USP Workshop On Particle Size

Welcome to the
USP Workshop on Particle Size:

Particle Detection and Measurement Day 2
December 8-10, 2010 USP Headquarters, Rockville, MD
USP Workshop on Particle Size: Particle Detection and Measurement December 8-10, 2010; USP Headquarters
Welcome and Opening Remarks

Desmond Hunt, Ph.D. USP Senior Scientific Liaison
Session III: USP General Chapters Track 1: Parenterals Spalding Auditorium Track 2: Aerosols Briggs/Parker/ Marshall/Wiley
Session III, Track 1: Parenterals - USP General Chapters <788> and <789>
Chair: Mr. Scott Aldrich Ultramikro
USP Workshop on Particle Size: Particle Detection & Measurement

8.1 Parenterals USP General Chapters <788> / <789>
Historical Perspective and Current Chapters Review
Scott Aldrich
Quality Standards for Medicines, Supplements, and Food Ingredients throughout the World
Outline
Introduction Background
History and Limits Evolution
History of liquid particle counting
Periods of Investigation Harmonization of <788> Aug 1 2007 Limits according to container volume and method used <1788> informational chapter official USP 34, 2011 <789> follows <788> discussion Chapter <1> Crossover
Current chapters

What is Particulate Matter? Light Obscuration Counting and Operations Membrane Microscopic Counting and Operations
Test Methods

Comparison of LO and MM methods
Introduction
Parenteral injections and ophthalmic solutions are sterile liquids, with expectations for physical appearance and stability. All released product must comply with two limit tests for particle content:
Must be essentially-free of visible particles USP General Chapter <1> Injections
See PF Stimuli Article Vol. 35(5) [Sept.-Oct. 2009], Visible Particulates in Injections - A History and a Proposal to Revise USP General Chapter Injections <1>
Must contain low amounts of sub-visible particles USP <788> Injections USP <789> Ophthalmic solutions

History of liquid particle counting
Periods of Investigation1
Cognitive Latent Definitive Evaluative Harmonization The Future
1Floyd
Benjamin, IES conference 1990

Cognitive Period 1905-1948
1905 First USP reference to parenteral solution as a compendial drug. No references to particulate matter content or clarity. 1916 NF IV includes 6 monographs for parenterals; however, no mention of appearance. 1936 NF VI includes definition of clarity, defining clearness:
aqueous solutions are to be clear, i.e., when observed over a bright light, they shall be substantially free from precipitate, cloudiness or turbidity. Specks or flecks; fibers or cotton hairs, or any undissolved material.
In 1942, USP XII and NF VII both attempt to further define the intention of the phrase substantially free, but only NF includes a published definition. 1946 USP now includes the term substantially free

Latent Period 1949-1965
No changes in compendial requirements, although many reports in literature regarding medical events from particulate matter injection. 1949 USP revision committee eliminates the clarity test and substitutes it with the requirement that every care should be exercised in the preparation of injections to prevent contaminationeach injection, in the final container, be subjected to visual inspection. 1965 - First Limit Standards (Canberra, Australia)
In 1965, the National Biological Standards Laboratory, Canberra Australia published so-called particulate standards. This stimulated compendia and regulatory activities in the US, leading to the Definitive Period.

Definitive Period I 1966-1973
1966 FDA symposium on the safety of large volume parenterals1 1971 LVPs found to contain microbial contamination 1972 Australian comparison of Coulter and HIAC counts for parenteral products2 Australian and English standards for particulate matter in LVPs Australia - ten containers must be less than 2x the following limits:
NMT 100 particles/mL 2m
NMT 5 particles/mL 20m
British Std: mean of 5 containers and individual
1973 Turco and Davis review the clinical consequences of injected particulate matter3
1. 2. 3. FDA - National symposium on safety of large volume parenteral solutions. U.S. Dept. of Health, Education and Welfare, Washington DC, 1966. Kendall and Tilley, Comparison of Coulter counter and HIAC counts for parenteral products Turco S. Davis NM. Hosp Pharm. 1973;8:137-40.

Definitive Period II 1974
1974 Revision of Foreign Particulate Standards Great Britain
NMT 1000 particles/mL 2m NMT 100 particles/mL 5m
Australia
NMT 1000 particles/mL 2m NMT 250 particles/mL 3.5m NMT 100 particles/mL 5m NMT 25 particles/mL 10m NMT 2 particles/mL 20m
PMA, USP, FDA meet to discuss further refinement of the standard for particulate matter in LVPs, to be evaluated by the microscopic method. In December 1974, USP proposed:
NMT 50 particles/mL 10m NMT 5 particles/mL 25m
Late 1974 USP publishes standards for LVPs following PMA and PDA studies and collaboration with AMA, ASHP, PMA:
Absence of particles 50m in a 10 sample mean NMT 1 particle/mL 5m in a 10 sample mean

Definitive Period III 1975-1986
1975 USP XIX chapter <788> used a membrane test to evaluate the particle load for large volume injections (over 100mL nominal volume). Particulate matter in Large Volume Injection
50 particles/ mL 10m 5 particles/mL 25m
1980 British Pharmacopeia issued new standards for LVPs, using either electrical zone sensing (Coulter) or light extinction (HIAC) instruments. 1984 USP revises <788> to differentiate LVPs using membrane microscopic and SVPs using HIAC with proposed limits:
10,000 particles/container 10m 1000 particles/container 25m
Throughout 1985, many meetings between PDA, PMA, FDA, industry and USP reviewed the limits and methods for all volumes A new method, light extinction (obscuration), was added in 1985, in USP XXI chapter <788> for small volume parenterals (SVP, 100mL and lower volume). Thus, LVP standards were established in the US as well, and initiated the discussions for SVPs.
Problems meeting particle standards for terminally sterilized dextrose-containing solutions were due to 5-hydroxymethylfurfural USP and FDA to insert a disclaimer into the definition of valid particulate matter.do not attempt to size or enumerate amorphous, semi-liquid, or otherwise morphologically indistinct materials, that is, material that has the appearance of a stain or discoloration on the membrane surface.
January 1, 1986, the SVP standard and test method become official.

Evaluative Period 1986 1995
The test methods for counting particles were continually improved through collaborative investigation and method revision.
1990 USP XXII defined new limits by membrane for large-volume injections (>100mL):
25 particles/mL 10m 3 particles/mL 25m
for small-volume injections (100mL):

10,000 particles per container 10m 1,000 particles per container 25m
1995 USP 23 <788> provided the most dramatic change.

The light obscuration method became the preferred or Method 1 approach.
Ease of method control, objectivity, and efficiency History of product experience and regulatory filing
Membrane method as Method 2 and an Improved Microscopic Assay for large-volume injections (>100mL):
LO 25 particles/mL 10m and MM 12 particles/mL 10m LO 3 particles/mL 25m and MM 2 particles/mL 25m LO 6000 particles per container 10m and MM 3000 particles per container 10m LO 600 particles per container 25m and MM 300 particles per container 25m
for small-volume injections (100mL):

Evaluative Period 1995 2006
In 2004, a new chapter for ophthalmic solutions <789> was published in USP 27 for particulate matter in three size categories:
LO and MM
50 particles/mL 10m 5 particles/mL 25m The same 10m and 25m limits and 2 particles/mL 50m
MM testing by membrane methodology
Two important non-U.S. compendial organizations, European Pharmacopoeia (Ph. Eur.) and Japan Pharmacopoeia (JP) included the USP change to include the SVI products in particle limits.
Ph. Eur. changed to include SVI products in 2005 JP updated to include them in 2006.

Harmonization Period 2007 2010
2007 all three organizations, through the Pharmacopeial Discussion Group, have harmonized the <788> methods, definitions and limits. This chapter is seen in the respective pharmacopeia as USP <788>, EP 5.5 and JP XIV, XV, and is the current standard. These standards monitor injectable solutions for the content of particulate matter undetected by visual inspection. However, the harmonized USP chapter provides much less method direction than in previous USP <788> versions, and is addressed by the planned publication of chapter <1788> in 2011.
Harmonization - Current USP Guideline

USP 33 <1>--Injections
Stipulates the expectations and tests for all injectable dose forms Foreign and Particulate Matter each final containershall be inspectedevery container whose contents show evidence of visible particulates shall be rejected Test methods concern quantification of particulate matter in: Pharmaceutical injectable products Ophthalmic solutions
USP 33 <788>--Particulate Matter in Injections

2 methods: LO and MM Limits depend upon product volume
USP 33 <789>--Particulate Matter in Ophthalmic Solutions

2 methods: LO and MM Limits are per mL
Exempt from <788> limits:

Radiopharmaceuticals Parenteral products for which labeling specified use of a final filter (provided available scientific data justify the exemption) Irrigating solutions
Current <788> Limits

The use of two methods is a "two-tiered approach. If LO results are suspicious or fail limits, the microscope method is run
Method 1 LO Parenteral Volume SVI LVI
100 mL and lower
Method 2 - Microscope 25m 10m 3000 per container 12 per mL 25m 300 per container 2 per mL
10m 6000 per container 25 per mL
600 per container 3 per mL
above 100 mL
Current <789> Limits

Since 2004 Chapter <789>--Particulate Matter in Ophthalmic Solutions
Official for the sub-visible particle limits of Ophthalmic products All limits on a per mL basis. Method 1 - LO 10m 50 per mL 25m 5 per mL Method 2 - Microscope 10m 50 per mL 25m 5 per mL 50m 2 per mL
<789> Methods are essentially <788> and Limits are tight
Particle Count by Membrane Microscopy Limits for Liquid Products

Comparison of Injectable & Ophthalmic Solution Product Particulate Load Limits As 5mL Fill Volumes.
Particle Size by Membrane Assay 10m 25m 50m USP Limits <788> 5mL Injectable Volume 3000 part./container 300 part./container No specification USP Limits <789> 5mL Ophthalmic Volume 250 particles in a 5mL container or 50 particles/mL 25 particles in a 5mL container or 5 particles/mL 10 particles in a 5mL container or 2 particles/mL

How will we characterize the next period of work?
USP Chapter Crossover

Two Particle Count Tests in USP <788> Guideline Chapter Light obscuration Microscopic Represent the baseline and standardized approach for particle measurement, with which many companies have compiled deep historical data. Provide a snapshot of particle load in final product form Are not necessarily optimal for every formula or dose form Visual Inspection detects particles there is crossover Note that Analytical methods and limits evolve with technical improvements Changes to methods and limits are influenced by regulatory, commercial and harmonization efforts
Size Domains
Where do the visible and sub-visible domains crossover?
Visible 150m Visible Gray zone
Sub-visible 25m
Sub-micrometer 10m 1m
Compendial Threshold
Increasing Probability of Detection
Visual Inspection
Product development and release must include both visual inspection and particle counting Commercial products must comply with Chapters <1> and <788> or <789> Size Crossover: Material may be visible down to ~50m Particles are visible in the upper end of the detectable size range by the primary sub-visible count method, light obscuration The microscopic method retains solids (some semi-solids) at membrane porosity and detects particles to many mms, well into the visible zone
Take Home
Size domain intersection for Chapter <1> visual inspection and Chapter <788>/<789> sub-visible methods LO results are truly sub-visible Membrane results span a wide size range, into the visible Product must meet all requirements from time of release to end of shelf life
What is Particulate Matter?

Particulate matter in injections and parenteral infusions is defined as extraneous, mobile, undissolved particles, other than gas bubbles, unintentionally present in the solution. Practically, any semi-solid to solid material, soft to hard, transparent to opaque may be counted as a particle and may be considered objectionable dependant upon the identity.
Such as air, liquid, gel, singular solid, aggregate, agglomerate, drug solid, salt, polymorph, lubricant, plasticizer
Particulate Matter Details

What forms do we see?
Insoluble, mobile solids/semi-solids Single entity alone or in aggregates one specie or multiple species chemical interactions yielding solids Formulation change yielding solids
Origins
Foreign to the Process: Presence due to Extrinsic Source Part of the Process/Product: Formation due to Intrinsic Source Process function failure Formulation/Package origin
Inherent to the formulation Presence in biologic products
Studying the Particle Load aids Refinement of the Final Product
Test Methods
Light Obscuration Membrane Microscopy
Current Test Methods

Two procedures are specified in both Chapters <788> and <789> <788> has particle limits according to test article volume:
Method 1 (Light Obscuration Particle Count Test)
1a. > 100mL per mL 1b. = 100mL per container 1b. < 100mL per container
Method 2 (Microscopic Particle Count Test)

2a. > 100mL per mL 2b. = 100mL per container 2b. < 100mL per container
<789> has limits per mL regardless of product volume. Method 1 is preferred when examining injections, parenteral infusions and ophthalmic solutions It may be necessary to test some preparations by the light obscuration particle count test followed by the microscopic particle count test to reach a conclusion on conformance to the requirements. It may be necessary to utilize membrane microscopic alone, based upon the nature of the formulation or package.
<788> <789> LO Particle Count Test

Equipment: an apparatus that sizes particles and counts by size using light obscuration (extinction) principle Not Included:

Instrument Standardization Tests Calibration Options
Typical Calibration
Mono-disperse spherical solids between 10m and 25m
System Suitability verified with USP Particle Count Reference Standard Blank Control
Particle-free water Particle-free water is water that has been passed through a 0.22m filter 5 x 5ml aliquots, total load 25 particles 10m
Product Samples
Volume 25mL
Pool 10 or more containers to allow sufficient aliquot
Necessary for LO/Unnecessary for MM
Volume 25-100mL
One container, minimally
Volume >100mL
One container, minimally
What is your statistical sampling consideration?
Light Obscuration Method

The product particulate matter content is determined by counting particles in pre-determined size bins, from small portions of product liquid Pools of sample are required for products <25mL volume Product may be sampled directly from the container Sample liquid passes between a light source and a sensor
Counts are generated when individual particles pass between them Height of each voltage spike depends on the size and shape of the particle causing the spike
Illustration per Groves
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Voltage response is recorded for a sphere of equivalent circular diameter The resultant particle size is generated from a sizevoltage response calibration curve constructed using spherical mono-disperse certified size standards
Per HachUltra
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Enumerates sub-visible solids, but also liquid and gas particles Samples test fluid via a metered system Vendor-specific analysis routines for calibration and daily sampling LO method has few operatorinduced effects Some articles cannot be tested meaningfully by light obscuration
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Blank Control
Degas the open container, by Sonication at 80 to 120 watts for approximately 30 seconds, or allowing the sample to stand to dissipate gas bubbles, or evacuate the sample to outgas (new in USP <1788) Suspend any particles by swirling the vessel by hand, or agitate by mechanical means Determine the particulate matter in 5 samples of particle-free water, each of 5 ml. Retain all counts. Goal: Not More Than 25 particles 10 m in the combined 25 mL sample (NMT 1 per mL)
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LO Instrument Standardization
Instrument qualification is essential to test performance Light Obscuration sections <788, 789> emphasize criteria rather than specific determination method:
Calibrate with spheres 10-25m System Suitability can be verified with USP PCRS
User is responsible for

Ensuring that the counter is used and operated according to the manufacturer's instructions Ensuring proper standardization methods are applied to instrument see Chapter <1788>
Instrument Standardization Tests Calibration Interim checks Daily Weekly 3 month
These principles must be followed ensure that instruments operate accurately within defined ranges
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LO Test Particle Limits

Reporting: average of 3 aliquots not to exceed particle limit
General Chapter
<788> Test 1.A. Large-Volume Injections (more than 100mL) <788> Test 1.B. Small-Volume Injections (less than and equal to 100mL) <789> Ophthalmic Solutions
10 m
25 per mL
25 m
3 per mL
1.A. > 100mL
1.B. 100mL
1.B. <100mL
Membrane Assay Method

Harmonized Compendial Methods USP/EP/JP
100 x 2% with graticule Two illumination paths USP Graticule verified at installation Not included:
Calibration day-of-use Blank control considerations Qualification
Training and Inter-Lab Certification Mono-disperse spherical solids from 10m and up USP Particle Count RS
Microscopic Particle Count Test

Blank Control for <788> <789>
Particle-free water USP Particle-free water is water that has been passed through a 0.22-m filter 50mL aliquot total load: NMT 20 10m and NMT 5 25m Considerations for <789>?
Sampling
LVP: may use single containers SVP: may use single containers Unless volume 25mL use 10 fills, pooled Dilution and Recon with particle-free water or suitable solvent 20x inversion to mix Entire volume is sampled Use wetted, full particle-free water rinses
Membrane Microscopy Method

Captures solid matter from liquid of one or more product containers onto a filter membrane Enumerates sub-visible to visible, solid to soft or semi-solid particulate matter on the membrane surface Retained particles are counted by scanning the retention surface at 100 with a compound binocular light microscope Method is highly operator-dependent Operator makes decisions regarding particle size and acceptability on-the-fly
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Membrane Microscopy Lab

Minimize traffic Separate HEPA areas for wet filtration and dry counting steps Dedicated glassware Dilution and pooling systems developed for product volumes, product types Establish lab controls Operator training essential Operator performance evaluation recommended
Microscopic Particle Count Test

Full Count particles in entire effective filtration area
Wet zone of liquid passage Inner diameter of funnel
Partial count is allowed; however <788> undefined <1788> defined

If 1000 or less particles are present, the membrane should be fully counted If the count difference between a GFOV at center and one at the edge is <2X, then Count 20 GFOV for 25mm (16mm EFA) membranes Count 100 GFOV for 47mm (37mm EFA) membranes
MM TestParticle Limits
Reporting: average count from units tested not to exceed particle limit
General Chapter
<788> Test 2.A. Large-Volume Injections (more than 100 mL) <788> Test 2.B. Small-Volume Injections (less than and equal to 100 mL) <789> Ophthalmic Solutions
10 m
12 per mL
25 m
2 per mL
50 m
Not applicable
3000 per container
300 per container
Not applicable
50 per mL
5 per mL
2 per mL
2.A. > 100mL
2.B. 100mL
2.B. <100mL
Informational Chapter <1788>

Provides LO calibration and control guidance removed from <788> after harmonization
Instrument Standardization Tests Flow Rate Volume Accuracy Calibration Sensor Resolution Particle Counting Accuracy
Provides microscopy setup and counting guidance Provides discussion regarding pharmaceutical development practices Previewed in Pharm Forum 35(6) Nov.-Dec. 2009 Official USP34, 2011
LO and MM: Compare and Contrast

Light Obscuration Provides a robust, vendor-supported, precise and accurate electronic particle count methodology It counts suspended particles that are solid and others that may be semi-solid, liquid or gas Membrane Microscopy Provides a count of particles by a 100 microscopic scan of a membrane surface Requires interpretation of some particles; prone to subjectivity Provides an orthogonal means of counting As a second-pass, is it a Referee method ? The two methods yield different perspectives of the particle population
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Light Obscuration Effectively counts all particles as equivalent circular diameters, over a wide range of concentration Skews resultant sizes relative to shape when deviating from the ideal spherical, or 1:1:1, axial ratio Counts air bubbles and immiscible oil droplets
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Membrane Microscopy Directly compares isolates to calibrated 10m and 25m circles and includes a linear scale Operator is directed to mentally transform the particle image onto the equivalent 10m and 25m circles while counting the membrane isolates Many users attempt to correlate the membrane count to that obtained by LO
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Ongoing Revision Process

Current methods and reliability are continually reviewed and improved by USP staff
Contribution from industry Assistance by industry specialists
Contributions from industry and by industry specialists are overseen by the USP-DF (Parenteral Products: Industry) Expert Committee USP publishes Stimuli Articles in Pharmacopeial Forum to allow perspectives, dissension, and initiatives from participants Certain formulations cannot be tested directly by either method:
Examples: sterile suspensions, nano/micro-suspensions, some emulsions, low-volume and special device products
USP Workshop Particles

December 8-10, 2010
USP Chapters <788> and <789> Methods - VS Alternative Methods for Investigation and Compliance
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Biotech products Vacines Novel treatments for Cancer Nanoparticles (overcoming insolubility) Controlled release microspheres Polymers Crystalline nanoparticles Liposomal formulations
USP Workshop on Particles
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Extrinsic Glass/stopper fragments Metals Silicon oil Filter/process particles Skin flakes Ordinary dirt particles Insect parts Fibers: clothing/hair/etc.
Intrinsic Ingredient degradation API / Excipient changes Active ingredients Protein aggregation Aggregation/foreign matter Silicon interaction Process related Ingredient anomalies Immiscible droplets
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Leachables Extractables Foreign particles Protein aggregation Glass delamination Increased use of plastics Pre-filled syringes New stopper materials/coatings
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Compliance
Detect manufacturing problems USP Particulate Matter, Lot release Formulation degradation over time/stress/stabil.
Research
Particle Size Analysis (especially sub-micron) Evaluation of processing methods Evaluation of ingredients New drug products are more complex and often require more characterization.
Investigational (methods, understanding)

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Provides 100% inspection of the batch. Method is probabilistic Provides a pass/fail from brief observation. Over 12 variables affect results. Interpretation of the term essentially free Some rejects may not be not counted. Particles in rejects may not be identified. Plays vital role with complex formulations
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What is the smallest particle size our inspectors see reliably ? What factors in sub-visible results, show correlation with V.I. results? What is the variability between our inspectors at this plant, and those in our other plants ? Same with <788> results ? How much does this variability affect our product reject and acceptance levels ? (cost) ? Do we understand the relations between Vis. Inspector Training, our actual inspection process, our rejection levels and USP<788> with each of our products ? What are the critical issues for our products when comparing human and automated inspection with Sub-Visible (USP<788>) results ?
Attend the annual PDA Visual Inspection Forum in Fall/2011.
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METHOD I Light Obscuration (LO) Laser (usually) light extinction (blockage). METHOD II Microscopic Membrane (MM) Sample is filtered using a small pore size (1um) membrane and examined by optical microscopy.
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Particle size instruments dont measure particle size. Different methods or instruments used to measure particle size (or count), produce different results. The current Compendial methods are LO and MM
Current (laser base) instruments for particle counting Investigational (orthogonal) method Other (analytical) complimentary principles
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Fibers & discrete particles
Gels
Immiscib droplets
Formulation
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USP <788> Particulate Matter Methods I and II
Single Particle Optical Sensing Test Results (Summation Mode) Particles per mL size (in microns) >0.5m
Sample
Microscopic Membrane Method > 25m > 10m > 25m
> 0.7m
> 0.8m
> 1m
> 2m
> 5m
> 10m
737995
A
562576
523208
460241
153786
36004
4441
4.7
2.6
1020601
B
768623
707751
603883
171300
41064
7145
203
2.3
0.7
1732076
C
1449868
1376814
1242247
497642
148685
22078
128
2.7
1.3
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SAMPLER
Measure Volume accurately ( 5%) Ensures consistent flow rate
Low flow rates are advantageous for investigations/etc.
Volume precision Commonly syringe based (1, 5, 10, 25 ml)

Accepts large or small containers
Flow rates are usually limited to syringe size (may not achieve 30ml/min with 1ml syringe)
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SENSOR Single detector for USP Minimum size sensitivity: 2 microns (or less) Flow rate range from 10ml/min Large cell opening (400 m) preferable Know coincidence values for 2 to 25 microns Remote sensor mounting may be helpful for
Use in a small Biosafety cabinet or clean hood Accommodates very large containers Allow sampling directly from experiment Easier to accommodate stirring boxes
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Software (must be part 11) Includes recipe structure Includes particle size distribution capability Export data to Excel Includes custom alarms for size Built-in PQ Check routine with trend monitor Converts output report to per/ml or other Graphic sample overlays for comparisons
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LO instruments with < 15 channels of size resolution.

Provide essential reporting Simple reports simplify training and SOPs Easier to calibrate since fewer sphere sizes used Data generally reported in a table Reports should provide easy to see
Individual runs, displayed to observe run-run informity Display all sizes reported in Cummulative mode Provide additional reports to graphically compare sample counts (per ml or per container) vs size
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Verify volume accuracy Verify flow rate Verify size accuracy Verify count accuracy Measure system drift Perform routinely Verification tests can overlap Use Polystyrene latex particles (PSL) Use standards certified for Size AND Count
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Date 3/2/2009 3/3/2009 3/4/2009 3/5/2009 3/6/2009 3/9/2009 3/10/2009 3/11/2009 3/12/2009 3/13/2009 3/16/2009 3/17/2009 3/18/2009 3/19/2009 3/20/2009 3/23/2009 3/27/2009 3/30/2009
Previous Method Total Particle Std Count > Initials Size (m) 10m AP AP AP AP AP AP AP AP AP AP AP AP AP AP AP AP AP AP 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 15.02 4092 4041 4049 4028 4169 5083 3925 5359 4383 4028 3885 3865 3867 3921 4011 4199 4356 3928 1.33 1.43 1.63 1.65 1.64 1.81 1.79 1.79 1.58 1.36 1.15
New Method Counts Within Mean Size 15.12 15.10 15.13 15.07 15.04 15.22 15.28 15.38 15.34 15.19 15.20 15.19 15.10 15.10 15.11 15.36 15.35 15.35 Std Dev 0.42 0.42 0.42 0.41 0.42 0.42 0.52 0.55 0.50 0.55 0.52 0.50 0.51 0.53 0.53 0.52 0.53 0.52 3.68 3.67 3.69 3.95 4.01 3.68 3.94 3.94 3.81 3.57 3.68 3.73 Dil Fac Peak 3285 3147 3361 3318 3215 2993 4166 4106 3924 4299 4202 2923 4076 4360 3884 4083 4331 4562
Size Error in percent 0.6658 0.5326 0.7324 0.3329 0.1332 1.3316 1.7310 2.3968 2.1305 1.1318 1.1984 1.1318 0.5326 0.5326 0.5992 2.2636 2.1971 2.1971 PQ Check Lot # JB16A JB16A JB16A JB16A JB16A JB16A JB16A JB16A JB16A JB16A JB16B JB16B JB16B JB16B JB16B JB16B JB16B JB16B
Ratio 10/15 1.54 1.48 1.51 1.68 1.73 1.33
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Electrozone Sensing (EZS) Optical Microscopy (human observation) Image Analysis (camera w/software analysis)
Static Dynamic
Single Particle Optical Sensing (SPOS) Nano Particle Tracking (NTA) Fluorescence NTA (FNTA) Archimedes, mass of individual particles Spectrex
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Syringes 1 ml, 10 ml, 25 ml Flow rate settings 10 100 ml Sensors (MC05 is added) Sampling Probes (added short-small bore probe)
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Interchangeable sampling probes, syringes, and sensors

Ensure you have one instrument to manage all applications New 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 Copy a recipe and make the changes!
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www.hachultra.com
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USP<788> LO Investigational 0.5 m Or FxNano 150 nm

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HACH HRLD150 2.0 to 400m PMS APSS 200 2.0 to 120m HACH HRLD150 1.0 to 150m Accusizer LE400-1 1.0 to 400m Accusizer Fx 0.7 to 400m * Accusizer 780 0.5 to 400m HACH MicroCount 05 0.5 to 400m PMS S05 0.5 to 20m PMS S03 0.3 to 20m PMS S02 0.2 to 2m Accusizer FxNano 0.15 to 10m * Nanosight LM20 0.03 to 1m * Coulter Counter 0.4 to 1,000m Dynamic Image Analyzers (next section) *Count determined via algorithm Contact Mfr for details
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http://www.malvern.com/common/downloads/MRK652.pdf
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www.nanosight.com
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www.nanosight.com
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Instrument Photo
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www.pssnicomp.com
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www.pssnicomp.com
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Dynamic Light Scattering (DLS) RAMAN spectroscopy Fluorescence microscopy Size Exclusion Chromatography AFM TEM Fluorescence Correlation Spectroscopy Scattering Flow Cytometry Separation methods Optical Microscopy
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- Determine particle concentration - Measure changes in essential particle size versus concentration over time - Compare results to other instruments and methods (SEC, UAC, etc.) to verify their results and gain better understanding - Measure changes with different excipients, surfactants, and ingredients. - Use destabilizing methods to develop a predictive model for aggregation.
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USP<788>, <789> now harmonized, so not likely to undergo major revisions. New Chapter Biologics makes sense . . . Unique issues can be present in ordinary Injectables Published data points to difficulties with the use of USP<788> to resolve several sample issues. USP should provide guidance/methods-compliance May result in several useful instrument/methods. Need wider industry data/experience in applications USP needs input from Regulatory & Industry . . . Perhaps in form of Advisory Committee, Forums, or partnering with PDA groups.
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Grateful appreciation to :
USP Committee for the opportunity to participate, and . .
To these Manufacturers for the use of their systems and their help. Nanosight: Jeremy Warren, Jim Munhall Brightwell Tech. Dave Thomas, Clark Fluid Imaging: Lew Brown, Mike Smith Occhio Image: Vincent Chapeau, Dr. Larry Unger Particle Sizing Systems: David Nicoli, Kerry Hasapidis Univ. CO, Denver: John Carpenter Affinity BioSystems: Ken Babcock Particle Measuring Systems: Dwight Beal, Ron Adkins Spectrex: John Hoyt
10 9
11 0
A Review of PF 30.6 Stimuli Article Particulate Contaminants of Sterile Injectable Drugs and Limits Set by USP General Chapter <788> B. Pillari, Ph.D. Microbiology Team Leader FDA, Office of Generic Drugs December 9, 2010
Goal of the Stimuli Article
Generate a discussion regarding the need and usefulness of tighter particulate contamination limits as set by USP <788>; Data mining over a 5 yr period suggests that the acceptance limits could be lowered
thus increasing drug safety without placing excessive burden on the industry.
112
Discussion Points
Particulates Particulate Limits - USP <788> ANDA Batch Records 1998 2002 Analysis
113
What type of particles? Why are they there? Where do they come from? What damage could they do?
114
Sources of Non-Viable Particulates

Rubber from stoppers Silicone from siliconization process Glass from vials Plastic from molding vials and bags Metal filings from filling machine Dust and pollen from air
115
Warning letter by NJ District of the FDA, September 29, 2003:

Failed to prevent release of 10 lots of Zemuron Injection containing metal fragments that were introduced into the product after plant engineer installed the wrong sized filling pump which caused pieces of metal to be scraped into the product.
116
USP 33 <788> Particulate Matter in Injections

Light Obscuration: Particle Size 25 m 10m SVP
NMT 600 NMT 6000 (Per Container) NMT 3 (Per mL)
117
LVP
NMT 25
USP <788> Two Test Methods
Method 1 (Light Obscuration Particle Count Test) Method 2 (Microscopic Particle Count Test)
118
USP <788> Two Test Criteria
SVP (<100 mL/container) LVP (>100 mL/container)
119
USP <788> Two Particle Sizes

>10 m to < 24 m >25 m to Visible (~ 50m)
120
USP 33 <1> Injections Particulate Matter in Injections

the injectable solution should be essentially free from visible particulates
121
Blood Cells & Capillary Bed
122
Relative Particle Sizes

Red Cells ~ 7m White Cells ~ 8 - 20m Platelets ~ 2m
1 millimeter = 1000m Human Hair is ~ 50-150m thick* HIV is ~0.1m
*(McCrone, W.C. and Delly, J.G. (1973). The Particle Atlas, Volumes I-IV, Ann Arbor Science Publishers, Ann Arbor, MI.).
123
Types of Risks

Thrombosis Granuloma Liver Damage Phlebitis Multiple Organ Failure Pulmonary Disease
124
SVP Particulate Counts by Year

SVP Lots Size
m
1998 97
1999 68
2000 99
2001 64
2002 78
10
259 326
25 10 25 10 25 10 25 10 25
214 14 587 44 194 16 291 51 274 15 635 54 153 19 238 39
Mean 11 28 SD
125
ANDAs 1998-2002
ANDA Type # of ANDAs # of Firms # of Drugs Injection "For-Injection"
1998 1999 2000 2001 2002 Total
64 15 25 85 12
52 7 23 50 18
68 17 30 80 19
53 17 33 53 11
58 17 42 66 12
295 73 153 334 72
# of Lots
126
SVP Mean Counts 1998 To 2002:

25m 10m 406
Number of Lots
Mean Counts (USP Limits)

Stand. Dev.
15 (600)
43
219
(6000)
415
127
Particle Counts by Dosage Form and Process

Dosage Form
Injections Lots Analyzed For Inject.
Sterilization
Aseptic
Terminal
334
72
294
112
25 m Particles
Mean Counts
Std. Dev. 10 m Particles Mean Counts Std. Dev.
13 38
P<0.05
24 44
17 45
P<0.05
10 24
181 384
P<0.01
375 401
245 438
P<0.01
154 289
128
Lots with Counts >1400

Firm Date FORM Vial Ster.
A 1998 A 1998 B 2000 B 2000 C 2000 D 2000 D 2000 D 2001 E 2002
INJ INJ FIJ FIJ INJ INJ INJ Inj INJ
GV GV GV GV GV GV GV GV Am
As As As As As T T AS As
25 m 10 m 28 5188 39 2292 265 3741 80 2330 377 2385 14 3856 116 1410 418 602 56 1892
129
Conclusions
Limiting Levels of Particulate in Injections is a safety issue. Current Manufacturing Processes Capabilities are far ahead of USP <788> limits. USP <788> acceptance limits could be lowered.
130
Acknowledgements

OGD Microbiology Team Document Room Staff Special Thanks To: Nrapendra Nath Bonnie McNeal Don Obenhuber Lisa Shelton
131
Rest in Peace Nrapendra Nath, Ph.D.

November 14, 2009
132
Session IV: Large Molecules and Emulsions

Chair: Dr. Satish Singh, Pfizer
Current State of Subvisible Particle Analysis in Protein Products

Linda O. Narhi, Shawn Cao and Yijia Jiang
outline
Introduction: Protein aggregates Challenges in sub-visible particle analysis of biologics Applications of modified LO method to protein product Other methods Future directions
Proteins can aggregate through different mechanisms

Assembly Structural Changes chemical modification
(e.g due to air-water
interface)
Silicone oil or Nano-particles
Nuclei
Structural Changes, chemical modifications
Adsorption/ Assembly
Thirumangalathu R et al 2009, J Pharm Sci (online) Bee J et al 2009, J Pharm Sci (online)
Krishnan S et al 2002, Biochemistry, 41: 6422-31 Chi EY et al 2003, Protein Science, 12: 903-13 Chi EY et al 2003, Pharm Research. 20: 1325-36
Thanks to Eva Chi (Univ of New Mexico)
Biopharmaceuticals encounter various stress conditions during manufacturing, storage and delivery that can cause aggregation
Fermentation & Harvesting Purification Formulation Drug Product Manufacture Storage Shipping Final Preparation Administration Stress Conditions Heat Freeze-thaw Exposure to light
Protein concentration Formulation change pH, salt Addition of extractables/leachables Chemical modification Mechanical Stress Surface effects and interfaces Nano-particle nucleation sites
Protein aggregates can have widely varying properties

Distribution of particle sizes (nm to m) Shape Reversibility Stability Chemical Modification Density Exposed T and B-cell epitopes Covalent vs. non-covalent bonds Morphology (fibrils, ordered/unordered structure, crystalline structure, amorphous, etc.)
Total amount of aggregate

Native vs non-native conformation
Protein aggregates represent an extremely low fraction by mass of the total protein, are typically dynamic and can be influenced by many factors, including the drug product container/closure.
Protein aggregates range in size (1 million fold)

Definitions by size (all are aggregates): Oligomers:10 nm 0.1 m Particles: - Submicron: 0.1 to 1 m - Subvisible: 1 to ~ 125 m - Visible: 125 m
0.001m
0.01m
0.1 m
1 m
10m
100m
1000 m
10,000 m
Currently there are no generally accepted definitions for particulates; these can be defined based on either size or mechanism of formation
Different techniques are needed for different size ranges. These also differ in sensitivity
oligomers
FFF-UV-RI-LS SECUV-RI-LS, AUC 0.001 m 0.01 m IgG Monomer 150 kD, 0.01 m Visual inspection
USP <788> Particle Matter = Subvisible Particles (~ 1 100 m)
sub-m particles
subvisible particles
visible particles
Flow microscopy
Light obscuration
0.05 m 1 m
50 m
600 m
Other technology: light scattering (static and dynamic), newer LO, coulter counter
molecules, mg/mL
particles, # of particles/mL
Applications of compendial method to protein aggregates
Lack of a protein particle standard has made the development of quantitative methods difficult
PS Latex Particle (15 m) Used for calibrating instrument Silicone oil droplet Protein particles
Disadvantage:
Lack of appropriate instrument calibration Inaccurate measurements under- or over- reporting Multiple types of aggregates size and properties
Challenges in Using USP <788> Method for Protein Solutions

Pertinence of the concentration limits (6000/600) to protein therapeutics
For small molecule parenterals:
Foreign material / contaminant Occlusion of capillaries Microbial infection Immunogenicity Historical data pool
For protein parenterals:

Foreign material
- Occlusion of capillaries - Microbial infection
Protein particles, product quality attribute

- Immunogenicity - Protein specific
No historical data pool
Importance of sample handling

Decreasing particle counts for reversible particles Generating particles by manipulation (stirring vigorously, evaporation
Challenges in Using USP <788> Method for Protein Solutions (contd.)

Importance of protein particles at smaller sizes
USP <788> requires 10 m and 25 m measurements Large amounts of small protein particles (e.g. 2 m) may be more immunogenic than small amounts of large protein particles Other utilities of quantifying smaller protein particles:
Could be predictive of larger particle formation with time For investigation and characterization applications For process and formulation development
Solutions at high protein concentration

Undercounting due to changes in optical properties (RI, OD) and increased background
Optical properties of protein aggregates are similar to those of the monomer Differentiating between protein and other particles Protein particles are often not captured by the filtration step in for the microscope method
Effect of Vacuum Degassing for Protein Solution

10 m particles/mL
Allow to stand
900 800 700 681 592 459 400 300 200 100 0 0 1 2 3 4 5 6 420 490 444 465
Vacuum (75 Torr) degas

900 800 700 681
500
Particles/mL
Particles/mL
600
600 500 400 300 200 100 51 0 0 1 2 3 4 5 6 71 75 86 87 86
Degas Duration (hour)
Degas Duration (hour)
Comparison of 1 mL vs. 5 mL Draw Volume: Protein Solution

10 m particles/mL
350 300 250
Particles/mL
Test 1
200 150 100 50 0 1 5
Test 2
Test 3
SVLO: 102 8 particles/mL 5 mL: 99 5 particles/mL
117
107107
103102102
99 10693
100
92 94
99 96 99
Sam ple Volum e (m L)
It might be possible to develop an even smaller volume method

15000
Run 1
Run 2
Run 3
Run 4
Run5
Run6
Run 7
Particles/mL
10000
5000
0 pre_buffer 4x0.2mL Tare 0.7mL pre_buffer 7x0.2mL Tare 0.1mL pre_buffer 7x0.2mL No Tare pre_water 7x0.2mL No Tare pre_water 7x0.2mL No Tare pre_water 7x0.2mL Tare 0.1mL pre_water 7x0.2mL Tare 0.1mL pre_water 7x0.2mL Tare 0.1mL
mAb Sample
How Many Vials to Test?

USP <788>: For large-volume parenterals, single units are tested. For small-volume parenterals less than 25 mL in volume, the contents of 10 or more units is combinedto obtain a volume of not less than 25 mL (and test). Small-volume parenterals having a volume of 25 mL or more may be tested individually USP <788>: The number of specimens (tested) must be adequate to provide a statistically sound assessmentstatistically sound sampling plans must be developed
1400 1200
Particles/mL
1 2 3 4 5 6 7 8 9
1400 1200
Particles/mL
1000 800 600 400 200 0
1000 800 600 400 200 0 1 2 3 4 5 Vial 6 7 8 9
Vial
How many samples to test depends on product history

20 20 18 16 14 14
Vials of Vials Number of Number
12 10 8 6 4 2 0 0 500 500 1000 1000 1500 1500 2000 2000 2500 2500 3000 3000 Mean 100% Mean Mean 100% ofof Mean
When a product is known to have low amounts of particles such as no more than 1000 particles per container for the 10 m particles, testing 1 unit per lot is sufficient to determine with confidence if the lot conforms to the compendial acceptance criteria. Conversely for a product known to have a high amount of particles such as 3000 3000 particles per container, testing more units may be needed. It may be necessary to set a limit of number of units such as 10 units per lot to test.
Pertinence of the 6000/600 per Container Limits

Instead of just pass/fail, product particle history is just as important.
Particles/mL
5 Product Lot
Particles/mL
5 Product Lot
Analytical Techniques for Measuring Subvisible Particles Applications to Protein samples
Subvisible particle levels vary and are dependent on the container closure and formulation
10000 2 m 5 m 10 m
10000 2 m 5 m 10 m
Type I Glass Vials

1000
Particles/mL
Particles/mL
Pre-filled Syringe
1000
100
100
10
10
1
Sample 1_T1 Sample 1_T2 Sample 1_T3 Sample 2_T1 Sample 2_T2 Sample 2_T3
1
Sample 3_T1 Sample 3_T2 Sample 3_T3 Sample 4_T1 Sample 4_T2 Sample 4_T3
Samples
Samples
10000
2 m
5 m
10 m
Particles measured in the absence of protein

1000
Particles/mL
100
10
1
In vials Placebo PFS
Samples
The majority of particles measured from product stored in a prefilled syringe are silicone oil
Product in PFS_
Product in vial
Smaller SbVP may be predictive of amount of larger particles

10000
Native Protein Light & Heat Stressed Protein
1000
100
10
A relationship exists between particles of different sizes, with the slope being productdependent
30
Particles/mL
1 0 5 10 15 Particle Size (um)

1000
20
25
100
Visible particles observed in 80% units
Particles/mL
10
No visible particles observed

1 1 10 Sizes (m ) 100
Information on subvisible particles below 10m can be useful for process characterization
Sample Particles/mL at 2 m 339 78 29 6 19 9 50 14 680 51 20 3 Particles/mL at 5 m 29 2 11 5 14 9 12 9 36 4 52 Particles/mL at 10 m Below LOQ Below LOQ Below LOQ Below LOQ Below LOQ Below LOQ Particles/mL at 25 m Below LOQ Below LOQ Below LOQ Below LOQ Below LOQ Below LOQ
Control (unfilt., Lot 1) 1x filtration Lot 1 2x filtration Lot 1 5x filtration Lot 1 Control (unfilt., Lot 2) 1x filtration Lot 2
Filtration by 0.2 m filter has removed particles at 2 m 1x filtration is effective in removing the particles Particle concentrations are below LOQ at 10 and 25 m
Analytical Techniques for Measuring Subvisible Particles Comparison of Techniques
The absolute quantity of particles varies between techniques

HIAC 100000 MFI
10000
Particles/mL
1000
100
10
1 2.00 5.00 10.00 15.00 ECD (m ) 20.00 25.00 50.00
HIAC, MFI and Coulter Counter Counting Results were Similar for 5 m PSL Standard in a Weak Electrolyte Buffer
5um PSL in a Buffer
60000 Coulter 50000
R2 = 1
MFI
HIAC
40000 P/mL
R2 = 0.9993
30000
R2 = 0.998
20000
10000
0 0 20 40 60 % Concentration 80 100 120
comparable counting results (Coulter MFI HIAC) good particle concentration vs. dilution linearity for all 3 techniques
HIAC, MFI and Coulter Counter Gave Different Particle Concentrations and Particle Size Distributions for Protein Particles in mAb Z DP at 30 mg/mL
SbVP_mAb Z at 50% DP
60,000 HIAC 50,000 40,000 30,000 20,000 10,000 0 0 10 20 30 Size (m) 40 50 60 MFI Coulter
Particles/mL
1,000,000 100,000 10,000 1,000 100 10 1
10
20
30
40
50
60
HIAC has the lowest counts of particles through all sizes and dilutions Coulter has highest counts of smaller particles (2-5 m) MFI has the highest counts of larger particles ( 5 m).
The Effect of Protein (mAb Z) Concentration on Counting Results by the 3 Techniques All Linear
80,000
2 m SbVP_mAb Z DP 30mg/mL Linearity

HIAC MFI Coulter
40,000

HIAC MFI Coulter
60,000
30,000
Particles/mL
R = 0.9879
40,000
R = 0.998
Particles/mL
R = 0.9988
20,000
R = 0.9971
10,000
20,000
R = 0.9647 R = 0.9825
0 0
20
40 % DP
60
80
100
20
40 % DP
60
80
100

20,000
HIAC
MFI
Coulter
3,000

HIAC MFI Coulter
15,000
Particles/mL
Particles/mL
R = 0.9974
10,000
2,000
R = 0.9932
1,000
5,000
R = 0.9974
0
R = 0.9833 R = 0.9642
0
R = 0.9824
20
40 % DP
60
80
100
20
40 % DP
60
80
100
Note: 100% DP = 30 mg/mL mAb Z
HIAC, MFI and Coulter Counter Gave Different Particle Concentrations and Particle Size Distributions for Protein Particles in mAb Y DS at 120 mg/mL
SbVP_mAb Y stressed DS at 120 mg/mL
300,000
HIAC
MFI
Coulter
1,000,000
Particles/mL
200,000
100,000 10,000 1,000 100
100,000
10 1
10
20
30
40
50
60
0 0 10 20 30 40 50 60
Size (m)
HIAC has the lowest counts of particles through all sizes and dilutions Coulter has highest counts of smaller particles (2-5 m) MFI has the highest counts of larger particles ( 5 m).
The Effect of High Protein ( mAb Y) Concentration on Counting Results by the 3 Techniques Non-linearity on HIAC and MFI
2 m SbVP_mAb Y 120mg/mL stressed DS Linearity
300000

40000
Coulter
MFI
HIAC R = 0.9556
30000
Coulter
MFI
HIAC
200000
R = 0.885 R = 0.6128 Particles/mL

20000
Particles/mL
100000
10000
R = 0.9445 R = 0.029
R = 0.4734
0 0
20
40 % DP
60
80
100
20
40 % DP
60
80
100
6000 5000 4000

Coulter MFI HIAC
R = 0.9959
3000 2000 1000
R = 0.725
0
R = -0.349 80 100
20
40 % DP
60
HIAC: not linear Coulter Counter: linear for 2& 5 m results MFI: linear only for 10 m results
Particles/mL
Linearity Improved for HIAC and MFI When High Protein (mAb Y) Concentration Results were Excluded
2 m SbVP_mAb Y stressed DS Linearity
150000 20000

HIAC R = 0.9939
15000
R = 0.9953
120000
Coulter
MFI
Coulter
MFI
HIAC
R = 0.9214 Particles/mL
90000
Particles/mL
10000
60000
5000 30000
R = 0.9841 R = 0.9552 R = 0.948

0
20
40 % DP
60
80
100
20
40 % DP
60
80
100

3000
Coulter
MFI
HIAC
2000
R = 0.9742
Particles/mL
1000
R = 0.9832 R = 0.9578 0 20 40 % DP 60 80 100
HIAC is NOT Linear as a Function of DP/Protein Concentration, PFS DP

5000 4500 4000 3500 10 um Particles/mL 3000 15 um 2500 20 um 2000 25 um 1500 50 um 1000 Poly. (2 um) 500 0 0 20 40 60 80 100 120 140 160
2 m particles
y = -0.3624x2 + 80.18x R = 0.9982 2 um 5 um
Concentration (mg/mL)
For Internal Use Only. Amgen Confidential.
164
HIAC is NOT Linear as a Function of Protein Concentration

DS stressed to increase particle counts
14000
2 m particles
12000 y = -1.0544x2 + 234.22x R = 0.9903 10000
2 5 10 15 20 25
Particles/mL
8000
6000
50 Poly. (2) Poly. (5) Poly. (10) Poly. (15) Poly. (20) Poly. (25)
4000
2000
0 0 20 40 60 80 100 120 140 160 Concentration (mg/mL)
165
HIAC is Linear as a Function of Particle Concentration When Protein concentration is kept constant (35 mg/mL)
14000
12000
y = 113.73x + 429.4 R = 0.9983

2 um 5 um 10 um
10000 Particles/mL
8000
15 um 20 um 25 um
6000
50 um Linear (2 um)
4000 y = 28.379x + 79.964 R = 0.9998 2000 y = 5.1208x + 23.7 R = 0.9998 0 20 40 60 Fraction 80 100 120
Linear (5 um) Linear (10 um)
166
Considerations for classification and monitoring aggregates and subvisible particles
0.001 m
Monomer Dimer
0.01 m
Sub-micron 1 5 million Da.
0.5 m 1 m
Sub-visible 1 E12 Da.
50 m
Visible
600 m
Characterization studies are performed to assess the potential impact to safety and efficacy
Generally, levels are low enough such that efficacy is unlikely to be impacted. Reversible forms have low risk of impacting safety Irreversible forms and impact of size have high uncertainty of impact to safety
Monitoring approach
Continuing to use compendial or product specific method for measuring 10 and 25 m size particles; monitor particle sizes < 10 m for information only Continue to collect data and perform characterization studies to enhance our understanding and assessment of impact
Different techniques can be used to characterize the SbVP all with specifc strengths and weaknesses
FFF, AUC, can be used in multiple buffers, including formulation buffer
Sensitivity in 0.1 to 100 micron level too low
Coulter counter has size range down to about 0.5 microns

Has buffer limitations, sample may need to be diluted Requires multiple runs and sample manipulations in order to measure the particle distribution across the full size range from 0.4 > 1000 um
Imaging systems provide information on morphology to differentiate between protein and silicone oil, etc
Not High throughput, variability
Hiac: Can be Qualified and validated, used with any buffer,

No information on particle type
For all techniques the sample handling appears to be the most important step in controlling variability of the method
Summary
An analytical gap in technology and sensitivity exists for measuring particles in the 0.1 and 2 m size Analytical results are complicated by lack of a protein standard Absolute particle counts are dependent on the technique; although relative amounts are consistent Sample handling and the nature of the sample are critical to optimizing the methods There are several orthogonal techniques available for SbVP quantitation or characterization The HIAC method (with modifications) has been demonstrated to be a reliable quantitative method for particles 2 m
Future Directions
Add biologics-specific SbVP method to compendia
Add new biologics-specific chapter Include sample handling discussion Somewhat flexiblechoose technique that is fit to purpose for specific molecule, stage of development, and aspect being monitored
Continue to develop techniques to quantitate and characterize particles

Collect 2 micron information FIO Eventually set specifications for each product based on historical data
Will be drug product specific, dependent on delivery device, formulation, protein and sample handling
Acknowledgements
Shawn Cao Nancy Jiao Joey Pollastrini Yijia Jiang Gerd Kleeman John Gabrielson Tony Mire-Sluis
Nomenclature for aggregates

Linda Narhi, Deepak Sharma, Jeremy Schmit, Karoline Bechtold-Peters
Breakout session at Protein aggregate and immunogenicity meeting in July

The goal was to come up with a consistent set of terms that can be used to specify what type of aggregate are being discussed in a non-ambiguous fashion, and to avoid using the terms soluble, insoluble, visible particle, etc. as these are very subjective.
How to define aggregates?

Does it refer to all self-associated species, or does it imply irreversible or non-native structures?
The definitions appears to be dependent on the field of biochemistry that is using it.
What about proteins and peptides where the active form can be a multimer of the monomeric subunit? Are these aggregates? The most inclusive definition would be that aggregate refers to all self-association of the monomeric subunit, with further descriptors used to describe the particular species being analyzed.
Includes folded and unfolded The monomeric subunit could be defined as the smallest functional unit. For IgG this would be the homo-dimer
Need to include heteroaggregates as well

If heterodimer is smallest functional unit, is this the monomer?
Category Classifications
Size
Submicron (includes oligomers, native association or not) 1-100 micron > 100 micron
Secondary/Tertiary structure
native-like partially unfolded unfolded amyloid
Reversibility
Reversible should be restricted to aggregates for which an equilibrium constant can be measured. That is, the disassociation of proteins may be observed on the experimental time scale simply by reverting to original conditions. irreversible Denaturant-reversible, etc. when conditions that disrupt structure, are different from original, are required to dissociate the aggregate
Covalent Modification
crosslinked intramolecular modification Chemical modification
Morphology
aspect ratio surface roughness internal morphology homo and heteroaggregates
optical properties: similar for all protein particles
Path Forward
Circulate definitions in newsletter for AAPS focus group on aggregates and biological effects
Seek feedback, examples. This was done in Oct. newsletter
Commentary in J. Pharm. Sci.

Seek feedback
Include in USP chapter

Work with IUPAC Encourage usage in papers submitted, reviewed, internal documents, etc.
USP Workshop on Particle Size: DETECTION AND MEASUREMENT

December 8 10, 2010
Injectable Products: USP Chapters <788> and <729>

IMPLICATIONS FOR BIOLOGICS
David F. Driscoll, Ph.D. Stable Solutions LLC, Easton, MA, USA

d.driscoll@stablesolns.com
Outline of Presentation
USP Chapter <788>
Indications Size Limits Medical Implications
USP Chapter <729>

Background and Indications Size Limits Medical Implications
Biologic License Applications

Indications Size Limits: Technical Challenges Medical Implications
Particle Size and Relevant Populations
0.5 m
5 m 100 m 200 m
USP <729> USP <788> USP <1> 0% 40% 95%

(Detection Rate of 1 particle in 10 mL vial)
Adapted from: USP 34 (2010); PF 35 (2009)
USP Chapter <788>

Background and Indications
Adopted in 1985 All aqueous injectable products Focuses on 2 sizes and 2 possible methods (LO + Microscopy)
Size Limits
10 m and 25 m Product-volume dependent
Medical Risk
Mechanical obstruction of microvasculature
Light Obscuration:
USP <788> template for <729>
Physicochemical stability of total nutrient admixtures Am J Health-Syst Pharm 1995;52(15):623-34
USP Chapter <729>

Background and Indications
Long overdue (U.S. since 1976; adopted 2007) Focuses on 2 methods and 2 key regions of globule size distribution <788> served as template
Size Limits
0.5 m and 5 m Applies to all [Lipid] (10, 20 & 30%)
Medical Risk
Mechanical obstruction of microvasculature
<788> and <729>

Focus on upper limits
Main pharmacopeial concern
Primary motivation of medical risk

Mechanical obstruction
Arbiter of medical risk

Single-particle optical sensing technique employing light obscuration/extinction is principle method of analysis
Light Obscuration or Extinction

Primary method for p.s. analysis in injectables:
Sizes AND Counts particles present Statistics are important, especially @ sizes 1 micrometer ( size, population)
Particle size and counting capabilities:

2 to 400 micrometers
Extinction Efficiency
Problems below 2 micrometers
Extinction Efficiency:
Size vs. Size + R.I.
Extinction Efficiency: Particle Size re: Aqueous Injections
Sizing Accuracy of LO
Peaks: 2.02 m; 5.09 m; 10.04 m; 25.25 m # of Particles/mL vs. Diameter
DFD-SSLLC-2010
SIZE: Standard LO Calibration Curve Using NIST Calibrator Microspheres

Calibration Table, (n = 9 points) Calibration Curve, Voltage vs. Diameter
Stnd-1,2
PARTICLE SIZE:
Recovery of NIST PSL Standards
Extinction Efficiency: Particle Size and Refractive Index re: Lipid Emulsions
Effects of Particle Size and n on Light (Obscuration) Extinction Efficiency

PF 2004;30:2244-53 Q vs. Diameter nP: 1.59 vs. nL: 1.47
Size Range: 1.4 20 m (in 0.1 m increments or 186 points per plot)
Effects of Particle Size and RI on Light (Obscuration) Extinction Efficiency

PF 2004;30:2244-53
Q vs. Diameter
(Speed-Bumps)
SB#1 SB#2
RI=1.59 RI=1.47
Size Range: 1.4 20 m (in 0.1 m increments)
Equivalence
Oscillating Zone
Size Range & Unity of Q-Ratios Quantifying the Speed-Bumps

Size Range 1.4 5.0 m 5.1 8.7 m Range of Q-ratios Q-ratio = 1.0 2.7 5.1, 8.7
0.41 2.05
0.83 1.00
(SB#1) 1.00
8.8 10.9 m
11.0 14.5 m
1.33 1.18
10.9
14.5
0.87 1.00
(SB#2) 1.00
14.6 16.7 m
16.8 19.3 m 19.4 20.0 m*
16.7
19.3 1.0
0.88 1.00 1.0
Q-Ratio Variability Quantifying the Speed-Bumps

Q-Ratio MeanSD 1) 1.4-5.0 1.250.54 2) 5.1-8.7 0.890.05 3) 8.8-10.9 1.190.10 4) 11.0-14.5 0.930.04 5) 14.6-16.7 1.110.06 6) 16.7-19.3 0.940.04 7) 19.4-20.0 1.000.00 Size (m) Range CV 43.2 5.3 8.7 4.0 5.1 4.5 0.0
CV vs. Size Range (1.4 5.0 m)
5.1 20.0 m
At PFAT5 Limit of <729>, the CV is < 10% in the Critical Size Range.
Summary of Effects of Refractive Index and Particle Size on LO + FAT

Stimuli Article: PF 2004;30:2244-53 Refractive Index is nil
Verifies Selection of PFAT5
Extinction Efficiency: Particle Size and Refractive Index re: Protein Injections
Recovery of PSL Count Standards

(2000 particles/mL)
10 m w/ 15 m PSL 2 m w/ 5 m PSL
Cao S, Jiang Y, Narhi L: PF 2010;36(3):824-34.
Summary of Effects of Refractive Index and Particle Size on LO + PRO
Refractive Index is nil
Method Precision vs. Absolute Accuracy Cao S, Jiang Y, Narhi L: PF 2010;36(3):824-34.
Summary of Compendial Methods

<729> (lipid injectable emulsions)
After adoption, current limits are now being met by all U.S. products.
<788> (aqueous injectable solutions)

Recent assessments by the Agency suggest significant in SVP limits.
<XXX> (injectable protein liquids)

Neither <788> nor <729> standards are applicable.
Summary of Technical Issues in P.S. Analysis of Protein Particles

SV-LO method fulfills the required performance objectives for protein therapeutics.
But
Are we looking at the correct population of particles responsible for clinically significant immunogenic reactions?
Technical Challenges PS < 1 m

Particle Physics
Particle Sizing
Particle Counting
Particle Statistics
Particle Profile
Session V: Large Molecules/Oral Formulations Track 1: Parenterals Track 2: Orals Spalding Auditorium Briggs/Parker/ Marshall/Wiley

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Welcome to the

USP Workshop on Particle Size:

Welcome and Opening Remarks

USP Workshop on Particle Size: Particle Detection & Measurement

Comparison of LO and MM methods

History and Limits Evolution

Benjamin, IES conference 1990

History and Limits Evolution

History and Limits Evolution

History and Limits Evolution

British Std: mean of 5 containers and individual

History and Limits Evolution

History and Limits Evolution

History and Limits Evolution

for small-volume injections (100mL):

1995 USP 23 <788> provided the most dramatic change.

for small-volume injections (100mL):

History and Limits Evolution

MM testing by membrane methodology

History and Limits Evolution

Harmonization - Current USP Guideline

USP 33 <788>--Particulate Matter in Injections

USP 33 <789>--Particulate Matter in Ophthalmic Solutions

Exempt from <788> limits:

Current <788> Limits

10m 6000 per container 25 per mL

600 per container 3 per mL

Current <789> Limits

<789> Methods are essentially <788> and Limits are tight

Particle Count by Membrane Microscopy Limits for Liquid Products

History and Limits Evolution

USP Chapter Crossover

Visible 150m Visible Gray zone

What is Particulate Matter?

Particulate Matter Details

Inherent to the formulation Presence in biologic products

Studying the Particle Load aids Refinement of the Final Product

Current Test Methods

Method 2 (Microscopic Particle Count Test)

<788> <789> LO Particle Count Test

Instrument Standardization Tests Calibration Options

What is your statistical sampling consideration?

Light Obscuration Method

Illustration per Groves

Light Obscuration Method

Light Obscuration Method

Light Obscuration Method

User is responsible for

LO Test Particle Limits

6000 per container 50 per mL

600 per container 5 per mL

1.A. > 100mL

Membrane Assay Method

Microscopic Particle Count Test

Membrane Microscopy Method

Membrane Microscopy Lab

Microscopic Particle Count Test

Partial count is allowed; however <788> undefined <1788> defined

3000 per container

300 per container

2.A. > 100mL

Informational Chapter <1788>