The Illumination of Structure using Light ... - ATA Scientific · Vitamin B12 1.35 kDa Cytochrome c...

The Illumination of Structure using Light Scattering

Michael CavesProduct Technical Specialist for Biophysical Characterisation

Light Scattering

Laser

Detector

Scattered light

› Scattered light intensity α size6

› Mean Average count rate measured

› All light scattering methods discussed measure this, in addition to the ‘technique-specific parameters’

Static Light Scattering (SLS)

Time

Inte

nsi

ty

(kcp

s) Mean Count Rate

125

120

› Theory

› Applications and specifications

Outline

› Dynamic Light Scattering

› SEC-LS

› Interaction Parameters

Outline - Theory

› Mean Average count rate measured at multiple concentrations and used to calculate absolute molecular weight and/or A2

› No resolution possible, average Mw is generated


Time

Inte

nsi

ty

(kcp

s) Mean Count Rate

125

120

› Fluctuation Frequency of count rate measured and used to calculate hydrodynamic size – Resolution Possible

Dynamic Light Scattering (DLS)

Time

Inte

nsi

ty

(kcp

s)

125

120

Small Particle = Fast Brownian motion = Fast Intensity Fluctuations

Large Particle = Slow Brownian motion = Slow Intensity Fluctuations

DLS Measures Fluctuations in the Scattered Light Intensity Caused by Brownian Motion

Small Particle = Fast Brownian motion = Fast Intensity Fluctuations

Large Particle = Slow Brownian motion = Slow Intensity Fluctuations

Hydrodynamic Diameter Definition

The diameter of a hard sphere that diffuses at the same speed as the particle or molecule

being measured

› Depends not only on the size of the particle “core”, but also on any surface structure

SmallParticles

Time

Inte

nsi

ty

Time

Inte

nsi

ty

LargeParticles

Laser

DLS Measures Fluctuations in the Scattered Light Intensity Caused by Brownian Motion

DLS Method Summary

Intensity Fluctuations Detected

Correlogram

Size(s) and Polydispersity

Correlation

Correlogram analysis

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

Time

Inte

nsi

ty

Time

Inte

nsi

ty

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

(Auto)Correlation

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

Time

Inte

nsi

ty

= 0

Time

Inte

nsi

ty

= 0

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

(Auto)Correlation

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

Time

Inte

nsi

ty

= 1

Time

Inte

nsi

ty

= 1

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

(Auto)Correlation

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

Time

Inte

nsi

ty

= 2

Time

Inte

nsi

ty

= 2

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

(Auto)Correlation

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

Time

Inte

nsi

ty

Time

Inte

nsi

ty

= 3

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

= 3

(Auto)Correlation

Time

Inte

nsi

ty

Time

Inte

nsi

ty

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

(Auto)Correlation

The Correlogram

Time

Inte

nsi

ty

Small Particles

Large Particles

Time

Inte

nsi

ty

0 TimeC

orr

elat

ion

Co

effi

cien

t

1

0

0 Time

Co

rrel

atio

nC

oef

fici

ent

1

0

correlate LOG

The Correlogram

Exponential decay lifetime indicates hydrodynamic size

Gradient indicates sample

polydispersity

Baseline quality used to assess contributions of Large particles

Intercept gives a measure

of S/N

› Particle size information is obtained by analysing the correlogram with various algorithms

DISTRIBUTION

Multi-exponential fit

Distribution of particle sizes (by intensity, volume or number)

CUMULANTS (ISO13321)

Single exponential fit

Mean size (z-avg diam.)Estimate of the width of the distribution (PDI)

Obtaining Size from the Correlogram

Number

Rel

ativ

e p

rop

ort

ion

Diameter (nm)5 50

1 1

Intensity(α d6)

Rel

ativ

e p

rop

ort

ion

Diameter (nm)5 50

1,000,000

1

Volume(α d3)

Rel

ativ

e p

rop

ort

ion

Diameter (nm)5 50

1

1,000

Intensity, Volume and Number DistributionsConsider a mixture of equal numbers of 5 and 50nm spheres

Intensity Distribution is calculated from the correlogram, Volume and Number are calculated from Intensity

Intensity, Volume and Number Distributions150 µM Lysozyme (14600 Da) and 50 mM Arginine (174 Da)

0

10

20

0.1 1 10

Rel

ativ

e p

rop

ort

ion

(%

)

Diameter (nm)

4.2 nm 94 %

3.7 nm 7 %

3.3 nm 0 %

IntensityVolumeNumber

0.67 nm 6 %

0.65 nm 93 %

0.64 nm 100 %

DLS Summary

› Measures the Brownian motion of particles and uses to calculate hydrodynamic size

› Highly sensitive to large particles

› Simple cuvette based method

› Large range - < 1 nm — > 1 µm


› SEC-LS

› Interaction Parameters

Outline - Theory

Resolution

› DLS is a relatively low resolution technique

› Growth of trace amounts of aggregate, even dimer and trimer can be detected - but not necessarily resolved

› Greater resolution can be achieved through separation

Resolution - SEC

› Batch light scattering methods may struggle to fully resolve and characterise smaller oligomericstates

› Malvern’s Viscotek SEC systems provide a separative solution to this problem

Resolution - SEC

› Separation based on the speed of flow through a porous column – based on size rather than molecular weight

Sample Solution

Large Component

Small Component

Small Particle = Slow FlowLarge Particle = Fast Flow

Traditional Concentration Detection

› Refractive Index and absorbance spectroscopy detectors

› Concentration detectors alone allow relative MW to be measured

› Retention volumes/times of sample components are compared with those of known standards

SEC Detection

› Traditionally, absorbance or refractive index detector used to analyse eluent as it flows off column

› Separate run of markers of known Mw used to calculate Relative Mw

Biorad Sigma-AldrichVitamin B12 1.35 kDa Cytochrome c 12.4 kDaEquine Myoglobin 17 kDa Carbonic anhydrase 29 kDaChicken ovalbumin 44 kDa BSA 66 kDaBovine Gam-Glob 158 kDa Alc. Dehydrogenase 150 kDaThyroglobulin 670 kDa β-Amylase 200 kDa

Blue Dextran 2000 kDa

Relative Mw

Parameter Hexamer Tetramer Dimer Monomer

Peak RV (ml) 13.2 14.2 16.0 16.9

RMW (kDa) 217 147 73 51

› Mean Average count rate measured at multiple concentrations and used to calculate absolute molecular weight and/or A2

› No resolution possible, average Mw is generated


Time

Inte

nsi

ty

(kcp

s) Mean Count Rate

125

120

Relative Mw

SLS – Molecular Weight

› Relationship of Mw with scattered light intensity:

› If C = 0 and scattering is isotropic:

25 °C

56 °C

C2APM

1

R

KC2

θ wθ

wθ M

1

R

KC

1/Mw


› Combination of concentration and light scattering detector gives absolute Mw from a single run

1/Mw


› Relationship of Mw with scattered light intensity:

› If C = 0 and scattering is isotropic:

25 °C

56 °C

C2APM

1

R

KC2

θ wθ

wθ M

1

R

KC

1/Mw

RALS – Isotropic Scatterers

› For isotropic scatterers:1/Pθ = 0

› No angular dependence

› Scattering can simply be measured at 90°

C2APM

1

R

KC2

θ wθ

› 1/Pθ – Different sized molecules scatter light in different directions with different intensity

› Small molecules of < ~ 30 nm diameter (< 1/20 of incident laser λ) scatter light evenly in all directions (isotropic scattering)

› Larger molecules scatter light in different directions with different intensities (anisotropic scattering)

Angular dissymmetry

Nointerference

LALS – Anisotropic Scatterers

› Light scattered at 0° cannot be directly measured

› Instead, we measure at 7° in order to minimise the effect of angular dependence

C2APM

1

R

KC2

θ wθ

MALS 20 – Anisotropic Scatterers

› Light scattered at 0° cannot be directly measured

› By plotting KC/Rθ as a function of sin2(θ/2) we can extrapolate back to 0°

› Mw can be calculated from the KC/Rθ at the intercept

› Rg can be calculated from the initial slope of the line

C2APM

1

R

KC2

θ wθ

MALS 20

› MALS-20 measures scattered light intensity over up to 20 angles and extrapolates back to zero in order to calculate molecular weight

› Allows accurate calculation of Radius of Gyration (Rg) for anisotropic scatterers

› Rg gives useful information on the morphology of aggregates

SEC-LS summary

› High-Resolution purity, DH and Mw analysis

› Using a SEC-MALS 20 detector allows accurate Mw analysis of impure samples

› Conformation analysis (Rg and Intrinsic Viscosity)

› Columnar interference can be a problem

› Separative Range: < 1 nm – 100 nm


› SEC-LS

› Zeta Potential

Outline - Theory

Zeta Potential (ZP) - Charge

› Zeta potential is the magnitude of charge at the slipping plane

› It is the zeta potential, not the surface charge, that determines inter-particle electrostatics

› Magnitude indicates the solution stability

› Electrophoretic Light Scattering: Measurement of electrophoretic mobility based on light scattering

› Particles are not separated, since the direction of the electric field switches continuously during measurement

› Particles analysed, therefore, under their formulation conditions

Measuring Zeta Potential (ELS)

Particle velocity V=0

Scattered light has same frequencyas incident laser

F1

Particle velocity V>0

vScattered light now has

greater frequencythan incident laser

F1

Laser Doppler Electrophoresis

Phase Analysis Light Scattering (PALS)

Interference produces modulated beam with frequency equal to difference between F1 and F2 – Beat Frequency

F2

F1

A AB

Electrophoretic Light Scattering

› High zeta potential High electrophoretic mobility Large difference between scattered and ref. frequencies High Beat Frequency

› Beat frequency is then combined with a modulated reference frequency, produced by a piezoelectric crystal, in order to generate a phase plot

Phase Plot

› Shows the phase difference between the beat frequency and the modulator reference frequency over time

› Phase/Time = Frequency

› Frequency α Zeta Potential -14

-12

-10

-8

-6

-4

-2

0

2

4

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Pha

se (

rad)

Time (s)

Phase Plot

Time (s)

Pha

se (

rad)

› Theory

› Applications and specifications

Outline

› Batch (DLS)› Separations (SLS)› Stability Prediction (interaction

parameters)

› Hardware

Outline – Applications and Specifications

Applications

› DLS is widely used for polymer analysis

› The sensitivity to larger particles makes it ideal for quality control

› The fundamentals of the measurement method makes the technique ideal for monitoring conformational changes

Pegylation Analysis

› Pegloticase has been reported to cause 92 % of patients to develop abs against it

› 10kDa PEG moiety may be cause of immunogenicity

› Attempts to conjugate Urc to smaller PEG moieties

Zhang et al. (2012) PlosOne7(6): e39659

PEG-UrcConjugate

Z-average Diameter

(nm) PDImPEG-rCU-1 17.7 0.062

mPEG-rCU-2 38.6 0.519

mPEG-rCU-3 22.4 0.287

Stability

› DLS extremely sensitive to protein aggregate formation

› To the right are the results of an IgG storage and stability study

› The aggregates formed upon freeze thaw, and also the larger aggregates formed at 25 °C, exist only in trace amounts.

Green = Freeze/thaw X 5Blue = 4 °C

Red = 25 °C

Polymer Conformation

› Mark Houwink relationship:

D = kM-α D = Diffusion CoefficientM = Molecular Weightα = Compactnessk = constant for a particular

polymer in a solvent

› Slope of plot of D/M gives compactness

› Since D α DH, slope of DH/M also gives compactness


› α = 1Rigid rod

› α = 0.5 – 0.67 Random Coil

› α = 0.3 Sphere

› Polystyrene in toluene adopts a spherical conformation

α = 0.31

Polystyrene in Toluene


› Peltier Temperature control allows highly precise thermal profiling

› Allows analysis of temperature-dependent phase transitions


› Phase Transition of Poly(N-isopropylacrylamide) at 32 °C

› Collapse of random coils into globules leads to increase in DH and RI

› Globule size is more uniform than coil size

PNIPAM in DI Water

10 °C : PDI = 0.49140 °C : PDI = 0.087


› Increase in DH

› Decrease in Mean Count Rate

› Particles are swelling as the temperature increases

Polymer Dispersion (confidential)

› Used to study silica nanoparticles modified with polymer brush

› Brush growth measured upon addition to aqueous solution

› 450 nm particle – early termination during production

› 120 nm particle – polymers assume coiled conformation

› 200 nm particle – polymers assume linear conformation


Cheesman et al. (2013) Langmuir 29: 6131-6140

Protein Aggregation

› Amyloid formation of PGK in 190 mM NaCl, pH 2, monitored using SLS, DLS and CD

› Data suggested a 2-step model for protofibril formation:

Step 1 - critical oligomer formationStep 2 - clustering of critical oligomers

› Aggregation is coupled with β-sheet formation

Modler et al. (2003) JMB 325: 135-148

Antibody Binding Studies

› Size changes upon binding of IgGwith GNP-protein A conjugate measured

› DLS allows binding to be followed over time after mixing of the two proteins (top)

› Alternatively, binding can be monitored as a function of component concentration (bottom)

› Quick, simple and inexpensiveJans et al. (2009) Anal Chem 81: 9425-9432

DLS Summary

› Quick analysis of size and purity (DH and PDI)

› DLS can be used to assess changes in polymer conformation

› High sensitivity means polymers can be assessed in solution or attached to other particles


parameters)

› Hardware


DLS – Low Resolution

› Applications so far have involved analysis of change/detection

› SEC-separation – necessary for high resolution analysis and quantification

› Range of detectors allows complete orthogonal analysis

› SLS can now be used to assess the molecular weights present in polydisperse samples

SEC Detection

UV or Refractometer ConcentrationRelative MW

RALS and LALS Absolute MW

MALS 20 Absolute MWRg

DLS DHAbsolute MW

Viscometer Intrinsic Viscosity

PMMA using Conventional Calibration

› Polystyrene standards used to build a calibration curve, used to calculate RMw of 95kDa Poly methyl methacrylate

› RMw = 88kDa

› The difference is due to the difference in structure between the PS and the PMMA

Poly (Methyl Methacrylate) (PMMA)

Parameter Parameter

Peak RV (ml) 18.91

Mw (kDa) 94.07

Mn (kDa) 45.82

Mw / Mn 2.053

Rg (nm) 10.2

› SEC-MALS gives absolute MW – independent of column retention volume

› Additionally, Rg can be measured across (most of) the peak – in this case Rhmust be relatively high

Uses of Relative Mw

Parameter Branched PS Linear PSRelative MW (kDa) 720 140

Absolute MW (kDa) 910 144

2005-10-08_19;37;32_AT_comb-ps_BPS_B108-1_01.vdt: Refractive Index

-43.03

-6.10

30.82

67.75

104.67

141.59

Re

fra

ctiv

e In

de

x (

mV

)

Retention Volume (mL)0.10 3.59 7.08 10.57 14.06 17.55 21.04 24.53 28.02 31.51 35.00

Conformation

› We saw earlier how comparison of Rh and MW can also be used to assess conformation and branching in batch mode – DLS can also be used as an SEC detector

› Comparison of relative MW and absolute MW can also be used to assess conformation/branching

› Use of a Viscometry detector also gives conformation information

Intrinsic Viscosity

Sample Mw ή (dl/g) DHWith Ca2+ 77.5 0.0531 4.0

Without Ca2+ 78.9 0.5134 8.6

Retention Volume (ml) Retention Volume (ml)

Conformation

› Intrinsic Viscosity (IV) α 1/Molecular Density (d)

› Knowledge of IV and Mw also allows calculation of Rh› We have, therefore, 2 methods with which to calculate the

hydrodynamic sizeDLS detector Viscometry and SLS detectors

0.0

80.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

Re

fract

ive

Ind

ex R

esp

on

se (

mV

)

0.400

1.900

0.500

0.600

0.700

0.800

0.900

1.000

1.100

1.200

1.300

1.400

1.500

1.600

1.700

1.800

Log

Rh

-0.300

1.900

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

Log

Ra

diu

s o

f Gyr

atio

n (

Tw

o A

ngle

)

-1.0

30.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

26.0

28.0

Vis

com

ete

r D

P R

esp

on

se (

mV

)

-1.0

240.0

15.0

30.0

45.0

60.0

75.0

90.0

105.0

120.0

135.0

150.0

165.0

180.0

195.0

210.0

225.0L

ow

An

gle

Lig

ht S

catte

ring

Res

po

nse

(m

V)

11.40 22.00

Retention Volume (mL)

12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0

Data File: 2005-07-15_13;10;21_PROBE_3_01.vdt Method: p-50-0002.vcm

Rh = 6 nm

Rg = 19 nm (1st point at which it can be measured here)

Conformation – Rh, Rg and IV

Rh = ~ 20 nm

IgG aggregation

Parameter Aggregates Dimer Monomer

Peak RV (ml) 13.23 14.00 15.80

Mw (kDa) 7661 309.2 147.4

Mn (kDa) 674.1 308.6 147.2

Mw / Mn 11.36 1.001 1.001

Rg (nm) 26.6 - -

Peak Wt % (RI) 1.4 6.5 92.1

IgG aggregation

Aggregate

Monomer

IgG aggregation

Parameter Aggregates Dimer Monomer

Peak RV (ml) 13.23 14.00 15.80

Mw (kDa) 7661 309.2 147.4

Mn (kDa) 674.1 308.6 147.2

Mw / Mn 11.36 1.001 1.001

Rg (nm) 26.6 - -

Peak Wt % (RI) 1.4 6.5 92.1

› Aggregates scatter anisotropically

› LALS or MALS must be used

Pepsin Degradation Products

Parameter Aggregates Monomer Degraded protein

Peak RV (ml) 10.84 18.33 20.92

Mw (kDa) 4431 34.7 6.4

Mn (kDa) 3892 34.4 4.7

Mw / Mn 1.138 1.01 1.4

Rg (nm) 69.9 - -

Peak Wt % (RI) 0.8 56.9 42.3

Pepsin aggregation

› Pepsin aggregates scatter anisotropically

› LALS or MALS must be used

Parameter Aggregates Monomer Degraded protein

Peak RV (ml) 10.84 18.33 20.92

Mw (kDa) 4431 34.7 6.4

Mn (kDa) 3892 34.4 4.7

Mw / Mn 1.138 1.01 1.4

Rg (nm) 69.9 - -

Peak Wt % (RI) 0.8 56.9 42.3

Bovine Serum Albumin

Parameter Aggregates Trimer Dimer Monomer

Peak RV (ml) 13.84 14.31 15.37 17.05

Mw (kDa) 379.72 206.7 135.3 66.8

Mn (kDa) 338.64 205.9 135.1 66.7

Mw / Mn 1.121 1.004 1.002 1.001

Rg (nm) - - - -

Peak Wt % (RI) 4 5.2 16.2 74.7

Bovine Serum Albumin

› All components scatter isotropically

› RALS will give just as accurate a MW as LALS and MALS

Parameter Aggregates Trimer Dimer Monomer

Peak RV (ml) 13.84 14.31 15.37 17.05

Mw (kDa) 379.72 206.7 135.3 66.8

Mn (kDa) 338.64 205.9 135.1 66.7

Mw / Mn 1.121 1.004 1.002 1.001

Rg (nm) - - - -

Peak Wt % (RI) 4 5.2 16.2 74.7

Thyroglobulin

Parameter Aggregates Monomer

Peak RV (ml) 11.60 12.50

Mw (kDa) 2588.0 686.0

Mn (kDa) 1601.0 681.5

Mw / Mn 1.617 1.007

Rg (nm) - -

Peak Wt % (RI) 16.7 83.3

Natural Polymer Comparison

› Lots of parameters, giving complete orthogonal analysis of polymers

› But how can we compare between polymers?

Parameter Pectin HPC Gum Arabic

Peak RV (ml) 17.03 9.51 17.57

Mw (kDa) 115.08 78.59 1015.00

Mn (kDa) 50.50 55.70 847.57

Mw / Mn 2.279 1.411 1.197

Rg (nm) 25.8 20.0 8.9

Rh 17.0 10.4 10.2

IV (dl/g) 3.673 1.072 0.158

Conformation plots – Rg vs. Log MW

Pullulan

Dextran

Gum Arabic

PectinHPC

› The Gum Arabic plot is below those of the other polymers

› Due to Gum Arabic being more compact and dense

Mark-Houwink plots – Log IV vs. Log MW

Pullulan

DextranGum Arabic

Pectin HPC

› The Gum Arabic plot is below those of the other polymers

› Due to Gum Arabic being more compact and dense

Ultr

a V

iole

t Re

spo

nse

(m

V)


10,82 11,73 12,64 13,55 14,46 15,37 16,28 17,19 18,10

149,28

133,92

118,56

103,20

87,84

72,49

57,13

41,77

26,41

164,64

11,05

9,91 19,01

Viscosity Detector

Light Scattering

RI-Detector

UV-Detector

PEGylated Protein

› Each detector responds to the sample differently

0,0

1,8 e-4

1,8 e-5

3,5 e-5

5,3 e-5

7,0 e-5

8,8 e-5

1,1 e-4

1,2 e-4

1,4 e-4

1,6 e-4

Co

nc A

0,0

2,1 e-4

2,1 e-5

4,2 e-5

6,3 e-5

8,5 e-5

1,1 e-4

1,3 e-4

1,5 e-4

1,7 e-4

1,9 e-4

Co

nc B

12,00 19,00


12,8 13,2 13,6 14,0 14,4 14,8 15,2 15,6 16,0 16,4 16,8 17,2 17,6 18,0 18,4

Retention Volume

Prot

ein

conc

entr

atio

n

PEG

con

cent

raio

n

Conc. ProteinConc. PEG

Protein-Aggregates

Protein-Monomer

PEG

Conjugation analysis

› The concentration of each component across the chromatogram can be solved

› They did not co-elute

0,0

5,5 e-4

5,5 e-5

1,1 e-4

1,7 e-4

2,2 e-4

2,8 e-4

3,3 e-4

3,9 e-4

4,4 e-4

5,0 e-4

Co

nc

A

0,0

3,5 e-4

3,5 e-5

7,0 e-5

1,0 e-4

1,4 e-4

1,7 e-4

2,1 e-4

2,4 e-4

2,8 e-4

3,1 e-4

Co

nc

B

10,00 16,00


10,5 10,8 11,1 11,4 11,7 12,0 12,3 12,6 12,9 13,2 13,5 13,8 14,1 14,4 14,7 15,0 15,3 15,6

Retention volume

Prot

ein

conc

entr

atio

n

PEG

con

cent

ratio

n

Conc. ProteinConc. PEG Protein-

Monomerwith PEG

Protein-Aggregateswith PEG

Conjugation analysis

› After further method development, the components co-elute

0,0

87,0

7,0

14,0

21,0

28,0

35,0

42,0

49,0

56,0

63,0

70,0

77,0

Re

fra

ctiv

e In

de

x R

esp

on

se (

mV

)

0,0

145,0

11,0

22,0

33,0

44,0

55,0

66,0

77,0

88,0

99,0

110,0

121,0

132,0

Ultr

a V

iole

t Re

spo

nse

(m

V)

11,90 18,80


12,4 12,8 13,2 13,6 14,0 14,4 14,8 15,2 15,6 16,0 16,4 16,8 17,2 17,6 18,0

Data File: 006A RXN SOLN (6.vdt) Method: PEG-Prot-0014.vcm

RI-Signal (sees Protein + PEG)UV-Signal (sees Protein only)

PEGylated Proteins – Compositional Analysis

RI-SignalUV-Signal

0,0

2,2 e-4

2,2 e-5

4,4 e-5

6,6 e-5

8,8 e-5

1,1 e-4

1,3 e-4

1,5 e-4

1,8 e-4

2,0 e-4

Co

nc

(Pro

tein

+P

EG

)

0,0

2,2 e-4

2,2 e-5

4,4 e-5

6,6 e-5

8,8 e-5

1,1 e-4

1,3 e-4

1,5 e-4

1,8 e-4

2,0 e-4

Co

nc

Pro

tein

0,0

2,2 e-4

2,2 e-5

4,4 e-5

6,6 e-5

8,8 e-5

1,1 e-4

1,3 e-4

1,5 e-4

1,8 e-4

2,0 e-4C

on

c P

EG

11,90 18,80


12,4 12,8 13,2 13,6 14,0 14,4 14,8 15,2 15,6 16,0 16,4 16,8 17,2 17,6 18,0


Conc. ProteinConc. PEGConc. Protein + PEG

Protein

PEG

Protein + little PEG



0,0

2,2 e-4

2,2 e-5

4,4 e-5

6,6 e-5

8,8 e-5

1,1 e-4

1,3 e-4

1,5 e-4

1,8 e-4

2,0 e-4

Co

nc

(Pro

tein

+P

EG

)

3,000

5,000

3,200

3,400

3,600

3,800

4,000

4,200

4,400

4,600

4,800

Lo

g M

ole

cula

r W

eig

ht

11,90 18,80


12,4 12,8 13,2 13,6 14,0 14,4 14,8 15,2 15,6 16,0 16,4 16,8 17,2 17,6 18,0


Conc. Protein + PEGlog MW

17kDaPure

Protein

3 kDaPure PEG

20 kDaprotein:PEG

1:1

23 kDaprotein:PEG

1:2

Polyethylene – HT-GPC

› Polyethylene is one of the worlds most widely used polymers

› Wide range of applications, from pipe lining and wire coatings to shopping bags

› Properties highly dependent on MW, analysis of which is essential for QC

› Insoluble at room temperature – must be analysed at 140 – 160 °C

Polyethylene – HT-GPC

LALSRALSViscosityRI

Param Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7

Mw (kDa) 141.8 141.8 140.8 141.0 140.9 140.4 141.7

Mn (kDa) 35.2 38.6 40.7 41.5 36.0 35.0 35.3

Mw / Mn 4.03 3.68 3.46 3.40 3.92 4.02 4.01

Rh 11.27 11.38 11.22 11.15 11.27 11.17 11.27

IV (dl/g) 0.95 0.94 0.93 0.91 0.94 0.94 0.94

Separations Summary

› High-Resolution purity, DH and Mw analysis

› Using a SEC-MALS 20 detector allows accurate Mw analysis of impure samples

› Conformation analysis (Rg and Intrinsic Viscosity)

› Columnar interference can be a problem

› Separative Range: < 1 nm – 100 nm


parameters)

› Hardware


Zeta Potential – Isoelectric Point Determination

-3

0

3

3 4 5 6 7 8 9 10

Mo

bili

ty (

µm

cm/V

s)

pH

HEPES, Malvern ZSP, Unpublished

NaCl, Malvern ZSP, Corbett and Jack (2011) - Colloids and Surfaces 376:31-41

› Derived from the slope of the Debye plot.

› Thermodynamic Interaction parameter

› Representative of the magnitude of particle-solvent interactions

Synapse Polymer

SLS – 2nd Virial Coefficient (A2)

SLS – 2nd Virial Coefficient (A2)

Ribonuclease (14.9 kDa)

Lysozyme (14.6 kDa)

Small ab fragment (20.7 kDa)

Ovalbumin (46.8 kDa)

BSA (71.4 kDa)Polystyrene standard (95.6 kDa)

› Also known as the DLS interaction parameter

› A measure of the dependence of the diffusion coefficient (Brownian Velocity) on the concentration.

› Thermodynamic component (dependent on A2)

› Hydrodynamic component

HSA

pH 7

KD = 8.33 ml/g

Concentration (mg/ml)

Diff

usio

n C

oeffi

cien

t (µm

/s)

pH 4

KD = 0.75 ml/g

Concentration (mg/ml)

Diff

usio

n C

oeffi

cien

t (µm

/s)

DLS – Dynamic Virial Coefficient (KD)

DLS/SLS Thermal Trends

› Monitor the Z-average diameter, or SLS count rate, whilst increasing temperature

› An accurate Tagg can be calculated

› Tagg is a stability predictor

Orthogonal Stability Prediction

› Tagg gives a direct measure of biopharmaceutical stability – at high temperatures

› Effect of formulation composition on biopharmaceutical stability can vary significantly with temperature

› For example, the effect of glycerol and NaCl on Uricaseinactivation is heavily temp-dependent (Caves et al. (2013) Biochemistry 52: 497-507)

› KD, A2 and ZP allow assessment under ambient conditions, but only of interactions involving native protein

IgG Formulation Development

Form-ulation

KD

(ml/g)

A2 (10-6 ml mol/g2)

ZP

(mv)

Tagg

(°C)KCl

Tween -5.2 -15 1.7 66KCl

NaCl -4.7 23 -1.1 70TweenLactose -9.7 104 3.4 72SucroseMannitol 31.9 1275 11.1 > 90

IgG Formulation Development

How Does Arginine Inhibit Aggregation? – SLS

› 50 mM arginine increases the temperature of aggregation onset by 2.5 °C

0

200

400

600

800

1000

1200

1400

1600

1800

50 55 60 65 70 75

Mea

n C

ou

nt

Rat

e (k

cps)

Temperature (°C)

PhosPhos + Arg

How Does Arginine Inhibit Aggregation? – DLS

25.0 °C71.5 °C76.5 °C84.0 °C

phos

phos + arg

How Does Arginine Stabilise Protein

› Leads to resistance to the initiation of aggregation (SLS)

› Alters the aggregation mechanism (DLS)

› Arginine’s aggregation-inhibition property is not due to it increasing surface charge of native protein (ELS)

Formulation (Native) Zeta Potential (mv)

Phos 7.0 ± 0.1

Phos + Arg 4.6 ± 0.5

Aggregate Prediction Summary

› A2, KD, and ZP allow prediction of aggregation propensity at ambient temperatures

› Tagg allows analysis of aggregation during denaturation

› Together, these parameters allow an orthogonal approach to aggregation prediction


parameters)

› Hardware


The Malvern Zetasizer Range

µV Nano APS

› DLS instruments – assess size (DH) and purity (PDI)

› SLS-capable – assess Mw for pure samples

› Aggregation prediction – Calculate KD, A2, ZP and TAgg

Zetasizer µV

› 60mW 830nm laser

› 90° Detection

› Low volume (2µL)

› Peltier Temperature control

› Perfect for use as a Flow mode detector for both Dhand Mw measurements

Zetasizer APS

› 60mW 830nm laser

› 90° Detection

› Automated Plate-Sampler

› Low Volume (20 µl)

› Peltier Temperature control

Zetasizer Nano

› 4mW or 10 mW 633nm He-Ne laser

› 173° (NIBS) or 13° Detection

› Peltier temperature control

› Flow mode capability

› ELS option

Zetasizer Nano

› DLS only (S) or ELS only (Z)

› DLS and ELS (ZS)

› ZSP (10 mW laser) –designed with protein applications in mind

› Can measure ZP of protein at low conc. (< 1 mg/ml)

Zetasizer Nano

› Folded Capillary Cell for ELS (ZP)

› Capillary design maximises inter-electrode distance

› This minimises the field strength produced by any given voltage

› Minimises stress on sample during measurement

The Malvern Zetasizer Range

µV Nano APS

Requires only 2µl sample

Perfect for flow mode

NIBS

ELS option

Flow mode capability

Automatedplate sampler

Perfect size-screening instrument

The Viscotek Range

› SEC systems and detectors (detectors compatible with other SEC systems)

› UV PDA detector –Simultaneous Measurement from 190 – 500 nm

› RI Detector – Allows conc. analysis of non-absorbing molecules

The Viscotek Range

› Viscometer – Calculate Intrinsic Viscosity for conformation analysis

› Zetasizer µV – DH

› RALS and LALS – Simple Mw calculation

› SEC-MALS 20 – Calculation of accurate Mw for proteins of all sizes and Rg

SEC-MALS 20

› Circular vertical flow cell –light always enters and exits the cell at 90°

› Minimises effects of flare and RI changes

› Maximises signal-to-noise

SEC-MALS 20

› Lateral flow cell –Detectors each measure different scattering volumes at ‘incorrect’ angles

› SEC-MALS 20 flow cell –Light always enters and leaves at a right angle

Feature Aggregates

20 Angles More angles than any other SEC system available.

Low Angle Sensitivity Lowest angles have comparable sensitivity to higher ones -lower concentrations can be measured with greater accuracy.

Absolute MwHigh res. Mw distribution – Mw calculated independently of retention volume

Accurate RgMeasurements of Rg using MALS – Size value based on mass distribution

Circular Vertical flow cell Minimal noise, especially at low angles (see point 2).

63 µl Flow Cell Smaller than other MALS flow cells

Versatility Connect to any third-party system or use with a Viscoteksystem

SEC-MALS 20 Features

The Viscotek Range

› Relative Mw› Absolute Mw

› Rg› DH› Intrinsic Viscosity

› All after separation – perfect for high res. Analysis of impure samples

Cuvette-based Size and Zeta Potential measurements without separation

Separation - high-res analysis, including accurate MW and Rg of impure samples

Summary

Thank You for Listening

Questions?

Michael CavesProduct Technical Specialist for Biophysical [email protected]

› Malvern’s NanoSight range provides data on particle size distribution, concentration and aggregation, with much higher resolution than conventional light scattering

› The addition of fluorescence options further extends the capabilities of the instruments, allowing truly multi-parametric characterisation of nanoparticles.

› NanoSight instruments provide scientists with detailed data and knowledge of nanoparticle systems that was previously unavailable.

2

Nanoparticle Tracking Analysis (NTA)

Particles are Visualised Directly, in Real Time

2

Microvesicles purified from serum by ultracentrifugation, sizes 100-500nm. This field of view is approximately 120 x 100 microns.

› Particles are too small to be imaged by the microscope

› The particles seen as light points moving under Brownian motion

› This is visualisation of scatter (not a resolved image)

› Speed of particles varies directly with particle size

3

Principle of Measurement

› Nanoparticles move under Brownian motion due to the random movement of solvent molecules surrounding them.

› Small particle move faster than larger particles.

› Diffusion Coefficient can be calculated by tracking the movement of each particle and then through application of the Stokes-Einstein equation particle size is calculated.

4

Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample.

analysistrackingcapture

Nanoparticle Tracking Analysis

5

The Nanoparticle Tracking Analysis software allows for captured video footage to be simultaneously tracked and analysed…

100+200nm nanoparticles being tracked and analysed by NanoSight NTA 2.3

Particle Sizing in action - Software Analysis

Concentration (Number Count)

Size in nm

6

Lower Detection Limit related to:› Material type › Wavelength and power of illumination

source › Sensitivity of the camera

Size Concentration

Minimum concentration related to:› Insufficient count for robust statistics

(requiring longer analysis time)

10 – 40 nm

1000 – 2000 nm

Upper Detection Limit related to:› Limited Brownian motion› Viscosity of solvent

Approx 106 / ml

Maximum concentration related to:› Inability to resolve neighboring particles› Tracks too short before crossing occurs

Approx 109 / ml

NTA Detection Limits

True size distribution profile

7

Mixture of 100nm and 200nm latex microspheres dispersed in water in 1:1 ratio

Particle distribution displays a number count vs particle size.

Size in nmConcentration (Number Count)

8

True size distribution profile

164

NTA accurately tracked 3

sub-populations

of nanoparticles

Mix of 3 populations

in a real sample

Size in nm

Concentration (N

umber C

ount)

Size in nm

Size in nm

nm

nm

nm

9

Number count of particle in a determined volume :

Particles Concentration – Number Count

Global concentration of all populations

Concentration of a population selected by user

Con

cent

ratio

n (

Num

ber

Cou

nt)

Size in nm

100 PS60nm Au

30nm Au

In this mixture of 30 nm and 60 nm gold nanoparticles mixed with 100 nm polystyrene, the three particle types can be clearly seen in the 3D plot confirming indications of a tri-modal given in the

normal particle size distribution plot. Despite their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS.

10

Resolving mixtures of different particle types through Scatter Intensity

11

NanoSight systems can be fitted with these lasers

Applications in:› Nanoparticle toxicity studies› Nano-rheology› Bio-diagnostics› Phenotyping specific exosomes

› Laser diode capable of exciting fluorophores and quantum dots› Choice of long pass or band pass filters allow suitably-labelled nanoparticles to be tracked in high backgrounds

405nm 488nm 532nm 635nm

Fluorescent Mode available

Analysis of 100 nm Fluorescence standard particles suspended in FBS*

Modal particle size peaks: 103 nm

Concentration: 9.5 x108 particles/ml

› 100 nm fluorescently labelled particles suspended in 100% FBS› Sample maintained at a constant temperature (37oC)› Sample viscosity was 1.33 cP

• Excitation wavelength - 532 nm* Fetal Bovine Serum

No filter Same sample, 565 nm Long pass optical filter

12

This allows selective visualising of

fluorescent/fluorescently labelled particles

Size in nm

13

Current methodologies for counting such as plaque assay only count infectious particles which often represent a small component in attenuated vaccines i.e. perhaps only 1% of product is infective.

The ability to count viruses in liquid suspension is essential for those working in vaccine development.

Particle aggregation and yield quality are factors which need to be understood when developing these viral vaccines.

Example : Purified Influenza Virus

› NanoSight technology has a unique application in the detection of early stage aggregation in protein therapeutics

› Protein monomer is too small to be individually resolved by this technique, but early stage aggregates are readily detected

› Protein monomer at high concentration causes high background noise in image, with the aggregate forming the resolvable particles

› Both size and number of aggregates can be calculated and studied, providing insight into product stability.

Data reproduced from Filipe, Hawe and Jiskoot (2010) Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2

14

Application: Protein Aggregation at 50°C

15

The ability to target drugs to a localised area in the body allows for lower concentrations to be used and provides optimal delivery concentration.

Other delivery vehicles can be analysed including degradable polymeric nanoparticles, liposomes, micelles, dendrimers, solid lipid nanoparticles and metallic nanoparticles.

liposomes used in drug delivery

Application: Drug Delivery

› Multi-walled Carbon nanotubes

› Cosmetics

› Foodstuffs

› Ink jet inks and pigment particles

› Nanobubbles

› Drug delivery

› Extracellular Vesicles (Exosomes and microvesicles)

› Nanoparticle Toxicology

› Protein Aggregates

› Virus and VLP samples

› Quantum dots

› Magnetic Nanoparticles

› Polymers and colloids

› Ceramics

› Fuel additives

NTA is proven on for a wide range of nanoparticles

Size

Number or concentration

Polydispersity

Relative Light Intensity

Fluorescence

NTA Summary – Sub-visible particles

17

Thank You for Listening

Questions?

Michael CavesProduct Technical Specialist for Biophysical [email protected]

RI detector

› Standard differential refractive index detector

› Calibration performed using a narrow polymer standard (i.e. polystyrene)

ii Cdc

dn

n

KRI

0

refractive index increment

K instrument constant

dc

dn

C concentration (g/L)

0n refractive index of solvent

UV-Vis Absorbance

› Beer-Lambert Law used to calculate concentration from absorbance signal

› Extinction coefficient can be calculated by measuring the absorbance at different concentrations:

› Malvern’s multi-channel detector allows detection of multiple wavelengths simultaneously

A = εlc

ε = dA/dc

A = absorbance (abs.)ε = extinction coefficientl = cell path lengthc = concentration

144

UV-Vis Absorbance

One of the most widely used analytical techniques

Applications for Organic and Inorganic species

Beer’s Law (Concentration Detector)

Conventional UV-Vis spectrophotometers contain singlechannel detectors

A = εlcε = dA/dc

A = absorbance (abs.)ε = extinction coefficientl = cell path lengthc = concentration

145

Advantages over traditional UV-Vis detectors

Multi-channel detector controlled by a microprocessor

Detects multiple wavelengths simultaneously

PDA can provide correlation between molecular weight and chemical composition when combined with Viscotek GPC instrumentation

UV-Vis Absorption PDA – Concentration Detector146

3D spectrum from the UV PDA

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