AUC and DLS probes for protein aggregation

1
AUC and DLS probes for protein aggregation
The National Physical Laboratory, Teddington 9th
December 2008

• Steve Harding Arthur Rowe
• National Centre for Macromolecular Hydrodynamics

2
Questions

• Aggregation state in response to
  bioprocessingn-mers present and relative amounts
• Conformation of the monomer species before and
  after bioprocessing

3
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
4
Stability Problems

• Aggregation or bits falling off during
  purification, sterilisation, shipping and storage
  processes.
• Temperature of storage and cycles of freeze thaw

5
Stability/aggregation Probes
Dynamic Light Scattering
Analytical ultracentrifugation
Viscometry
SEC/MALLs
6
Stability/aggregation Probes
Analytical ultracentrifugation
Dynamic Light Scattering
Viscometry
SEC/MALLs
7
Multi-angle DLS
8
Fixed angle DLS
Cuvettes
Malvern nanozetasizer 90
9
q
Correlator-Computer correlates fluctuations in
intensity at angle q due to the Brownian motion
of the macromolecules.
Detector/ Correlator-Computer
An intensity autocorrelation function g(2)(t) is
calculated and its decay with time gives us the
diffusion coefficient D
10
(No Transcript)
11
(No Transcript)
12
Optima XLA/ XLI
13
(No Transcript)
14
Sedimentation Velocity

Sedimentation Equilibrium
Centrifugal force ?
Centrifugal force ? ?Diffusion
Air Solvent

Solution
conc, c
?
STEADY STATE PATTERN FUNCTION
ONLY OF MOL. WEIGHT PARAMETERS
conc, c
Rate of movement of boundary ? sed.
coeff
?
distance, r
so20,w 1S10-13sec
distance, r
15
Data analysis g(s) plot
16
Ultracentrifuge Analysis IgG4 preparation
17
Ultracentrifuge Analysis IgG4 preparation