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Liquidborne Particle Counting using Light Obscuration and Light Scattering Methods

Liquidborne Particle Counting using Light Obscuration and Light Scattering Methods

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Liquidborne Particle Counting using Light Obscuration and Light Scattering Methods

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  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. 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 Talc Alumino-silicate with K and Ti

  12. Sizing Particles by Microscope Martin’s Diameter Largest Dimension Area A d d Area B Projected Area Ferret’s Diameter 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 3. Sample Handling Aggregation Settling Cavitation 2. Sample Preparation Contamination - Particles - Gases - Liquids 1. System Preparation Initial Cleanliness Calibration

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

  23. 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

  24. 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

  25. Detection Ranges 0.1µm 1µm 10µm 100µm Light Obscuration Light Scattering nm

  26. Light Obscuration Light Obscuration Sensors and system • also known as Light Extinction • also known as Light Blocking

  27. Detector Output Principles: Light Obscuration

  28. Detector Output Principles: Light Obscuration

  29. Principles: Light Obscuration

  30. Particle Detection Like an air particle counter, the larger the particle, the larger the pulse that is created

  31. Principles: Light Scattering Detector Output Detector Light Trap Laser Diode Mirror

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

  33. Advantages: Light Scattering Good sensitivity from 0,1µm to 50µm Wide range of sample concentration Good rejection of false counts High sample flow rates

  34. Disadvantages: Light Scattering More complicated construction = higher cost Characteristics of particle surface (shiny, color) affect response

  35. Dark Light Shiny Effect of colors and surfaces on Light Scattering

  36. Talc Alumino-silicate with K and Ti

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

  38. 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

  39. 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

  40. 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

  41. 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

  42. 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]

  43. 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.”

  44. 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

  45. Sample Preparation Contamination • Particles • Gases • Liquids

  46. 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

  47. 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

  48. 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

  49. 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

  50. Sample Handling Aggregation Settling Entrained gases