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

Contaminants Reported in IV Solutions Extrinsic

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

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

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

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

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

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

Proposed: USP 787, USP 1787 USP <787> Small sample volume - 1 mL ?? Concerns with variability - within production lots - in analytical methods

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

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

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

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

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

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

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

II. Sample Handling

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

Consistency of sample characteristics Temperature Settling Probe position

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

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)

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

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

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

Light Obscuration Light Obscuration Sensors and system also known as Light Extinction also known as Light Blocking

Principles: Light Obscuration Detector Output

Principles: Light Obscuration Detector Output

Principles: Light Obscuration

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

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

Principles: Light Scattering Detector Particle Light Trap Laser Diode Mirror

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

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

Effect of colors and surfaces on Light Scattering Dark Light Shiny

Alumino-silicate with K and Ti Talc

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

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

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

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”

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

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]

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.”

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

Sample Preparation Contamination Particles Gases Liquids

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

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

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

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

Sample Handling Aggregation Settling Entrained gases

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

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>

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?

Patent Pending

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)

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

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

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

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

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

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

New Sampling Probe 3 probes available Tare New small / short probe 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!

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

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

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

Liquidborne Particle Counting using Light Obscuration and Light Scattering Methods