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AUC and DLS probes for protein aggregation Steve Harding & Arthur Rowe National Centre for Macromolecular Hydrodynamics The National Physical Laboratory, Teddington 9th December 2008

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Questions Aggregation state in response to bioprocessing n-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 Probes Analytical ultracentrifugation Dynamic Light Scattering ViscometrySEC/MALLs

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Stability/aggregation Probes Analytical ultracentrifugation Dynamic Light Scattering ViscometrySEC/MALLs

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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

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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 s o 20,w 1S= sec 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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