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Introduction to Archimedes& to RMM (Resonant Mass Measurement)

Stéphane RouquetteEuropean sales manager, Biosciencesstephane.rouquette@malvern.com

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

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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!