Solution Properties of antibodies:

1

• 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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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 1S10-13sec
distance, r
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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 1S10-13sec
distance, r
6

• Solution Properties of antibodies
• Purity

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Ultracentrifuge Analysis IgG4 preparation
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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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Bead model, s7.26 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
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Consistent with function.
Bead model, s7.26 Svedbergs, Rg 6.8nm
High Affinity Receptor
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Consistent with function.
High Affinity Receptor
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Better approach is is to use shell models!
Conventional Bead model
Bead-shell model
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• 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
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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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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
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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
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Experimental measurement of f/fo for IgGFab
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Experimental measurement of f/fo for IgGFab
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• Estimation of time-averaged hydration, dapp for
  the domainswhole antibody
•  
• dapp (f/fo)/P3 - 1rovbar
• Fab' domain
• P(bead model) 1.023
• f/fo (calculated from so20,w and M) 1.220.01
• dapp 0.51 g/g
• Fc domain
• P(bead model) 1.039
• f/fo (calculated from so20,w and M) 1.290.02
• dapp 0.70 g/g
• Intact antibody 2 Fab's 1 Fc.
• Consensus hydration dapp 0.59 g/g

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we can now estimate P(experimental) for
the intact antibody   P(experimental)
f/fo x (1 dapp/rovbar)-1/3  
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IgGs all these compact models give Ps
lower than experimental
P1.107
P1.118

P1.112
P1.121
P1.122
P1.143
so we rule them out!
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Models for IgG2 IgG4. Experimental P1.220.03
(IgG2) 1.230.02 (IgG4)
P 1.217
P 1.230
Carrasco, Garcia de la Torre, Davis, Jones,
Athwal, Walters Burton Harding, Biophys. Chem.
2001
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(Fab)2 P(experimental) 1.230.02
P1.208
(Fab)2
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Open models for IgG1 (with hinge)
P(experimental) 1.260.03
P 1.263
P 1.264
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A
P1.215
P1.194
B
C
P1.172
These are coplanar models for a mutant hingeless
antibody, IgGMcg. P(experimental) 1.230.03
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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 h
viscosity increment n Radius of gyration Rg
Mittelbach factor G And work is ongoing in the
NCMH in conjunction with other laboratories