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 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 18-24 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.
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 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 18-24 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.
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 18-24 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.
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 18-24 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.
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. Detector/ Correlator-Computer An intensity autocorrelation function g(2)(t) is calculated and its decay with time gives us the diffusion coefficient D
Optima XLA/ XLI
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
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.
Ultracentrifuge Analysis: IgG4 preparation
Ultracentrifuge Analysis: IgG4 preparation