measuring heat transfer during binding ITC can be used in drug discovery, to
enables accurate determination of binding guide hit validation, lead optimization, and
constants (KD), reaction stoichiometry mechanism of action studies for small
(n), enthalpy (∆H) and entropy (ΔS), thus molecule drugs, fragment-based drug discovery,
providing a complete thermodynamic profile as well as biopharmaceutical drug discovery.
of the molecular interaction. ITC goes ITC is also used in food analysis
beyond binding affinities and can elucidate laboratories. According to one US-based
the mechanisms underlying molecular professor using the Affinity ITC system: “I use
interactions. This deeper understanding of the ITC because its versatility enables me
structure-function relationships enables more to remain at the forefront of food science
confident decision making in hit selection and research. On the ITC I can measure enzyme
lead optimization. kinetics, do a pH titration, or measure protein
In practice, ITC can be the primary assay binding interactions, all critical to optimizing
for KD determination and can also be used to food production processes.”
validate KD data from other assays like SPR, The Affinity ITC measures interactions under
BLI, MST, ELISA, and other binding assays. native conditions without immobilization.
Data from ITC provides insights into the The affinity, in particular, is able to measure
binding mechanisms of an interaction, without binding interactions of highly viscous
structure-activity relationships (SAR) (Figure 8). solutions because of its unique delivery
During protein engineering, ITC for mutant mechanism that separates the injection
and wild type proteins can give the scientist a syringe from the stirring paddle.
© Malvern Panalytical

Figure 8: Schematic highlighting the process of ITC from sample injection to standard curve calculation

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2018 Biophysical Characterization Technology
3.4 Microscale Thermophoresis (MST)
Microscale thermophoresis (MST)
measures binding interactions utilizing the
characteristics of thermophoresis combined
with temperature related intensity change
(TRIC).
It is a versatile, rapid and easy-to-use
method and can precisely measure binding
affinity for virtually every type of biomolecular
interaction, even for difficult targets that
are challenging to evaluate by other binding
affinity methods.
With MST, you can measure the binding
affinity of a variety of biomolecules types
(proteins, small molecules, fragments, DNA,
RNA, etc.) and sizes (101 – 107 Daltons), in
close-to-native conditions and in any buffer
or bioliquids. For example, MST has been
used to characterized solubilized membrane
proteins2 and has also been used in mimicked
membrane conditions3 . It can even be used
to make anaerobic measurements4.
Figure 9: Application note from Nanotemper Technologies highlighting how MST can be used in
binding characterization
Analysis using MST also uses very little
sample and has the highest throughput of
any of the techniques, with measurements
typically taking 15 minutes to complete.
One example of an MST technology is the
Monolith NT.115 from NanoTemper, and it has
been used to characterize binding affinity
for a broad range of biomolecules and
applications. Some examples are fragment-
based and small molecule screening,
characterization of protein -RNA interactions,
aptamers, membrane proteins, and the list
goes on.
In this application note (Figure 9), discover
how MST technology can be used to
specifically label His-tagged proteins for
MST binding studies directly in cell lysates.
For researchers performing biophysical
analysis of proteins, this overcomes a
common hurdle encountered in having
sufficient amounts of materials with the
appropriate purity to perform detailed
analysis.
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2018 Biophysical Characterization Technology

4. Comparison of Biomolecular
Interaction Analysis
Below is a comparative table for the four techniques outlined in this guide.

SPR – Surface ITC -Isothermal

BLI – Bio-Layer MST – Microscale
Plasmon Titration
Interferometry Thermophoresis
Resonance Calorimetry

Signal read-out Refractive index Wavelength shift Temperature Fluorescence

intensity

Kinetics +++++ ++++ No No

Affinity +++++ ++++ +++++ ++++

Affinity range pM - mM pM - mM nM - µM pM - mM

Precision of read-out High Medium Medium Low

Sample consumption Medium Low High Very-low

Thermodynamics Yes Limited Yes Yes

Stoichiometry Yes Yes Yes Yes

Analyte concentration ++ +++++ No No

Throughput Limited High Low Limited

Sample types Very flexible Maximum flexibility Few Intermediate

Table continued on next page →

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2018 Biophysical Characterization Technology

SPR – Surface ITC -Isothermal

BLI – Bio-Layer MST – Microscale
Plasmon Titration
Interferometry Thermophoresis
Resonance Calorimetry

Sample types Very flexible Maximum flexibility Few Intermediate

Cleaning / Maintenance High effort and Very low N/A N/A

cost intensive

Advantages Information Information No immobilization Low sample

content content No labeling consumption
Label-free Low sample
Real-time consumption
Fragments and Label-free
small molecule Real-time
measurement Microfluidics-free
Reference method Reference method
for FDA/ICH/EMA for FDA/ICH/EMA
Cell based BLI

Disadvantages Immobilization of Immobilization of High sample Cannot discern

one binding partner one binding partner consumption second binding site
required required Limited or non-specific
Trained personnel Evaporation applicability binding
for high-quality Analyte has to be Labeling with
data is mandatory ≥ 150 Da hydrophobic
Cleaning and fluorophores
maintenance mostly required

CHART COLOR KEY FAVORABLE MODERATE UNFAVORABLE N/A

PERFORMANCE PERFORMANCE PERFORMANCE

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2018 Biophysical Characterization Technology
5. Summary & Acknowledgements
This guide provides an overview of four label-
free biomolecular interaction techniques. As
highlighted above, it is key to ensure you look
at the whole picture when determining which
tool to use.
Other methods to consider include NMR for
low molecular weight drug discovery, LC-
MS, quartz crystal microbalance analysis and
X-ray crystallography for binding interaction
confirmation.
In the coming years, look out for assays
that are faster, are more sensitive, use less
sample, and give reliable and quantitative
results. As targets become more complicated,
the reliance on multiple technologies will
undoubtedly increase, and, as such, being
able to integrate this data to speed up
decision making will be a critical step.
Some final words from an expert: “It would
be interesting to see how these technologies
could evolve to get an even deeper
understanding of biological processes in
humans. An orthogonal approach to combine
phenotypic and target-based screening
technologies might increase success rate and
speed up commercialization of new drugs.”
- Christina Burtsoff Asp - Product Marketing
Manager, Life Sciences, GE Healthcare
5.1 Acknowledgements
This guide was written with the help and
guidance from the following individuals:
•R  enee Tobias – Senior Product Manager at
ForteBio
•C  hristina Burtsoff Asp - Product Marketing
Manager, Life Sciences, GE Healthcare
• Verna Frasca, Ph.D. - Field Applications
Manager Bioscience, Malvern Panalytical Inc.
•C  olette Quinn, Ph.D. - Microcalorimetry
Product Manager, TA Instruments-Waters LLC
•P  atricia Piatti - Product Marketing Manager,
Nanotemper
5.2 References
1. F
 DA Draft Guidance for Industry, Quality
Considerations in Demonstrating Biosimilarity
to a Reference Protein Product (2012).
2. R
 oche JV et al, J Biol Chem. 2017 Sep
1;292(35):14636-14648. doi: 10.1074/jbc.
M117.788364. Epub 2017 Jul 14.
3. K
 och S et al, J Biol Chem. 2016 Oct
21;291(43):22534-22543. Epub 2016 Sep 9.
4. T
 se ECM et al, J Am Chem Soc. 2017
Sep 13;139(36):12784-12792. doi: 10.1021/
jacs.7b07230. Epub 2017 Aug 29.
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6. Editor’s Picks

Pioneer Octet Biacore 8K

Pall ForteBio Pall ForteBio GE Healthcare

“We get great results on this
instrument. It’s a fundamental part
of our research.”
Dr. Anders Bach,
University of Copenhagen
“Has become the most popular
instrument in our busy biophysics
facility”
Dr. Katherine Stott,
University of Cambridge
“Excellent data generation
capabilities and highly flexible
method set-up options.”
Rupesh Nanjunda,
Janssen

MicroCal Affinity ITC Monolith NT.115

Malvern Panalytical TA Instruments Nanotemper

“This is a bread and butter
instrument for our lab.”
Katherine Hoffmann Ph.D.,
California Lutheran University
“Easy to use, great software,
outstanding application support.”
William Wittbold,
Ajinomoto Althea
“The instrument is indispensable
for us to complement our SPR
kinetic measurements.”
Karoly Liliom Ph.D.,
Department of Biophysics,
Semmelweis University,
Budapest, Hungary

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