The webinar focus is on how Reichert's Surface Plasmon Resonance (SPR) systems can generate additional valuable information that is not attainable on other commercial SPR platforms. SPR has traditionally been used as a research tool to characterize biomolecular interactions including the kinetics, affinity, and thermodynamics of the molecules under study. Reichert SPR systems can routinely carry out these traditional applications but have the added capability to interface with other analytical techniques and instruments. A recent example is that we have developed a system called SPR-PLUS that employs a gradient pump that can accelerate the discovery process by changing experimental variables such pH or buffer concentration in one experiment. Furthermore, this SPR-PLUS system can also easily combine a small chromatography column in series, so that additional separations before or after the initial binding experiment can be carried out in a single setup. Lastly, Reichert's open architecture creates the ability to combine SPR with other techniques such as Mass Spectrometry (MS) or Electrochemistry (Echem) so users can discover more about what is binding (MS) and also how changing potentials affects binding (Echem) to name a few potential applications.

1. Combining Reichert’s SPR Systems with
Other Informative Techniques

2. Reichert Life Science Legacy
• 100+ year optics history
– Instruments with leading sensitivity, robustness and efficiency
• 20 years of SPR expertise
• >250 publications on Reichert SPR
• Cover the full spectrum of bio molecular interactions
– Protein-protein
– Protein-small molecule
• Ensuring success through service
– SPR support staff helps researchers solve problems
– Methods development, high-volume experiments, feasibility studies
A history of exceptional performance, value and support.

3. Current Customer Sampling

4. Advanced SPR Technology
• Innovative two and four channel systems
– Ideal sensitivity for low molecular weight analysis
• Noise and sensitivity performance
required for challenging applications
• More applications and sample types
– Robust enough for crude samples,
cell lysates, aggregates
• High sample capacity
– Two 96- or 384-well plates
– Up to 768 samples—or any combination
of plates and vials
• Scalable to meet your needs now and later
– Solutions grow as you do
– Professional services available

5. SPR TECHNOLOGY

6. Surface Plasmon Resonance
Metal Surface
Plasmons (Electron
Waves)
Surface Plasmon Resonance
Angle
Intensity
> qc
Resonance
(Energy Transfer)

7. Mass Sensor
Angle
Intensity
Time
Response
Raw Data
Response Data

8. Response Units
Time
Response
Units are in mRIU (10-6 refractive index units)
1 mRIU = 1 pg/mm2 of mass binding
A very precise refractometer
A very precise mass sensor

9. Versatile Technique
Time
Response
Binding Reponse
Baseline
Is there an interaction? (Yes/No Binding)
How strong is the interaction? (Affinity)
How quickly do they interact and dissociate? (Kinetics)
Why? (Thermodynamics) (DH, DS, DG)
How much? (Concentration)
Regeneration

10. The Importance of Kinetics
0.0001 0.001 0.01 0.1 1
102
103
104
105
106
107
kd (s-1)
ka(M-1s-1)
10 pM 100 pM 1 nM 10 nM
1 mM
10 mM
100 mM
1 mM
100 nM
KD
KD(M) =
kd (s-1)
ka (M-1s-1)

11. All Classes of Biomolecules
<100 Da to Proteins to Cells
• Proteins
• Lipids
• Carbohydrates
• Nucleic Acids
• LMW Molecules
• Whole Cells
• Bacteria, Viruses

12. Reichert SPR – More than Traditional SPR
• Application 1 – SPR-PLUS
• Application 2 – Combining SPR with Electrochemistry
• Application 3 – Combining SPR with Photochemistry
• Application 4 – Combining SPR with Mass Spectrometry

13. Application 1 - SPR-PLUS System
• System implements a gradient pump so user can apply a gradient of pH or salt to
accelerate method development and characterization
• A programmable valve manifold is included to give the user the ability to divert the
flow path to another instrument such as a chromatography column or mass spec
• Fraction collector provides the ability to automate the collection process to perform
post-analysis on the collected fractions at timed intervals

14. SPR-PLUS Example 1: Combining Reichert SPR
with Chromatography

15. Combining SPR with Chromatography
See also
(1) ] Deshpande et al. (2009). The use of self‐interaction chromatography in stable formulation and crystallization of proteins. Biotechnology journal, 4(9), 1266-
1277.
[2] Cleland, J. et al. (1993). The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Critical reviews in
therapeutic drug carrier systems, 10(4), 307-377.
[3] Ahamed, T. et al. (2005). Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior. Journal of Chromatography A,
1089(1-2), 111-124.
SPR Flow Path
Including 2
chromatography
columns:
Left schematic
Right actual setup

16. Combining SPR with Chromatography

17. SPR-PLUS Example 2: Using a Gradient Pump to
Differentiate Stressed Proteins from Wild Type

18. Gradient Pump - Isocratic Dissociation of Stressed Proteins

19. Gradient Dissociation – Separate Stressed Proteins
Most oxidized
sample has
dissociation at
highest pH
– wild type
needs lowest pH
to dissociate

20. SPR-PLUS Example 3: Combining SPR with Mass
Spectrometry via Fraction Collection

21. See also Schlothauer, T. et al. (2013, July). Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies. In
MAbs (Vol. 5, No. 4, pp. 576-586). Taylor & Francis.
Schematic of Fraction Collection Setup

22. .
(a) Schematic sensorgram of antibody
association and dissociation phase.
(b) Staged mass
spectra of the
FC-Fc part
Mass Spec Analysis on Fractions

23. Application 2: Electro-Switchable Sensor for Neutravidin using
Reichert’s E-CHEM Flowcell
Chun L. Yeung et.al. Adv. Funct. Mater. 2010, 20, 2657.

24. Electro-Switchable Oligopeptide Surfaces
Chun L. Yeung et.al. Adv. Funct. Mater. 2010, 20, 2657.
• Biorecognition element
• Switching element (NH3
+)
• Attachment site for Au

25. Neutravidin Binding with Applied Potential
Chun L. Yeung et.al. Adv. Funct. Mater. 2010, 20, 2657.

26. Switchable and Reversible System
Chun L. Yeung et.al. Adv. Funct. Mater. 2010, 20, 2657.

27. Ulrich Jung et.al. Langmuir 2010, 26, 13913.
Application 3: Photoisomerization of an Azobenzene
Derivative

28. Role of Linker Molecule
• Azobenzene-containing alkanethiols
• trans-cis photoisomerization
• Induced by irradiation with either 365
nm or 465 nm.
• Investigate role of molecular
structure specifically the linker
between the azobenzene moiety and
the alkanethiol
Ulrich Jung et.al. Langmuir 2010, 26, 13913.

29. Ester Linker and mSAM
• Ether group linker exhibited pronounced
photoisomerization
• Mixed SAM with n-butanethiol to reduce
steric hinderance
3-(4-(4-hexyl-phenylazo)-phenoxy)-propane-1-thiol (2b)
STM Image of mSAM
Ulrich Jung et.al. Langmuir 2010, 26, 13913.
• Stripe-like structure
• Homogenously distributed protrusions
attributed to 2b molecules coadsorbed
within the butanethiol SAM.

30. Photoisomerization Response & Kinetics
trans-cis cis-transPure 2b
Pure n-butanethiol
• Follows first-order kinetics
Ulrich Jung et.al. Langmuir 2010, 26, 13913.

31. Rate Constants and Efficiencies
Ulrich Jung et.al. Langmuir 2010, 26, 13913.
• Strong dependence on the molecular structure of the adsorbates
• m-SAMs to reduce steric hinderance
• Photoisomerization follows first-order kinetics
• Time constants suggest high quantum efficiencies on the same
order of magnitude as those of the molecules in solution

32. Application 4: Combining Reichert SPR in-line with
Mass Spectrometry

33. SPR-Mass Spec Coupling Schematic

34. Epitope Determination
• Aß- autoantibody Immobilized on a
Dextran sensor chip
• Tryptic mixture of Aß-peptide fragments
injected over immobilized antibody
• Sensorgrams of the epitope peptide
binding to Aß- autoantibody
• ESI-MS identification by online SPR-
MS of the epitope Aß(17-28) eluted
from the Aß-antibody upon proteolytic
extraction

35. Applications of SPR-MS Analyzer
• Affinity-based biomarker evaluation
• Identification of protein and peptide epitopes
• Precise antibody affinity characterization
• Direct label-free antigen quantification

36. The Reichert SPR Advantage
• Your partner every step of the way
– Unmatched customer service and support solutions
– Maximum uptime drives better results
• Solve your research bottlenecks
– Scalable to research and lab needs
• Reliable binding, kinetics, concentration
and thermodynamic data
– Helping you answer questions quantitatively
• Increase your sample flexibility
– Broader application options
– Robust fluidics
– Combine SPR with other Techniques
• Reduce your equipment and maintenance costs