Eunah Lee, Ph.D. and Jeff Bodycomb, Ph.D - Particle Characterization Raman Spectroscopy - HORIBA
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HORIBA Scientific
Particle Characterization
Raman Spectroscopy
Size, Chemistry, and More: Raman and Laser
Diffraction for Pharma Particle Analysis
Eunah Lee, Ph.D. and Jeff Bodycomb, Ph.D.
March 9, 2021
© 2021 HORIBA, Ltd. All rights reserved.
HORIBA Products for Biopharmaceuticals
SPRi Fluorescence Raman XRF Particle
Analysis
Binding Analysis Chemical Molecular ID, Elemental Particle size
Identification Protein Analysis distribution
and Analysis Secondary
Structure
© 2021 HORIBA, Ltd. All rights reserved.
Pharmaceutical Process
Drug Discovery Pre-Clinical Clinical Submission Launch
© 2021 HORIBA, Ltd. All rights reserved.
Submission – Technology Transfer
Drug Discovery Pre-Clinical Clinical Submission Launch
• Formulation per drug delivery system. For example
‐ Peroral and topical: Verify API form, concentration and distribution.
Particle size distribution depending on solubility and for dose uniformity
‐ Inhalation and parenteral: Particle counts, size, shape and chemistry
• Formulation for scale up and manufacturing
© 2021 HORIBA, Ltd. All rights reserved.
Submission – Technology Transfer
Drug Discovery Pre-Clinical Clinical Submission Launch
Particle analysis Raman microscopy
Scale Up
Formulation development
– OINDP
© 2021 HORIBA, Ltd. All rights reserved.
Pharmaceutical Process
Drug Discovery Pre-Clinical Clinical Submission Launch
• QC/QA tests including
‐ Raw material screening
‐ Failure tests
‐ Content uniformity
‐ Root cause analysis
• Surveillance is critical to monitor
‐ Potential drug interactions and long term effects
‐ Counterfeit drugs and patent infringement
• Research and investigation by regulatory entities
© 2021 HORIBA, Ltd. All rights reserved.
Launch – Continuous Verification
Drug Discovery Pre-Clinical Clinical Submission Launch
Particle analysis Raman microscopy
Vaccine Endpoint
Polymorph
Characterization
Rapid Vaccine Endpoint Determination
– 15 min vs. days
(a) Raman spectra of α, β, δ forms of mannitol;
(b) Images showing the spatial distribution of
mannitol forms in powder blends
© 2021 HORIBA, Ltd. All rights reserved.
Particle Characterization Techniques
Laser Diffraction
• Scattering technique – large particles scatter intensely at narrow angles and small
particles scatter weakly at wide angles
• Two color systems enable accurate measurement of small particles
Dynamic Light Scattering
• Scattering technique – Brownian motion induces changes in scattered light intensity
• Intensity is measured as a function of delay time and scattering angle
• Static light scattering
Image Analysis
• Static image analysis uses a microscope and digital camera to collect images of deposited
particles
• Dynamic image analysis drops particles between light source and camera – projected
shadows are recorded
© 2021 HORIBA, Ltd. All rights reserved.
Laser Diffraction
Laser diffraction overview
LA960 overview
A simple example – raw material analysis – one API, one excipient
© 2021 HORIBA, Ltd. All rights reserved.
Perspective
© 2021 HORIBA, Ltd. All rights reserved.Laser Diffraction
Convert scattered light as a
function of angle to a
particle size distribution
• Quick, repeatable
• Powders, suspension
• Most common technique
© 2021 HORIBA, Ltd. All rights reserved.LA-960 Optics
© 2021 HORIBA, Ltd. All rights reserved.Path Length Difference
Diffraction effects
arise due to
scattering from
various points in the
particle (and, in the
large particle limit
only at the edges).
© 2021 HORIBA, Ltd. All rights reserved.How much sample (wet)?
It depends on sample, but here are some examples: Volume
Sample Handlers
Larger, broad distributions require larger sample volume (mL)
Lower volume samplers for precious materials or solvents.
Aqua/SolvoFlow 180 - 330
MiniFlow 35 - 50
Fraction Cell 15
Small Volume
10
Fraction Cell
© 2021 HORIBA, Ltd. All rights reserved.Instrument to instrument variation
4 instruments
(real sample)
© 2021 HORIBA, Ltd. All rights reserved.Excipient: Mg Stearate
Measured as a dry
powder, note tight
repeatability.
Run Median Size,
microns
1 8.25
2 8.20
3 8.21
4 8.24
Mean 8.22
Std. Dev. 0.024
CoV 0.3%
© 2021 HORIBA, Ltd. All rights reserved.Nanoemulsion Vaccine Adjuvant
Smaller particle
lead to better flow
through a filter (e.g.,
filtration
sterilization).
Monitoring size
helps downstream
processing steps.
© 2021 HORIBA, Ltd. All rights reserved.Nanoemulsion Vaccine Adjuvant
Squalane
processed
with
Microfluidizer
Effective measurements of particles over 1 micron
to give consistent results from start to finish.
© 2021 HORIBA, Ltd. All rights reserved.PLA for drug delivery
Polylactide
nanoparticles, good
good vs bad
batch.
D10, d50, d90 the
same. Used
volume mean as bad
criteria.
© 2021 HORIBA, Ltd. All rights reserved.An example – Children’s tylenol
Chewable in Bubble gum Suspension in Grape flavor:
flavor: Active ingredient:
Acetaminophen 160 mg in each
Active Ingredient: 5 mL
Acetaminophen 160 mg in each Inactive ingredients: anhydrous
tablet citric acid, D&C red no. 33,
FD&C blue no. 1, flavors,
Inactive ingredients: anhydrous glycerin, high fructose corn
citric acid, cellulose acetate, syrup, microcrystalline cellulose
crospovidone, D&C red no. 7 and carboxymethylcellulose
calcium lake, dextrose, flavor, sodium, purified water, sodium
benzoate, sorbitol solution,
magnesium stearate, povidone, sucralose, xanthan gum
sucralose
© 2021 HORIBA, Ltd. All rights reserved.Laser diffraction analysis results
Red and Green: Chewable in Bubble gum flavor
Black: Suspension in Grape flavor
© 2021 HORIBA, Ltd. All rights reserved.Sample Preparation for Image Analysis
Powders
Vacuum-induced dry Vacuum filtration with Dispense particulate
Particulate in suspension
Aerosols/Sprays/Inhalers
powder dispersion filter insert/holder directly on substrate
(glass/metal)
© 2021 HORIBA, Ltd. All rights reserved.Adding Chemical ID to Particle Characterization
Raman spectroscopy
• Inelastic light scattering
• Probe molecular vibrations within a sample
• Non-invasive, non-destructive technique
• No sample preparation
© 2021 HORIBA, Ltd. All rights reserved.XploRA confocal Raman microscope
• Excellent Performance • Technical Evolution
• High sensitivity • Multivariate analysis
• Ultimate spatial resolution • SWIFT/SWIFT XS
• Multimodal optical microscopy • Particle Finder
• Compact design • User Account Control
• Multiwell module
• KnowItAll database searching
• Extreme Extension • Advanced Automation
• AFM-Raman • OneClick Raman operation
• Photoluminescence • Laser switching
• Macro accessory • Autocalibration
• Remote sampling • Extended video montage/mosaic
• EMCCD options • EasyNav
© 2021 HORIBA, Ltd. All rights reserved.Soleil confocal Raman microscope
© 2021 HORIBA, Ltd. All rights reserved.LabSpec 6 Spectroscopy Suite
© 2021 HORIBA, Ltd. All rights reserved.Particle Correlated Raman Spectroscopy (PCRS)
Acquire Optical Image Auto Detect Particles Particle morphology
Statistical analysis Chemical ID Spectral acquisition
220
210 1 000
200
190 800
Intensity (counts)
180
170
160
600
150 1 000
140
400
130 800
Number (counts)
Intensity (counts)
120
110 200 600
100
90
80
0 400
70
1 000 2 000
60
200
Raman shift (cm-¹)
50
40
30 0
20
10 500 1 000 1 500 2 000
Raman shift (cm -¹)
0
40 60 80 100 120 140 160 180 200 220 240 260 280
Diameter (µm)
© 2021 HORIBA, Ltd. All rights reserved.An example – Nasal Spray
• Spray action defines aerosol droplet size and dictates
deposition area
• Particle size (single particle/agglomerate) effects API
uptake and bioavailability
API
cellulose
© 2021 HORIBA, Ltd. All rights reserved.Particle Diameter
90
A 700
A
80
70 600
60 1 µm 1 µm
500
Number (counts)
Number (counts)
50
400
40
30
API Brand A Brand B 300 Cellulose Brand A Brand B
200
20 Mean 4.6 3.6 Mean 4.4 3.6
10 100
St dev 1.9 1.2 St dev 2.2 1.2
0 0
5 10 15 20 5 10 15 20
Median
Diameter (µm)
4.2 3.4 Diameter (µm)
Median 3.8 3.4
350
160
10% (D10) 2.6 2.3 10% (D10) 2.4 2.3
140 300
50% (D50) 4.2 3.4 50% (D50) 3.8 3.4
120
250
Number (counts)
Number (counts)
100 90% (D90) 7.0 5.2 90% (D90) 6.9 5.1
200
80
150
60
100
40
20 1 µm 50 1 µm
B B
0 0
5 10 15 20 5 10 15 20
Diameter (µm) Diameter (µm)
© 2021 HORIBA, Ltd. All rights reserved.Ellipse Ratio
Brand A Brand B
350
400
350 300
300 250
Number (counts)
Number (counts)
250
200
200
150
150
100
100
50
50
0 0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Ellipse ratio (a.u.) Ellipse ratio (a.u.)
1 µm 2 µm 1 µm 1 µm 1 µm 2 µm 1 µm 1 µm
1 µm 2 µm 2 µm 1 µm 1 µm 1 µm 1 µm 1 µm
© 2021 HORIBA, Ltd. All rights reserved.An example – Paracetamol in suspension
Micrograph
transmitted, brightfield
Raman spectrum from library – Paracetamol
Particles highlighted in green
Raman spectrum of green particles
© 2021 HORIBA, Ltd. All rights reserved.Particle characteristics and distribution
Size, shape and location of individual particles
12
10
8 Size distribution by diameter
Number (counts)
6
4
2
0
20 40 60 80 100 120
Diameter (µm)
© 2021 HORIBA, Ltd. All rights reserved.An example – Vitamin C in tablet
Micrograph, reflected, brightfield
Raman image, 10 ms/spectrum
© 2021 HORIBA, Ltd. All rights reserved.Particles embedded in matrix
Raman image, 10 ms/spectrum Representative spectra
1.0
Ascorbic acid
0.8
Intensity (counts)
Citric acid
0.6
0.4
Carotene
0.2
Sorbitol
0.0
500 1 000 1 500 2 000 2 500 3 000 3 500
Raman shift (cm-¹)
© 2021 HORIBA, Ltd. All rights reserved.Particle characteristics and distribution
Raman image, ascrobic acid
Size distribution by area*
*particles smaller than 100 μm 100 μm are excluded
Size, shape and location of individual particle
© 2021 HORIBA, Ltd. All rights reserved.Other Pharmaceutical and Cosmetic Examples
Raman Chemical Imaging – Tablet
Topical Creams - Sunscreen
1000 µm
140
25
120
20
Number (counts)
100
Number (counts)
80 15
60
10
40
5
20
0 0
50 100 150 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Diameter (µm) Diameter (µm)
© 2021 HORIBA, Ltd. All rights reserved.Particle Characterization: LA960 vs. XploRA PLUS
LA960 XploRA PLUS
Technology Laser diffraction Static imaging + Raman imaging
Size range 10 nm to 5 mm 500 nm to 1 mm
Sample form Good for dry or wet Prefers dry, stationary
Shape Possible Possible
Chemical ID No information Yes
Speed 60 s per run, zillion particle per run Moderate
Drum -> sampling from different parts, mix
them well -> subsample -> run
Mixture -> sampling from different parts -> Run the same samples on Raman and see if
run them one -> compare statistics -> chemical ID matches size distribution
mixing quality variation
Tandem analysis Screening for outliers in batches Root cause analysis
© 2021 HORIBA, Ltd. All rights reserved.© 2021 HORIBA, Ltd. All rights reserved.
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