1. 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

3. Contaminants Reported in IV Solutions
Extrinsic

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

5. 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 , 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

8. 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

11. 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

12. Alumino-silicate with K and Ti
Many shapes and sizes
Alumino-silicate with K and Ti
Talc

13. Sizing Particles by Microscope
Largest Dimension d
Martin’s Diameter
Area A
Area B d
Ferret’s Diameter d
Projected Area d 13

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

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
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. Issues with Sampling Particles in Liquids
Sampling Errors Account for most problems
Accidental Contamination or Alteration by Technician
1. System Preparation
Initial Cleanliness
Calibration
2. Sample Preparation
Contamination
- Particles
- Gases
- Liquids
3. Sample Handling
Aggregation
Settling
Cavitation

23. Particle Counter vs. Microscope
Sizing of Particles
Microscope: Subjective size measurement
Electronic: consistent for specific particle type
Particle orientation and movement
Electronic: large, long particles can be mis-read
Calibration
How to establish accurate sizing and counting
Consistency (repeatability)

24. Sizing Particles by Microscope
Largest Dimension d
Martin’s Diameter
Area A
Area B d
Ferret’s Diameter d
Projected Area d 24

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
Reproducibility
Repeatability

27. Light Obscuration Light Scattering
Detection Ranges
0.1µm
1µm
10µm
100µm
Light Obscuration
Light Scattering 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. Principles: Light Obscuration
Detector Output

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
Detector
Detector Output
Light Trap
Laser Diode
Mirror

34. Principles: Light Scattering
Detector
Particle
Light Trap
Laser Diode
Mirror

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
Martin’s Diameter
Area A
Area B d
Ferret’s Diameter d
Projected Area 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 40

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 41

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

43. Sampling Particles in Liquids
Good, consistent results depend on:
Well-trained operators
Careful technical methodology
Constant effort needed:
Review ways to avoid or reduce errors due to sampling methods
Usually cause “false positives”

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 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

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 False 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>

56. 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

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 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

66. New Sampling Probe 3 probes available Tare New small / short probe
¼” ID =1.2 ml tare volume
1/16” ID = ml tare volume
New small / short probe
1/16” ID = 0.09 ml tare volume
Tests can be performed on 1 ml of product!

67. 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
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