Steve HardingNational Centre for Macromolecular Hydrodynamics

A combined approach to size,heterogeneity, conformation & flexibility

of bio-macromolecules

Size (mol wt.) & heterogeneity:

• SEC-MALLs – mol wt distributions

• AUC sedimentation equilibrium analysis – M* & distributions

• AUC sedimentation velocity analysis – g*(s) & distributions

• Size (mol wt.) and heterogeneity: SEC-MALLs

• Size (mol wt.) and heterogeneity: SEC-MALLs

mucin

• Size (mol wt.) and heterogeneity: SEC-MALLs

• Size (mol wt.) and heterogeneity: Analytical ultracentrifuge

Sedimentation Velocity Sedimentation Equilibrium

Air Solvent

Solution

conc, c

distance, r

Top view, sector ofcentrifuge cell

Rate of movement ofboundary sed. coeff

Centrifugal force

conc, c STEADY STATEPATTERN

FUNCTION ONLYOF MOL. WEIGHTPARAMETERS

distance, r

Centrifugal forceDiffusion

so20,w

1S=10-13sec

• Size (mol wt.) and heterogeneity: Analytical ultracentrifuge

M* analysis of sedimentation equilibrium

Creeth JM & Harding SE (1982) J. Biochem. Biophys. 7, 25-34

Mw,app

cell bottom

Chitosan G213

Sedimentation equilibrium M* plot

Ball, Harding & Mitchell (1988)

SEC - sedimentationequilibrium mol. wtdistribution: alginate

Sedimentation velocity g*(s) plot

Numerical solutions tothe Lamm equation byClaverie et al (1975) &implemented by Todd &Haschemayer (1981)

Lamm (1923) equation:

DLS analysis: Contin plot

R. Tester, T Patel, S. Harding, Carbohydrate Research (2006)

Sedimentation velocity g*(s) plot: starch

……consider antibodies used in therapies, and theirstate of aggregation after bioprocessing

SAN02 Freeze-thaw (1.16mg/mL)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 10 20 30 40 50

s(S)

g*(

s)

Control

10cycles

20cycles

25cycles

Size distribution by sedimentation velocity of a bioprocessed IgG4 antibody

SAN03 pure

0

5

10

15

20

25

0.1 1 10 100 1000 10000

Vo

lum

e(%

)

Size (d.nm)

Size Distribution by Volume

Record 108: san 003 pure Record 109: san 003 pure Record 110: san 003 pure

Record 111: san 003 pure Record 112: san 003 pure Record 113: san 003 pure

size, d (nm)

Am

ou

nt

(%)

Size distribution by DLS of a bioprocessed IgG4 antibody

…provides an excellent assessment of the quality/ the extent of heterogeneity

…this preparation is even worse!

Also, changes in sedimentation coefficient of monomer may reflectchange in conformation

Molecular conformation & flexibility:

• general conformation – conformation zoning

• flexibility – persistence length using global methods

• whole body or ellipsoid representations

• bead model representations

Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33

so20,w~ M0.4-0.5so

20,w~ M0.15so20,w~ M0.67

[] ~ M0.5-0.8[] ~ M1.8[] ~ M0

CoilRodSphere

• General molecular conformation: Haug triangle and power law coeffs

Mark-Houwink-Kuhn-Sakurada Power law plot

Galactomannansa=0.74+0.01

Picout et al (2003) Biomacromolecules 2,1301-1309

Rollings J (1992) in Laser Light Scattering inBiochemistry (Harding, Sattelle & Bloomfield eds)

Change in Conformation

Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33

so20,w~ M0.4-0.5so

20,w~ M0.15so20,w~ M0.67

[] ~ M0.5-0.8[] ~ M1.8[] ~ M0

CoilRodSphere

ks/[] ~1.6ks/[] <1ks/[] ~1.6

Conformation Zoning:

Pavlov et al. (1997). Trends inAnalytical Chemistry, 16, 401-405.

0.5 1.0 1.5 2.0 2.5-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5lo

g(1

0-11 k sM

L)

log (1012

[s]/M L)

A

B

C

D

E

0.5 1.0 1.5 2.0 2.5-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5lo

g(1

0-11 k sM

L)

log (1012

[s]/M L)

A

B

C

D

E

A: very stiff rod

B: with limitedflexibility

C: semi-flexible

D: random coil

E: globular orbranched

0.5 1.0 1.5 2.0 2.5-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

log

(10-1

1 k sML)

log (1012

[s]/ML)

A

B

C

D

E

Bovine glycogen ●

Pectins ■

Pullulans ▲

A: very stiff rod

B: with limitedflexibility

C: semi-flexible

D: random coil

E: globular orbranched

Measure of flexibility: persistence length Lp

Theoretical limits: 0 (random coil) → ∞ (perfect rod)

Practical limits ~ 2nm → 200nm

2/1

2/1

3/10

3/10

3/12 2w

L

p

Lw M

M

LBMA

M

....22

843.13

12/1

32

2/1

0

00

pL

w

pL

w

A

L

LM

MAA

LM

M

N

vMs

“Bushin-Bohdanecky” relation

“Yamakawa-Fujii” relation

• Molecular conformation: flexibility analysis

“Hydfit” or Global analysis: Garcia de la Torre &Ortega, Biomacromolecules (2007)

Konjac glucomannan, Lp ~ 13nm (Kok et al, 2009)

Conformation Zoning:

Pavlov et al. (1997). Trends inAnalytical Chemistry, 16, 401-405.

0.5 1.0 1.5 2.0 2.5-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5lo

g(1

0-11 k sM

L)

log (1012

[s]/M L)

A

B

C

D

E

0.5 1.0 1.5 2.0 2.5-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5lo

g(1

0-11 k sM

L)

log (1012

[s]/M L)

A

B

C

D

E

A: very stiff rod

B: with limitedflexibility

C: semi-flexible

D: random coil

E: globular orbranched

• Molecular conformation: ellipsoid representation

ELLIPS algorithms www.nottingham.ac.uk/ncmh

Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations of hydrationindependent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)

Structure and heterogeneity of

gliadin: a hydrodynamic evaluationS. Ang et al, Eur. Biophys. J. (2009)

• Molecular conformation: ellipsoid representation

ELLIPS algorithms www.nottingham.ac.uk/ncmh

Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations of hydrationindependent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)

• Molecular conformation: ellipsoid representation

ELLIPS algorithms www.nottingham.ac.uk/ncmh

Universal_Param: Calculates shape parameter from s, D, [], BELLIPS1 Evaluates a/b for prolate or oblate ellipsoid fromshape parameterELLIPS2 Evaluates shape parameter from (a,b,c) or (a/b, b/c)ELLIPS3 Evaluates (a/b, b/c) from combinations ofhydration independent shape functions.ELLIPS4 Evaluates (a/b, b/c) from electro-optic decaycombined with other hydrodynamic data.ELLIPSDRAW 3D plot of ellipsoid from (a/b, b/c)COVOL Evaluates B from (a/b, b/c)

R

a/b

b/c

a/b

b/c

R

Shape parameters R and are fromsedimentation, viscosity andfluorescence measurements

(a/b) = 4, (b/c) = 1

ELLIPS3 applied to neurophysin monomers

dimersb/c b/c

a/b

a/b

(a/b)= 4, (b/c)= 1 (a/b)=2.5, (b/c)= 3

monomers

Neurophysin dimerises – here’s what happens

……whole-body ellipsoids won’t do for complicatedshapes like antibodies……

……whole-body ellipsoids won’t do for complicatedshapes like antibodies…… so use bead modelling

• Molecular conformation: bead modelling

1st demonstrationthat IgE is cuspshaped, 1990

Bead model: s=7.26S,Rg= 6.8nm

• Molecular conformation: bead modelling

Consistent with function….

Bead model, s=7.26 Svedbergs, Rg= 6.8nm

High AffinityReceptor

A model of chimeric IgG3 wild type

A model ofchimeric IgG3 m15with 15aa in hinge.

A model of chimerichinge deleted IgG3HM5.

Conformation of engineered antibodies from s, Rg,Dmax, [] and crystal structure of the domains

Challenge: conformation determination in mixed systems

… here’s our heterogeneous bioprocessed antibody again

Thanks to:

Professors Arthur Rowe, JoseGarcia de la Torre, Simon Ross-Murphy, Georges Pavlov & Drs.Dave Scott & Gordon Morris