Transcript NPL_Dec08_ProteinAggregation.ppt

AUC and DLS probes for protein
aggregation
The National Physical Laboratory,
Teddington
9th December 2008
Steve Harding & Arthur Rowe
National Centre for Macromolecular Hydrodynamics
Questions
• Aggregation state in response to bioprocessing
n-mers present and relative amounts
• Conformation of the monomer species before
and after bioprocessing
Conformation of Engineered antibodies
A model of
chimeric IgG3 m15
with 15aa in hinge.
A model of chimeric
hinge deleted IgG3
HM5.
A model of chimeric IgG3 wild type.
Lu et al, Biophys. J. 2007
Stability Problems
• Aggregation or bits falling off during purification,
sterilisation, shipping and storage processes.
• Temperature of storage and cycles of freeze
thaw
Stability/aggregation Probes
Analytical ultracentrifugation
Viscometry
Dynamic Light Scattering
SEC/MALLs
Stability/aggregation Probes
Analytical ultracentrifugation
Viscometry
Dynamic Light Scattering
SEC/MALLs
Multi-angle DLS
Fixed angle DLS
Cuvettes:
Malvern nanozetasizer 90
q
Correlator-Computer correlates
fluctuations in intensity at angle q
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
Detector/ CorrelatorComputer
kBT
D
6 R
Optima XLA/ XLI
Sedimentation Velocity
Sedimentation Equilibrium
Centrifugal force

Air
Centrifugal force 
Diffusion
Solvent
Solution
conc, c
conc, c
of movement of
 Rate
boundary  sed. coeff
distance, r
so20,w
1S=10-13sec
STATE
 STEADY
PATTERN
distance, r
FUNCTION ONLY OF
MOL. WEIGHT
PARAMETERS
Data analysis: g*(s) plot
Ultracentrifuge Analysis: IgG4 preparation
Ultracentrifuge Analysis: IgG4 preparation