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TRANSCRIPT
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Introduction to Archimedes& to RMM (Resonant Mass Measurement)
Stéphane RouquetteEuropean sales manager, [email protected]
Archimedes fills a gap
SEC/GPC
AUC
LO
Microscope
DLS*
Visible
Laser Diffraction*
nm 10 100 µm 10 100 mm cm (107 nm)
*Qualitative
• FDA identified potential immunogenicity concerns surrounding protein aggregates 0.2µm – 5 µm
• Orthogonal techniques required to support/expand on existing methods
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ARCHIMEDES Preview
› Measures physical properties of individual particles in fluid
50 nm - 5 µm (250 nm to 5 µm for protein aggregates)
Mass, Size, Density
› Also measures sample content: # particles/ml, particle mass/ml
› High resolution, precision, and accuracy
› Fast, NIST-traceable calibration
› Intuitive ParticleLab software
› Key applications:
Protein formulations Nanoparticles
Living cells, bacteria
Pigments
Particle density
How ARCHIMEDES Works
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Technology: Resonant Mass Measurement
Microchannel Resonator
• Microfluidic channel embedded inside resonant structure
• Particle passing through the resonator changes the mass and shifts the resonant frequency
• Excursion in resonant frequency gives an accurate and precise measure of particle’s buoyant mass
resonatorresonator
bypa
ss c
hann
elby
pass
cha
nnel
resonator
bypa
ss c
hann
elMEMS Sensor
Measuring Particle Mass in Fluid
› Embed a microfluidic channel *inside* the resonant structure
› A particle passing through the channel changes the mass and shifts the resonant frequency
› The excursion in resonant frequency gives a precise measure of particle mass
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ARCHIMEDES’ Instrumentation
Frequency Resolution: 25 parts per billion @ 1 kHz
Mass Limit of Detection: < 1 femtogram = 10-15 g
PC
Signal Processing
Frequency Measurement
andFeedback
Sensor
Optics
PneumaticsFluidics
sample
waste
pressuresource
MB=f*SM =
MB /(1-f/p)
D =(6M/ (p))1/3
Particle Properties and StatisticsA clear connection between the raw signal and useful data
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Limits of detection
Material Density Buffer density Ratio Limite of detection
Gold nano particles 19.00 1.00 1900% about 50 nm
Nano bubbles 0.0012 1.00 ‐99.9% about 150 nm
Protein aggregates 1.30 1.00 30% about 250 nm
Silicon oil 0.97 1.00 ‐3% about 500 nm
Superior Particle Metrology1% precision and accuracy
Mixture of NIST-traceable standards
Ultra-high resolutionEasily distinguishes particles< 2% different in size
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ARCHIMEDES for Protein Formulations
ARCHIMEDES Addresses a Key Size Range
10 nm 100 nm 1 µm 10 µm
#/ml
ARCHIMEDES Flow Microscopy
Aggregate Size
SEC
* “One of the highest risks for induction of neutralizing antibody appears to be associatedwith highly repetitive arrays of native protein – those that potentially include subvisibleparticulates (SVP) between 0.1 to 10 microns in therapeutic protein products.”
Dr. Barry Cherney, former US FDA Deputy Director Division of Therapeutic Proteins
› Subvisible and submicron aggregates
› Extends size range below flow microscopy
› Growing regulatory interest*
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Archimedes: Characterising Protein Aggregates
›Assess impact of stress of SubVis particles
› Impact of shear stress on protein aggregates
›Obtain particle size distribution profile
›Compare different stress conditions – Force Deg
ControlShear-stress
Archimedes: Characterising Protein Aggregates
›Compare materials, manufacturing / application equipment etc
›Comparison of two different syringe manufacturers
›Process understanding, process design, selection of appropriate components
Manufacturer AManufacturer B
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ARCHIMEDES distinguishes silicone oil from protein by buoyancy
OilOilOil ProteinAggregates
ProteinAggregates
ProteinAggregates
AggregateAggregate
Aggregates
Oil Droplets10 Hr1 Hr 2 Hr
#/
ml
µg
/ml
pp
m
#/
ml
Measurement of nano-suspensions & protein formulations
LN5
Dia 16
LN5 move axis labels so that they are clearerLisa Newey-Keane; 3-10-2012
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Development of Aggregates in Stressed Protein Formulations
Stirred IgG, 10 mg/ml, filtered 100 nmSample drawn intermittently, measured continuously
ARCHIMEDES’ Advantages for Proteins› Addresses a key size range
› Distinguishes protein aggregates from oil
› Quantitative measures of mass, size, content (µg/ml)
› Excellent repeatability, reproducibility
› No sample preparation needed: No dilution
No electrolyte/salt
No filtering (rare cases only)
Sip directly from vial or syringe
› Minimal sample consumption: 100 µl
› Accepts viscosities up to 100 cP
› No optical artifacts (no shape information)
› Fast and thorough cleaning, no cross-contamination
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Other Applications of ARCHIMEDES
Determining Particle DensityMeasure buoyant particles in pre-filled syringes using solutions of different densities
Intercept 0.98 g/cc indicates silicone oil (0.97 g/cc)
from Genentech, Patel et al, J. Analytical Chem. 2012
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Thank You!