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 bioprocessing n-mers present and relative amounts
Conformation of the monomer species before and after bioprocessing
Protein stability: particular relevant issue in pharmaceutical field and will continue to gain more importance as the number of therapeutic protein products in development increases. If a therapeutic protein cannot be stabilized adequately, its benefit to human health will not be realized.
The shelf life required for economic viability of a typical pharmaceutical protein product is months.
Aggregation is particularly problematic because it is encountered routinely during refolding, purification, sterilisation, shipping and storage processes. In this project, we mainly focus on the stability study of some antibody samples on storage.
It has been found that when the Ab. is boiled in presence of SDS, the half antibody could be detected that is believed to be held together by non-covalent bonds.

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
Protein stability: particular relevant issue in pharmaceutical field and will continue to gain more importance as the number of therapeutic protein products in development increases. If a therapeutic protein cannot be stabilized adequately, its benefit to human health will not be realized.
The shelf life required for economic viability of a typical pharmaceutical protein product is months.
Aggregation is particularly problematic because it is encountered routinely during refolding, purification, sterilisation, shipping and storage processes. In this project, we mainly focus on the stability study of some antibody samples on storage.
It has been found that when the Ab. is boiled in presence of SDS, the half antibody could be detected that is believed to be held together by non-covalent bonds.

5. Stability/aggregation Probes
Analytical ultracentrifugation
Dynamic Light Scattering
Viscometry
SEC/MALLs
Protein stability: particular relevant issue in pharmaceutical field and will continue to gain more importance as the number of therapeutic protein products in development increases. If a therapeutic protein cannot be stabilized adequately, its benefit to human health will not be realized.
The shelf life required for economic viability of a typical pharmaceutical protein product is months.
Aggregation is particularly problematic because it is encountered routinely during refolding, purification, sterilisation, shipping and storage processes. In this project, we mainly focus on the stability study of some antibody samples on storage.
It has been found that when the Ab. is boiled in presence of SDS, the half antibody could be detected that is believed to be held together by non-covalent bonds.

6. Stability/aggregation Probes
Analytical ultracentrifugation
Dynamic Light Scattering
Viscometry
SEC/MALLs
Protein stability: particular relevant issue in pharmaceutical field and will continue to gain more importance as the number of therapeutic protein products in development increases. If a therapeutic protein cannot be stabilized adequately, its benefit to human health will not be realized.
The shelf life required for economic viability of a typical pharmaceutical protein product is months.
Aggregation is particularly problematic because it is encountered routinely during refolding, purification, sterilisation, shipping and storage processes. In this project, we mainly focus on the stability study of some antibody samples on storage.
It has been found that when the Ab. is boiled in presence of SDS, the half antibody could be detected that is believed to be held together by non-covalent bonds.

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

12. Optima XLA/ XLI

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
1S=10-13sec
distance, r

15. Data analysis: g*(s) plot
The distribution function of apparent sedimentation coefficient values g*(s) was modelled as a collection of Gaussian distributions for individual species.

16. Ultracentrifuge Analysis: IgG4 preparation

17. Ultracentrifuge Analysis: IgG4 preparation