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Solution Properties of antibodies: Purity Conformation

Solution Properties of antibodies: Purity Conformation. Text book representation of antibody structure:. Main tool: Analytical Ultracentrifuge. 2 types of AUC Experiment:. Sedimentation Equilibrium. Sedimentation Velocity. Centrifugal force . Centrifugal force  Diffusion.

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Solution Properties of antibodies: Purity Conformation

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  1. Solution Properties of antibodies: • Purity • Conformation

  2. Text book representation of antibody structure:

  3. Main tool: Analytical Ultracentrifuge

  4. 2 types of AUC Experiment: Sedimentation Equilibrium Sedimentation Velocity 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

  5. 2 types of AUC Experiment: Sedimentation Equilibrium Sedimentation Velocity 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

  6. Solution Properties of antibodies: • Purity

  7. Ultracentrifuge Analysis: IgG4 preparation

  8. Ultracentrifuge Analysis: IgG4 preparation

  9. Solution Properties of antibodies: • Conformation – “Crystallohydrodynamics”

  10. Single Ellipsoids won’t do…

  11. So use the bead model approximation … Developed by J. Garcia de la Torre and co-workers in Murcia Spain 2 computer programmes: HYDRO & SOLPRO (please refer to D2DBT7 notes – see the example for lactoglobulin octamers)

  12. Conventional Bead model Bead-shell model

  13. Bead model, s=7.26 Svedbergs, Rg= 6.8nm 1st demonstration that IgE is cusp shaped Davies, Harding, Glennie & Burton, 1990 …by comparing hydrodynamic properties with those of hingeless mutant IgGMcg

  14. Consistent with function…. Bead model, s=7.26 Svedbergs, Rg= 6.8nm High Affinity Receptor

  15. Consistent with function…. High Affinity Receptor

  16. Better approach is is to use shell models! Conventional Bead model Bead-shell model

  17. We call this approach “Crystallohydrodynamics” Crystal structure of domains + solution data for domains + solution data for intact antibody = solution structure for intact antibody Bead-shell model: Human IgG1

  18. Take Fab' domain crystal structure, and fit a surface ellipsoid…. PDB File: 1bbj 3.1Å Fitting algorithm: ELLIPSE (J.Thornton, S. Jones & coworkers) Ellipsoid semi-axes (a,b,c) = 56.7, 35.6, 23.1. Ellipsoid axial ratios (a/b, b/c) = (1.60, 1.42) Hydrodynamic P function = 1.045: see d2dbt8 notes

  19. Now take Fc domain crystal structure, and fit a surface ellipsoid…. Do the same for Fc PDB File: 1fc1 2.9Å

  20. Now fit bead model to the ellipsoidal surface Fab’ Fc P(ellipsoid)=1.045 P(bead) = 1.023 P(ellipsoid)=1.039 P(bead) = 1.039 Use SOLPRO computer programme: Garcia de la Torre, Carrasco & Harding, Eur. Biophys. J. 1997 Check the P values are OK

  21. The TRANSLATIONAL FRICTIONAL RATIO f/fo (see d2dbt8 notes) f/fo =conformation parameter x hydration term f/fo = P x (1 +d/rovbar)1/3 Can be measured from the diffusion coefficient or from the sedimentation coefficient   f/fo = constant x {1/vbar1/3} x {1/ M1/3} x {1/Do20,w} f/fo = constant x {1/vbar1/3} x (1-vbar.ro) x M2/3 x {1/so20,w}

  22. Experimental measurement of f/fo for IgGFab

  23. Experimental measurement of f/fo for IgGFab

  24. Estimation of time-averaged hydration, dapp for the domains+whole antibody dapp = {[(f/fo)/P]3 - 1}rovbar Fab' domain P(bead model) = 1.023 f/fo(calculated from so20,w and M) = 1.22+0.01 dapp = 0.51 g/g Fc domain P(bead model) = 1.039 f/fo(calculated from so20,w and M) = 1.29+0.02 dapp = 0.70 g/g Intact antibody = 2 Fab's + 1 Fc. Consensus hydration dapp ~ 0.59 g/g

  25. we can now estimate P(experimental) for the intact antibody P(experimental) = f/fo x (1 +dapp/rovbar)-1/3

  26. IgG’s: all these compact models give P’s lower than experimental P=1.107 P=1.118 P=1.112 P=1.121 P=1.122 P=1.143 …so we rule them out!

  27. Models for IgG2 & IgG4. Experimental P=1.22+0.03 (IgG2) =1.23+0.02 (IgG4) P = 1.217 P = 1.230 Carrasco, Garcia de la Torre, Davis, Jones, Athwal, Walters Burton & Harding, Biophys. Chem. 2001

  28. (Fab)2 : P(experimental) = 1.23+0.02 P=1.208 (Fab)2

  29. “Open” models for IgG1 (with hinge) P(experimental) = 1.26+0.03 P = 1.263 P = 1.264

  30. A P=1.215 P=1.194 B C P=1.172 These are coplanar models for a mutant hingeless antibody, IgGMcg. P(experimental) = 1.23+0.03

  31. UNIQUENESS PROBLEM: Although a particular model may give conformation parameter P in good agreement with the ultracentrifuge data, there may be other models which also give good agreement. This is the uniqueness or “degeneracy” problem. To deal with this we need other hydrodynamic data: Intrinsic viscosity [h] – viscosity increment n Radius of gyration Rg – Mittelbach factor G And work is ongoing in the NCMH in conjunction with other laboratories

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