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?

10. Characterization of host rDNA
Cell Culture

11. Environmental Conditions
Effect Cell Stability and Viability

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

30. Aspherical Objects: Preview

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.