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Liquidborne Particle Counting using Light Obscuration and Light
Scattering Methods
2
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
4
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
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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
6
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
7
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
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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
9
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
10
Proposed: USP 787, USP 1787
USP <787>
Small sample volume
- 1 mL ??
Concerns with variability
- within production lots
- in analytical methods
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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
12
Alumino-silicate with K and Ti
Talc
Many shapes and sizes
13
Sizing Particles by Microscope
Largest Dimension
d
Ferret’s Diameter
d
Projected Area
d
Martin’s Diameter
Area A
Area B
d
14
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
15
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
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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
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
18
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
19
II. Sample Handling
20
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
21
Consistency of sample characteristics
Temperature
Settling
Probe position
22
Sampling Errors Account for most problems Accidental Contamination or Alteration by Technician
Issues with Sampling Particles in Liquids
1. System Preparation
Initial Cleanliness
Calibration
3. Sample Handling
Aggregation
Settling
Cavitation
2. Sample Preparation
Contamination - Particles
- Gases
- Liquids
24
Sizing Particles by Microscope
Largest Dimension
d
Ferret’s Diameter
d
Projected Area
d
Martin’s Diameter
Area A
Area B
d
25
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
26
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
• Addresses non-compendial applications, e.g. R&D and other low frequency, small sample volume applications
ReproducibilityRepeatability
27
Detection Ranges
1 10 100 1000 10000 100000 1000000
Light Scattering
Light Obscuration
1µm0.1µm 10µm 100µm
nm
28
Light Obscuration
Light Obscuration Sensors and system
• also known as Light Extinction
• also known as Light Blocking
29
Principles: Light Obscuration
Detector Output
30
Detector Output
Principles: Light Obscuration
31
Principles: Light Obscuration
32
Particle Detection
Like an air particle counter, the larger the particle, the larger the pulse that is created
33
Principles: Light Scattering
Laser Diode
Detector
Mirror
Light Trap
Detector Output
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Laser Diode
Detector
Mirror
Particle
Light Trap
Principles: Light Scattering
35
Advantages: Light Scattering
Good sensitivity from 0,1µm to 50µm
Wide range of sample concentration
Good rejection of false counts
High sample flow rates
36
Disadvantages: Light Scattering
More complicated construction = higher cost
Characteristics of particle surface (shiny, color) affect response
37
Effect of colors and surfaces on Light Scattering
Dark Light Shiny
38
Alumino-silicate with K and Ti
Talc
39
Sizing Particles by Microscope
Largest Dimension
d
Ferret’s Diameter
d
Projected Area
d
Martin’s Diameter
Area A
Area B
d
40
General Comments on Liquid Counting
Particle Counters Report Size – But measure an Optical Response
– Difference in reported size compared to microscope
Calibration Relates the Optical Signal to Size
– Difference between calibration material characteristics and “real world” particles
Projected Area
d
41
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
42
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
44
System Preparation
2-step Verification - optional:• Run 2 test solutions
– Blank (“particle-free” water)– Aqueous solution containing known counts
at 10 µm or 15 µmIn the range of 1000 to 3000 counts per mL
• Frequency – based on risk analysis– Each morning– Shift change– Change of operator– Other interval
45
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]
46
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.”
47
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
48
Sample Preparation
Contamination
• Particles
• Gases
• Liquids
49
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
50
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
51
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
52
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
53
Sample Handling
Aggregation
Settling
Entrained gases
54
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
55
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>
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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
57
Questions?
58
Patent Pending
59
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)
60
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
61
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
62
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
63
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
64
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
65
HIAC 9703+ Flexibility
Interchangeable sampling probes, syringes, and sensorsEnsure you have one instrument to manage all
applications
Now supports MC05 sub-micron sensor
Change configuration with no impact to instrument validation
Customized reportingCustomize the number of reviewers and approvers
for compendial test reports
Add company logo, user-defined descriptors
Customized test recipesProcedure Builder enables the development of
unique test recipes for your application
Enables testing to marketing license-specific applications
66
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!
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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
68
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 re-qualification that can occur if instrument is moved
Future developments to expand 9703 applications
69
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
70
Liquidborne Particle Counting using Light Obscuration and Light
Scattering Methods