Concentration and Size of Viruses and Virus-like Particles

© 2020 HORIBA, Ltd. All rights reserved. 2© 2018 HORIBA, Ltd. All rights reserved. 2
Sizing and Counting Viruses
and Virus-like Particles
HORIBA Scientific
Particle Analysis
Jeff Bodycomb, Ph.D.

Background
Technology overview
Results
Fluorescence
Concluding Comments
Outline

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Background

Established technologies
1. Dynamic Light Scattering (DLS)
2. Laser Diffraction
3. Nanoparticle Tracking Analysis (NTA)
4. Transmission Electron Microscopy (TEM)

Laser Diffraction
•Particle size 0.01 – 3000 µm
•Converts angular variations in scattered light to
particle size distribution
•Quick, repeatable
•Most common technique
•Suspensions & powders
Laser diffraction
Silica
~ 30 nm

Unmet needs
• Visualization of polydisperse particles
• Accurate & reproducible measurement of:
 Particle number concentration
 Particle size distribution

Technology

Particles in suspension undergo Brownian motion due to solvent molecule
bombardment in random thermal motion
 Brownian Motion
 Random
 Related to Size
 Related to viscosity
 Related to temperature
Brownian motion

Visualization of Brownian motion
microscope + camera
light sheet
scattered light
light sheet thickness
investigated volume
light source

Instrument Schematic
Video
Camera
(CCD)
Objective
Lens
Cuvette
Scattered
light
Laser
445 nm
(Blue)
Laser
520 nm
(Green)
Laser
635 nm
(Red)

Hydrodynamic size
Gives the diameter of a sphere that moves
(diffuses) the same way as your sample.
Dh Dh
Dh

Problem: intensity vs. size
100M X
Scattered
Light
Intensity
450 nm laser on polystyrene beads
Diameter [nm]
Angularscatteringcoefficient[1/m]
10 1000100
1E-13
1E-23

Eight orders of magnitude
• DLS – large particles skew results (small not detected) or
mask small particles
• cNTA – different sized particles can’t be seen simultaneously
(highly irregular images for large particles, dim for smallest)
• cNTA – interrogated volume depends on particles size and
their refractive index (similar to FC problem when sizing)

Problem is well known
“Sample polydispersity affects the ability to track and therefore
analyze different size fractions in the particle number-size distribution.
[…]
In a polydisperse sample large particles scatter a lot more than small
particles making it difficult to detect or track small size particles.”

ViewSizer solution
100 1000
1E-19
1E-17
1E-15
1E-13
10
1E-21
1E-23
(Multispectral Advanced Nanoparticle Tracking Analysis)
Angularscatteringcoefficient[1/m]
Diameter [nm]

Why three colors?

Concentration (counts per volume)
• Observed volume depends on intensity of scattered light
• Calibration of interrogated volume is done using standards
(various sizes and refractive indices) → lookup table
• Volume factor is calculated from average intensity of
scattered light for each tracked particle (takes laser power
& camera gain into account)
• Particle size distribution is calculated with variable volume
factor for each size bin

Light sheet thickness
Intensity
Thickness
“Top hat”
Gaussian
Vo
V>Vo
V<Vo

Volume calibrationExamples (Poly = extrapolation)
0
2
4
6
0 200 400 600 800 1,000
Diameter [nm]
silica n=1.4
Poly n=1.5
PSL n=1.6
silver
Effectivevolume[nL]
Silica - Blue laser (210 mW),
Camera gain 30 dB
0
1
2
3
4
5
6
7
8
0 200 400 600 800 1000 1200
Volumefactor[a.u.]
Diameter [nm]Diameter (nm)
Diameter (nm)
VolumetricFactor(u.a.)EffectiveVolume[nL]
Volume
Diameter
Intensity
d
I
V
Lookup surface
Look up
surface
Diameter
Intensity
How volume calibration is performed:
• Measure concentrations for patterns of different sizes
and made of different materials (various refractive
indices)
• Determine the effective volumes
• Create volume look up surface
• Extrapolate using intensity of individual tracks and
applying the Mie dispersion formula
US patent 9,857,283

Operation
+
insert cuvette
+
stir bar
ready
Load sample.
Components are readily separated
for easy and thorough cleaning.

Operation
Place in analyzer.
Press record
To prevent cross contamination, separate for thorough cleaning.
+

Results

Gold mixes: DLS vs. MANTA

NIST exploratory sample
Diameter [nm]
50 100 200 500 1000
DensityofPSD[counts/mL/nm]
1.0e+3
1.0e+4
1.0e+5
1.0e+6
1.0e+7
Initial effort to simulate protein aggregate mixture.

Plant virus data
Single frame from video.
Points correspond to scattering from
individual virus particles and aggregates.
Size DistributionTobacco Mosaic Virus (TMV)

Livestock virus data
Single frame from video.
Points correspond to scattering from
individual virus particles and aggregates.
Size Distribution

Comparison
TMV
Livestock

Human viral vector
Screen shot of purified viral vector
(aka Jeff’s favorite photo from Europe last year)

Human viral vector
Compare size distribution at harvest and after a
purification step.

Human viral vector
Compare concentration. Purification also concentrates virus
for next step.

Infectious Titer Methodologies
• Viral Plaque Assay – Time-tested Gold Standard of
Virology
–Inexpensive, effective, time-consuming, user-
dependent results (manual counting)
• Quantitative Polymerase Chain Reaction (qPCR)
–Expensive, accurate, requires virus specific primers
–Tracks DNA or RNA, not entire virus particles

Viral Plaque Assay
• Viral Plaque Assay
• Serially dilute viral preparations, infect
plates of confluent cells
–Incubate (that is, wait)
–Apply agar gel slow diffusion of virus
–Count the number of plaques (groups of
dead cells) for each serial dilution
• Pretty simple but there are some
drawbacks
–User to user variability
–Not all viruses cause drastic disruptions
to cell morphology or cell death
–TIME
Credit: Wikimedia user Y tambe

Virus with ViewSizer
Takes days
ViewSizer takes 15 minutes
Correlation means saving time and money during
virus manufacture for gene therapy.
ViewSizer tracks all particles, including empty virus particles and
infectious aggregates.
There is correlation between particle count and titer…
Bacteriophage. 2011 Mar-Apr; 1(2): 86–93

Virus life cycle
1. Enter cell
2. Use cell to
reproduce
3. Shed
BUT… The
result is very poor. There are
lots of aggregates, partially
formed, and empty virus
particles.
To get correct titer correlation,
you need to look at a range of
sizes properly.
Single laser won’t do…
Image: Wikipedia user YKTimes

Virus Life Cycle
brokenAggregates (infectious)
empty
Wide size range means you need 3 lasers to properly characterize all the virus particles.

Exosomes
6x10
5
5
4
3
2
1
0
NumberDistributionDensity,p/mL/nm
5 6 7 8 9
100
2 3 4 5 6 7 8 9
1000
2
Diameter, nm
Human Preadipocyte Exosomes
diluted 1000x
Concentration: 5.7e07 p/mL
Average size 148 nm

Lysozyme before heatingPBS after heating Lysozyme after heating
Lysozyme heated to 60 C

Fluorescence

Solid State Lasers
Mirror
Beam CombinersSample
Scattered &
Fluorescent
Light All
Wavelengths
Simultaneously Objective
Video Camera
No
Filter
ON ON ON
Use normal MANTA method to measure all particles
Normal mode: no filter

Solid State Lasers
Mirror
Beam CombinersSample
Scattered &
Fluorescent
Light
Objective
Video Camera
Filter
blocks
blue
Fluorescent
Light Only
ON OFF OFF
Detect green fluorescing materials excited by blue laser.
Use one laser and filter

Fluorescence - challenges
Photobleaching (reduced emission intensity from
overexposure)
Lasers pulsed in synch with camera shutter minimizes excitation
energy
Laser power levels can be adjusted individually
Sample stirring introduces fresh aliquots of unbleached sample
Measure concentrations only if applicable (size measurements take
longer)
Detection limits
Primarily a question of particle size
How much fluorescent material can be attached to, or included in, a
nanoparticle?
Very much application specific (fluorophore to sample optimization)

Analyzing a mixture

More mixtures
Mix of Fluoro-Max beads 140 nm dia with 102 nm and 203 nm dia PSL
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
8.0E+05
9.0E+05
0 100 200 300 400
DensityofPSD[part/mL/nm]
Diameter [nm]

Multiple fluorophores
Mix of Two Carboxylate Fluorescent Beads ( both nominally 100 nm dia)

Concluding comments

NTA - Disadvantages
Samples: Suspensions Range: 10 nm – 40 µm
• Slow (≈15 min )
• Does not perform morphology analysis
• Method development (dilution)

NTA - Advantages
Samples: Suspensions
• Particle concentration
• Good representation of particles throughout the whole
measurement range
• Particle counter
• Determination of kinetic processes
• Fluorescence analysis
• Absolute method (no calibration required)
• Visualization
Range: 10 nm – 40 µm

Key benefits of ViewSizer
– Individual particle method, not ensemble average
– Accurate PSD for polydisperse samples
– Concentration measured, not estimated
– Absolute method (no calibration needed)
– Particle visualization

* Sample dependent
Specifications
Range of Particle Sizes Measured * 10 nm to 2 µm, 15 µm with settling
Minimum Sample Volume 0.4 mL
Number of Lasers
3: red-635 nm, green-520 nm, blue-450
nm
Camera Type Scientific color CCD
Typical Sample Concentration 5 x 106 to 2 x 108 particles/mL
Sample Temperature Range
(Controlled)
5 °C to 50 °C, +/- 0.1 °C (-15 °C to 110 °C
available)
Dimensions 55 cm W x 66 cm D x 35 cm H
Weight 27 kg
Operational Environment 15 °C to 30 °C with < 85% RH

cuvette w/insert
ViewSizer 3000

Summary
Breakthrough technology
New & better particle characterization
High resolution distribution
Particle concentration/count
Diameter [nm]
50 100 200 500 1000
DensityofPSD[counts/mL/nm]
1.0e+3
1.0e+4
1.0e+5
1.0e+6
1.0e+7

Danke
Grazie
Σας ευχαριστούμε
감사합니다
Obrigado
谢谢
ขอบคุณครับ
ありがとうございました
धन्यवाद
நன்ற
Cảm ơn
Dziękuję
Tack ska ni ha
Thank you
Merci
Gracias
Большое спасибо

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