The Illumination of Structure using Light Scattering. Michael Caves Product Technical Specialist for Biophysical Characterisation

The Illumination of Structure using Light Scattering Michael Caves Product Technical Specialist for Biophysical Characterisation

Light Scattering Laser Scattered light Detector Scattered light intensity α size 6

Static Light Scattering (SLS) Mean Average count rate measured 125 Intensity (kcps) Mean Count Rate 120 Time All light scattering methods discussed measure this, in addition to the technique-specific parameters

Outline Theory Applications and specifications

Outline - Theory Dynamic Light Scattering SEC-LS Interaction Parameters

Static Light Scattering (SLS) Mean Average count rate measured at multiple concentrations and used to calculate absolute molecular weight and/or A 2 125 Intensity (kcps) Mean Count Rate 120 Time No resolution possible, average M w is generated

Dynamic Light Scattering (DLS) Fluctuation Frequency of count rate measured and used to calculate hydrodynamic size Resolution Possible 125 Intensity (kcps) 120 Time 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

DLS Measures Fluctuations in the Scattered Light Intensity Caused by Brownian Motion Laser Small Particles Large Particles Intensity Intensity Time Time

DLS Method Summary Intensity Fluctuations Detected Correlation Correlogram Correlogram analysis Size(s) and Polydispersity

1 0 Time Correlation Coefficient Time Time 0 1 0 Time Intensity Intensity Correlation Coefficient (Auto)Correlation 0

1 0 Time Correlation Coefficient = 0 Time = 0 Time 0 1 0 Time Intensity Intensity Correlation Coefficient (Auto)Correlation 0

1 0 Time Correlation Coefficient = 1 Time = 1 Time 0 1 0 Time Intensity Intensity Correlation Coefficient (Auto)Correlation 0

1 0 Time Correlation Coefficient = 2 Time = 2 Time 0 1 0 Time Intensity Intensity Correlation Coefficient (Auto)Correlation 0

1 0 Time Correlation Coefficient Time = 3 Time 0 1 0 Time Intensity Intensity Correlation Coefficient (Auto)Correlation = 3 0

Time Time 1 0 Time Intensity Intensity Correlation Coefficient 0 1 0 Time Correlation Coefficient (Auto)Correlation 0

The Correlogram Small Particles 1 Intensity Time 0 Time Large Particles Intensity Correlation Coefficient 0 correlate LOG 1 Correlation Coefficient Time 0 0 Time

The Correlogram Intercept gives a measure of S/N Exponential decay lifetime indicates hydrodynamic size Gradient indicates sample polydispersity Baseline quality used to assess contributions of Large particles

Obtaining Size from the Correlogram Particle size information is obtained by analysing the correlogram with various algorithms CUMULANTS (ISO13321) DISTRIBUTION Single exponential fit Multi-exponential fit Mean size (z-avg diam.) Estimate of the width of the distribution (PDI) Distribution of particle sizes (by intensity, volume or number)

Intensity, Volume and Number Distributions Consider a mixture of equal numbers of 5 and 50nm spheres Intensity (α d 6 ) Volume (α d 3 ) Number Relative proportion 1 1,000,000 Relative proportion 1 1,000 Relative proportion 1 1 5 50 Diameter (nm) 5 50 Diameter (nm) 5 50 Diameter (nm) Intensity Distribution is calculated from the correlogram, Volume and Number are calculated from Intensity

Intensity, Volume and Number Distributions 150 µm Lysozyme (14600 Da) and 50 mm Arginine (174 Da) Relative proportion (%) 20 10 Intensity Volume Number 0.67 nm 6 % 0.65 nm 93 % 0.64 nm 100 % 0 0.1 1 10 Diameter (nm) 4.2 nm 94 % 3.7 nm 7 % 3.3 nm 0 %

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

Outline - Theory Dynamic Light Scattering SEC-LS Interaction Parameters

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 oligomeric states 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 Small Particle = Slow Flow Large Particle = Fast Flow Small Component Large Component Sample Solution

Traditional Concentration Detection Refractive Index and absorbance spectroscopy detectors Concentration detectors alone allow relative M W 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 M w used to calculate Relative M w Biorad Vitamin B12 1.35 kda Equine Myoglobin 17 kda Chicken ovalbumin 44 kda Bovine Gam-Glob 158 kda Thyroglobulin 670 kda Sigma-Aldrich Cytochrome c 12.4 kda Carbonic anhydrase 29 kda BSA 66 kda Alc. Dehydrogenase 150 kda β-amylase 200 kda Blue Dextran 2000 kda

Relative M w Parameter Hexamer Tetramer Dimer Monomer Peak RV (ml) 13.2 14.2 16.0 16.9 RM W (kda) 217 147 73 51

Relative Mw

SLS Molecular Weight Relationship of M w with scattered light intensity: KC R θ 1 M w P θ 2A 2 C 1/M w 25 C If C = 0 and scattering is isotropic: KC R 1 θ M w 56 C

SLS Molecular Weight Combination of concentration and light scattering detector gives absolute M w from a single run 1/M w

SLS Molecular Weight Relationship of M w with scattered light intensity: KC R θ 1 M w P θ 2A 2 C 1/M w 25 C If C = 0 and scattering is isotropic: KC R 1 θ M w 56 C

RALS Isotropic Scatterers For isotropic scatterers: 1/P θ = 0 KC R θ 1 M w P θ 2A 2 C No angular dependence Scattering can simply be measured at 90

Angular dissymmetry 1/P θ 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) No interference

LALS Anisotropic Scatterers Light scattered at 0 cannot be directly measured KC R θ 1 M w P θ 2A 2 C Instead, we measure at 7 in order to minimise the effect of angular dependence

MALS 20 Anisotropic Scatterers Light scattered at 0 cannot be directly measured KC R θ 1 M w P θ 2A 2 C By plotting KC/R θ as a function of sin 2 (θ/2) we can extrapolate back to 0 Mw can be calculated from the KC/R θ at the intercept Rg can be calculated from the initial slope of the line

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 (R g ) for anisotropic scatterers R g gives useful information on the morphology of aggregates

Size?

SEC-LS summary High-Resolution purity, D H and M w analysis Using a SEC-MALS 20 detector allows accurate M w analysis of impure samples Conformation analysis (R g and Intrinsic Viscosity) Columnar interference can be a problem Separative Range: < 1 nm 100 nm

Outline - Theory Dynamic Light Scattering SEC-LS Zeta Potential

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

Measuring Zeta Potential (ELS) 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

Laser Doppler Electrophoresis Particle velocity V=0 F 1 Scattered light has same frequency as incident laser Particle velocity V>0 F 1 v Scattered light now has greater frequency than incident laser

Phase Analysis Light Scattering (PALS) F 1 F 2 A B A Interference produces modulated beam with frequency equal to difference between F 1 and F 2 Beat Frequency

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 Phase Plot Shows the phase difference between the beat frequency and the modulator reference frequency over time Phase/Time = Frequency Phase (rad) Phase (rad) 4 2 0-2 -4-6 -8-10 Frequency α Zeta Potential -12-14 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (s) Time (s)

Outline Theory Applications and specifications

Outline Applications and Specifications Batch (DLS) Separations (SLS) Stability Prediction (interaction parameters) Hardware

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) PlosOne 7(6): e39659 PEG-Urc Conjugate Z-average Diameter (nm) PDI mpeg-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 Green = Freeze/thaw X 5 Blue = 4 C Red = 25 C 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.

Polymer Conformation Mark Houwink relationship: D = km -α D = Diffusion Coefficient M = Molecular Weight α = Compactness k = constant for a particular polymer in a solvent Slope of plot of D/M gives compactness Since D α D H, slope of D H /M also gives compactness

Polymer Conformation α = 1 Rigid rod Polystyrene in Toluene α = 0.5 0.67 Random Coil α = 0.3 Sphere α = 0.31 Polystyrene in toluene adopts a spherical conformation

Polymer Conformation Peltier Temperature control allows highly precise thermal profiling Allows analysis of temperature-dependent phase transitions

Polymer Conformation Phase Transition of Poly(Nisopropylacrylamide) at 32 C PNIPAM in DI Water Collapse of random coils into globules leads to increase in D H and RI Globule size is more uniform than coil size 10 C : PDI = 0.491 40 C : PDI = 0.087

Polymer Conformation Increase in D H Polymer Dispersion (confidential) Decrease in Mean Count Rate Particles are swelling as the temperature increases

Polymer Conformation 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 formation Step 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 IgG with 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 inexpensive Jans et al. (2009) Anal Chem 81: 9425-9432

DLS Summary Quick analysis of size and purity (D H 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

Outline Applications and Specifications Batch (DLS) Separations (SLS) Stability Prediction (interaction 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 Concentration Relative M W RALS and LALS Absolute M W MALS 20 Absolute M W R g DLS D H Absolute M W Viscometer Intrinsic Viscosity

PMMA using Conventional Calibration Polystyrene standards used to build a calibration curve, used to calculate RM w of 95kDa Poly methyl methacrylate RM w = 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 M w (kda) 94.07 M n (kda) 45.82 M w / M n 2.053 R g (nm) 10.2 SEC-MALS gives absolute M W independent of column retention volume Additionally, Rg can be measured across (most of) the peak in this case R h must be relatively high

Uses of Relative M w 141.59 104.67 R e fr a c tiv e In d e x ( mv) 67.75 30.82-6.10-43.03 0.10 3.59 7.08 10.57 14.06 17.55 21.04 24.53 28.02 31.51 35.00 Retention Volume (ml) 2005-10-08_19;37;32_AT_comb-ps_BPS_B108-1_01.vdt: Refrac tiv e Index Parameter Branched PS Linear PS Relative M W (kda) 720 140 Absolute M W (kda) 910 144

Conformation We saw earlier how comparison of R h and M W can also be used to assess conformation and branching in batch mode DLS can also be used as an SEC detector Comparison of relative M W and absolute M W can also be used to assess conformation/branching Use of a Viscometry detector also gives conformation information

Intrinsic Viscosity Retention Volume (ml) Retention Volume (ml) Sample M w ή (dl/g) D H With Ca 2+ 77.5 0.0531 4.0 Without Ca 2+ 78.9 0.5134 8.6

Conformation Intrinsic Viscosity (IV) α 1/Molecular Density (d) Knowledge of IV and M w also allows calculation of R h We have, therefore, 2 methods with which to calculate the hydrodynamic size DLS detector Viscometry and SLS detectors

Conformation R h, R g and IV Data File: 2005-07-15_13;10;21_PROBE_3_01.vdt Method: p-50-0002.vcm 80.0 75.0 30.0 28.0 240.0 225.0 1.900 1.800 1.900 1.800 70.0 26.0 210.0 1.600 1.700 Refractive Index Response (mv) 65.0 60.0 55.0 50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 Viscometer DP Response (mv) 24.0 22.0 20.0 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0-1.0 Low Angle Light Scattering Response (mv) 195.0 180.0 165.0 150.0 135.0 120.0 105.0 90.0 75.0 60.0 45.0 30.0 15.0-1.0 R g = 19 nm (1 st point at which it can be measured here) R h = ~ 20 nm R h = 6 nm 1.400 1.200 1.000 0.800 0.600 Log Radius of Gyration (Two Angle) 0.400 0.200 0.000-0.200-0.300 1.600 1.500 1.400 1.300 1.200 1.100 1.000 0.900 0.800 0.700 0.600 0.500 0.400 Log Rh 11.40 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 22.00 Retention Volume (ml)

IgG aggregation Parameter Aggregates Dimer Monomer Peak RV (ml) 13.23 14.00 15.80 M w (kda) 7661 309.2 147.4 M n (kda) 674.1 308.6 147.2 M w / M n 11.36 1.001 1.001 R g (nm) 26.6 - - Peak Wt % (RI) 1.4 6.5 92.1

IgG aggregation Aggregate Monomer

IgG aggregation Aggregates scatter anisotropically LALS or MALS must be used Parameter Aggregates Dimer Monomer Peak RV (ml) 13.23 14.00 15.80 M w (kda) 7661 309.2 147.4 M n (kda) 674.1 308.6 147.2 M w / M n 11.36 1.001 1.001 R g (nm) 26.6 - - Peak Wt % (RI) 1.4 6.5 92.1

Pepsin Degradation Products Parameter Aggregates Monomer Degraded protein Peak RV (ml) 10.84 18.33 20.92 M w (kda) 4431 34.7 6.4 M n (kda) 3892 34.4 4.7 M w / M n 1.138 1.01 1.4 R g (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 M w (kda) 4431 34.7 6.4 M n (kda) 3892 34.4 4.7 M w / M n 1.138 1.01 1.4 R g (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 M w (kda) 379.72 206.7 135.3 66.8 M n (kda) 338.64 205.9 135.1 66.7 M w / M n 1.121 1.004 1.002 1.001 R g (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 M W as LALS and MALS Parameter Aggregates Trimer Dimer Monomer Peak RV (ml) 13.84 14.31 15.37 17.05 M w (kda) 379.72 206.7 135.3 66.8 M n (kda) 338.64 205.9 135.1 66.7 M w / M n 1.121 1.004 1.002 1.001 R g (nm) - - - - Peak Wt % (RI) 4 5.2 16.2 74.7

Thyroglobulin Parameter Aggregates Monomer Peak RV (ml) 11.60 12.50 M w (kda) 2588.0 686.0 M n (kda) 1601.0 681.5 M w / M n 1.617 1.007 R g (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 M w (kda) 115.08 78.59 1015.00 M n (kda) 50.50 55.70 847.57 M w / M n 2.279 1.411 1.197 R g (nm) 25.8 20.0 8.9 R h 17.0 10.4 10.2 IV (dl/g) 3.673 1.072 0.158

Conformation plots R g vs. Log M W Pectin Dextran Pullulan HPC Gum Arabic 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 M W Pectin HPC Dextran Pullulan Gum Arabic The Gum Arabic plot is below those of the other polymers Due to Gum Arabic being more compact and dense

PEGylated Protein Each detector responds to the sample differently 164,64 Ultra Violet Response (mv) 149,28 133,92 118,56 103,20 87,84 72,49 RI-Detector Viscosity Detector 57,13 41,77 26,41 11,05 Light Scattering UV-Detector 9,91 10,82 11,73 12,64 13,55 14,46 15,37 16,28 17,19 18,10 19,01 Retention Volume (ml)

Conjugation analysis The concentration of each component across the chromatogram can be solved They did not co-elute Protein concentration 1,8 e-4 1,6 e-4 1,4 e-4 1,2 e-4 Conc A 1,1 e-4 8,8 e-5 7,0 e-5 5,3 e-5 3,5 e-5 Conc. Protein Conc. PEG Protein- Aggregates PEG Protein- Monomer 2,1 e-4 1,9 e-4 1,7 e-4 1,5 e-4 1,3 e-4 Conc B 1,1 e-4 8,5 e-5 6,3 e-5 4,2 e-5 PEG concentraion 1,8 e-5 2,1 e-5 0,0 0,0 12,00 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 19,00 Volume (ml) Retention Volume

Conjugation analysis After further method development, the components coelute Protein concentration 5,5 e-4 5,0 e-4 4,4 e-4 3,9 e-4 Conc A 3,3 e-4 2,8 e-4 2,2 e-4 1,7 e-4 Conc. Protein Conc. PEG Protein- Aggregates with PEG Protein- Monomer with PEG 3,5 e-4 3,1 e-4 2,8 e-4 2,4 e-4 2,1 e-4 Conc B 1,7 e-4 1,4 e-4 1,0 e-4 PEG concentration 1,1 e-4 7,0 e-5 5,5 e-5 3,5 e-5 0,0 0,0 10,00 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 15,616,00 Retention Volume (ml) Retention volume

PEGylated Proteins Compositional Analysis Data File: 006A RXN SOLN (6.vdt) Method: PEG-Prot-0014.vcm Refractive Index Response (mv) 87,0 77,0 70,0 63,0 56,0 49,0 42,0 35,0 28,0 21,0 14,0 7,0 0,0 RI-Signal (sees Protein + PEG) UV-Signal (sees Protein only) 11,90 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 18,80 Retention Volume (ml) 145,0 132,0 121,0 110,0 99,0 88,0 77,0 66,0 55,0 44,0 33,0 22,0 11,0 0,0 Ultra Violet Response (mv)

PEGylated Proteins Compositional Analysis 2,2 e-4 2,0 e-4 1,8 e-4 2,2 e-4 2,0 e-4 1,8 e-4 RI-Signal 2,2 e-4 UV-Signal Conc. Protein 2,0 e-4 Conc. PEG Conc. Protein + PEG 1,8 e-4 Data File: 006A RXN SOLN (6.vdt) Method: PEG-Prot-0014.vcm PEG Protein Conc (Protein+PEG) 1,5 e-4 1,3 e-4 1,1 e-4 Conc Protein 1,5 e-4 1,3 e-4 1,1 e-4 Conc PEG 1,5 e-4 1,3 e-4 1,1 e-4 Protein + little PEG 8,8 e-5 8,8 e-5 8,8 e-5 6,6 e-5 6,6 e-5 6,6 e-5 4,4 e-5 4,4 e-5 4,4 e-5 2,2 e-5 2,2 e-5 2,2 e-5 0,0 0,0 0,0 11,9012,4 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 18,80 Retention Volume (ml)

PEGylated Proteins Compositional Analysis Data File: 006A RXN SOLN (6.vdt) Method: PEG-Prot-0014.vcm 2,2 e-4 2,0 e-4 Conc. Protein + PEG log MW 5,000 4,800 Conc (Protein+PEG) 1,8 e-4 1,5 e-4 1,3 e-4 1,1 e-4 20 kda protein:peg 1:1 3 kda Pure PEG 4,600 4,400 4,200 4,000 Log Molecular Weight 8,8 e-5 6,6 e-5 4,4 e-5 2,2 e-5 23 kda protein:peg 1:2 17kDa Pure Protein 3,800 3,600 3,400 3,200 0,0 3,000 11,90 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 18,80 Retention Volume (ml)

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 M W, analysis of which is essential for QC Insoluble at room temperature must be analysed at 140 160 C

Polyethylene HT-GPC LALS RALS Viscosity RI Param Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 M w (kda) 141.8 141.8 140.8 141.0 140.9 140.4 141.7 M n (kda) 35.2 38.6 40.7 41.5 36.0 35.0 35.3 M w / M n 4.03 3.68 3.46 3.40 3.92 4.02 4.01 R h 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, D H and M w analysis Using a SEC-MALS 20 detector allows accurate M w analysis of impure samples Conformation analysis (R g and Intrinsic Viscosity) Columnar interference can be a problem Separative Range: < 1 nm 100 nm

Outline Applications and Specifications Batch (DLS) Separations (SLS) Stability Prediction (interaction parameters) Hardware

Zeta Potential Isoelectric Point Determination 3 HEPES, Malvern ZSP, Unpublished NaCl, Malvern ZSP, Corbett and Jack (2011) - Colloids and Surfaces 376:31-41 Mobility (µmcm/vs) 0-3 3 4 5 6 7 8 9 10 ph

SLS 2 nd Virial Coefficient (A 2 ) Derived from the slope of the Debye plot. Synapse Polymer Thermodynamic Interaction parameter Representative of the magnitude of particle-solvent interactions

SLS 2 nd Virial Coefficient (A 2 ) Small ab fragment (20.7 kda) Ribonuclease (14.9 kda) Lysozyme (14.6 kda) Ovalbumin (46.8 kda) BSA (71.4 kda) Polystyrene standard (95.6 kda)

DLS Dynamic Virial Coefficient (K D ) 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 A 2 ) Hydrodynamic component Diffusion Coefficient (µm/s) Diffusion Coefficient (µm/s) HSA ph 4 ph 7 K D = 0.75 ml/g Concentration (mg/ml) K D = 8.33 ml/g Concentration (mg/ml)

DLS/SLS Thermal Trends Monitor the Z-average diameter, or SLS count rate, whilst increasing temperature An accurate T agg can be calculated T agg is a stability predictor

Orthogonal Stability Prediction T agg 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 Uricase inactivation is heavily temp-dependent (Caves et al. (2013) Biochemistry 52: 497-507) K D, A 2 and ZP allow assessment under ambient conditions, but only of interactions involving native protein

IgG Formulation Development

IgG Formulation Development Formulation K D (ml/g) A 2 (10-6 ml mol/g 2 ) ZP (mv) T agg ( C) KCl Tween -5.2-15 1.7 66 KCl NaCl -4.7 23-1.1 70 Tween Lactose -9.7 104 3.4 72 Sucrose Mannitol 31.9 1275 11.1 > 90

How Does Arginine Inhibit Aggregation? SLS 1800 Mean Count Rate (kcps) 1600 1400 1200 1000 800 600 400 200 Phos Phos + Arg 0 50 55 60 65 70 75 Temperature ( C) 50 mm arginine increases the temperature of aggregation onset by 2.5 C

How Does Arginine Inhibit Aggregation? DLS phos 25.0 C 71.5 C 76.5 C 84.0 C phos + arg

How Does Arginine Stabilise Protein Leads to resistance to the initiation of aggregation (SLS) Alters the aggregation mechanism (DLS) Formulation (Native) Zeta Potential (mv) Phos 7.0 ± 0.1 Phos + Arg 4.6 ± 0.5 Arginine s aggregation-inhibition property is not due to it increasing surface charge of native protein (ELS)

Aggregate Prediction Summary A 2, K D, and ZP allow prediction of aggregation propensity at ambient temperatures T agg allows analysis of aggregation during denaturation Together, these parameters allow an orthogonal approach to aggregation prediction

Outline Applications and Specifications Batch (DLS) Separations (SLS) Stability Prediction (interaction parameters) Hardware

The Malvern Zetasizer Range µv Nano APS DLS instruments assess size (D H ) and purity (PDI) SLS-capable assess M w for pure samples Aggregation prediction Calculate K D, A 2, ZP and T Agg

Zetasizer µv 60mW 830nm laser 90 Detection Low volume (2µL) Peltier Temperature control Perfect for use as a Flow mode detector for both D h and M w 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 interelectrode 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 Automated plate sampler Perfect sizescreening 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 D H RALS and LALS Simple M w calculation SEC-MALS 20 Calculation of accurate M w for proteins of all sizes and R g

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

SEC-MALS 20 Features Feature Aggregates 20 Angles More angles than any other SEC system available. Low Angle Sensitivity Absolute M w Accurate R g Lowest angles have comparable sensitivity to higher ones - lower concentrations can be measured with greater accuracy. High res. Mw distribution M w calculated independently of retention volume Measurements 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 Viscotek system

The Viscotek Range Relative M w Absolute M w R g D H Intrinsic Viscosity All after separation perfect for high res. Analysis of impure samples

Summary Cuvette-based Size and Zeta Potential measurements without separation Separation - high-res analysis, including accurate M W and R g of impure samples

Thank You for Listening Questions? Michael Caves Product Technical Specialist for Biophysical Characterisation Michael.Caves@Malvern.com

Nanoparticle Tracking Analysis (NTA) 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

Particles are Visualised Directly, in Real Time 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) Microvesicles purified from serum by ultracentrifugation, sizes 100-500nm. This field of view is approximately 120 x 100 microns. Speed of particles varies directly with particle size 2

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. 3

Nanoparticle Tracking Analysis Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample. capture tracking analysis 4

Particle Sizing in action - Software Analysis Concentration (Number Count) The Nanoparticle Tracking Analysis software allows for captured video footage to be simultaneously tracked and analysed Size in nm 100+200nm nanoparticles being tracked and analysed by NanoSight NTA 2.3 5

NTA Detection Limits Lower Detection Limit related to: Material type Size Wavelength and power of illumination source Sensitivity of the camera 10 40 nm Concentration Minimum concentration related to: Insufficient count for robust statistics (requiring longer analysis time) Approx 10 6 / ml Upper Detection Limit related to: Limited Brownian motion Viscosity of solvent Maximum concentration related to: Inability to resolve neighboring particles Tracks too short before crossing occurs 1000 2000 nm Approx 10 9 / ml 6

True size distribution profile Mixture of 100nm and 200nm latex microspheres dispersed in water in 1:1 ratio Particle distribution displays a number count vs particle size. Concentration (Number Count) Size in nm 7

True size distribution profile nm Concentration (Number Count) 164 nm NTA accurately tracked 3 subpopulations Size in nm Size in nm of nanoparticles nm Size in nm Mix of 3 populations in a real sample 8

Particles Concentration Number Count Global concentration of all populations Concentration (Number Count) Concentration of a population selected by user Number count of particle in a determined volume : Size in nm 9

Resolving mixtures of different particle types through Scatter Intensity 100 PS 60nm 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

Fluorescent Mode available NanoSight systems can be fitted with these lasers 405nm 488nm 532nm 635nm 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 Applications in: Nanoparticle toxicity studies Nano-rheology Bio-diagnostics Phenotyping specific exosomes 11

Analysis of 100 nm Fluorescence standard particles suspended in FBS* 100 nm fluorescently labelled particles suspended in 100% FBS Sample maintained at a constant temperature (37 o C) Sample viscosity was 1.33 cp Size in nm No filter Modal particle size peaks: 103 nm Concentration: 9.5 x10 8 particles/ml Same sample, 565 nm Long pass optical filter This allows selective visualising of fluorescent/fluorescently labelled particles Excitation wavelength - 532 nm * Fetal Bovine Serum 12

Example : Purified Influenza Virus 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. 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. 13

Application: Protein Aggregation at 50 C 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. 14 Data reproduced from Filipe, Hawe and Jiskoot (2010) Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2

Application: Drug Delivery liposomes used in drug delivery 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. 15

NTA is proven on for a wide range of nanoparticles Drug delivery Extracellular Vesicles (Exosomes and microvesicles) Nanoparticle Toxicology Protein Aggregates Virus and VLP samples Multi-walled Carbon nanotubes Cosmetics Foodstuffs Ink jet inks and pigment particles Nanobubbles Quantum dots Magnetic Nanoparticles Polymers and colloids Ceramics Fuel additives

NTA Summary Sub-visible particles Number or concentration Polydispersity Size Relative Light Intensity Fluorescence 17

Thank You for Listening Questions? Michael Caves Product Technical Specialist for Biophysical Characterisation Michael.Caves@Malvern.com

RI detector Standard differential refractive index detector Calibration performed using a narrow polymer standard (i.e. polystyrene) RI K dn i C i n0 dc K dn dc n C 0 instrument constant refractive index increment concentration (g/l) refractive index of solvent

UV-Vis Absorbance 144 Beer-Lambert Law used to calculate concentration from absorbance signal Extinction coefficient can be calculated by measuring the absorbance at different concentrations: A = εlc A = absorbance (abs.) ε = extinction coefficient l = cell path length c = concentration ε = da/dc Malvern s multi-channel detector allows detection of multiple wavelengths simultaneously

UV-Vis Absorbance 145 One of the most widely used analytical techniques Applications for Organic and Inorganic species Beer s Law (Concentration Detector) A = εlc ε = da/dc A = absorbance (abs.) ε = extinction coefficient l = cell path length c = concentration Conventional UV-Vis spectrophotometers contain single channel detectors

UV-Vis Absorption PDA Concentration Detector 146 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

3D spectrum from the UV PDA