1. Introduction to Multi Angle (MALS) and Dynamic (DLS) Light Scattering
Dr. Dierk Roessner
Next generation strategies & insights on large molecule characterization

2. Table of content Introduction to Static and Dynamic Light Scattering
Experimental setup, results
Example 1: SEC MALS
Example 2: DLS
Take home message

3. Information from scattered light
Why light scattering?
Information from scattered light

4. What is Light Scattering?
blue sky and clouds
In nature…
Notes:
Light is scattered from the Atmosphere and we have a wavelength dependence, why the sky is blue and sunset red
Blue light scattered stronger than red light -> remaining light at sun set is seen as beautiful red
red sunsets

5. Information from scattered light
Molar mass Mw
RMS Radius
Second virial coefficient A2
How?
through analysis of the amount of scattered light at
different scattering angles and
different concentrations, option
Static Light Scattering
Multi Angle Light Scattering
Monomer or oligomeric state
Structure-property-relationships
Size of aggregates
Polymer conformation
Protein-protein interaction
Polymer theta conditions
We bring LS to the lab…

6. Static or Multi Angle Light Scattering
Zimm equation:
Regression of the scattered light to angle 0 °
rms radius is determined from angular dependency
Light Scattering measures directly molar mass, radius and interaction, by measuring intensity of scattered light
Now more specific explanation
Why do we measure at different angles.
Short constant K* describtion
A2 shows interaction between molecules (do not explain no)
K* = 4p2 (dn/dc)2 n02NA-1l0-4

7. Angular Intensity Varies with Size
rms radius = 150nm
At every point of the chromatogram:
Regression to scattering angle zero
Intercept with y-axis is molar mass * c
Initial slope is the root mean square radius
Intensity
Elution Volume
Scattering Angle
rms radius = 25nm

8. Static or Multi Angle Light Scattering
Degasser
SEC
Column
HPLC Pump
DAWN
Autosampler
Only explain a simple setup and go not into details.
HPLC (different vendors) coupled to our detectors. UV
Optilab
SEC-MALS interfaces to standard U/HPLC-SEC instrumentation
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9. Static or Multi Angle Light Scattering
BSA LS RI
Molar mass 1231 data slices
Dextran LS RI
Molar Mass
Notes:
The molar mass distribution for samples with discrete species, e.g. monomer, dimer, trimer and higher oligomers follows a step-type pattern. This is generally the case for proteins, such as Bovine Serum Albumin (BSA, monomer molar mass 66 kDa).
The molar mass distribution of polydisperse samples (such as the dextran above) is generally continuous. The light scattering and RI traces will not overlay if the sample is polydisperse!
Explain, monomer molar mass and the traces one can see.
We measure constant molar masses which would not be possible with conventionel calibration
Dextran is polydisperse and we see a continuous change in molar mass
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10. Information from scattered light – part 2
Why light scattering?
Information from scattered light – part 2
DLS

11. Information from scattered light – part 2
Hydrodynamic radius Rh
Size distribution of Rh
Interaction parameter kD
How?
through time analysis of the fluctuation of scattered light at
one scattering angle and
different concentrations
Dynamic Light Scattering
Constant temperature
Temperature ramping, Tagg
Analysis of aggregates
Polydispersity of monomer peaks
Experiments at different concentration reveal interactions
Explain the topics

12. Dynamic Light Scattering

13. Autocorrelation Function
9 nm latex spheres t b h D T k R  6 =
kB – Boltzmann’s constant
T – temperature (Kelvin)
η – viscosity of solvent
Rh – hydrodynamic radius
Inflection point ~ Dt
Width ~ Polydispersity
Stokes-Einstein
Relationship
Hydrodynamic Radius
“Molecular Size”
Autocorrelation function:

14. Dynamic Light Scattering
Distribution of particle sizes – batch DLS
Cumulant fit (fitting to a single exponential) yields Rh = 20 nm!
9 nm + 50 nm radius particles
Explain the differences of the fit with the shown autocorrelation

15. Dynamic Light Scattering
9 nm + 50 nm radius particles
Regularization result:
Peak 1: Rh = 7 nm, width = 1 nm
Peak 2: Rh = 43 nm, width = 10 nm
Explain that size distribution is calculated, so

16. Information from scattered light
Comparing MALS and DLS
Information from scattered light

17. Different Types of Light Scattering
Dynamic Light Scattering
Static Light Scattering
Diffusion!
kBT
RH =
6πηDt
One instrument can do both
Brownian motion of sub-micrometer particles causes intensity fluctuations in the scattered light. The rate of fluctuation is analyzed to determine the diffusion coefficient DT and calculate particle size.
Static light scattering determines molar mass and second virial coefficient from the scattering intensity, concentration and optical parameters (wavelength, refractive index, etc.).

18. Example 1 Multi Angle Light Scattering coupled to SEC
Heat shock protein GrpE, actively preventing the aggregation of stress-denatured proteins

19. Conflicting quaternary structures in literature: GrpE
SEC = hexamer
SEC MALS = ?
Differences between SEC and SEC MALS?
Lit report different oligomeric states of the protein in solution
How is that possible and can we solve the problem with LS
With SEC we find hexamer
What do we find with SEC MALS
Schönfeld and Behlke (1998), Methods Enzymol. 290, pp

20. Conflicting quaternary structures in literature: GrpE
Molar mass determination by size exclusion chromatography (SEC)
Column or conventional calibration with proteins, e.g. BioRad Mix
Superdex /10
Mobile phase = PBS
Flow rate 0.5 mL/min
BioRad Standard Mix
Thyroglobulin
670 kg/mol
γ-Globulin
150 kg/mol
Ovalbumin
42.8 kg/mol
Myoglobin
17 kg/mol
GrpE Ve from SEC = 11.9 mL
MM from conventional calibration = 140 kDa
140 kDa corresponds roughly with a hexamer (132 kDa)
GRPE molar mass is determined with conventionell calibration with protein standards of globulaire proteins
We get a high molar mass ->hexamer

21. Conflicting quaternary structures in literature: GrpE
Molar mass determination by Multi Angle Light Scattering (MALS) coupled to SEC
Adds online absolute molar mass (Mw) determination to the SEC
Molar mass from SEC
GrpE MM from SEC = 140 kDa, wrong! MM from SEC MALS = 42 kDa
MM from theory = 43 kDa
Molar mass from SEC MALS
With MALS we get, see slide

22. Conflicting quaternary structures in literature: GrpE
Column or conventional calibration of SEC
SEC separates by hydrodynamic volume, not by molar mass
Applicable if same molecular conformation as (globular) calibration proteins
Ideal steric separation, no charge or hydrophobic interactions
SEC is insufficient for reliable characterization
Multi Angle Light Scattering coupled to SEC
Adds absolute molar mass determination to the SEC
Identifies the oligomeric state of GrpE in solution
But why elutes the GrpE dimer so fast?
The GrpE protein has an elongated structure
Radius of GrpE is Rh = 3.9 nm
Radius of a 43 kDa globular would be Rh = 3.0 nm
GrpE dimer
Explain why the difference is observed.
SEC only works if no interaction with stationairy phase happens.
Correct molar mass only possible with LS
GrpE elutes different because of structure with Helices which makes it elongated

23. Example 2 Dynamic Light Scattering
Characterization of aggregates after lyophilization

24. Characterization of aggregates after lyophilization
Before Lyophilization
After Lyophilization
ACF before and after lyophilisation in different buffers.
Lyophilisation leads to shift in autocorrelation and a kind of bumb in the curve due to aggregates
Monoclonal Antibody .. .

25. Characterization of aggregates after lyophilization
Before Lyophilization
After Lyophilization
Lyophilized MABs contain significant amount of aggregates
% intensity shows nicely the aggregates very sensitive . ..

26. Characterization of aggregates after lyophilization
Batch DLS is highly sensitive to the presence of a small amount of aggregates.
Aggregates
% mass is trying more to show the real amount of aggregates which is quite small. Nether the less, the sample changed by lyophilisation and one can see this by looking at the different parameter of the DLS results.
-> DLS is a good tool to measure aggregates very sensitive in protein solutions.
Sample
Radius
(nm)
%PD
Peak1 Radius (nm)
Peak 1
% Intensity
Peak1
% Mass
MAB1PBS pre-lyophilized
6.1
3.9
5.9
100.0
MABB1PBS lyophilized
7.0
23.9
5.8
81.9
99.6
MAB2Sucrose pre-lyophylized
6.3
MABB2 Sucrose lyophilized
8.6
multimodal
68.3
98.8

27. Multi Angle (MALS) and Dynamic (DLS) Light Scattering
Take home message
Multi Angle (MALS) and Dynamic (DLS) Light Scattering

28. Take home message Dynamic Light Scattering
Measures size Rh, size distribution and interaction parameter kD
Often used stand alone, typical experiment duration is between 20 and 50 seconds, high throughput possible
Static and Multi Angle Light Scattering
Absolute determination of molar mass Mw, RMS radius and second virial coefficient A2
Often used online coupled to SEC or other types of separation techniques
Dynamic LS: Fluctuations in scattered light intensity
Static LS:
Averaged scattered light intensity