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The Coulter Principle for Cellular & Biological Applications

Kira Shapiro
Kira Shapiro
Kira ShapiroParticle Characterization Consultant

The Coulter Principle for Cellular & Biological Applications

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The Coulter Principle for Cellular and 
      Biological Applications




             Multisizer™ 4
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.
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
Current Users of the Multisizer

     •   Georgia Tech   •   UC Berkeley   •   UCLA
     •   MIT            •   Leiden U.     •   Lehigh U.
     •   Harvard        •   McGill U.     •   RIT
     •   U. Texas       •   U. Chicago    •   Johns Hopkins
     •   UC London      •   NYU           •   Columbia
Studying Cell Death
(Apopotosis)
 • 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




        http://www.microbiologybytes.com/virology/kalmakoff/baculo/pics/Apoptosis.gif
Cell Shrinkage is a prerequisite 
for Apopotosis




               Maeno, et al. PNAS 2000, 97, 17, 9487

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The Coulter Principle for Cellular & Biological Applications

  • 1. The Coulter Principle for Cellular and  Biological Applications Multisizer™ 4
  • 2. 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.
  • 3. 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
  • 4. Current Users of the Multisizer • Georgia Tech • UC Berkeley • UCLA • MIT • Leiden U. • Lehigh U. • Harvard • McGill U. • RIT • U. Texas • U. Chicago • Johns Hopkins • UC London • NYU • Columbia
  • 5. Studying Cell Death (Apopotosis) • 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 http://www.microbiologybytes.com/virology/kalmakoff/baculo/pics/Apoptosis.gif
  • 6. Cell Shrinkage is a prerequisite  for Apopotosis Maeno, et al. PNAS 2000, 97, 17, 9487
  • 7. Determining Fertility  based on Sperm Volume Hossain, et al, Human Reproduction, 13. 6. 1578-1583. 1998 • Depending on the time of year, farm animals display  high or low fertility (counts) • Volume changes can be indicative of disorders (size)
  • 8. Effect of Quinine on Sperm Yeung, et al, J. of Andrology, 23. 4. 522-528 2002
  • 9. Characterization of Cell Culture Industrial scale cell cultures require constant    monitoring and analysis Coulter 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?
  • 12. 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
  • 13. 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.
  • 14. Cell Banking:  Real Time  Measurement of Changes in Cell Size  • 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 Mukherjee, et al, Cryobiology, 55(1):10-18. 2007
  • 15. 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.
  • 16. Corollary I: Which Bead  Formulations? • Micron Sized Beads are used in a variety of  cellular and biological applications Typically coated with recognition molecules Can 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
  • 17. Corollary I: Which Bead  Formulations? Formulation A ~6 Formulation B ~12 ~18 ~24 The volume peaks increase by multiples of 6, indicating singlets, doublets, triplets, etc.
  • 18. Corollary II: Microbubble  Characterization • Large beads (2‐20 micron) Hollow and filled with imaging agents Most typically applied in ultrasound Heavy focus for biomedical research • The MultisizerTM allows researchers to  characterize these emerging imaging agents
  • 19. Particulates and Aggregates in Protein  Formulations Beckman Coulter Particle Characterization
  • 20. Protein Aggregates: Overview • Problem statement • The opportunity • How the Coulter Principle competes • Voice of the customer • Frequently asked questions • References • The Coulter Principle
  • 21. 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
  • 22. Genzyme Case Study: Importance of Particles in Biologics Genzyme says FDA will oversee its factory By ANDREW POLLACK NEW YORK TIMES, Published: March 24, 2010 November 2009 FDA 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 Key Message FOREIGN OR PROTEINACEUOS PARTICLES HAVE THE POTENTIAL TO MAKE PEOPLE SICK
  • 23. The Opportunity: Regulatory pressure is forcing manufacturers to  account for particulates of smaller and smaller  sizes.   Current lower limit =10 micron Likely 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.
  • 24. 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 µm Two current, acceptable techniques for foreign  particulates greater than 10 µm ‐ Technique 1: Light Obscuration ‐ Technique 2: Microscopy USP: United States Pharmacopeia EMEA: European Medicines Agency
  • 25. The Opportunity: • Competing techniques being evaluated for  particulates less than 10 micron: The Coulter Principle (BEC) Light Blockage (HIAC) Flow Imaging (MFI, Flowcam)
  • 26. Opportunity Matrix: Particulate Less than Greater than Characteristics 10 micron 10 micron Foreign Covered by current Opportunity for MS4 Particulates USP & EMEA Methods Protein Opportunity for MS4 Opportunity for MS4 Aggregates USP: United States Pharmacopeia EMEA: European Medicines Agency
  • 27. Coulter Principle: Advantage MultisizerTM 4 Data from Actual Protein Therapeutic 1.20E+05 Pre-filtration Approx. flow Post-Filtration Number per mL imaging lower limit (~1.5 μm) 6.00E+04 Approx. light blockage lower limit (~2 μm) 0.00E+00 0.4 1.9 3.4 Diameter (mm) Diameter (μm) Other techniques miss the majority of particulates
  • 28. Technique Comparison: Handles Min Operating Min Max Transparent Sample Principle Particles? Size Concentration Volume Light 2.0 18,000 Light Based Questionable 2 mL Blockage micron particles/mL Flow 1.5 750,000 Light Based Questionable 2 mL Imaging micron particles/mL Coulter 0.4 >1,000,000 Principle Impedance Excellent 4 mL micron particles/mL MultisizerTM
  • 29. How can we estimate aggregate mass? Mass = Density x Volume
  • 31. Voice of the Customer: “We WANT…” 1. Accurate counts below 10 micron, and  ideally as low as 0.1 micron 2. The ability to run samples neat 3. Total sampling volumes between 2 and 5 mL 4. Extremely linear and reproducible data
  • 32. 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 
  • 33. Frequently Asked Questions “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
  • 34. 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.