Liquidborne Particle Counting using
Light Obscuration and Light
Scattering Methods
What has been . . .
Focus has been on injectable liquids
• Possibility to block capillaries and arteries
– Red Blood cells are about 5 µm
– Capillary (5 to 10 µm)
– Large veins (10 to 50 µm)
• Threat of microbial infection
• Allergic reaction to foreign substances
2
Definition of Particulate
Contaminants
Unwanted insoluble matter that exist as “randomly-sourced
extraneous substances”
• Excludes homogeneous monotonic materials that exist as a
precipitate or suspension
• i.e. colloids, drug degradation or otherwise derived from a
defined source and can be analyzed by chemical means
Regarded as “contamination” and “adulteration” under Food and
Drug Act
• the chemical composition of the particulate is varied, and
would not be declared on the label
– Examples: bits of paper fiber, fragments of filler material, etc
4
Liquid Particle Counting
Applications
Final Product Testing – USP <788>
• SVP or SVI (Small Volume Parenteral/Injectable)
– Ampoules, Vials
• LVP or LVI (Large Volume Parenteral/Injectable)
– IV (Intravenous) solutions
Process contamination studies
Decomposition studies (stability)
DI or WFI Water
Precision Cleaning – Medical Devices
• Aqueous
• Other Chemicals
5
Other Applications for
Particle Counting
Medical Devices
• Cleanliness of manufacturing environment
• Cleanliness of device before implantation
– pacemakers, stents, artificial arteries
• Cleanliness of reclaimed devices
Design of particulate-based medicines
• Inhalation therapies
• Intentional occlusion of blood flow to cancers
• Time-based dosages
• Transdermal absorption
6
Global Regulations:
Particles in Liquids
USP <788>, EP 2.9.19, JP XV, KP, CP
Primary method
• Optical Particle Counter [OPC]
– Light Obscuration Counter
Secondary method
• Optical microscope
– Subjective
– Labor intensive
– Requires more time to process samples
7
Proposed:
USP 787, USP 1787
USP <787>
Under discussion
Focused on reducing necessary test
volumes due to concerns of
biotechnology manufacturers of cost
for test
Expensive and often very small dose
factory
– for example: 500 uL pre-filled syringe
8
Proposed:
USP 787, USP 1787
USP <787>
Primary method ?
• Optical Particle Counter [OPC]
– Light Obscuration Counter
Secondary method ?
• Optical microscope
– Subjective
– Labor intensive
– Requires more time to process samples
9
Proposed:
USP 787, USP 1787
USP <787>
Small sample volume
- 1 mL ??
Concerns with variability
- within production lots
- in analytical methods
10
Optical Particle Counter
Optical Instrument
•
•
•
•
Must move fluid through sensor
Can quantify particles from 100 nm to 5000 µm
Counts particles individually (one at a time)
Cannot tell you composition
• But results are immediate
11
Many shapes and sizes
Alumino-silicate with
K and Ti
Talc
12
Sizing Particles by Microscope
Largest Dimension
Martin’s Diameter
Area A
d
d
Area B
Ferret’s Diameter
Projected Area
d
d
13
Challenges of Protein-based Products
Handling can change material !!!
•
•
•
•
Agitation
Heat and Light
Contaminates
Container: Vials versus syringes/cartridges
• Shear forces
Key concern is Aggregation
• Reduction of native form (impacts efficacy)
• Introduction of homogeneous aggregates
• Introduction of heterogeneous aggregates
14
Challenges of Protein-based Products
Transparency of most proteineous entities
• Refractive index
• NIST working on calibration material
Not “contamination” but instead a shift from native form
• Not a solution as with small-molecule therapeutics
• Formation of quaternary structures [dimers, etc.]
• Protein complexes
Reconstitution of lyophilized product
15
Refractive Index
Key is the ability to distinguish between the
particle and the surrounding fluid
- needs to be great enough
Optical response is proportional to
comparative index
16
Refractive Index
Key is the ability to distinguish between the
particle and the surrounding fluid
- needs to be great enough
Optical response is proportional to
comparative index
17
Refractive Index
NIST working on protein-like calibration
material
• Probably 2 years away
• Exploring 2 methods of manufacture
• Need thread-like material
• Indices near water
• Stable over reasonable period
18
II. Sample Handling
19
Settling/Agitation
Entrained gas
- sonication probably not ideal with protein
structures
- light vacuum seems to work OK
Settling
Limits collection of particles
- especially of greater mass
- dependent on time and viscosity
- improved collection with slanted containers
20
Consistency of sample
characteristics
Temperature
Settling
Probe position
21
Issues with Sampling Particles in
Liquids
Sampling Errors Account for most problems
Accidental Contamination or Alteration by Technician
1. System
Preparation
2. Sample
Preparation
3. Sample
Handling
Initial Cleanliness
Contamination
Aggregation
Calibration
- Particles
- Gases
- Liquids
Settling
Cavitation
22
Sizing Particles by Microscope
Largest Dimension
Martin’s Diameter
Area A
d
d
Area B
Ferret’s Diameter
Projected Area
d
d
24
HIAC Liquid Particle Counters
Example: HIAC 9703
• The industry standard liquid
particle counter since 1997
• USP <788> was written
specifically around HIAC
technology
• Every major manufacturer of
particle calibration standards
uses the HIAC 9703
25
HIAC Liquid Particle Counters
Example: HIAC 9703+
• Improved sample mounting
method for small vials or
containers
• Detection of usual conditions
such as bubbles or
contamination
• Proven syringe sampler
• SVI and LVI sampling
Reproducibility
Repeatability
• Addresses non-compendial
applications, e.g. R&D and
other low frequency, small
sample volume applications
26
Detection Ranges
0.1µm
1µm
10µm
100µm
Light Obscuration
Light Scattering
1 nm
10
100
1000
10000
100000 1000000
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Light Obscuration
Light Obscuration Sensors and system
• also known as Light Extinction
• also known as Light Blocking
28
Principles: Light Obscuration
Detector
Output
29
Principles: Light Obscuration
Detector
Output
30
Principles: Light Obscuration
31
Particle Detection
Like an air particle counter, the larger
the particle, the larger the pulse that
is created
32
Principles: Light Scattering
Detector
Detector
Output
Light Trap
Laser Diode
Mirror
33
Principles: Light Scattering
Detector
Particle
Light Trap
Laser Diode
Mirror
34
Advantages: Light Scattering
Good sensitivity from 0,1µm to 50µm
Wide range of sample concentration
Good rejection of false counts
High sample flow rates
35
Disadvantages: Light Scattering
More complicated construction = higher cost
Characteristics of particle surface (shiny, color) affect
response
36
Effect of colors and surfaces on Light Scattering
Dark
Light
Shiny
37
Alumino-silicate with
K and Ti
Talc
38
Sizing Particles by Microscope
Largest Dimension
Martin’s Diameter
Area A
d
d
Area B
Ferret’s Diameter
Projected Area
d
d
39
General Comments
on Liquid Counting
Particle Counters Report Size
– But measure an Optical Response
– Difference in reported size
compared to microscope
Projected Area
d
Calibration Relates the Optical
Signal to Size
– Difference between calibration
material characteristics and “real
world” particles
40
General Comments
on Liquid Counting
Particle Counters Report Size
• But measure an Optical
Response
• Differences in reported size
compared to microscope
Calibration Relates the Optical
Signal to Size
• Difference between calibration
material characteristics and
“real world” particles
41
LO results versus LS results
Light Obscuration [LO]
•
Good immunity to variations of surface and morphology
•
Very stable
•
Limit of quantitation circa 1.2 – 1.3 microns
Light Scattering [LS]
Results affected by surface characteristics and coloring
Good stability
Limit of quantitation sub-micron
Problem can occur in the attempt to correlate results of these two
methods above 1 micron
42
System Preparation
2-step Verification - optional:
• Run 2 test solutions
– Blank (“particle-free” water)
– Aqueous solution containing known counts
at 10 µm or 15 µm
In the range of 1000 to 3000 counts per mL
• Frequency – based on risk analysis
–
–
–
–
Each morning
Shift change
Change of operator
Other interval
44
System Preparation
Check for bubbles in sample lines and syringe
• Affects flow rate and calibration
Verify correct calibration curve installed
• Different flow rates for same sensor
• Change of syringe size
• Some companies have multiple sensors
Verify calibration is current
• Sensor resolution and response curve
• IST tests conducted [USP, JP]
45
System Preparation
Instrument Standardization Tests [IST]
• Five tests of system
–
–
–
–
–
Volume accuracy
Flow rate accuracy
Calibration of sensor
Resolution
Count accuracy
• Required by USP and JP but not EP
– USP <788> 31
“…at intervals of not more than six months.”
– JP <24>
“…at least once a year.”
46
Sample Preparation
Removing residue of previous sample
• If previous sample contained particles, may be good plan
to run a “particle-free” blank before the next sample
• Use liquid that is compatible with sample fluid
– An aqueous blank could trigger false counts in
an oil-based sample by causing immiscible
droplets
– Potential residue from previous sample can
cause change of counts
Data from first run of series is often discarded
47
Sample Preparation
Contamination
• Particles
• Gases
• Liquids
48
Sample Preparation
True Particle Sources
•
•
•
•
•
•
•
Residue on glassware and equipment
Ambient air
Paper dust
Glass
Diluent
Residue from previous sample
Colloidal suspensions
False Particle Sources
• RF signals or other electronic interference
• Bubbles from entrained gases
49
Sample Preparation
Work in controlled environment
Use particle-free gloves
Let water flow for 200 mL or more after opening a valve / tap
•
Opening / closing valve generates particles
Wipe the outside of containers before sampling
•
Particles on surface of vials or ampoules
Open vials and ampoules away from beaker or flask
• Particles from activity can fall into open container
• Wash outside of containers to reduce potential particle source
50
Degassing sample
Three common methods
• Allowing to stand in ambient air
Risk of large particles settling
• Sonification [ultrasound]
80 to 120 watts [USP <788>]
30 seconds [USP <788>]
• Vacuum
Bell jar or dessicator
0.6 – 0.8 atmospheres for 2 to 10
minutes
51
Sample Preparation
Possibility of decreasing true particle counts
• Settling
• Lack of agitation
• Position of probe in sample vessel
• Remaining material from previous sample run
– Sample with lower counts
– Blank
52
Sample Handling
Aggregation
Settling
Entrained gases
53
Sample Handling
Aggregation
• Samples held over time or at extremes of temperature
can develop aggregates of smaller particles
• Exposure to light can also trigger this reaction
• Sub-micron particles can thus add to particle counts
above 10 µm
• Suggestion:
– Profile counts under 10 µm [e.g. at 2 or 5 µm] in addition
to standard count sizes at 10 and 25 µm
54
Sample Handling
Settling
• Undercounting caused by
– Gravitational settling
– Failure to suspend particulate matter adhering to
walls or stopper of container
• Standards have recommended procedure for
agitation
– Multiple inversions of original container before
decanting
– Constant motion of liquid during sampling
“Gently stir the contents of the container by hand-swirling or by
mechanical means…” USP <788>
55
Optical Particle Counter
Instrument & sensor
•
•
•
•
Must move fluid through sensor
Can quantify particles from 100 nm to 5000 µm
Counts particles individually (one at a time)
Cannot tell you composition
• But results are immediate
56
Questions?
57
Patent Pending
58
New Hardware: 9703+
Key features
Auto stop for sensor elevator arm
Small vial holding clamp
Sample probe with reduced dead volume
Back-flush and forward flush from front panel
Supports MC-05 sensor (0.5 micron sensitivity)
59
Software: PharmSpec 3
Key new features
Compendial test support continues
USP, EP, JP, KP looks same as previous
PharmSpec versions
Uses same log on as for Windows
Improved Report format
Improved Error Detection and Display
60
Still the HIAC 9703 you know and trust –
only better!
Syringes
• 1 ml, 10 ml, 25 ml
Flow rate settings
• 10 to 100 ml
Sensors
• MC-05 is added
Sampling Probes
• added shorter small-bore probe
Instrument size / shape
• 50%+ of instruments are placed in
laminar flow cabinets.
• Smooth, curved surfaces create less
turbulence for the air flow
61
Easier, Faster and Confident
Sampling
Use less sample, save valuable time
- protect your investment
• Small vial clamp ensures that
sample does not spill during
testing
• Probe needle safety switch
prevents probe damage
• New small needle probe with
industry’s smallest tare volume
62
Easier, Faster and Confident
Sampling
Reduce uncertainty from data
anomalies
• Bubble alarm notification
• Contamination alarm notification
• Advanced notification when
service or calibration is due
• Invalid configuration notification
63
HIAC just got easier!
Less time needed for clean-up
• Automated flushing and cleaning routines
• Push a button, walk away and return to a
clean sensor
Export your data with ease
• Select one, several or all of your historical
data records with our batch export utility
• Select PDF, Word, Excel, or text files
Save time with electronic signature
• Stricter interpretation of 21CFR Part 11
electronic signature process…. WITHOUT
more manual inputs
• Remembers user Login ID
64
HIAC 9703+ Flexibility
Interchangeable sampling probes,
syringes, and sensors
Ensure you have one instrument to manage
all applications
Now supports MC05 sub-micron sensor
Change configuration with no impact to
instrument validation
Customized reporting
Customize the number of reviewers and
approvers for compendial test reports
Add company logo, user-defined descriptors
Customized test recipes
Procedure Builder enables the development
of unique test recipes for your application
Enables testing to marketing license-specific
applications
65
New Sampling Probe
3 probes available
• Tare
¼” ID =1.2 ml tare
volume
1/16”
ID = 0.172 ml tare
volume
• New small / short probe
1/16”
ID = 0.09 ml tare
volume
Tests can be performed
on 1 ml of product!
66
Small Vial Clamp
Small Vial Clamp Platform
• Can be retrofitted
• Can be removed
Ease of Use
• Use one hand to
compress lever arms
• Use other hand to place
sample in central location
• Decompress hand
• Clamp auto-centers and
holds sample container
67
Docking Module
Docking Module
• Enables removal/disabling
of the stir bar mechanism
• Enables field installation of
small vial clamp outside of
the laminar flow cabinet
–Avoid potential of requalification that can
occur if instrument is
moved
Future developments to
expand 9703 applications
68
Sampling Safety Switch
Sampling safety switch
• Ensures the sampling probe
does not crash (and bend or
break) into the docking
module
• Ensures the probe does crash
into or tip the sample
container
69
Liquidborne Particle Counting using
Light Obscuration and Light
Scattering Methods
70