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![Page 1: AUC and DLS probes for protein aggregation Steve Harding & Arthur Rowe National Centre for Macromolecular Hydrodynamics The National Physical Laboratory,](https://reader035.fdocuments.in/reader035/viewer/2022062511/5515d1a3550346dd6f8b465f/html5/thumbnails/1.jpg)
AUC and DLS probes for protein aggregation
Steve Harding & Arthur RoweNational Centre for Macromolecular Hydrodynamics
The National Physical Laboratory, Teddington
9th December 2008
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Questions
• Aggregation state in response to bioprocessingn-mers present and relative amounts
• Conformation of the monomer species before and after bioprocessing
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A model of chimeric IgG3 wild type.
A model of chimeric IgG3 m15 with 15aa in hinge.
A model of chimeric hinge deleted IgG3 HM5.
Conformation of Engineered antibodies
Lu et al, Biophys. J. 2007
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Stability Problems
• Aggregation or bits falling off during purification, sterilisation, shipping and storage processes.
• Temperature of storage and cycles of freeze thaw
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Stability/aggregation ProbesAnalytical ultracentrifugation Dynamic Light Scattering
Viscometry SEC/MALLs
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Stability/aggregation ProbesAnalytical ultracentrifugation Dynamic Light Scattering
Viscometry SEC/MALLs
![Page 7: AUC and DLS probes for protein aggregation Steve Harding & Arthur Rowe National Centre for Macromolecular Hydrodynamics The National Physical Laboratory,](https://reader035.fdocuments.in/reader035/viewer/2022062511/5515d1a3550346dd6f8b465f/html5/thumbnails/7.jpg)
Multi-angle DLS
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Fixed angle DLS
Cuvettes:
Malvern nanozetasizer 90
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Detector/ Correlator-Computer
Correlator-Computer correlates fluctuations in intensity at angle due to the Brownian motion of the macromolecules.
An intensity autocorrelation function g(2)(t) is calculated and its decay with time gives us the diffusion coefficient D 6
Bk TD
R
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0
2
4
6
8
0.1 1 10 100 1000 10000
Inte
nsity
(%)
Size (d.nm)
Size Distribution by Intensity
Record 108: san 003 pure Record 109: san 003 pureRecord 110: san 003 pure Record 111: san 003 pureRecord 112: san 003 pure Record 113: san 003 pure
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0
10
20
30
0.1 1 10 100 1000 10000
Vo
lum
e (
%)
Size (d.nm)
Size Distribution by Volume
Record 108: san 003 pure Record 109: san 003 pureRecord 110: san 003 pure Record 111: san 003 pureRecord 112: san 003 pure Record 113: san 003 pure
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Optima XLA/ XLI
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Sedimentation Equilibrium
Air Solvent
Solution
conc, c
distance, r
Rate of movement of boundary sed. coeff
Centrifugal force
conc, c
distance, r
Centrifugal force Diffusion
so20,w
1S=10-13sec
STEADY STATE PATTERN
FUNCTION ONLY OF MOL. WEIGHT PARAMETERS
Sedimentation Velocity
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Data analysis: g*(s) plot
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Ultracentrifuge Analysis: IgG4 preparation
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Ultracentrifuge Analysis: IgG4 preparation