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Solution Properties of antibodies: Purity Conformation

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Text book representation of antibody structure:

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Main tool: Analytical Ultracentrifuge

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Sedimentation Velocity Sedimentation Equilibrium 2 types of AUC Experiment: 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=10 -13 sec STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

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Sedimentation Velocity Sedimentation Equilibrium 2 types of AUC Experiment: 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=10 -13 sec STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

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Solution Properties of antibodies: Purity

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Ultracentrifuge Analysis: IgG4 preparation

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Solution Properties of antibodies: Conformation – Crystallohydrodynamics

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Single Ellipsoids wont do…

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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)

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Conventional Bead model Bead-shell model

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

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

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Consistent with function…. High Affinity Receptor

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

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

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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

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

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

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The TRANSLATIONAL FRICTIONAL RATIO f/f o (see d2dbt8 notes) f/f o =conformation parameter x hydration term f/f o = P x (1 + o vbar) 1/3 Can be measured from the diffusion coefficient or from the sedimentation coefficient f/f o = constant x {1/vbar 1/3 } x {1/ M 1/3 } x {1/D o 20,w } f/f o = constant x {1/vbar 1/3 } x (1-vbar. o ) x M 2/3 x {1/s o 20,w }

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Experimental measurement of f/f o for IgGFab

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Estimation of time-averaged hydration, app for the domains+whole antibody app ={[(f/f o )/P] 3 - 1} o vbar Fab' domain P(bead model) = 1.023 f/f o (calculated from s o 20,w and M) = 1.22+0.01 app = 0.51 g/g Fc domain P(bead model) = 1.039 f/f o (calculated from s o 20,w and M) = 1.29+0.02 app = 0.70 g/g Intact antibody = 2 Fab's + 1 Fc. Consensus hydration app ~ 0.59 g/g

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we can now estimate P(experimental) for the intact antibody P(experimental) = f/f o x (1 + app o vbar) -1/3

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P=1.107 P=1.112 P=1.118 P=1.121P=1.122P=1.143 IgGs: all these compact models give Ps lower than experimental …so we rule them out!

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

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

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

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

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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 [ ] – viscosity increment Radius of gyration R g – Mittelbach factor G And work is ongoing in the NCMH in conjunction with other laboratories

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