AUC and DLS probes for protein aggregation

Steve Harding & Arthur RoweNational Centre for Macromolecular Hydrodynamics

The National Physical Laboratory, Teddington

9th December 2008

Questions

• Aggregation state in response to bioprocessingn-mers present and relative amounts

• Conformation of the monomer species before and after bioprocessing

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

Stability Problems

• Aggregation or bits falling off during purification, sterilisation, shipping and storage processes.

• Temperature of storage and cycles of freeze thaw

Stability/aggregation ProbesAnalytical ultracentrifugation Dynamic Light Scattering

Viscometry SEC/MALLs

Stability/aggregation ProbesAnalytical ultracentrifugation Dynamic Light Scattering

Viscometry SEC/MALLs

Multi-angle DLS

Fixed angle DLS

Cuvettes:

Malvern nanozetasizer 90

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

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

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

Optima XLA/ XLI

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

Data analysis: g*(s) plot

Ultracentrifuge Analysis: IgG4 preparation

Ultracentrifuge Analysis: IgG4 preparation