The Coulter Principle for Cellular & Biological Applications
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Transcript of The Coulter Principle for Cellular & Biological Applications
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The Coulter Principle for Cellular and Biological Applications
Multisizer™ 4
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The Coulter Principle
• The Coulter Principle, which uses electrical impedance to measure particulate volume was developed over 60 years ago for counting and sizing red blood cells.
• It is the subject of numerous ASTM and ISO standards for sizing and counting.
• It is independent of particle refractive index, chemistry, etc. It will detect any particle that displaces liquid.
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The Coulter Principle in Practice
Particles, liquid and electric current are pumped through an orifice of an exact diameter The particles cause changes in the electric current & these changes are monitored to count and size particles
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Current Users of the Multisizer
• Georgia Tech• MIT• Harvard• U. Texas• UC London
• UC Berkeley• Leiden U.• McGill U.• U. Chicago• NYU
• UCLA• Lehigh U.• RIT• Johns Hopkins• Columbia
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http://www.microbiologybytes.com/virology/kalmakoff/baculo/pics/Apoptosis.gifhttp://www.microbiologybytes.com/virology/kalmakoff/baculo/pics/Apoptosis.gif
• Apopotosis involves the swelling and breaking apart of cellular structure
• Carefully designed studies can use the Coulter Principle to understand the role of various chemicals, proteins, etc. in the process
Studying Cell Death(Apopotosis)
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Cell Shrinkage is a prerequisite for Apopotosis
Maeno, et al. PNAS 2000, 97, 17, 9487Maeno, et al. PNAS 2000, 97, 17, 9487
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Determining Fertility based on Sperm Volume
• Depending on the time of year, farm animals display high or low fertility (counts)
• Volume changes can be indicative of disorders (size)
Hossain, et al, Human Reproduction, 13. 6. 1578-1583. 1998
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Effect of Quinine on Sperm
Yeung, et al, J. of Andrology, 23. 4. 522-528 2002Yeung, et al, J. of Andrology, 23. 4. 522-528 2002
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Characterization of Cell Culture
Industrial scale cell cultures require constant monitoring and analysisCoulter Counter provides answers to many common questions
How big are the host cells?How uniform are the host cells?Have any of the host cells broken apart?What is the relative percentage of debris to desired cell type?
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Characterization of host rDNA Cell Culture
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Environmental Conditions Effect Cell Stability and Viability
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Filtration Efficiency
• After growing up cell culture, filtration is used to remove debris• Key parameters are efficiency and fouling‐ batch differences in media culture can cause filter fouling
• The Coulter Principle can be used to help select filters that perform the best
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Data from Major Pharma Company
Company was interested in removing the cellular debris/fragments prior to pumping the supernatant into downstream processing steps. First two curves are as harvested (showing debris/fragment concentrations). The remaining curves are samples set aside in beakers etc that were then sampled. Two
different clarification filtering systems were used.
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Cell Banking: Real Time Measurement of Changes in Cell Size
Mukherjee, et al, Cryobiology, 55(1):10-18. 2007Mukherjee, et al, Cryobiology, 55(1):10-18. 2007
• To permanently preserve cell lines, cryoprotectants are used prior to freezing
• Cryoprotectants themselves cause cell damage• Studying the rate of this damage and changes in cell size as a function of time allows users to make better decisions about preservation methods
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Multi‐Tube Overlap: Bacterial Aggregation in a Culture
Bacterial aggregates can reduce the effectiveness of antimicrobial agents. A combination of detergents and filters can be used to decrease the amount of 'clumps'. The percentage of 'clumps' relative to single cells can be determined by using two different apertures. The Multi‐Tube Overlap function merges the results into a single continuous distribution.
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Corollary I: Which Bead Formulations?
• Micron Sized Beads are used in a variety of cellular and biological applications
Typically coated with recognition moleculesCan be dye‐loaded, magnetic, or other…Optimization of formulation conditions is key
• The Coulter Principle is the only technique that can provide the resolution necessary for these studies
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~6
~12~18 ~24
Formulation B
Formulation A
Corollary I: Which Bead Formulations?
The volume peaks increase by multiples of 6, indicating singlets, doublets, triplets, etc.
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Corollary II: Microbubble Characterization
• Large beads (2‐20 micron)Hollow and filled with imaging agentsMost typically applied in ultrasoundHeavy focus for biomedical research
• The MultisizerTM allows researchers to characterize these emerging imaging agents
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Particulates and Aggregates in Protein Formulations
Beckman Coulter Particle Characterization
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Protein Aggregates: Overview
• Problem statement• The opportunity• How the Coulter Principle competes• Voice of the customer• Frequently asked questions• References• The Coulter Principle
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Problem Statement:
• Protein scientists have identified an overlooked region of product quality data.
Currently, techniques such as AUC, CE, and chromatography provide information up to 100 nm particles in size.Other technologies, such as light blockage provide information above 10 micron in size.
• A “blind spot” exists with particles between 100 nm and 10 micron.
Particles in this range may cause immunogenicity.Active studies of this size range began in mid-2008.Regulators are demanding data in this range.
Journal of Pharmaceutical Science-2009 Journal of Pharmaceutical Science-2009
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Genzyme Case Study:Importance of Particles in Biologics
November 2009FDA reports that Cerezyme, Fabrazyme and three other enzyme drugs that are put into vials at the factory were contaminated with PARTICLES of steel, rubber or fiber.Financial analyst predicts the overall cost to the Genzyme will be in the $200 million to $300 million range
Genzyme says FDA will oversee its factoryBy ANDREW POLLACK
NEW YORK TIMES, Published: March 24, 2010
Key MessageFOREIGN OR PROTEINACEUOS PARTICLES
HAVE THE POTENTIAL TO MAKE PEOPLE SICK
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The Opportunity:
Regulatory pressure is forcing manufacturers to account for particulates of smaller and smaller sizes.
Current lower limit =10 micronLikely future lower limit = 1 micron
Currently used technologies do not perform well below 10 micron
The Multisizer™4 has been shown to be very effective in this size range.
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The Opportunity:
• FDA & EMEA require data for particulates less than 10 micron, but do not specify the technique that should be used.
USP and EDQM pharmacopeias DO establish acceptable techniques for FOREIGN particles greater than 10 µmTwo current, acceptable techniques for foreign particulates greater than 10 µm‐ Technique 1: Light Obscuration‐ Technique 2: Microscopy
USP: United States PharmacopeiaEMEA: European Medicines Agency
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The Opportunity:
• Competing techniques being evaluated for particulates less than 10 micron:
The Coulter Principle (BEC)Light Blockage (HIAC)Flow Imaging (MFI, Flowcam)
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Opportunity Matrix:
Particulate Characteristics
Less than 10 micron
Greater than 10 micron
Foreign Particulates Opportunity for MS4 Covered by current
USP & EMEA Methods
Protein Aggregates Opportunity for MS4 Opportunity for MS4
USP: United States PharmacopeiaEMEA: European Medicines Agency
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Coulter Principle: Advantage
0.00E+00
6.00E+04
1.20E+05
0.4 1.9 3.4
Diameter (mm)
Num
ber p
er m
L
Pre-filtrationPost-Filtration
Approx. light blockage lower limit (~2 μm)
Approx. flow imaging lower limit (~1.5 μm)
Diameter (μm)
MultisizerTM 4 Data from Actual Protein Therapeutic
Other techniques miss the majority of particulates
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Technique Comparison:
OperatingPrinciple
Handles Transparent Particles?
MinSampleVolume
MinSize
MaxConcentration
Light Blockage Light Based Questionable 2 mL 2.0
micron18,000
particles/mL
Flow Imaging Light Based Questionable 2 mL 1.5
micron750,000
particles/mL
Coulter Principle
MultisizerTMImpedance Excellent 4 mL 0.4
micron>1,000,000particles/mL
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How can we estimate aggregate mass?
Mass = Density x Volume
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Aspherical Objects: Preview
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Voice of the Customer:
“We WANT…”1. Accurate counts below 10 micron, and
ideally as low as 0.1 micron2. The ability to run samples neat3. Total sampling volumes between 2 and 5 mL4. Extremely linear and reproducible data
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How We Meet Customer Needs:
Accurate counts below 10μm, ideally as low as 0.1μm The MS4 is the only instrument that can count particulates under 1 μm and down to 0.400 μm.
The ability to run samples neat The MS4 can count proteins in a wide variety of native buffers without dilution
Sampling volumes between 2 and 5 mL Our new sample procedure and adaptor allow s volumes as small as 4 mL
Extremely linear and reproducible data The MS4 is highly sensitive and reproducible Typical CV's are below 2% The Coulter Principle has been used as the #1 method for counting red blood cells for over 50 years
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“Do you have to dilute?”Not necessarily. Depends on sample characteristics and smallest size range needed. Protein scientists often dilute for other analyses (size exclusion chromatography, CE, AUC)
“Can you provide morphology information?”Ask why this is important. Customers are most concerned about counts and current flow image techniques and rely upon image aspect ratios for morphology
Frequently Asked Questions
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Frequently Asked Questions
“Can you count down to 100 nm?”How low can you count now? The MultisizerTM is the only instrument with accurate and reproducible data below 1 micron.
“What sample volumes are required?”The standard set up accommodates 10 mL. However, we have a special technique for proteins that can use as little as 4 mL.
“What is the Coulter Principle?”It uses electrical impedance to count and size particles. It has been around for more than 55 years and widely used as a counting standard.