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Holographic Characterization of Protein Aggregates Size, morphology - - PowerPoint PPT Presentation

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Holographic Characterization of Protein Aggregates Size, morphology and differentiation one particle at a time (and fast) D. G. Grier, C. Wang, X. Zhong, & M. D. Ward New York University D. B. Ruffner & L. A. Philips Spheryx, Inc

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SLIDE 1

Holographic Characterization

  • f Protein Aggregates

Size, morphology and differentiation … one particle at a time (and fast)

  • D. G. Grier, C. Wang, X. Zhong, & M. D. Ward

New York University

  • D. B. Ruffner & L. A. Philips

Spheryx, Inc

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SLIDE 2

Holographic Characterization

Particle-resolved measurements of

 Radius: subvisible range

200 nm to 10 mm

 Refractive index: proxy for composition

Useful for differentiation

 Morphology

Useful for differentiation Useful for analyzing aggregation mechanism

 Concentration

Divide observed counts by measured sample volume

 Real-time processes

30 measurements/second Complete analysis in 10 minutes

2016 UMB-CERSI 2

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SLIDE 3

So, what’s the problem?

2016 UMB-CERSI 3

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SLIDE 4

Conventional Characterization (DLS)

2016 UMB-CERSI 4

500 1000 40

PDMS spheres in water

Malvern Zetasizer Nano ZS

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SLIDE 5

Holographic Characterization

2016 UMB-CERSI 5

0.6 0.4 0.8 1.0 1.2 1.37 1.38 1.39 1.40 1.41 1.42

  • ne sphere

Radius Refractive Index

5,000 spheres 0.5 1.0

Relative Probability

Wang, et al., Soft Matter 11, 1062 (2015)

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SLIDE 6

Holographic Tracking & Characterization

Lorenz-Mie Microscopy

2016 UMB-CERSI

Lee et al., Optics Express 15, 18275 (2007)

6

Parameters 3D Position 𝒔𝑞(𝑢) Radius 𝑏𝑞 Refractive Index 𝑜𝑞 Tracking Characterization

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SLIDE 7

Holographic Tracking & Characterization

How Lorenz-Mie Microscopy works

2016 UMB-CERSI

Lee et al., Optics Express 15, 18275 (2007) Focal plane

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Incident plane wave: Scattered field: : position : radius : refractive index

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SLIDE 8

2016 UMB-CERSI

Lee et al., Optics Express 15, 18275 (2007)

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Holographic Tracking & Characterization

How Lorenz-Mie Microscopy works

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SLIDE 9

2016 UMB-CERSI

Lee et al., Optics Express 15, 18275 (2007)

Lorenz-Mie scattering coefficients for a sphere:

  • refractive index:
  • relative radius:

9

Holographic Tracking & Characterization

How Lorenz-Mie Microscopy works

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SLIDE 10

Lorenz-Mie Microscopy

2016 UMB-CERSI

Lee et al., Optics Express 15, 18275 (2007)

10

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SLIDE 11

Property Precision Range 3D Position Δ𝒔𝑞 ≤ 3 nm 100 × 100 × 100 𝜈m3 Radius Δ𝑏𝑞 ≤ 2 nm 200 nm − 20 𝜈m Refractive Index Δ𝑜𝑞 ≤ 10−3 1 − 5 Concentration Δ𝑑𝑞 ≈ 𝑑𝑞 103mL−1 − 108mL−1

Holographic Tracking & Characterization

Independently verified performance

2016 UMB-CERSI

Krishnatreya et al., Am. J. Phys. 82, 23 (2014)

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NIST Traceable Particles 𝑒𝑞 = 2 𝑏𝑞 = 1.54 ± 0.05 𝜈m 𝑜𝑞 = 1.598 ± 0.005 Bangs Labs #12035

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SLIDE 12

Property Precision Range 3D Position Δ𝒔𝑞 ≤ 3 nm 100 × 100 × 100 𝜈m3 Radius Δ𝑏𝑞 ≤ 2 nm 200 nm − 20 𝜈m Refractive Index Δ𝑜𝑞 ≤ 10−3 1 − 5 Concentration Δ𝑑𝑞 ≈ 𝑑𝑞 103mL−1 − 107mL−1

Holographic Tracking & Characterization

Independently verified performance

2016 UMB-CERSI

Krishnatreya et al., Am. J. Phys. 82, 23 (2014)

12

NIST traceable PS Measured flow volume (no calibration)

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SLIDE 13
  • 1. Particles pass through

laser beam in microfluidic channel

Automated Holographic Characterization

Size/refractive index distribution: for 2,500 spheres acquired in 10 minutes

  • 5. Build statistics:

Each point characterizes

  • ne particle
  • 2. Microscope records

interference pattern

  • 3. Compare

measurement to scattering theory

  • 4. Comparison yields

: radius : refractive index : 3D position

2016 UMB-CERSI 13

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SLIDE 14

Characterizing Colloidal Mixtures

2016 UMB-CERSI 14

silica polystyrene

Yevick, Hannel & Grier, Optics Express 22, 22864 (2014)

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SLIDE 15

Characterizing Protein Aggregates

2016 UMB-CERSI 15

Wang et al., J. Pharm. Sci. 105, 1074 (2016) BSA in Tris + added salt

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SLIDE 16

Differentiating Silicone Oil Droplets

2016 UMB-CERSI 16

Wang et al., J. Pharm. Sci. 105, 1074 (2016)

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SLIDE 17

Characterizing Protein & Contaminants

2016 UMB-CERSI 17

Wang et al., J. Pharm. Sci. 105, 1074 (2016)

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SLIDE 18

Effective Medium Theory

Refractive index: Volume fraction: Refractive index: Porous particle Effective refractive index: Lorentz-Lorenz factor: Material: Medium:

2016 UMB-CERSI 18

Cheong et al., Soft Matter 7, 6816 (2011)

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SLIDE 19

Fractal Aggregates

Volume fraction: Effective Medium Theory:

2016 UMB-CERSI 19

Wang et al., Soft Matter 12, 8774 (2016)

Size-Index Scaling:

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SLIDE 20

Fractal Scaling of Protein Aggregates

Bovine Serum Albumin Bovine Insulin Filamentary clusters Cluster-cluster aggregates

2016 UMB-CERSI 20

Wang et al., Soft Matter 12, 8774 (2016)

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SLIDE 21

10 1 10-4 10-3 10-2 10-1

Holographic Characterization of Human IgG

2016 UMB-CERSI 21

10 1 silicone Large fractal dimension: compact clusters Differentiation between protein aggregates and silicone droplets Ruffner et al., unpublished (2016)

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SLIDE 22

Holographic Characterization

 Detects, counts and characterizes

subvisible protein aggregates in situ

 Differentiates by composition & morphology  Fast, time-resolved measurements  Independently verified precision & accuracy  Minimal calibration

2016 UMB-CERSI 22

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SLIDE 23

2016 UMB-CERSI 23

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SLIDE 24

Comparison with Industry Standard

DLS (Malvern Zetasizer Nano)

Cheong et al., Opt. Express 17, 13071 (2009) Cheong, Xiao, Pine & Grier, Soft Matter 7, 6816 (2011)

  • 1. Consistent mean radius results in common range
  • 2. HVM also works for larger particles
  • 3. HVM results have smaller (better) instrumental spread
  • 4. HVM also yields refractive index (porosity)

2016 UMB-CERSI 24

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SLIDE 25

So, what do our clusters look like?

2016 UMB-CERSI 25

hologram fit

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SLIDE 26

So, what do our clusters look like?

2016 UMB-CERSI 26

hologram fit Dixon et al., Opt. Express 19, 16410 (2011) Chen et al., J. Pharm. Sci. 105, 1074 (2016) Holographic Deconvolution Microscopy

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SLIDE 27

So, what do our clusters look like?

2016 UMB-CERSI 27

hologram fit

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SLIDE 28

So, what do our clusters look like?

2016 UMB-CERSI 28

hologram fit

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SLIDE 29

So, what do our clusters look like?

2016 UMB-CERSI 29

hologram fit