P Saws Day 1 Master

Welcome to the
USP Workshop on Particle Size:

Particle Detection and Measurement
December 8-10, 2010
USP Headquarters, Rockville, MD

December 8-10, 2010; USP Headquarters
Welcome and Opening Remarks

Anthony DeStefano, Ph.D.
USP Vice president, General Chapters

Workshop Overview
Desmond Hunt, Ph.D.
USP Senior Scientific Liaison

Session I: Particle and Product Size

Fundamentals
Chair: Richard Meury
Eli Lilly
Particle Size Fundamentals

Rich Meury
Eli Lilly and Company
General Chapters Physical Analysis Expert Committee
USP Workshop on Particle Size: Detection and Measurement
08 December 2010
Outline
What are particles?
What is particle size?
Particle size detection and measurement
08 Dec 2010 USP Workshop
Company Confidential Copyright 2000 Eli Lilly and Company
Particle size detection and

measurement
Diversity of applications
Bulk / contaminents
Parenteral / oral
Commonality
Understanding of particle size
Measurement techniques
Potential effects of API and excipient

particle size
Manufacturability flow
Content uniformity
Dissolution
Bioavailability
Stability
Appearance
Parenteral and Ophthalmic solutions

Regulatory / GMP / Compendial requirements for
visible and subvisible particles
What is a particle?
a small object that behaves as a whole unit in
terms of its transport and properties (Wikipedia)
a relatively small or the smallest discrete portion
or amount of something (Merriam-Webster)
a small discrete unit of matter (ASTM)
10
A particle may be:

Solid in a gas
Solid in liquid
Liquid in gas
Liquid in liquid
Gas in liquid
11
What is a particle?
Anything that produces a response in the
measurement.
12
What is particle size

(and why is it so misunderstood)?
Shape
Distributions
Sample
Measurement technique
13
Shape
For non-spherical particles, size is not sufficiently
defined by a single number
Leads to measurement variability
Leads to dependence on measurement technique
Pharmaceutically relevant particles exhibit a wide
variety of shapes
14
15
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16
Variability of Size Measurements of a

Single Particle
Shape
Flake
Trapezoid
Sphere
Mean
SD
Range
427
489
427
76
33
11
66%
26%
8%
Powder Technology, 57, 143.
17
Equivalent Sphere
Same min. length
dmin
dw
dmax
Same max.
length
Sphere of same
weight
dv
Same
Volume
dsed
ds
Same sedimentation
rate
dsieve
Same surface
area
18
Equivalent Spherical Diam.

100 m
Volume = p
r h
= 10000
d = 20
20 m
39 m
h = 100
Volume = 4/3
pr
= 10000
r = 39.1
19
Distributions
Particle populations are distributions of both size and
shape.
Not adequately described by a single number
Variety of ways to describe and display distribution

information
Need to define the parameter or statistic used to

describe the population
20
Distributions
Descriptors / Statistics
Basis (y axis)
volume, count, other
Scale (x axis)
linear or logarithmic
Bins
size, resolution
21
Size without distribution information

or definition of reported parameter
22
Actual size distribution:

normal or log-normal
23
Actual size distribution

180
Volume distribution density
160
140
120
100
80
60
40
20
0
10
100
Diameter (microns)
24
Cumulative size distribution plot

100
90
80
Cumulative
70
60
50
40
30
20
10
0
10
100
Diameter (microns)
25
Log-probability plot
99.9
Cumulative %
99
90
70
50
30
10
1
0.1
10
100
Diameter (microns)
26
Log-probability plot
99
Cumulative %
90
70
50
30
10
1
10
100
Diameter (microns)
27
Count and volume distributions

Count
Volume
10
Diameter
28
Relative quantities
1 m
10 m
100 m
count
surface
100
10,000
volume /
mass
1,000
1,000,000
29
Descriptors
Position
means, median, mode
Dispersion
GSD, span, width, range
Quantiles / Percentiles
X50, D90, Q20
Most relevant parameter depends on purpose of

measurement.
30
Descriptors
Means and variances
generally used with counting techniques

treat all particles in bin as mid-point
for cumulative, treat 1/2 as below mid-point
Percentiles and quantiles
generally used where bins defined by limits

interpolated from cumulative distribution
31
Definitions
Dn or Xn
nth percentile size, the size which

n % of the distribution is below.
Qy
yth quantile, the % below size y
GVM
geometric volume mean diameter
GSD
geometric standard deviation
Width
(D84.13/D15.87)1/2, estimate of
GSD
VMD
volume median diameter, X50
Da,b
da/ db (D4,3 is arithmetic

volume mean diameter)
32
Definitions
d
particle size, diameter of a sphere
particle size, diameter of a sphere

subscripts may be used to denote measured
quantity, e.g. volume, surface, or Feret diameter
qr(x) density distribution (linear)

where r indicates the dimension,
e.g. r = 0 for number distribution,
r = 3 for volume distribution
q*r(ln x)
density distribution (logarithmic)
Qr(x)
cumulative distribution (general)
33
Analysis and Display of

Particle Size Data
Bins defined by either mid-point or upper and
lower limits.
depends on technique
determines what parameters are used
Treat distribution plots as histograms
distribution density
normalize AUC
more correct representation of relative amounts
34
Distribution Density
Linear scale:
q*r,i = Qr,i / xi
Logarithmic (geometric) scale:

q*r,i = Qr,i / log(xi/xi-1)
35
Interpolation
100
90
80
Q20
70
60
50
40
10
D50
D90
100
36
Interpolation: Percentile sizes

Linear
x l Du Dl
Dx Dl
u l
Geometric
x l
Dx Dl exp
lnDu / Dl
u l
where
l = % below bin, u = % above bin

Dn = size at n %
37
0.18
0.15
0.8
0.12
0.6
0.09
0.4
0.06
0.2
0.03
0
Cumulative Fraction
Volume Fraction
Lognormal Distribution
0
10
100
1000
Diameter
GMD (D50) = 100

GSD = (Sqrt[D84/D16]) = 2.0
38
Useful equations for dealing with lognormal distributions
Dn x g S
xi x j e
( i j ) ln 2 S
( i j ) ln 2 S
x i x j e x x e(i j ) ln
i
j
i.e. GCM = GVM *exp(-3ln2S)
39
Sample
Effects / limitations of sampling
Effects of sample preparation
Matrix
40
Sampling
Actual measured sample is (usually) extremely small
fraction of original
Multiple sample divisions chances for bias / segregation
Lot, containers, composite sample, lab sample, sample preparation,

analytical sample, measured sample
For best results, use riffler or sample splitter
41
Golden rules of sampling

(T. Allen)
Sample from material in motion

Multiple cuts from whole stream
42
Sample size and container

mg to kg
request appropriate amount for analysis
request appropriate packaging
container not more than about 1/2 full

easy to mix and sample
43
Sample preparation
Matrix / packaging / handling

Dispersion
Dissolution
Reactions, form changes

Dependent on sample characteristics
44
Key thoughts:
Particle size analysis results depend

on the sample characteristics and
preparation
The analysis is only as good as the

sample
45
Measurement Technique
All techniques measure different size related
properties
affected by shape and material properties
RI, density, etc.

Within a technique, depends on measurement
conditions
experimental design
software, algorithms
data handling and reporting
46
Summary of Methods
Microscopy
Image analysis - static and dynamic
Sieving
Electrical sensing zone
Light Diffraction
Light Extinction / Scattering (SPOS)
Dynamic Light Scattering
Aerodynamic methods
Sedimentation
Others
47
Microscopy
10
20
Best way for shape

Number distribution
Slow
Subjectivity
Representative?
Which diameter to
use?
48
Image analysis
Quantitation of images
Allows more objective use of microscopy
Other uses as well
49
Sieving
Wide overall and dynamic ranges
limited at low end, about 50 microns
Few sample restrictions
Relatively large sample required
Slow
Low resolution
Direct mass measurement
50
Electrical sensing zone

(Coulter method)
Moderately limited overall and dynamic ranges
High resolution
Requires electrolyte
Direct counting
Moderately fast
51
Light diffraction
Relatively wide overall and dynamic ranges
Moderate resolution
Few sampling restrictions
Indirect measurement
Fast
52
Light Extinction / Scattering

(single particle optical sensing)
Moderate dynamic and overall ranges
High resolution possible, not always utilized
Single particle counting, direct
Main uses in contamination monitoring
53
Dynamic light scattering
Sub-micron range
limited resolution
54
Aerodynamic Methods
Impinger
Cascade Impactor
Time of Flight
55
Sedimentation
gravity and centrifugal
photo and X-ray detection
slow
56
Others
Scanning methods
time of transition of scanned beam (FBRM)

chord measurement
Separation methods
FFF
Ultrasonic attenuation
Laser Doppler velocimetry
57
Summary
Not necessarily only one correct answer
Depends on underlying question
Particle size measurement depends on:
Sample and preparation

Measurement technique and conditions
Accurate communication of results depends on:
Understanding of these effects

Use of appropriate parameter or statistic
58
Acknowledgements
Some of the slides were provided by Malvern Instruments.
59
Desirable Formulations Properties

and Delivery Strategies
API / Dry Systems
Stephen W. Hoag, Ph.D.
University of Maryland, Baltimore
School of Pharmacy
20 N. Pine St.
Baltimore MD 21201
Phone 410-706-6865
Email shoag@umaryland.edu
Lecture Objectives and Outline

Objective:
To make a successful dosage form there are critical

quality attributes (CQA) that the final dosage form
must have. To obtain the desirable CQA the API
must have desirable physical properties. This talk
will examine the how particle properties can affect
the CQA of the final dosage from and strategies for
developing a formulation.
Outline
Formulation requirements
Particle properties and powder behavior
Formulation Strategies
Dosage Form Requirements

Main dosage form design criteria
Accuracy/Precision dosing
Drug delivery
Manufacturability
Patient acceptability
Stability
Raw Material
Inputs
Manufacturing
Process
Processing
Conditions
Final Product
Outputs
Tablet Press Operation
Source: Wikipedia - Tablets
Capsule Filling
Dosator Method
Dosing Disc Principle
Capsule
Plug
Powder Flow Significance

Powder must flow from hopper to tablet die
1,000 3,000 kg Lot
100 300 mg Tablet
Low density
or fluidized
powder bed
High density
powder bed
Highly consolidated
powder bed
Drug Delivery
Bioavailability can depend on particle size
E.g. Folic acid
Amount Released 75% of labeled claim in 1 hr

Gut Wall
Disintegration
Dissolution
Steps depend on formulation & particle size
Formulation Attributes:
Minimum running characteristics
Compactibility
Fluidity
Lubricity
Minimum delivery characteristic
Release
Particle properties and powder

behavior
Particle Properties
PS & PSD
Distribution Type
Particle Shape
Image Source: PharmTech, Dec 2001, p38-45
Forces Between Particles

Particle-particle Adhesion
H
Av
x
2
24 a
van der Waals
10Q 2
2
Aq
x
1 a / x 2
Electrostatic
AL x FA
Capillary
Particle Contact & Size
Represents adhesive force at contact point

Smaller particles have many more contact
points/volume than larger particles
Percolation Segregation
Smaller particles tend to filter down
through larger particles
Bed Vibration
Net affect larger particles on top of bed
Dissolution Noyes Whitney Eq.

Surface
to
Volume
Ratio
dm SD
(C s Cb )
dt
h
Particle Diameter
S = Surface Area
m = Mass
t = Time
D = Diff. coef.
h = Bndry. layer
C = Conc.
Property Constraints
Flow Rate
Dissolution Rate
Minimum
Flow Rate
Particle Size
The best formulation strategy is?
It depends on circumstances
Need to do risk assessment to decide:
Other excipients, i.e. formulation
Best process to be used
Particle Ranges
Formulation Type
Nano Particles
Size Range
Properties
200 500 nm Flow: Need specialize formulation techniques

to get dispersion of particle in dissolution
medium
Dissolution: Typically excellent
Micronized API
10 40 m
Flow: Very cohesive, poor flow, hard to mix,

but doesnt easily segregate
Dissolution: Typically excellent
Typical API
50 150 m
Flow: Less cohesive, average flow, easy to

mix, but may easily segregate
Dissolution: Depends
Granules
250 400 m Flow: Non cohesive, excellent flow, typically

dont segregate
Dissolution: Depends
Beads
400 750 m Flow: Need specialized formulating

techniques, hard to make into tablet or
capsule
Dissolution: Often CR formulation
Consideration of the Dose of Drug is

the Starting Point...
LOW DOSE (<25 mg)
Most of the tablet will be excipients

Content Uniformity
High DOSE (>250 mg)
Most of the tablet will be drug

Compactibility
Fluidity
Low Dose Formulation Strategy
Flow, lubricity and compactability less important

Poor compactability
Poor fluidity
Overcome by including lubricant
Poor dissolution
Overcome by including a glidant
Poor lubricity
Over come with filler-binders
Overcome by adding disintegrant

Use water soluble filler-binders
Reduce API particle size
Risks Content Uniformity
Need specialized mixing procedures like geometric dilution

Controlling Particle size
%CV Coefficient of Variation

np (1 p )
s
%CV 100 100
np
X
100
np
X np
Assuming p is small thus (1-p) = 1
np (1 p)
%CV - Example
For example:
Tablet 400 mg total

10 mg active
Density = 1.5 g/cc
ca 6% Max allowed %CV

30
25
% CV
20
15
10
5
0
0
250
500
750
Particle Size (um)
1000
High Dose Formulation Strategy
Flow, lubricity and compactability very

important
Poor compactability
Granulation: wet or dry
Poor lubricity
Granulation: wet or dry
Poor fluidity
Limits to how much can bulk up with excipients
Overcome by including lubricant
Poor dissolution
Overcome by adding disintegrant

Reduce API particle size
Water soluble excipients and granulation binders
Summary
First principle models relating particle

properties to finished product CQA dont
exist
Can be guided by general principles and
empirical experience
To develop formulation need to understand
how particle size affects formulation and
how to use this info when developing
formulation
References
Pharmaceutical dosage forms: tablets, Hoag and Augsburger, 3rd
Ed, vol. 1-3 Marcel Dekker, Inc. (2008)
The theory and practice of industrial pharmacy; Lea & Febiger,

Lachman, Lieberman & Kanig (1986) pp 3-21
Principles of powder technology; John Wiley and Sons, New York,
Martin Rhodes, (1990) pp 227-298
Carstensen, JT. Solid pharmaceutics: mechanical properties and
rate phenomena. Academic press, NY (1980).
Muhammad E. Fayed, Lambert Otten, Handbook of powder science
and technology, 2nd edition (1997) Chapman & Hall, New York
Characteristics and Requirements for an

Ideal Parenteral Liquid Dosage Form
USP Workshop on Particle Size
Rockville, MD
December 8-10, 2010
Presenter
Gregory A. Sacha, Ph.D.
Parenteral Dosage Form
Preparations administered by circumventing the

bodys most protective barriers and, therefore, are
prepared to meet Pharmacopeial requirements for
sterility, pyrogens/endotoxins, and particulate matter.
2010 Baxter International Inc. Baxter is a registered trademark of Baxter International Inc.
87
Categories of Injections (USP)

Solutions ready for injection
Dry, soluble products ready to be combined with a
solvent just prior to use.
Suspensions ready for injection
Dry, insoluble products ready to be combined with a
vehicle just prior to use.
Emulsions
Liquid concentrates ready for dilution prior to
administration.
88
Parenteral Product Packaging

Ampoules
Vials
Pre-filled Syringes
Glass and Plastic Bottles
Plastic Bags and Bottles
Add-Vantage
Dual Chamber Vials/Syringes
Cartridges, Delivery Pens, and
Autoinjectors
89
Parenteral Packaging
90
Route of Administration
Intravenous
Direct Injection
Infusion
Subcutaneous
Intramuscular
Intradermal
Intraspinal
Intrathecal
Intra-arterial
Others
91
Characteristics and Requirements
92
Regulatory
Meets SISPQ Safety, Identity, Strength, Purity, Quality
Includes sterility, free from pyrogens, toxicologically safe,
essentially particle free
User Requirements
Ease of administration
Pharmaceutical elegance
Comfort
Manufacturing Requirements
Ease of manufacturing
No interactions with processing equipment

Toxicologically Safe
Formulations contain the drug and excipients, e.g.
Solubilizing agent(s) / vehicle
Buffers, other stabilizers
Anti-microbial preservatives
Excipients can be a source of impurities
Many potential formulation additives are not sufficiently
safe for injectable drug administration
Limited number of preservatives available for multidose vials due to toxicity
Limited concentration ranges for surfactants
93

Isotonic
Typically about 280 mOsm/kg
All routes of injectable administration and topical
ophthalmic application should be isotonic
Only possible exception: small volume IV injections
Possible outcomes of administering non-isotonic
formulations include:
Hemolysis
Phlebitis
Muscle irritation
94

Stability
Physically, chemically, and microbiologically
No interaction with packaging components
Appropriate container type and formulation
components to prevent glass delamination.
Formulation variables conducive to delamination
include pH > 8 and chelating agents
Ammonium sulfate treatment may increase
potential for flaking
Eprex incident polysorbate in the formulation
leached a curing agent from the rubber stopper that
caused pure red cell aplasia (PRCA)
95

Compatible with IV admixtures if needed
For example, adding heparin, insulin, and other
components to a daily parenteral nutrition infusion
Combining any small volume injectable product with an
infusion solution requires knowledge of product
compatibility with infusion plus with any other drug
products part of the admixture
Hospital pharmacists rely on the Trissels Handbook on
Injectable Drugs
96
Requirements
Sterility
Free from microbial contamination for entire shelf life of
product
Tested on a small number of samples compared to the
entire batch size
Methods are Membrane Filtration and Direct Transfer
Tests are limited by:
Sampling and statistical representation
Potential for accidental contamination
Potential for interactions between media, conditions,
and drug that may inhibit microbial growth.
97
Requirements
Pyrogen Free
Fever producing agents plus, depending on amount,
can produce a cascade of more serious physiologic
problems including death (sepsis)
Endotoxins: lipopolysacharides from gram negative
microbial contamination
All endotoxins are pyrogens but not all pyrogens
are endotoxins
Limulus Amebocyte Lysate Test (LAL) and rabbit
pyrogen test
98
Requirements
Essentially Free from Visible Particulate
Matter
Sources are from throughout the
manufacturing process
Material preparation and handling
Tubing used throughout the process
especially if using a peristaltic pump after
the filtration process
Material used to cover manufacturing
equipment during the autoclave process
Fragments of packaging material used to
contain stoppers & pre-sterilized syringes
Extractables from Bioshield paper used to
cover equipment for autoclaving
99
Requirements
Essentially Free from Visible Particulate Matter
Manufacturing
Metal fragments from mixing units
Metal fragments from stopper insertion tubes for prefilled
syringes
Fragments from magnetic stir bars
Metal fragments from abrasion during cleaning processes
Erosion of gaskets
Packaging Materials
Silicone on syringes and stoppers
Glass from breaking ampoules
Coring from puncturing stopper with needle
Removing plastic covering from containers
Dislodging stoppers
10
0
10
1
Requirements
Limits for Subvisible Particulate Matter
1st Test by Light Obscuration Method
If the numbers exceed the requirements:
2nd Test by Microscopic Particle Count Method
Test
Product
10 m
25 m
L.O.
SVI
6000
600 / container
LVI
25
3 / mL
SVI
3000
300 / container
LVI
12
2 / mL
Micro
User Requirements
Pharmaceutical elegance
Clear, colorless solution
Flows easily through the needle
Little pressure required for injection
Fast rate of administration
Prefer not to stand and slowly administer to patient by
hand
10
2
User Requirements
Easy to prepare and stable for user requirements
Prefilled needles ready to inject
Prefilled IV bags
Over pressure in the vial so that it is easier to remove
Syringes and IV infusions may not always be prepared
in laminar flow hoods. This can be a source of
microbial and particle contamination.
Dosages my be prepared 24 hours or more in
advance.
10
3
Manufacturing Requirements
Easy to prepare
Requires a limited number of weighing, addition, and pH
adjustment steps
Requires few to no calculations
Requires no in-process release testing
Does not interact with manufacturing equipment
No sticky residues affecting flow through filling needles
M-cresol is absorbed by silicone tubing and will lead to
losses during hold times
Easy to filter
Easy to clean
10
4
10
5
Summary
Simple injectable solutions have many rigorous
requirements to ensure patient safety.
Also have user and manufacturing specific requirements.

Manufacturing methods, packaging and formulation
components, and administration methods all present
challenges to meeting sterility, stability, and particle free
specifications.
Desirable Formulations Properties and

Delivery Strategies
Injectable Dosage Forms: Large Molecules
USP workshop on Particle Size: Particle Detection &

Measurement
Rockville, Maryland
Sandeep Nema, PhD

Executive Director, Pfizer
What is Biologics?
Biologics = Biopharmaceuticals (in Europe) ~ Biotherapeutics
Biologics comprise a heterogenous group of pharmaceutical products which are derived from living
organisms, e.g.:
Recombinant therapeutic proteins

Immunoglobulins (antibodies)
Peptides
Oligonucleotides
Vaccines
Blood and blood components
Somatic cells
Gene therapy
Biologics can be composed of:
Sugars
Proteins
Nucleic acids
Complex combinations of these substances
May be living entities such as cells and tissues
Biologics
Produced by biotechnology methods and other technologies

Isolated from a variety of natural sources (microorganisms, animals, humans, plants)
US Regulatory Definition of a Biologics
Public Health Service (PHS) Act

PHS Act 351(i); 42 U.S.C. 262(i)
Biological product means a virus, therapeutic serum, toxin,

antitoxin, vaccine, blood, blood component or derivative,
allergenic product, or analogous product, or arsphenamine or
derivative of arsphenamine (or any other trivalent organic
arsenic compound)
applicable to the prevention, treatment, or cure of a disease or
condition of human beings
Regulatory review based on 1991 CDER/CBER Inter-center

Agreement and 2003 CDER/CBER Reorganization
No Single Universal Classification of BioTx

BioTx can be grouped or classified based on a broad variety of attributes:
Molecular Weight (MW):
MW < 500 small molecules
MW >1,000 medium molecules
MW > 5,000 - 10,000 large molecules
Small molecules SAR may follow the Lipinski Rule of Five; BioTx no analogs of the Rule of Five
Chemistries and the level of structural complexity:
BioTx are usually (not always) biopolymers built from amino acids, nucleotides, saccharides
Conjugates of large molecules with medium size (CovX) or small molecules (PEG)
Manufacturing or source of the material:
Small molecules are made by well understood and controlled chemical synthesis
Proteins (including antibodies) are made by less controlled fermentation
Cell components
Cells or tissues
Regulatory cutoff in the US (may be different in other countries):
Peptides and oligonucleotides fall under NDA
Proteins and antibodies under BLA
Functional class
Enzyme
Antibodies
Hormones
Others
IMS classification
Small Molecules vs. Therapeutics
Small Molecules
Chemical entity
Biotherapeutics
Small molecule, most NCEs
Mostly therapeutic proteins: MAb, Usually

prepared by genetic recombinant methods in cells
MW
< 1 kDa
Large molecular weight e.g. MAb MW ~ 150 kDa
Half-life
Usually short (< 24 h)
Generally long, e.g. 21 days in some MAb
Route of administration
Oral usually
IV or sc usually
Immunogenicity
Usually low - anaphylactic reactions uncommon
Can be immunogenic even if fully human:

anaphylactic/oid reactions
Toxicology data
Animal toxicology/efficacy data useful for human
Animal models (AEs and tox) not always

predictive
(Complexity)
studies
Drug interactions
Possible, e.g. via CYP450 enzymes
Very uncommon
Selectivity
Can act on other receptors at higher doses
Selective for target regardless of dose (larger

functional effects?)
Volume of distribution
May be much higher than ECF
Limited to plasma volume for Ab
Active metabolites
Possible and can be many
None
110
Typical Small Molecule vs Biologic Therapeutics

Structure and Size Comparison
Small molecule*
Structure
Compound/
function
Coumadin:
BMS;
Biologic*
MW
308.33
MW
(Da)
Compound/
function
204.22
Tryptophan:
Amino acid
Structure
HC
C
C
HC
Anticoagulant
NH
C
HC
CH
NH2
OH
CH
C
CH2
C
O
Amoxil: GSK;
419.45
5808
Bacterial
infection
Singulair:
Merck;
Insulin
(Humulin:Lilly)
Diabetes
608.18
22124
GH deficiency
Lipitor:Pfizer;
Lipid lowering
agent
1209.42
~150000
Representation of
insulin hexamer
structure from
www.biotopics.co.uk/
as/insulinproteinstruc
ture.html
hGH
(Pfizer:
Genotropin)
Asthma
Included only
as a molecular
size reference
(CFK drawing)
mAb
(Humira:
Abbott)
RA
Representation
of human growth
hormone
structure from
Expasy
Representation
of a general
monoclonal
antibody
structure (Pfizer
drawing)
DIA
*Reference: Molecule information from Physicians Desk Reference, Thomson

LARC/
EIRGM
Publishing, unless otherwise noted.
2008
What is considered a Large Molecule?

Biologics
What about complex synthetic molecules, with or w/o modifications, where there
is heterogeneity?
Oligonuceotides
RNAi
Product-related substances
Molecular variants of the desired product formed during manufacture and/or storage
which are active and have no deleterious effect on the safety and efficacy of the drug
product. These variants possess properties comparable to the desired product and
are not considered impurities.
Example in many mAbs 20-40% deamidated species; various glycosylated forms
Molecular variants Heterogeneity
Glycoforms
113
IgG Heterogeneity Simplified Example
The # of variation sites in each half-antibody the number of possible

variations at each site= 2 6 4 4 5 5 2 = 9600 possible states.
(9600)2 108 possible states (assume each half independent)

Ref: Kozlowski and Swann, Advanced Drug Delivery Review, 58(5-6), 707-722, 2006
114
Generic Formulation Ingredients

Limited excipients are used in the formulation of injectables and only a subset for
biologics
Approximately 50:50 Liquid vs lyophilized dosage forms
Ingredient
Purpose
Examples
Surfactant
Reduce aggregate
and particle
formation
Tween 80, Tween

20, Pluronic
Cryo/lyo-protectants
Protect damage
during freezing or
drying
Sucrose, trehalose
Buffers
Maintain pH
Histidine, succinate,
citrate, phosphate
Others as needed
Salts, Antioxidant,
chelators,
preservatives
NaCl, Methionine,
EDTA, phenol/cresol
Manufacturing Processes Drug Product

High level overview
www.germes-online.com
Excipient addition
Small molecule
API
Mixing/blending
Biologic
Sterile filtration
Compression
Fill/Lyophilize /Finish
Fill/Finish
Oral Tablet
Clip art: www.aperfectworld.org
Sterile
Liquid
DIA
LARC/
EIRGM
2008
Sterile Lyophile
Typical Protein Sterile Biologic Drug Product

Manufacturing Process
API stored at low temp
Bulk thawing
Prepare diluent
Clarifying filtration (0.45m)
Sterile filter (0.2m)
Mix
Sterile holding vessel

IPC: protein
concentration
Add diluent
Mix
IPC: protein conc,
bioburden & pH
Sample: endotoxin, bioburden
Bioburden
reduction
IPC: prefiltrarion
bioburden, conc
Sterile
filter
(0.2m)
Holding
vessel
IPC: Bulk Sterility
Fill &
finish
DIA
LARC/
EIRGM
2008
Example: Release & Stability Testing, Sterile Solution Injectable DP

Attribute
Biotherapeutics Method
Small Molecule Method
Color
Clarity or Degree of
Opalescence
Visual appearance
Visual Appearance
Visual appearance
Visual Appearance
Visual Inspection
pH
Osmolality
Volume in container
Label Claim
Identity
Purity
Visible Particles
pH
Osmometry
Volume
Absorbance at 280 nm or HPLC
Peptide Mapping(Primary ID method)
CGE (Reducing) / SDS-PAGE
(Reducing) - fragments
CGE (non-Reducing) / SDS-PAGE
(non-Reducing) - aggregate
SEC - aggregate
Binding ELISA
Cell Based Bioassay
Purity
Purity
Potency/Activity
pH
Osmometry
Volume
HPLC* or spectroscopy (typically a

single method)
Safety
Endotoxin
Endotoxin
Safety
Safety
Sterility
Sub-visible Particulate Matter
Sterility
Particulate Matter
Acidic species
Isoelectric Focusing, cIEF
(deamidation)
Oxidation
Lys-C map followed by RP-HPLC
Coloration
Spectrophotometer or using a 96 well plate method at fixed wave-lengths
Comparing to standards
- Munsell color standard
- Gardner solution color standard
- Tristimulus
- Ph. Eur. 2.2.2
Can be an indication of potential interactions
What Opalescence can or cant tell?

Opalescence monitored and quantiated
Comparison to reference (Ph. Eur. 2.2.1 Clarity and degree of
opalescence of liquids). No analogous USP test
Nephelometry
Turbidimetry
Light Scattering using 96 well plate
High through put adaptations
Colored solutions present a challenge for measurement of opalescence
since even moderately colored solutions provide a negative interference.
should be evaluated with ratio turbidimeters or nephelometers with ratio
selection.
Opalescence can be an early sign of problems
Benign Opalescence especially at high concentration protein
formulations
IgG1 24 mg/mLin a buffer, 150 mM NaCl, and PS-80, pH 6.0 in glass vials
Reference III standard [1830 nephelometric turbidity units (NTU)]
Following filtration through a 0.22 m sterilizing filter, the opalescence
remained unchanged
Characterization of Opalescent Solution
-No change in dimer conc

- Zav hydrodynamic radius slightly higher
with increase in protein conc
- Viscosity increases f(conc.)
- Non-ideal behavior
negative B22 values net attractive forces between
Protein molecules
- No changes in biophysical characteristics

- Ionic strength dependent
Increase in opalescence correlates to molecular self-association of mAb

Attributed opalescence of an IgG1 formulation to Rayleigh scatter and indicated
that opalescence is not caused by noncovalent association
N. Wang et. al., Opalescence of an IgG1 Monoclonal Antibody Formulation
is Mediated by Ionic Strength and Excipients, BioPharm International, April 2009
121
Opalescence and Turbidity
Mahler et al, EJPB, 59, 407 (2005)
Opalescent Solution and Particles

Ph.Eur.
IV
Stir
shake
USP
Stir
shake
IV
Mahler et al, EJPB, 59, 407 (2005)
Utilization of Orthogonal Techniques

like FFF, AUC, DLS
No PS80
0.45um filter
+PS80
Visible Particles
Intrinsic
Reversible or irreversible aggregation
Precipitation (solubility, degradants, interactions)
Extrinsic
Environmental particles
Shedding from plastic, stoppers or flakes from glass
Pharmacopoeial Requirements: Extrinsic Particles
USP <1> Injection: essentially free from visible particulates or essentially free from
particles of foreign matter that can be observed on visual inspection
Ph Eur. Injections: Solutions for injection should be clear and practically free from
particles
JP: Injections should meet the requirements of the Foreign Insoluble Matter Test
<6.06> and Insoluble Particulate Matter Test <6.07>. clear and free from readily
detectable foreign insoluble matter
Focus has been on extraneous particles. USP and EP define particulate matter as
extraneous mobile undissolved particles, other than gas bubbles, unintentionally
present in the solutions.
Ph. Eur. Monograph on Monoclonal Antibody

Monoclonal Antibodies for Human Use
monograph requires such products to be

without particles.
Inconsistent
No threshold size or number provided
What is Essentially or Practically Free of Particles?
- DAC-5
- PF Proposal
Characterization - Visibility
Clear & essentially particulate-free
Possible presence of non-visible aggregates
Presence of particulates
Size distribution (light obscuration)
Morphological evaluation
Composition analysis after separation
Hazy/turbid
UV
Turbidimeter
May use standard solutions

NTU
~0
<2
<20
<200 <4k
<10k
127 Wei
Wang
Ophthalmic Particles
The USP and JP describe topical ophthalmic product requirements while the Ph.
Eur. does not.
USP 32 <789>: Particulate matter consists of mobile, randomly sourced, extraneous
substances, other than gas bubbles, that cannot be quantitated by chemical analysis
because of the small amount of material they represent and because of their
heterogeneous composition. Ophthalmic solutions should be essentially free from
particles that can be observed on visual inspection. Light obscuration or
microscopic particle count test as per USP <788> are required with more stringent
limits applied.
Drops (per mL basis vs per contianer)
Ointments
Injections
JP XV General Test 18 <6.08>: Ophthalmic solutions prepared as aqueous solution
and aqueous vehicles attached to Ophthalmic Solutions to be prepared before use
should be clear and free from foreign insoluble matter when inspected with the
unaided eye. A limit for Insoluble Particulate Matter is described in JP <6.08>.
Sub-visible particles
Major parts are harmonized across USP, EP and JP though some key
differences remain
1. USP defines large volume injection as those containing >100mL of DP
while Ph. Eur. and JP consider 100 mL product as large volume
injection and requiring a more stringent sub-visible PM limit.
2. Currently, USP exempts injection products intended for subcutaneous
(sc) and intramuscular (im) injection from the requirements of <788>. No
such exemptions are allowed in Ph. Eur and JP, but Ph. Eur. recognizes
that a higher limit may be appropriate for these products.
3. Radiopharmaceutical preparations are exempt from these requirements
and so are the preparations for which the label states that the product is
to be used with a final filter provided that the filter delivers a solution
that complies with the Ph. Eur. sub-visible particles test (2.9.19)
Pharmacopeial Forum article suggesting that the current PM limit
should be more restrictive (not based on extensive biologics product
analysis)
Should one monitor other Sub-visible sizes?

For protein drug products, particles in the size range of 1 to 10
microns (in addition to 10 and 25 micron size range) should also
be quantitated as they have the potential to cause
immunogenicity.
Immunogenecity depends on Route of administration
ID >SC / IM > IV
Little evidence between particles and immunogenecity / nAb or
safety
FDA is asking this information for IND and BLA
Good practice during development (e.g. 2, 5, 8 micron sizes)
130
Scientific Data to Address Following

Questions regarding 2-10 micron particles?
What are the various aggregation pathways under different stress conditions
of heat, light, freeze-thaw, agitation, etc? Are different aggregates generated
and what is the influence when various stresses are combined?
What is the definition of aggregate? Is 5-mer an aggregate?
Is there a connection between the type of aggregate and immunogenicity or
other adverse events (AE)?.
Understanding the relationship between the amount of aggregate and AE. In
most cases the expected amount of aggregate of size 0.1 to 10 micron is
expected to be in the range of 0.0002 to 0.01% (ref)
Improving the analytical methodology to measure and quantitate particles
below 10 micron. Current instruments like HIAC, Coulter Counter, Micro
Flow Imaging (MFI), Particle Insight, Flow Particle Imaging Analyzer, etc
give variable and different results likely due to differences in the operational
principles.
Identifying and distinguishing extraneous particles like silicone oil, and
foreign matter from the protein particles so that relative proportions of each
type of particles can be determined. Instruments like Raman microscopy
(RapID), FTIR microscopy, electron microscopy with elemental analysis, etc
are available but they have significant limitations in terms of throughput.
131
Identification of Particles is Important

Important to understand the cause of particle formation
October 2010, Procrit (Epoetin Alfa) Injection

Presence of Particulate Matter: Extremely thin glass flakes (lamellae)
have the potential to be present in Procrit (epoetin alfa) vials. Flakes
result from interaction of the formulation and the glass over the shelf
life of the products
132
Protein and Other Particles - ID
MFITM uses morphology

Nanoparticle Tracking Analysis
RAP ID (Berlin) uses Raman Spectroscopy
Isolation at smaller sizes difficult
- FTIR or Raman can be used on isolates
Other techniques for isolated particle ID (or on filter)
- EDX
- NMR
- XPS
- TEM, SEM, AFM
- CD, DSC
- Protein specific dyes
Protein Particles Pictures using MFI

D. K. Sharma et al, Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate
Analysis in Protein Formulations, The AAPS Journal, online published 2June 2010
Impact of Container/Closure
HIAC Counting of particles

Significant difference (due to silicone oil droplets ?)
What is a relevant count / limit for particulates ?
S.K. Singh et. al., An industry perspective on the monitoring of subvisible particles
as a quality attribute for protein therapeutics, J Pharm Sci., 99 (8), 3302, 2010
Processing and Particles

Potential Particle Formation During Biologics DP Manufacturing
Freezing and thawing

Stirring during formulation
Transfer of solution via pump
Filtration
Filling through Needle
Transport and shipping
Contact with various material of construction (SS, plastic, teflon, filter
membrane)
Residual disinfectant or sterilant like peroxide
Container closure interactions
Glass flaking; silcone oil protein interactions; metal protein interactions
Air-water or ice interface

Cavitation
Shear?
Compatibility of formulation with material
Intrinsic Particles
At least 12 products have been approved in US and EU where visible protein
particles are generated over time but their amount in relation to the total drug
(protein) is negligible.
Arzerra (ofatumumab) : Colorless solution and may contain a small amount of
visible translucent-to-white, amorphous, ofatumumab particles. However, the
solution should not be used if discolored or cloudy, or if foreign particulate matter is
present.
Vectibix (panitumumab): solution may contain a small amount of visible translucentto-white, amorphous, proteinaceous, panitumumab particulates
Erbitux (cetuximab): solution may contain a small amount of easily visible, white,
amorphous, cetuximab particulates
Stelara (ustekinumab):colorless to light yellow product that may contain a few small
translucent or white particles.
We can expect more!
Dosage and Administration

Qualify filters - Demonstrating that filtration of the solution (via in-line IV filters)
results in removal of these protein particles when product ages
- Included in Dosage and Administration Instruction
- Shipping and other stresses during handling taken into account
All approved products validated that dose of drug can still be administered
How does one perform visual inspection of these products?
- visual inspection standards have to be created so that the inspectors can
qualitatively compare the number and size of these particles.
Concerns associated with the presence of particles that develop over time, while
the product is in distribution or storage, must be addressed during development
via formulation selection and stability testing. In some cases, precautions and
specific instructions-for-use may be included product labeling, for example use of
in-line filters at time of administration.
What happens to product In-vivo?

Upon administration to the patient, the protein is
In a different environment (pH, buffer, ionic strength)
Protein binding
Higher temp (37C) for minutes to days to months depending on the half-life
What changes can occur?
Aggregation, particle formation, ppt
Oxidation, acidic species formation
Data from Genentech (Ref. J. Liu)
Time in
Serum at
37C
SEC Results
Weeks
% HMWS
% Main
% LMWS
2.0
97.4
0.6
4.4
92.9
2.7
7.8
86.0
6.2
What do we know?
This is just beginning to be studied. There is lot to learn
Hard to study in-vivo
Need complex analytical (separation, detection) techniques
Can monitor gross changes only
How do these changes link to safety and efficacy?
Challenges and Unanswered questions?

Is there a relationship between intrinsic protein particles and safety? Is
there a particle size influence
No to maybe to potentially to yes
Safety of the marketed products (in terms of nAb)
If the intrinsic particles are present in a product , is validating in-line filter
or a syringe filter OK?
How do you determine and quantitate intrinsic protein particles in
suspension / dispersed products?
Many studies formulation, processing done at lab scale not in an
aseptic environment, where it is hard to avoid extrinsic particles.
Not covered are challenges with the visual inspection, what is
considered essentially free, validation of automated inspection systems
where there product is foams / has bubbles?
Material limitations, hence a Stage appropriate controls
Acknowledgements
Satish Singh
Carol Kirchhoff
Typical mAb Analytical Methods: Release and Characterization

DRUG SUBSTANCE
Appearance
pH
UV
IEF
Peptide Map
SDS-PAGE, reducing
SDS-PAGE, nonreducing
HPLC- SEC
Carbohydrate Size Profiling
DNA
rProtein A ELISA
HCP ELISA (commercial kit)
SDS-PAGE, silver stain
Binding ELISA
Bioburden
Endotoxin
Mycoplasma
Virus Testing
Functional Bioassay
HCP ELISA (cell line specific)
CHARACTERIZATION
DRUG PRODUCT
Appearance
Strength (UV)
IEF
Peptide Map
SDS-PAGE, reducing
SDS-PAGE, nonreducing
HPLC- SEC
Endotoxin
Particulate Matter
Sterility
Binding ELISA
Functional Bioassay
Additional Impurity Assays as needed:
Oxidation
Deamidation (iCE)
Fragment
MALDI-TOF Mass Spec

Electrospray Mass Spec
LC/MS, MS/MS
AUC
CD
Biacore
Fluorescence
Impurity Isolation/Enrichment
Impurity Bioactivity
N-Terminal Sequence
C-Terminal Sequence
Free Thiol
Western Blotting
Alternative HPLC Assays
Additional CE Assays
Amino Acid Analysis
2D Electrophoresis
Carbohydrate Charge Profiling
DSC
Phase appropriate methods not all methods available at early stages
Biologics PHS Act Definition

Monoclonal antibodies for in-vivo use in the prevention or treatment of disease.
Proteins intended for therapeutic use, including cytokines (e.g. interferons), enzymes (e.g.,
thrombolytics), and other novel proteins.
Immunomodulators (non-vaccine and non-allergenic products) intended to treat disease by
inhibiting or modifying a pre-existing immune response
Growth factors, cytokines, and monoclonal antibodies intended to mobilize, stimulate,
decrease or otherwise alter the production of hematopoietic cells in vivo
Combination products comprise of a biological product component with a device and/or drug
component.
Cellular products, including products composed of human, bacterial or animal cells (such as
pancreatic islet cells for transplantation), or from physical parts of those cells (such as whole
cells, cell fragments, or other components intended for use as preventative or therapeutic
vaccines)
Vaccines
Allergenic extracts used for the diagnosis and treatment of allergic diseases and allergen
patch tests
Antitoxins, antivenins, and venoms
Blood, blood components, plasma derived products (for example, albumin, immunoglobulins,
clotting factors, fibrin sealants, proteinase inhibitors), including recombinant and transgenic
versions of plasma derivatives, (for example clotting factors), blood substitutes, plasma
volume expanders, human or animal polyclonal antibody preparations including radiolabeled
or conjugated forms, and certain fibrinolytics such as plasma-derived plasmin, and red cell
reagents
143
Identification of Particles
Important to understand the cause
E.g. generation of particles during freeze-thaw
Photo-degradation of the product and generation of particles
Particles
Excipient / drug interactions
When the B2-coated stopper was first used, the xxx drug product formulation
contained polysorbate 80. After the formulation change that removed this surfactant,
fiber-like particles were seen in some final container lots. An investigation into the
phenomenon revealed that a silicon oil coating applied to the stoppers at Mfg site
caused the particles to form in the absence of polysorbate 80. Two development
runs were filled on to establish whether unsiliconized stoppers could be used for
manufacture of drug product that did not contain polysorbate 80. No particles were
observed for these two lots, and the product passed the critical release
specifications. Based on this evidence, the use of unsiliconized stoppers for product
xxx final container production was implemented for all subsequent lots.
Aggregates Classification based on Size
146
USP Workshop on Particle Characterization, 8-10

December 2010
Analytical Techniques for the

Forensic Analysis of Particulate
Matter
Ronald G. Iacocca PhD

Senior Research Advisor
Analytical Sciences R&D
Product Research and Development
Outline of Presentation
New technologies for measuring foreign particulate
matter
Advantages and challenges of imaging technologies
Advanced spectroscopic techniques for looking at
isolated particles
Case study
3/25/2011
File name/location
148
Copyright 2010 Eli Lilly and Company
Disclaimer
This information is not intended to suggest
regulatory policy or QC procedures for the release
of clinical trial or market product
3/25/2011
File name/location
149
Current Regulatory Policy

USP <788>
6000 particles 10-25 m

600 particles >25 m
No visible particles, essentially particle free
Technology used
HIAC or light obscuration techniques

Membrane microscopy particle counting
Manufacturing automated vision systems combined with human
inspection
3/25/2011
File name/location
150
Current Techniques
Advantages
Very robust for the defined size ranges

Well established and understood
Disadvantages
No shape information with light obscuration

Particles may be a gel or semi-solid, and will not be visible on a
membrane filter
Particles may be transparent
Difficult to determine root cause
3/25/2011
File name/location
151
Scenario: Subvisible Count is Exceeded, or

Visible Particulates are Observed
New technology that can provide insight into the root
cause of particle generation
Dynamic Image Analysis
3/25/2011
File name/location
152
Dynamic Image Analysis

Key
1 dispersed particles
2 device for control of particle
motion
3 measurement volume
4 light source
5 optical system
6 depth of field
7 image capture device
8 image analyser
9 display
Detection
Zone
3/25/2011
File name/location
153
Typical Images Obtained
3/25/2011
File name/location
154
Foreign Particulate Matter
3/25/2011
File name/location
155
Protein Aggregates
Equivalent Circular Diamter

Measurement Range: 1m to
1827m
Cell Depth: 100m
3/25/2011
File name/location
156
Foreign Particle
3/25/2011
File name/location
157
Prior to Compositional Identification

Particulates
Observed
Are images
available
Particle
Density
Many particles or
one singular
occurrence
Multiple
Lots
3/25/2011
File name/location
Persistent?
Formulation
Instability?
Glass
incompatibility
Materials
shedding
particles (MOC)
Filters?
Particle
Shape
Sharp
corners: no
solubility
Fluffy
Corrosion
product or
ppt.
Particle
Size
Expect:
Small more
numerous
Large one or
two
Structural
Rigidity
Structural Material
(glass, metal,
polymer, API)?
Residue?
Color
Helpful, but may

be misleading.
Color = f(size)
158
Investigation Portion of Particulate

Investigation: Identification of Composition
Characterization portion of particle investigation
Use more advanced tools
Working with single particles rather than ensemble
techniques
3/25/2011
File name/location
159
Solid State Characterization of Materials
3/25/2011
File name/location
160
Problem Statement
Development formulation tank was swabbed.
Particulates were found
Analyze the swab to determine composition

Find source of slight residue
3/25/2011
File name/location
161
FTIR Analysis
Preliminary FTIR
analysis
of the particles
showed that
the unknown
material
was PTFE
Swabs were
submitted
for additional
analysis
3/25/2011
File name/location
162
Scanning Electron Analysis

Morphology and Elemental Composition
3/25/2011
File name/location
Element
Wt %
At %
CK
OK
FK
MgK
AlK
SiK
PK
MoL
SK
ClK
KK
TiK
CrK
FeK
NiK
44.32
14.24
06.85
00.53
01.57
05.47
00.67
01.05
00.32
01.94
00.16
01.83
04.96
12.60
03.48
64.34
15.52
06.29
00.38
01.02
03.39
00.38
00.19
00.17
00.96
00.07
00.67
01.66
03.93
01.03
163
So, In the Midst of the Unknown,

Analyze More!
L01336p49B001.spe: blank swab
2005 Apr 5 Al mono 49.8 W 194.0 45.0 224.00 eV
3.1569e+004 max
Company Name
L01336p49I001.spe: swab from Alimta tank
9.34 min
2005 Apr 5 Al mono 49.8 W 194.0
Su1s/Point3: blank swab/1 (SG3)
L01336p49B001.spe
x 10
-F1s
Atomic %
C1s
58.0
F1s
25.2
O1s
13.9
Si2p
2.7
Cl2p
0.2
-C1s
Atomic %
C1s 71.5
O1s 28.5
L01336p49I001.spe
x 10
-O1s
2.5
-Cl2s
1200
1000
800
600
Binding Energy (eV)
400
200
0
1400
1200
1000
800
600
Binding Energy (eV)
400
-Cl2p
-Si2s
-Si2p
-F2s
0.5
0
1400
-C1s
c/s
-O KLL
-C KLL
c/s
-O1s
-O KLL
-O KLL
-F KLL2
-F KLL1
-F KLL
1.5
9.34 min
Su1s/Point4: swab from Alimta tank/1 (SG3)
3.5
Company Name
5.1673e+004 max
200
Elemental analysis shows C, Cl, O, Si, F but no metallic

elements
3/25/2011
File name/location
164
Final Analysis Time of Flight

Secondary Ion Mass Spectroscopy
Summary of analytical
data:
Only showed
elements of PTFE
3/25/2011
File name/location
165
Conclusion of Case Study

Multiple particle present, which is always found.
Whenever stainless steel is swabbed, stainless steel
particles are detected
Stainless steel particles were coated with an approved
lubricant that had a structure similar to PTFE, hence
different answers were obtained with instruments that
had different sampling depths.
All of the data were correct, one had to realize that
this was a multivariate problem.
3/25/2011
File name/location
166
Summary
New technologies will enable more detailed particle
analysis
Numbers of particles in size bins is insufficient for todays drug
development/drug manufacturing arena
Must look at aspects such as:
Frequency of occurrence
Shape
Color
Kinetics where to they appear in the process
For chemical analysis of particles
Spectroscopic techniques can provide a wealth of

information on composition and root cause if the
operating principles are understood
3/25/2011
File name/location
167
The Development of
Particle Size Reference Standards
G Rideal and J Storey

Whitehouse Scientific Ltd, Waverton, Chester, CH3 7PB, UK
Summary
1. The evolution of particle size standards
2. The importance of producing representative standards
Spatula sampling versus spin riffling
3. The certification of BCR Mirror standards
4. Early laser diffraction results manufacturers and end users
5. A new 19 190 micron opaque microsphere standard
(a) Primary particle size results
(b) Laser results (manufacturers and end users)
6. Multimodal standards image analysis and sub-micron

7. Sieve calibration microspheres
8. Conclusion
Obtaining a representative sub sample

Sampling a 15 150 micron bottle by spatula
First sample from 10g bottle
Second sample
Producing single shot reference standards

The spinning riffler
Masterbatch in
Sub samples out

Subdivisions from 8 - 100
A continuous 100 stage spinning riffler
Contra-rotating
powder disperser
Rotating feeder
Collecting pots
Size distribution in the sub-samples is independent

of the position on the riffler
(100 stage batch riffler)
Subdivision strategy for a 1000Kg

Clear glass standard (15 150 microns)
1000 Kg
320 x 2.5g samples for analysis
Up to 4 million bottles @ 0.25g
25 Kg
1/100 = 250g
1/100 = 2.5g
Series 1
Series 2
Series 3
Series 4
Series 5
Validation of the spinning riffler

2.5g bottle-to-bottle variation
(15 150m clear glass standard)
Series Number
1
2
3
4
5
6
Averages
Uncertainty* (+/-)
d (0.1)
37.33
37.63
37.58
37.70
37.54
37.52
37.55
0.26
d (0.5)
63.17
63.36
63.17
63.16
63.08
62.93
63.14
0.28
d (0.9)
92.23
92.25
92.02
92.00
91.87
91.79
92.03
0.36
* Confidence level not less than 95%
Courtesy of Malvern Instruments
Tests
57
54
57
55
57
53
Certification of the BCR Mirror standards
3 30 micron standard
10 - 100 micron standard
For clear glass microspheres, laser diffraction

results depend of diffraction theory used
1994 laser diffraction data for
the 3 - 30 micron standard
Fraunhofer
theory
Mie theory
Errors increase with decreasing size
Comparison of Mie and Fraunhofer scattering

theory for 3 30m clear glass microspheres
Results for Mie theory
Results for Fraunhofer theory
Note. Both theories give identical results for opaque standards

(Courtesy of Malvern Instruments)
Laser diffraction data for a 3 30 micron

clear glass reference standard
- round robin test
(a) Benchmark results
(2006)
(b) Results from 60 labs (PACQS)
NB. Participating laboratories used Mie scattering theory

The large scatter of results suggests that insufficient attention to
dispersion was paid by many of the laboratories
Laser diffraction data for a 10 100 micron

clear glass reference standard
- PACQS round robin test
(2006)
Much closer results than for the 3 30 micron standard

- indicates good sampling and dispersion
New 19 190 micron opaque microsphere standard

spin riffling results from 550kg master batch
Table 1. Subdivision accuracy: 19 190 micron

opaque standard (Sympatec Helos)
Percentile
10
25
50
75
90
Size (microns)
37.4
46.9
61.0
74.7
87.8
1.0
0.9
0.8
0.7
0.8
Uncertainty (+/-%)
54 tests (Courtesy of LGC)
Results for 1g bottles analysed dry
Fine structural detail introduced in the

19 190m opaque standard
High resolution image analysis
Reducing resolution replicates laser
Evidence of fine structure from laser analysis
19 190 micron standard primary analysis results

Image analysis - ShapeSizer
Coulter Counter 100 tests
Image analysis - Malvern
Mean and uncertainty values for 19 190mm silver coated microsphere standard
Percentile
5
10
25
50
75
90
95
ShapeSizer Size (m)1
Uncertainty (+/-%)
36.02
10.55
40.51
8.49
49.31
5.64
65.80
6.47
72.91
3.20
85.75
4.66
92.65
8.27
Coulter2 (av. 50 tests)

Uncertainty (+/-%)
34.3
4.52
39.3
4.78
49.1
7.66
64.5
6.98
74.7
6.93
85.6
9.14
92.4
9.59
Malvern Morphologi3
Uncertainty (+/%))
33.9
1.3
38.1
0.84
47.3
1.10
64.6
0.67
73.2
1.69
87.4
1.40
100.4
2.28
1. 5 x 10,000 particles,
2. 5 x 60,000 counts,
3. 5 x 95,000 particles
Notes.
1. The uncertainty levels can be reduced by increasing the particle count
2. Sub-sampling such low corresponding weights may also lead to errors
19 190 micron standard - laser diffraction results
Laser diffraction analysis of a 19 190 micron opaque standard

Percentile
10
25
50
75
90
95
Sympatec
(Dry)
Beckman (wet)
32.2
37.4
46.9
61.0
74.7
87.8
97.0
34.5
38.8
49.3
63.0
75.7
88.3
97.6
Horiba (wet)
38.1
42.2
50.5
61.3
74.7
88.6
100.1
Horiba (dry)
40.8
44.8
51.9
61.6
73.8
87.2
97.7
Fritsch (wet)
34.1
38.8
48.1
61.1
76.3
91.4
100.6
Malvern (wet)
33.3
38.2
48.6
61.7
75.6
88.2
95.7
PACQS1 (15 labs)
37.9
1. A quality audit scheme run by LGC
62.2
89.2
19 190 micron opaque standard

repeatability and reproducibility
(laser diffraction analysis)
(a) Repeatability
5 repeats - Sympatec
(b) Reproducibility
15 labs - PACQS
Remarkably consistent results whether analysed on the same

instrument or in 15 labs using a range of instruments
19 190m Opaque Standard comparison of methods
Excellent agreement with the primary methods
A 500 2000m Multimodal Image Analysis Standard
Comparison of Retsch CAMSIZER and Haver CPA cumulative data with the certificated values
from a 500 2000mm Image Analysis Standard (Multistandard)
Percentile
Certificate data (microns)
Haver CPA (microns)
Retsch CAMSIZER (microns)
5
715
730
721
10
780
790
781
25
897
905
897
50
1101
1125
1123
75
1334
1335
1346
90
1652
1660
1670
95
1816
1827
1846
Performance in analysing the individual peaks in a 500 2000mm Image Analysis

Standard (Multistandard)
Peak #
1
2
3
4
5
6
7
8
592
758
858
1098
1301 1449 1640
1884
Certificate (m)
610
750
860
1100
1300 1400 1650
1860
Haver CPA (m)
600
760
865
1110
1310 1430 1660
1865
Retsch CAMSIZER m
500 2000m Multimodal Image Analysis Standard

Effect of sample weight/number on results
(Haver CPA)
(a) Reducing weight
(b) Repeatability for 20g samples
Reducing count from 150,000 to 15,000 does not significantly affect the results
Sub-micron multimodal standard

CPS Disc Centrifuge
The line start principle: 3 latex blend
900nm
380nm
190nm
Inject sample
Sub-micron multimodal standard (continued)

CPS Disc Centrifuge
Separation and analysis of the 3 latex blend
190nm
380nm
900nm
Sub micron challenge test standard

10 peak multimodal standard
- from 1.5 microns to 0.1 microns
Single peak analysis aligns with Multistandard

Ensures NIST traceability of the results
Sieve calibration microspheres

Microspheres certified
Calibration graph produced
For NIST traceable certification of sieves from 20 3350 microns
Conclusions
1. Particle size standards have to evolve to meet the demands
of the latest technology
2. Monosized latex standards for calibrating single channels in a
Coulter Counter are not so suitable for laser sizers or sieves
.
3. Irregular shaped standards, eg BCR quartz, give different results

depending on the method of analysis
4. Spherical reference standards should give the same results

irrespective of the method of analysis
5. However in lasers, the diffraction theory used can influence the
size, especially for clear glass microspheres at small sizes
6. Opaque spherical standards do not depend on diffraction theory
7. High resolution methods eg. image analysis or centrifugation
require multimodal standards to challenge the resolution
8. To guarantee long term supplies and minimise subsampling, large
master batches and single shot bottles are to be preferred
NIST Perspective on Standards for Subvisible Particles

(with an Emphasis on Protein Therapeutics)
Dean Ripple
Process Sensing Group
NIST, Gaithersburg

Particle Detection & Measurement
December 8, 2010
Outline
Overview of reference materials & their properties
Candidate reference materials
Instrumental effects & relationship to reference material
choice
Pathways to accurate measurements
Planned activities
Acknowledgments
Josh Wayment
Germarie Sanchez-Pomales
Michael Carrier
Dick Cavicchi
Rebecca Zangmeister
Michael Tarlov
Vision
Goal 1. Establish repeatability for each instrument type for lowcontrast protein aggregates or surrogates
Test surrogate systems

Round robin comparisons
Identify need for reference materials or other instrument standardization
Develop protein-based standards
Promote consensus through appropriate standards
Goal 2. Understand differences among instruments
NIST-built light obscuration/light scattering apparatus

Compare light obscuration and flow imaging in the 1 to 50 m range.
Extend comparison to Coulter counters, particle diffusion
Document biases to enable informed changes to USP <788> <789>, etc.
Goal 3. Support efforts in particle identification, correlation with

immunogenicity
Desirable Properties of Reference Standards

1. Irregular structure
2. Variable aspect ratio (width to length ratio) ranging from fiber to
near-spherical
3. Small refractive index difference between particles & matrix fluid
4. Properties stable over long periods (years)
5. Properties stable with expected variations in use:
mixing prior to use
exposure to flow field shear
variations in matrix liquid
leaching from storage container
6. Viscosity of matrix liquid is in range of water to typical protein
formulations (or variable)
7. Proper use is simple
Existing NIST Reference Materials

Particle Size Distribution
Number
Particle Type
1021
Glass bead
1003c
Glass bead
1004b
Glass bead
659
SiN
1978
ZrO grains
1982
ZrO spheroids
1984
WC/Co grains
1985
WC/Co spheroids
8632
Road dust
Size Range (m)

2 to 12
20 to 45
40 to 150
0.2 to 10
0.3 to 2
10 to 150
9 to 30
18 to 55
1 to 20
Limitations of existing standards:

No particles with low aspect ratios
No particles with low optical contrast
Existing standards have high density
Diameter
Number
1690
1961
Particle Type
Polystyrene sphere
Polystyrene sphere
Size (m)
1
30
Multiple diameter standards from

other sources as well.
Optical Properties Space

Validate instruments over a range of optical particle properties that
bracket optical properties of protein particulates
Refractive
Index
Polystyrene
Spheres
Precip. CaF2 in
CaCl2 solution
Artificial
Particles
Aerogel
fragments
Length / Width
Ratio
Abraded
ETFE
Roughness
Interaction of Diameter & Concentration

Particle counts may be in error due to either:
direct errors in counting particles (e.g., particle too transparent to
trigger count)
errors in particle size gives wrong distribution of concentration vs.
size
A single calibration with a concentration vs. size curve will only work
provided the variation of concentration with size for the reference
material is similar to that of the tested material
OR the size uncertainty is much smaller than that of concentration
Interaction Proportional to Slope of Concentration vs. Size Curve
Particle concentration
1.10
Apparent error in
concentration, c
1.05
1.00
0.95
0.90
0.90
Error in
size, l
0.95
1.00
1.05
1.10
1.15
Particle size
Typical protein particulates: 10% error in size propagates to

25% error in concentration
Candidates for Particulate Standards

Particle type
Characteristic shape
Typical size (m)
Precipicated calcium fluoride
Irregular crystals
1 to 40
Aerogel
Irregular crystals
1 to 20
Irregular, aggregated
polymers
Routinely <1, larger?
Aggregate of
nanoparticles
Routinely <1, some

larger
Self-assembled polyions
Sintered fumed silica
Natural fibers
Long cylinders, straight Diameters of 1 m or

or serpentine
larger
Engineered particles
Any 2D shape
<1 to >500
Abraded polymer
Irregular forms
<1 to 100
T or pH induced protein
aggregates
Irregular aggregates;
irregular filaments
<1 to >100
Artificially fixed protein

aggregates
as above
as above
Engineered Fibers
Borrow from semiconductor manufacturing technology:
Fabricated polymer films

A single 150 mm wafer can produce
2x108 particles of area 40 m2
Candidate system: SU-8
Highly biocompatible
Submicron to mm length scales
Index of refraction 1.6
(Cavalli et al,J. of Comb. Chem., 2007, Vol. 9, No. 3)
Advantages
Disadvantages
Any 2D shape
Variable optical density
Variable particle flexibility
Particles are well defined
Possibly expensive
No irregular 3D shapes?
Requires index matching liquid?
An interesting possibility for standards for visible particles, as well
NIST Approach for Engineered Fibers

Recipe:
Spin coat and bake release layer on
silicon wafer
Spin on 2 m of SU-8 photoresist
Expose SU-8 layer through optical mask
Develop & bake SU-8
Release
Initial work: large, 2D particles
Goal: small, textured particles
NIST Center for Nanoscale Science

and Technology (CNST)
NanoFab Facility
Optical Images of Particles

Optical microscopy of SU-8 particle on Si wafer
340 m
Particles in solution via microscopic flow imaging
Abraded Polymer Particles: NIST Recipe

ETFE polymer (alternating tetrafluoroethlyene & ethylene copolymer)
has desirable properties:
1. Refractive index of 1.40 (very similar to protein adsorbed on wall
from solution)
2. Very durable & tough
3. Can we make it look like a
protein?
ETFE roofing panels
Recipe for preparation:

(Eden Project, wikicommons picture)
Mechanically abrade to make wet slurry
Dilute with water + 0.1 % (w/v) surfactant
Filter & size selection by gravimetric
sedimentation
ETFE in water + 24 %
sucrose (n = 0.03)
ETFE in water (n = 0.07)
Agitated IgG
Protein Particulates
1. Induce particulate formulation in protein solution by varying
temperature, pH, agitation, etc.
2. Stablize particulates by protein fixative, surfactants, buffer
choice
Are they stable?
Does the particle geometry mimic natural particulates?
Which morphology do we mimic?
(variable scale)
NIST Approach
1. Form aggregates by overnight agitation
2. Cross link with gluteraldehyde

3. Quench cross-linker
4. Remove excess cross-linker & initial
buffer by dialysis
5. If necessary, filter or sediment out large
aggregates
6. Formulate to stabilize aggregates:
Choice 2: Freeze dry with added sugar.
Optimal surfactant to add? What happens to hydrated
aggregates?
Choice 1: Very high sucrose concentration: 60% sucrose by
weight is soluble at room temperature; yet will form a glass of the
solution (not the lyophilized product) at -80 C
Optical Method 1: Flow Microscopy
Commonalities of instruments:
digital capture and analysis of particulates
flat flow cells
typical range of 1 to 100 m
Proprietary features:
particle identification algorithm
optical light source, camera, objectives, apertures
Contrast & spatial resolution depend on proprietary choices
Brightfield Images: Phase Contrast of Out-of-Focus Images

An out of focus image will be shaded either light or dark due to diffraction
of light by a particlesimilar to phase contrast microscope.
Image contrast due to out-of-focus condition may be greater than inherent
image contrast for in-focus image!
Apparent size of object will depend definition of particle edge as dark or
light contour.
Detection will depend on choice of threshold value and whether phasecontrast effect favors detection or not.
Particles imaged in flow cells will have this effect to some extent
Same particle, different focus (20x, 7.2 m shift in focus):
20 m
Optical Method 2: Light Obscuration

Particle passing through light beam
reduces optical transmission
Diffraction effects for small particles must
be accounted for
Scattering cross section depends on
particle morphology
Existing instruments:
well validated & key part of USP <788>
question of sensitivity to particles of low
refractive index mismatch
Improvements in sensitivity possible?
NASA Kepler Missionfind

extrasolar planets by
obscuration of star light
Sensitivity: 10-5 change in
transmission
Price: $600 Million
Light Obscuration: Refractive Index of Particle

4
Aspect ratio = 4 (prolate spheroids),
Wavelength 633 nm
n = 0.1
0.05
0.02
0.01
Extinction efficiency
0
1
10
Equivalent diameter, m
100
Light Obscuration: Effect of Wavelength

4
Aspect ratio = 4 (prolate spheroids)

n = 0.02
Wavelength = 450 nm
633 nm
800 nm
Factor of 2.3 difference in

efficiency for 10 m particles
0
1
10
Equivalent diameter, m
100
Light Obscuration: Effect of Roughness

1.0000
Sphere
4th deg. Chebyshev, 0.05 roughness

0.1000
Mildly rough particles

give very little change in
extinction efficiency
0.0100
0.0010
0.1
1
Equiv. spherical diameter, m
10
Light Obscuration Summary

Refractive index matters: factor of 2 drop in sensitivity if n= 0.02
for 10 m particles
Wavelength matters: factor of 2 drop in sensitivity for long
wavelengths + small n for 10 m particles
Very elongated particlesdrop in sensitivity
Roughnessno indication of significant effect
Not at all clear why light obscuration undercounts for >20 m
1. We need direct measures of particulate refractive index or density
2. NIST spectral light scattering/obscuration apparatus will attempt to
exploit wavelength dependence of scattering in 1 to 20 m region
to distinguish proteins from non-proteins. Can we also find
effective extinction efficiency?
Particle Refractive Index

Refractive Index
of Particle
Best measure of protein

mass bound in aggregates
Possible Methods:
Immersion in index matching fluidsdifficult because the particles are

quite fragile and sensitive to the matrix liquid
Quantitative Phase Microscopy(Nugent and Paganin) good choice,

but the method is patented and software presently hard to obtain. Variants
may be outside of patent.
Digital holographypromising method
Spectral light scatteringNIST is assembling a system to do this.
Through-focus Scanning Optical Microscopy (TSOM)Extended

version of QPM; needs lots of modeling
Gravimetric SedimentationMeasure of average density of particles;

works for aspect ratio near 1
Gravimetric Measurement of Particulate Density

Sedimentation velocity vs proportional
to /d2
Measure diameter d and velocity vs

with flow imaging microscope in no
flow condition
Special care needed to suppress

convection (vs 1 m/s for d = 10 m)
Only works for particles that are close
to spherical
300
Distance, m
200
100
0
0
20
40
Time, s
Track of a polystyrene bead
Initial results for IgG particulates: 10 kg/m3
60
Electrical Sensing Zone Method: Coulter Counter

Flow of ions
V/I = R
Perturbing the electrical
resistance R of the channel
... perturbs measured
voltage to current ratio, V/I
Nominal measurement of particle volumewhat methods would we use to

certify volume of highly non-spherical reference materials?
Volume may be difficult to interpret or relate to optical measurements for
extended, fibrous, or porous particles
Do proteins aggregate in optically invisible, extended structures (gel-like) with
reduced ionic conductivity?
Conclusions
Instrumental effects could introduce sizable errors in particle

concentration count for protein particulates
Several promising surrogates:

Next stepsusability studies, comparison with industrially
developed particles, stability studies
Measurement of protein-particulate refractive index/density a high

priority to guide reference standards & to understand light
obscuration/scattering
Coulter counter promising, but what are we measuring?
Calibration/validation of light obscuration & flow imaging:

Monodisperse bead size standards + polydisperse surrogate

Reference Standards: USP

Perspective
Barb Jones, Ph.D.
USP Vice President, Reference Standards Evaluation
Current Standard
Particle
Used
Count Reference Standard (Lot K0H089)
in the Following General Chapters:
<788>
Particulate Matter in Injections (Light Obscuration)
<789>
Particulate Matter in Ophthalmic Solutions (Light Obscuration)
Each set contains 2 units of a dilute suspension of monosized

polystyrene Spheres (15 m) and 2 units of suspending fluid to be
used as blanks.
Collaborative Testing
12
collaborating Laboratories
Each
laboratory analyzed 4 containers of the suspended

particles and 4 blanks.
Samples
were mixed, degassed, and three portions from each

container were analyzed.
The
particle counts were measured at both 10 m and 15 m.
Only
the data from the 2nd and 3rd portions were used.
Data Analysis and Value Assignment
A statistical
12
analysis was performed and limits were assigned.
laboratories contributed data
Counts
were corrected (using the data from the preceding

blanks) using the method specified in <788>
On
collaborator failed the particle County Accuracy Test and

that data was excluded.
Corrected Counts at 10 and 15 m

Figure 1. Corrected Counts at 10 and 15 m
4500
4000
3500
Corrected count per ml
3000
2500
10 um
15 um
2000
1500
1000
Collaborator 6
500
0
0
10
20
30
40
50
60
Code
70
80
90
100
110
120
Ratio of Counts at 10 m to Those at 15 m

Figure 2. Ratio of Counts at 10 m to Those at 15 m
4
3.5
Collaborator 6
10:15 ratio
2.5
1.5
0.5
0
0
10
20
30
40
50
60
Code
70
80
90
100
110
120
Differences between 2nd and 3rd Portions

Figure 3. Differences between 2nd and Comparing
3rd Portions 2nd
Sample
Vialsnumbers for Standard
and 3rd
Code
0
10
20
30
40
50
60
70
80
90
100
110
120
1000
Difference of Counts (3rd - 2nd)
500
0
10 um
15 um
-500
-1000
Collaborator 6
-1500
Differences between 2nd and 3rd Portions

Comparing 2nd and 3rd numbers for Blank
Blank Vials
Code
0
10
20
30
40
50
60
70
80
90
100
110
120
60
Difference of Counts (3rd - 2nd)
40
20
Collaborator 6
-20
-40
-60
10 um
15 um
Lower limit
Upper limit
Particle Size Standard

The
USP Particle Count Reference

Standard is not suitable for Particle
Size determination
USP
would like your opinions on

what is needed for a Particle Size
Reference Standard(s)

Session II: Industry Perspectives

Track 1: Solutions
Spalding Auditorium
Track 2: Aerosols
Briggs/Parker/
Marshall/Wiley

Session II, Track 1:

Industry Perspectives for Solutions
Chair: Dr. Michael Mulkerrin
Oncomed

Industry Perspectives for Solutions

Historical Perspective
Russell E. Madsen, M.S.
President, The Williamsburg Group, LLC
Chairman, USP Visual Inspection of Parenterals Expert Panel
Introduction
This presentation provides a history of visual

inspection practices and requirements for
parenteral products in the United States
Discusses a proposal to demonstrate a batch of
the product is essentially free as currently
specified in USP <1>
The Need to Inspect
Visual inspection of parenteral products is driven

by the need to minimize the introduction of
unintended particulate matter
Inspection also offers the opportunity to reject
non-integral units (e.g., those with cracks or
incomplete seals)
Current expectation for inspection of each
finished unit (100% inspection)
Human Visual Performance
Threshold for human vision is generally accepted to

be about 50 m
Detection is probabilistic
Probability of detection increases with increasing
particle size
probability of detection for a single 50 m particle
in clear solution in a 10 mL vial with diffuse
illumination between 2000-3000 lux is slightly
greater than 0%
probability increases to approximately 40% for a
100 m particle and becomes greater than 95%
for particles 200 m and larger
Particle Effects on Patients
Most studies have focused on sub-visible particles,

with a diameter of less than 50 m
Smallest particles (approximately 1 m in
diameter) are often trapped in the liver, lungs and
spleen
IV infusion of particles larger than the internal
diameter of capillaries may be clinically
significant, increasing the risk of an embolic
syndrome
No reports of adverse events associated with the
injection of individual visible particles have been
found
Essentially Free
Zero defects is the desired goal

ZD is not a workable specification for visible
particulate matter
current packaging components
processing capability
Thus the terminology essentially free - but what
does it mean?
More precise definition is desirable to prevent
misunderstanding and to aid in communication
History of Inspection Standards
In 1915, USP IX described the need for

injectable compounds to be true solutions
In 1916, NF IV included six monographs for
parenteral products detailing the method of
preparation
Neither provided guidance with respect to
solution clarity
The first appearance of solution clarity and

freedom from contaminants for parenterals
occurred in 1936 in NF VI
A requirement for clarity in injectable solutions
specified, Aqueous ampule 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.
The requirement for visual clarity of parenteral

products was coordinated November 1, 1942,
appearance of NF VII and USP XII
These compendia used the term
substantially free to describe the need for
control of particle contamination and the
perceived lack of ideal quality in the injectable
material inspected
NF VII (1942)
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 un-dissolved material. Substantially free
shall be construed to mean a preparation which
is free from foreign bodies that would be readily
discernable by the unaided eye when viewed
through a light reflected from a 100 watt Mazda
lamp using as a median a ground glass and a
background of black and white
USP XII (1942)

Appearance of Solutions or Suspensions
Injections which are solutions of soluble
medicaments must be clear, and free of any
turbidity or un-dissolved material which can
be detected readily without magnification
when the solution is examined against black
and white backgrounds with a bright light
reflected from a 100 watt Mazda lamp or its
equivalent
Both the USP and the NF used the same test

procedure; however the USP procedure was
more rigorous in that it omitted the qualifying
adjective, substantially.
These compendial requirements were used by
the FDA in its role as the Quality Control Office
for all pharmaceuticals purchased by the Armed
Forces during World War II
USP XIII (1947)

Clarity of SolutionsWater for Injection, pharmacopeial Injections or
pharmacopeial Solutions of medicament, intended for parenteral
administration, unless exempted by individual monographs, must be
substantially free of any turbidity or undissolved material which can be
detected readily without accessory magnification (except for such
optical correction as may be required to establish normal vision), when
the solution is examined against a black background and against a light
which at a point ten inches below the source provides an intensity of
illumination not less than 100 and not more than 350 foot candles. This
intensity of illumination may be obtained from a 100 watt, inside frosted
incandescent lamp operating at rated voltage, or from fluorescent
lamps, or from any equivalent source of light.
Additional information on page 628 provided details of the light sources
described and equated the light from three 15 watt fluorescent lamps to
that from the 100 watt bulb
United States vs. Bristol Labs, Inc. (1949)

FDA inspector found particle-contaminated ampules in the
accepted stock of six Bristol Laboratories injectable products
The company was served on September 25, 1948, with FDA
Injunction #198
Bristol Laboratories challenged the results of the test by preparing a
blinded test group of 150 ampules containing 1 mL sterile saline
(the test group included 38 ampules that the FDA inspector had
rejected as contaminated with particles)
At the conclusion of the testimony for the government, on January
25, 1949, the court granted the defendants motion for dismissal
the FDA expert witness passed 36 out of 38 previously rejected
containers on the witness stand
The case was dismissed on the grounds 1) that the standards
involved were indefinite and 2) that the evidence was insufficient to
show such violation of the Act as would warrant the granting of the
relief prayed for (destruction of the ampules)
USP XV through XVIII (1955-1970)

USP XV: Every care should be exercised in the preparation of
injections to prevent contamination with micro-organisms and
foreign material. Good pharmaceutical practice also requires
that each Injection, in its final container, be subjected individually
to visible inspection.
USP XVI and XVII: Every care should be exercised in the
preparation of injections to prevent contamination with microorganisms and foreign material. Good pharmaceutical practice
also requires that each Injection, in its final container, be
subjected individually to visible inspection whenever the nature
of the container permits.
Fed. Std. 142 (1959-1970)

Int. Fed. Std. No. 00142, Parenteral Preparations, was issued by
the United States Navy-BuMed (August 1, 1959)
Mandatory on all Federal agencies, issued pursuant to the
Federal Property and Administrative Services Act of 1949
Applicable to sterile parenteral preparations in final containers,
intended for human consumption
The standard was superseded on October 31, 1966, by Fed.
Std. No. 142a
Provided requirements for clarity of solutions as well as limits for
visible particulate matter (the standard was applicable as a final
test to a sample of finished product, not to 100% on-line
inspection, and the sampling was in accordance with MIL-STD105)
Fed. Std. 142a

Section S6.2.1 Clarity of solutions. Applicable to type I, class 1; type
II, class 1; type II, class 3; and solutions of dry solids (type IV, class 1).
Solutions of parenteral preparations shall be clear and free from
undissolved or particulate matter within the limits permitted in the
classification of defects and the applicable acceptable quality level
(AQL), when examined without accessory magnification (except for
such optical correction as may be required to establish normal vision)
against a black background and against a white background and
illumination from a light which at a point 25.4 centimeters (10 inches)
from its source, provides an intensity of illumination of not less than 100
and not more than 350 foot-candles. Some biological products need
not be clear and entirely free from turbidity, provided this is
characteristic of the product. The clarity standards for such products
shall be judged on an item for item basis with the characteristic
properties of the product considered in each case.
Fed. Std. 142a

For aqueous solutions (type I, class 1), the solution not
clear defect was classified as Major A, Inspection Level II
and the AQL (percent defective) as 1.0
For example, if there were 30,000 units in the batch, the
clarity of solution test would pass if upon inspection a
sample of 315 units, 7 or fewer contained visible
particulate matter
Thus, under Fed. Std. 142a, agencies of the Federal
government, including FDA, would deem this level to be
acceptable and to comply with the meaning of the USP
term essentially free.
The significance of Fed. Std. 142a is that it provided
government-endorsed acceptance limits for the presence
of visible particles
USP XIX, Supplement 1 (1975)

Solutions of medicament, intended for parenteral
administration, unless exempted by individual
monographs, must be substantially free of any turbidity
or un-dissolved material which can be detected readily
without accessory magnification (except for such optical
correction as may be required to establish normal vision).
Every care should be exercised in the preparation of
injections to prevent contamination. Good pharmaceutical
practice also requires that each Injection, in its final
container, be subjected individually to a physical
inspection, whenever the nature of the container permits,
and that every container whose contents show evidence
of contamination with visible foreign material be rejected.
USP XX (1980)
Every care should be exercised in the
preparation of injections to prevent
contamination. Good pharmaceutical practice
also requires that each injection, in its final
container, be subjected individually to a
physical inspection, whenever the nature of
the container permits, and that every
container whose contents show evidence of
contamination with visible foreign material be
rejected. [Same as XIX Supplement 1]
USP XXIII (1995)

Use of the term substantially free was replaced by the
term essentially free
Every care should be exercised in the preparation of
all products intended for injection, to prevent
contamination with microorganisms and foreign
material.
<788> Particulate Matter In Injections
Particulate matter consists of mobile, randomly sourced,
extraneous substances . . . that cannot be quantitated by
chemical analysis due to the small amount of material that it
represents and to its heterogeneous composition. Injectable
solutions, including solutions constituted from sterile solids
intended for parenteral use, should be essentially free from
particles that can be observed on visual inspection.
Current USP, General Chapter <1> Injections

Each final container of all parenteral
preparations shall be inspected to the extent
possible for the presence of observable
foreign and particulate matter (hereafter
termed visible particulates) in its contents.
The inspection process shall be designed and
qualified to ensure that every lot of all
parenteral preparations is essentially free
from visible particulates. No inspection
method is specified.
Japanese Pharmacopoeia, Fifteenth Edition,

General Rules for Preparations, 11. Injections
(2006)
Unless otherwise specified, Injections meet
the requirements of the Foreign Insoluble
Matter Test for Injections <6.06>.
Two inspection methods are described
JP Method 1 (applied to injections either in solutions,

or in solution constituted from sterile drug solids)
Clean the exterior of containers, and inspect
with the unaided eyes at a position of light
intensity of approximately 1000 lx under an
incandescent lamp: Injections must be clear and
free from readily detectable foreign insoluble
matters. As to Injections in plastic containers for
aqueous injections, the inspection should be
performed with the unaided eyes at a position of
light intensity at approximately 8000 to 10,000 lx,
with an incandescent lamp at appropriate
distances above and below the container.
JP Method 2 (applied to injections with constituted

solution)
Clean the exterior of the containers, and
dissolve the contents with constituted solution or
with water for injection carefully, avoiding any
contamination with extraneous foreign
substances. The solution thus constituted must
be clear and free from foreign insoluble matters
that is clearly detectable when inspected with the
unaided eyes at a position of light intensity of
approximately 1000 lx, right under an
incandescent lamp.
European Pharmacopoeia 6.0, Parenteral

Preparations, Injections (2008)
Solutions for injection, examined under
suitable conditions of visibility, are clear and
practically free from particles. The inspection
is described as follows. Gently swirl or invert
the container . . . and observe for about 5
s[econds] in front of the white panel. Repeat
the procedure in front of the black panel.
Record the presence of any particles.
Stimuli to the Revision Process, Pharmacopeial

Forum Vol. 35(5) [Sept.Oct. 2009]
ESSENTIALLY FREE [Insert at the end of the
Definitions section of <1> Injections]:
Where used in this Chapter, the term
essentially free means that when the batch of
Injection is inspected as described herein, no
more than the specified number of units may
be observed to contain visible particulates.
Stimuli to the Revision Process, Pharmacopeial Forum Vol.

35(5) [Sept.Oct. 2009]
Visible Particulates in Injections [Insert as a subheading
under Foreign and Particulate Matter]:
This test is intended to be applied to product that has
been 100% inspected as part of the manufacturing
process; it is not sufficient for batch release testing alone,
and a complete program for the control and monitoring of
particulate matter remains an essential prerequisite. This
includes dry sterile solids for injection when reconstituted
as directed in the labeling. Other methods that have been
demonstrated to achieve the same or better sensitivity for
visible particulates may be used as an alternative to the
one described below.
Stimuli to the Revision Process, Pharmacopeial Forum Vol.

35(5) [Sept.Oct. 2009]
Visible Particulates in Injections (cont.)
Injections shall be clear and free from visible particulates
when examined without magnification (except for optical
correction as may be required to establish normal vision)
against a black background and against a white
background with illumination that at the inspection point
has an intensity between 2000 and 3750 lux. This may be
achieved through the use of two 15-W fluorescent lamps
(e.g., F15/T8). The use of a high-frequency ballast to
reduce flicker from the fluorescent lamps is
recommended. Higher illumination intensity is
recommended for examination of product in containers
other than those made from clear glass.
Stimuli to the Revision Process, Pharmacopeial Forum Vol. 35(5)

[Sept.Oct. 2009]
Visible Particulates in Injections (cont.)
Before performing the inspection, remove any adherent labels
from the container and wash and dry the outside. The unit to be
inspected shall be gently swirled, ensuring that no air bubbles
are produced, and inspected for approximately 5 s against each
of the backgrounds. The presence of any particles should be
recorded.
For batch-release purposes, sample and inspect the batch using
ANSI/ASQ Z1.4 General Inspection Level II, single sampling
plans for normal inspection, AQL 0.65. Not more than the
specified number of units contains visible particulates.
For product in distribution, sample and inspect 60 units. Not
more than one unit contains visible particulates.
PF Stimuli Proposal
Based on Fed. Std. 142a, updated AQL from date
captured by PDA Survey of Visual Inspection
Practices 2008
Attempts to harmonize USP, JP and Ph. Eur.
Stakeholders comments captured at May 13,
2010, meeting of USP Visual Inspection of
Parenterals Advisory Panel
Comments are being addressed and will likely
result in several changes, including the proposed
AQL and sampling levels

DETECTION AND MEASUREMENT
December 8 10, 2010
Medical Impact on Particle Size

David F. Driscoll, Ph.D.
Stable Solutions LLC, Easton, MA, USA
d.driscoll@stablesolns.com
Parenteral Particles
(Physical Characteristics)
Soft Particles
refers to droplets, as, e.g., emulsions, such as
injectable oil-in-water dispersions
flexible, malleable, deformable
LOW embolic risk
Hard Particles
refers to solids, particulates, precipitates in
injectable liquids
inflexible, rigid, obstructing
HIGH embolic risk
Medical Risk: Mechanisms

Embolic Risk (USP788 + USP729)
Blood flow from the right side of the heart
into the pulm. artery pulm. capillaries
For truly embolic (hard) particles (> 5 m),
the embolic dose is relatively low
For similarly sized, but deformable (soft) globules,
the embolic dose is much greater
Metabolic Risk
Blood flow from the left side of the heart
into the aorta main arteries to vital organs
Can be small-diameter (< 5 m) rigid (hard)

particulates or deformable, large-diameter, (soft) fat
globules (> 5 m), involving the RES
The liver comprises 90 to 95% of RES function and is a
target organ for injury from intravascular particles
Medical Risk: Mechanisms

continued
Immunological Risk
Intravenous route for allergen increases the
risk for systemic anaphylactoid response,
irrespective of particle hardness or softness
Pharmacological Risk
Alteration of homogeneity of dosage form can
result in therapeutic failures from either a
subtherapeutic (under-delivery) or toxic
(over-delivery) response.
MEDICAL RISKS:
Particulates and Embolic
Phenomena
1996;20:81-87
(CaHPO4)
Report Summary
Paroxysmal respiratory failure and death
occurred in two young adult females
autopsy revealed an amorphous material
containing containing calcium [and
phosphorus] obstructing the pulmonary
microvasculature of each patient both
received an identical [intravenous]
admixture.
DFD-SSLLC-2010
Autopsy Specimen from Case-1
Chemical analysis of the precipitate isolated from [amorphous debris extracted

from the pulmonary microvasculature of the autopsy specimens] revealed the
presence of calcium and phosphorus
MEDICAL RISKS:
Lipids and Embolic Phenomena
Report Summary
The pathophysiological effects of infusing
large amounts of stable lipid emulsions
have been shown to cause RES
dysfunction in animals and humans. This
study investigated the effects on vital RES
organs, i.e., liver and lungs, from the
infusion of a stable (PFAT5 < 0.05%) vs.
highly unstable (PFAT5 ~ 50x higher) fat
infusion over 24 hours.
Oil-Red-O Staining of Lung Specimens in

Fat Infusion Groups Receiving Stable A
vs. Unstable B Admixtures
Characterization
FAT DEPOSITS:
Normal (absent); Abnormal (limited, moderate or diffuse)
TISSUE STRUCTURE:
Normal (intact); Abnormal ( cellularity, infiltration, and/or necrosis)
Human Evidence of Pulmonary Fat Emboli

(15/30 infants receiving i.v. fat;
median duration 2 weeks)
The location of fat, predominantly in small
pulmonary capillaries, and the absence of

lipid emboli in other organs, suggests that
lipid coalescence takes place before death
and is not a postmortem artifact.
Puntis JWL, Rushton DI. Pulmonary intravascular lipid in
neonatal necropsy specimens.
Archives of Disease in Childhood 1991;66:26-28.
MEDICAL RISKS:
Lipids and Injury
to Vital Organs
Report Summary
The pathophysiological effects of infusing
large amounts of stable lipid emulsions
have been shown to cause RES
dysfunction in animals and humans. This
study investigated the effects on vital RES
organs from the infusion of a stable
(PFAT5 < 0.05%) vs. modestly unstable
(PFAT5 ~ 10x higher) fat infusion over 24
hours.
Biochemical Analyses of Liver and Metabolic

Effects of Infusion of Stable (s-TNA) vs.
Unstable (u-TNA) Lipid Infusions
Human Evidence of Liver Injury

Associated with I.V. Fat for 2 Weeks
The association between liver dysfunction
and hypertriglyceridemia (TG > 150 mg/dL)
demonstrated by the present study is
consistent with the important role played
by the liver in the triglyceride clearance in
neonates.
Toce SS, Keenan WJ. Lipid intolerance in newborns is
associated with hepatic dysfuntion but not infection.
Archives of Pediatric and Adolescent Medicine 1995;149:1249-53.
MEDICAL RISKS:
Lipids and Impaired Metabolic
Clearance
J Pediatrics 2008;152:232-36
(Lipid infusions for 1st week of life.)
Plots of Serum Triglycerides (TG)

in Critically Ill Premature Infants
Coarse, PFAT > 0.05% --- Fine, PFAT < 0.05%

5
TG Level, Upper Limit of Normal: 150 mg/dL

(All serum TG levels obtained on DOL- 4)
(All TG levels, n = 122)
(TG levels > 150 mg/dL, n = 22)
MEDICAL RISKS:
Proteins and Particulates and
Immunogenic Phenomena
Human Evidence of the

Immunogenicity of Biologicals
Safety-Related
Regulatory Actions:
41/174 = 23.6%
Most Common Issue:

Immunomodulatory
JAMA 2008;300:1887-96
Medical Impact of Parenteral Particles

1. Compliance with USP Chapters <729> and
<788> reduce medical risks (except immunogenic).
2. All particles (soft or hard) should be controlled
in injectable formulations.
3. Increasing particle populations are associated
with the following medical risks:
Embolic
Metabolic
Immunogenic
Pharmacologic
Foreign Particulate Matter in

OINDP
General Considerations
James R. Coleman
John A. Robson

OINDP
USP <788> PARTICULATE MATTER IN
INJECTIONS
Containers > 100 mL
Particles 10 m
12 particles /mL
Particles 25 m
2 particles/mL
Foreign Particulate Matter in OINDP: General Considerations
25 March 2011
295

OINDP
USP <788> PARTICULATE MATTER IN
INJECTIONS
Containers 100 mL
Particles 10 m
3000 particles/container
Particles 25 m
300 particles/container
25 March 2011
296
Foreign Particulate Matter in OINDP
USP<789> PARTICULATE MATTER IN

OPHTHALMIC SOLUTIONS
Containers > 100 mL
Particles 10 m
50 particles /mL
Particles 25 m
5 particles/mL
25 March 2011
297
USP<789> PARTICULATE MATTER IN

OPHTHALMIC SOLUTIONS
Containers 100 mL
Particles 10 m
50 particles/mL
Particles 25 m
5 particles/mL
Particles 50 m
2 particles/mL
25 March 2011
298
.limits for foreign particulate matter

less than 10 micrometers (m),
greater than 10 m,
and greater than 25 m.
Composition of particles
25 March 2011
299
Composition:
Health: Safety assessment
Specific compositions
Particle number
Source: identification for control
25 March 2011
300
1.Foreign Particles Testing in Orally Inhaled and Nasal

Drug Products. Blanchard, J. et al. Pharmaceutical
Research Vol.31, No.12 December 2004.
2.Considerations for Foreign Particulates in a Quality by
Design Environment. Sundahl, M. IPAC RS 2006
Conference.
3.Best Practices for Managing Quality and Safety of
Foreign Particles in Orally Inhaled and Nasal Drug
Products, and an Evaluation of Clinical Relevance.
Blanchard, J. et al. Pharmaceutical Research Vol. 24, No.
3 January 2007.
25 March 2011
301

OINDP
USP <788> defines:
Particulate matter in injections and parenteral infusions
consists of mobile undissolved particles, other than gas
bubbles, unintentionally present in the solutions.
25 March 2011
302

OINDP
Three types of FPM:
Component: Occurs prior to filling
Environmental: Originating during filling, closure,
packaging
Functional: Originating from operation of the device
25 March 2011
303

OINDP
These distinctions are critical because control of each
type requires very different actions:
Component: supply chain control, cleanliness.
Environmental: process control.
Functional: control of materials.
25 March 2011
304

OINDP
Component: Wash the components and analyze the
particulate content
Environmental: Open the unused device and analyze
particulate content.
Functional: Use the device, analyze the particulate
content.
25 March 2011
305
Consistent with Quality by Design principles,

during development:
Compile an analytical reference library of all components
And all materials encountered as FPM
25 March 2011
306
Consistent with Quality by Design principles,

during development,
preliminary or interim specifications for action or
acceptance can be established.
These should be based on:
Safety assessment of the FPM
Stability batches
Process batches
Scale-up batches
Statistical analysis for Control limits
25 March 2011
307
Capability of analytical methods

Establish a practical lower size limit:
As particle size decreases, analytical capability
diminishes
Isolation of smaller particles becomes more
challenging
Below 2 m, uncertainty increases due to
higher background noise.
Sampling problems
25 March 2011
308

OINDP
Agencies have expressed interest in three ranges of
particle size:
25 m
< 25 and > 10 m
10
25 March 2011
309

OINDP
Blanchard et al. suggest
General:
25 m - 100 m
10 m 25m
2 m 10 m
For nasal products:
2 m 20 m
> 20 m
25 March 2011
310

OINDP
Sampling:
For Parenterals: sample the container
For DPI: What is in the device prior to use
What comes out of the device
25 March 2011
311

OINDP
Sampling:
For MDI: What is in the device prior to use
What comes out of the device
Through the mouthpiece
Through the valve
25 March 2011
312

OINDP
For OINDP: Through the mouthpiece
Uncertainty about effect of mouthpiece in patients
25 March 2011
313

OINDP
For OINDP: Through the valve
Largest dose that a patient might receive
One type of worst case
25 March 2011
314

OINDP
USP <788> defines:
Particulate matter in injections and parenteral infusions
consists of mobile undissolved particles, other than gas
bubbles, unintentionally present in the solutions.
25 March 2011
315

OINDP
Challenges:
Separation / Isolation / Capture
Measuring/analyzing only foreign particulates
Measuring/analyzing all foreign particulates
Preparation for sizing and composition determination
25 March 2011
316

OINDP
Challenges:
Validation when no FPM standards or reference materials
Analyze particles individually
Analyze a sufficient number to be statistically significant
25 March 2011
317

OINDP
Challenges:
Analyze a sufficient number to be statistically significant
Number of
particles
analyzed = N
N ~ Std. Dev.
% RSD
10
3.16
31.62%
100
10.00
10 %
1,000
31.62
3.16
25 March 2011
318

OINDP
ICH, USP, Pharm. Eur. and other regulatory bodies require
validation of analytical methods.
Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Accuracy
25 March 2011
319

OINDP
(Eur) Pharmaceutical Analytical Sciences Group (1999)
Position Paper on Particle Sizing: Sample Preparation,
Method Validation and Data Presentation
Accuracy: Calibration or verification with reference
materials for size and for composition
Precision: Repeatability equivalent results for repeated
analysis, including sample preparation
Intermediate Precision equivalent results with
different personnel and instrumentation
Reproducibility equivalent results when carried
out at different sites, with different personnel
and different instrumentation
25 March 2011
320

OINDP
Pharmaceutical Analytical Sciences Group (1999)
Position Paper on Particle Sizing: Sample Preparation,
Method Validation and Data Presentation
Specificity: Demonstrated capability to distinguish
identity or composition
Detection Limit: Demonstrated range of the instrument
Robustness: Demonstrated sensitivity to changes in
procedure
25 March 2011
321

OINDP
Whatever method for sizing is chosen,
Light Obscuration, Light Scattering, Image Analysis,
Microscopy
25 March 2011
322

OINDP
Aerodynamic Diameter: Diameter of a sphere with a
density of 1gm/cm that has the same inertial properties
as the measured particle.
Stokes Diameter: Diameter of a spherical particle with the

same density and inertial properties as the measured
particle.
Stokes Diameter = Aerodynamic Diameter / Density
25 March 2011
323

OINDP
Stokes diameter microns
Aerodynamic diameter vs. Stokes diameter

12.00
Polypropylene density 0.92
10.00
8.00
Aluminum density 2.7
6.00
Titanium dioxide density
4.20
4.00
2.00
0.00
0.00
Ferric oxide density 5.3

10.00
20.00
30.00
Stainless steel density 7.8
Aerodynamic Diameter microns
25 March 2011
324

Reported
as
foreign particulates
Acrylonitrile butadiene styrene
Polybutylene Terephthalate
Aluminum
Polycarbonate
Bacterial fragments
Polychlorotrifluoroethylene
Bromobutyl rubber
Polyester
Clay
Polyethylene
Copper
Polyimide
Elastomer
Polyoxymethylene
Ethylene Propylene Diene

Polymer
Polypropylene
Glass
Stainless steel
Iron
Talc
Kaolin
Transparent synthetic fibers
25 March 2011
325
For counting and sizing, many suitable technologies

For speciation of individual particles:
Organics: Infrared
Raman
Inorganic: SEM/EDS
25 March 2011
326
For counting and sizing, many suitable technologies

For composition:
Organics: Infrared
Raman
Standard and collected spectra
Inorganic: SEM/EDS
Elemental standards and collected spectra
25 March 2011
327
IPAC-RS Working Groups Best Practices:

1. Enumeration of particles in size classes for manufacturing Quality
control
1. Rapid
2. Portable
2. When trending toward limits , or when exceeding limits then

investigate using composition to identify and control source.
1. Slow
2. Requires personnel and equipment
25 March 2011
328
The End
25 March 2011
329
The Beginning
of an interesting journey
25 March 2011
330

OINDP
Regulatory Agencies are

interested in knowing:
Number of particles especially in
PM10, respirable size range
Composition of particles
25 March 2011
331
For both solution and suspension nasal

sprays,
there should be validated tests and
associated acceptance criteria for particulate
matter.
The acceptance criteria should include limits
for
foreign particulate matter less than 10
micrometers (m),
greater than 10 m, and greater than 25 m.
25 March 2011
332

OINDP
EPA: Airborne particulate matter
Two major size classes (EPA):
PM 10 particles generally less than or
equal to 10 micrometers
PM 2.5 generally referring to particles
less than or equal to 2.5 micrometers in
diameter
respirable range
25 March 2011
333

OINDP
For OINDP: After use:

Nasal: through the valve
Oral: Through the mouthpiece
Most like what the patient will
receive
Uncertainty about effect of the
mouthpiece
25 March 2011
334

OINDP
For OINDP:
Prior to use
Open the device
(carefully)
To distinguish functional
source
25 March 2011
335

OINDP
These distinction are critical

because control of each type
requires very different actions:
Control of Component particulates
occurs in the supply chain; Control
of component cleanliness.
Control of Environmental
particulates occurs in filling,
25 March 2011
336

OINDP
Ideal: collect all and only FPM burden
with minimal general or selective loss
of FPM
However do not know how much is there so
do not know when this has been achieved.
There is no right answer
No or minimal gain of FPM
Clean room conditions
25 March 2011
337
Foreign Particulate Matterin

OINDP
All glassware must be scrupulously
cleaned,
Only distilled, deionized, filtered water
must be used and plenty of it, with
several rinses
Glassware dried in a HEPA filtered hood
All reagents must be filtered,
Extensive use of blanks is essential
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338

OINDP
Under the Clean Air Act, EPA is required to review
and, if appropriate, revise the air quality criteria
for the primary (health-based) and
secondary (welfare-based)
national ambient air quality standards (NAAQS)
every 5 years. On October 17, 2006, EPA published
a final rule to revise the primary and secondary
NAAQS for particulate matter to provide increased
protection of public health and welfare.
25 March 2011
339

OINDP
Three major differences:

1. Method validation
2. Sampling/Sample Preparation
3. Speciation
25 March 2011
340

OINDP
Many suitable methods for

particle sizing.
Most are for providing particle
sizes that are particles of the
same material
FPM are by definition
heterogeneous and may differ in
25 March 2011
341

OINDP
Pharmaceutical Analytical Sciences Group (1999)
Position Paper on Particle Sizing: Sample
Preparation, Method Validation and Data
Presentation
Specificity: Not strictly applicable
Detection Limit: Range; Instrument
specifications
Quantification Limit: Not considered useful
Linearity: Effect of sample concentration, mass,
other variables
Range: Instrument specifications
Robustness: Sensitivity to changes in the
25 March 2011
342

OINDP
Precision: Repeatability of entire

technique, including sample
preparation
Intermediate Precision with
different personnel and
instrumentation
Reproducibility Operation at
different sites
25 March 2011
343
Foreign Particulate Issues:

Safety Aspects and
Considerations for Setting Limits
Jim Blanchard, Ph.D.
Principal Scientist
Aradigm Corp.
For the International Pharmaceutical Aerosol
Consortium on Regulation and Science (IPACRS) Foreign Particulate Working Group
Scope
Focus on foreign particulate in aerosolized drug
products
Safety evaluation
Considerations for establishing safety limits
Sources and types
Clinical safety
Slide 345
Safety of Foreign Particulate in

Aerosolized Drug Products
Dependent on:
Size
Mass and number
Composition
Slide 346
Safety of Foreign Particulate in

Inhaled particles 10 m in aerodynamic
diameter can penetrate beyond the upper
airways and deposit in the lungs
Larger particles have less deposition in the
lungs.
Therefore, control of foreign particulate 10 m

should include both safety and quality
considerations
Control of larger particulate can be based
primarily on quality considerations.
Slide 347
Particle Deposition in Respiratory Tract:

Oral Breathing at Rest
Deposition Fraction
Total
0.8
Head
0.6
0.4
Tracheobronchial tree
0.2
Pulmonary
0
0
10
15
20
Aerodynamic Diameter (m)

Multiple-Path Particle Dosimetry Model V2.0
Safety Evaluation of Foreign Particulate

in Aerosolized Products
Conduct standard inhalation toxicology studies
of the formulation
The dose of foreign particulate penetrating to
the animals lungs is adequate relative to the
patients dose of foreign particulate.
Safety assessment following identification and
enumeration of foreign particulate
Any particles with safety concerns should be
eliminated, if possible, or minimized.
Action or safety limits can be developed.
Slide 349
Safety Evaluation of Foreign Particulate

in Aerosolized Products
To develop safety limits, the levels of foreign
particulate can be compared to the levels
allowed for particulate in ambient air
Use safety limits for foreign particulate 10 m
based on the U.S. Environmental Protection
Agencys (EPA) National Ambient Air Quality
Standard (NAAQS) for particulate matter with an
aerodynamic diameter 10 m (PM10).
Slide 350
Aerodynamic Diameter Definition

Aerodynamic diameter (Da) = diameter of a
sphere with density of 1 g/cm3 that has the same
settling velocity as the particle
Da = DStokes (sphere) x (density)1/2
Stokes diameter = diameter of a sphere that
has the same settling velocity and density as
the particle
Da = Dgeometric x (density/shape factor)1/2
Shape factor = 1 for sphere, = 1.71 for glass
fiber (L/D=5); 2.04 for talc
Slide 351
Advantages of NAAQS PM10 Standard

Designed to protect both healthy and sensitive
populations, e.g., asthmatics or patients with
other lung diseases, children, and the elderly
over their lifetime.
Therefore, it is appropriate to protect patients
receiving acute or chronic aerosolized
therapy.
Slide 352

More appropriate than occupational exposure
standards
Occupational standards are designed to
protect nearly all workers exposed repeatedly
8 hrs/day, 40 hrs/week over a working
lifetime, therefore these are higher and
inappropriate for sensitive people
For inert dusts, the US Occupational Safety
and Health Administration (OSHA)
permissible exposure limit is 15 mg/m3 total
particulate and 5 mg/m3 for respirable
particulate (<3.5 m) vs. 150 g/m3 for PM10
Slide 353

Based on particle mass and is independent of
particle composition, therefore it is applicable to
all foreign particulates regardless of
composition.
Allows assessment of the overall safety of
foreign particulates without necessarily
identifying all the particles.
Slide 354
Use of NAAQS PM10 Standard

To provide added safety, the IPAC-RS Working
Group recommends using limits for foreign
particulates that are only 1-5% of the NAAQS
PM10
The patients exposure to the foreign
particulates in the formulation is small
compared to their allowable exposure to other
particulates in the ambient air.
Slide 355

NAAQS for PM10 is 150 g/m3 (24-hr average).
EPA assumes a person breathes 20 m3 of air
per day
PM10 standard corresponds to 3 mg/day
1-5% level corresponds to 30-150 g/day of
foreign particulate.
Poses little added risk to the patient
Slide 356

In December 2006, EPA revoked the PM10
standard determined as the annual arithmetic
mean, which was 50 g/m3
EPA cited lack of evidence of health problems
from chronic long-term exposure to these
particles
The 50 g/m3 was the PM10 standard used in
the two IPAC-RS foreign particulate papers in
2003 and 2007
Slide 357

EPA has lower standards for PM2.5 ( 2.5 m)
15 g/m3 annual arithmetic average
35 g/m3 24-hr average
However, in ambient air, these particles are
mainly from smog and combustion sources and
have at different chemical composition and
safety profile than foreign particulate in inhaled
drug products
Therefore, the IPAC-RS Working Group
considered that the PM2.5 standard was
inappropriate
Slide 358

Mass of particles = number of particles x density
To relate 30-150 g/day to the number of foreign
particles in a drug product, an aggregate density
of particles must be determined
Measured analytically (e.g., SEM/EDX and
FTIR/Raman)
Assume a worst-case maximum density for all
foreign particles based on the materials that
the formulation contacts, e.g. stainless steel
with density of 8 g/cm3
Slide 359

Based on 5% safety level (150 g/day) maximum
acceptable number of foreign particulate (spherical)
Density, g/cm3
Diameter, m
1
3.6 x 107
4.5 x 106
1.1 x 107
1.3 x 106
4.5 x 106
5.6 x 105
1.3 x 106
1.7 x 105
5.6 x 105
7.0 x 104
10
2.9 x 105
3.6 x 104
Not all particles with density of 8 g/cm3 have

aerodynamic diameter <10 m (and be subject to
PM10), however the worst-case density approach is
both practical and provides a conservative safe level
Slide 360

The IPAC-RS Working Group found for two drug
products the mass of foreign particles from 2-10
m were >100x less than that allowed by the 5%
PM10 standard
These data support that the limit is a feasible
starting point to develop safety limits on levels of
foreign particulate
Slide 361
Sources of Foreign Particulate
Active pharmaceutical ingredient (API)

Excipients
Container/closure system components
Delivery devices
Processing equipment
Slide 362
Typical Foreign Particulate in

The IPAC-RS Working Group also surveyed
IPAC-RS member companies to identify foreign
particulates typically found in aerosolized drug
products
Slide 363
Typical Foreign Particulate in

acrylonitrile-butadienestyrene
aluminum
bromobutyl rubber
polytetrafluoroethylene
glass
iron
polychlorotriflouroethylene
paper/cardboard/cellulose
polyamide (nylon)
polyacetal
polycarbonate
Slide 364
polybutylene terephthalate
polyethylene
polyester
polyoxymethylene
polyimide
polystyrene
polypropylene
stainless steel
skin cells
talc
transparent synthetic fibers
Clinical Safety of Typical Foreign

Particulate in Aerosolized Drug Products
Possible types of clinical responses
Clinically inert
Non-immunologic response, e.g., polyolefins,
talc, aluminum
Immunologic response, e.g., nickel (as a
component of stainless steel)
The determining factor is whether the particles
are inhaled in a sufficiently high concentration.
Slide 365
Clinical Safety: Polyolefins

Polyolefins, such as polypropylene, polyethylene and
nylon, when inhaled as microfibers or flock, have been
reported to induce an interstitial lung disease, termed
flock workers lung.
20-26% of workers exposed to polypropylene flock (8
hr/day x 6 days/week) had abnormalities in lung function
compared with 4.4% of unexposed controls (Atis 2005).
Inhalable and respirable dust concentrations were 4.4 mg/m3 and
<0.2 mg/m3, dose = 44 - <2 mg/day, respectively; mean fiber
diameter = 6.9 2.1 (SD) m; mean length = 96.7 35.2 m.
Therefore, a foreign particle exposure limit of 1-5% of the

PM10 (30-150 g/day) is well below that encountered in
occupationally exposed individuals.
Slide 366
Atis S et al, Eur Res J, 2005; 25:110-17
Clinical Safety: Talc

Talc is hydrated magnesium sulfate
Pulmonary disease has been reported in highly exposed
workers or following obsessive inhalation of talcum
powder
However, clinical and pathological evidence suggests talc
itself does not cause pulmonary fibrosis
Most pulmonary abnormalities reported for talc are in fact
due to contaminating substances, e.g., asbestos and
silica (Fraser et al, 1999).
Therefore, talc itself appears to a low toxicity profile
Slide 367
Fraser RS et al, Fraser and Pars Diagnosis of Diseases

of the Chest, 1999; 2449-2450
Clinical Safety: Iron

Workers exposed to high concentrations of iron, usually
as iron oxide (Fe2O3) , may develop siderosis.
The only pathological finding is iron-laden
macrophages in the peribronchovascular interstitium
and alveoli
Not associated with pulmonary fibrosis or functional
impairment (Harding et al, 1958; Nemery 1990)
However, when the iron is admixed with silica, exposed
workers can develop silicosiderosis, which can lead to
significant pulmonary fibrosis and disability
Therefore, iron itself appears to a low toxicity profile
Slide 368
Harding HE et al, Lancet 1958; 2: 394-8

Nemery B, Eur Res J 1990; 3: 202-19
Clinical Safety: Aluminum

There are rare reports of pulmonary fibrosis following
high exposure to aluminum dust and its oxides
The number of fibrous and nonfibrous aluminum
particles in an aluminum pot worker who died from
respiratory insufficiency were 1000-fold greater than in
the general population (Gilks and Churg 1987) .
Slide 369
Gilks B and Churg A, Am Rev Respir Dis 1987; 136: 176-9
Clinical Safety: Aluminum (2)

Eklund et al (1989) measured pulmonary function and
performed bronchoalveolar lavage (BAL) in 14 aluminum
pot room workers.
The mean duration of employment was 12.9 9.0 years
with a mean exposure (8-hour samples) of 1.77 mg/m3
(range 0.49-4.50); dose = 49-450 mg/day.
The workers all had normal chest x-rays and normal vital
capacity, total lung capacity and diffusing capacity.
There was no evidence of an alveolitis in BAL.
Thus, using the 1-5% PM10 standard (30-150 g/day)
should further minimize any potential risk from aluminum
foreign particles.
Slide 370
Eklund A et al, Br J Ind Med 1989; 46: 782-86
Clinical Safety: Nickel and Stainless

Steel
Disease has been recognized with nickel and nickel salts
used in stainless steel and other metal alloys.
Workers refining nickel ore had increased risk of
malignancy in both the lungs and nasal mucosa related
to exposure to nickel sulfate and the combination of
nickel sulfides and oxides.
In contrast, workers involved in the manufacture of nickel
alloys did not seem to be at an excess risk for pulmonary
carcinoma (Steenland et al, 1996).
Therefore, patients exposed to stainless steel in inhaled
drug products should not be at an increased risk for
respiratory tract malignancy
Slide 371
Steenland K et al, Am J Ind Med 1996; 29: 474-90
Clinical Safety: Nickel (2)

Nickel allergy, i.e. to nickel sulfate, is one of the most
common forms of allergic contact dermatitis, with
prevalence rates of 10-15%.
Yet, nickel has only rarely been associated with the
development of bronchial asthma. (Dolovich et al, 1984;
Nemery 1990)
Asthma appears to be IgE mediated whereas contact
dermatitis is a delayed hypersensitivity reaction.
(Nieboer et al, 1984)
Slide 372
Dolovich J et al, Br J Ind Med 1984; 41: 51-5

Nemery B, Eur Res J 1990; 3: 202-19
Nieboer E et al, Br J Ind Med 1984; 56-63
Clinical Safety: Nickel (3)

A large fraction of the inhaled dose may be swallowed,
which may lead to an endogenous nickel reaction.
However, nickel is found in many foods; the average
human intake of nickel is 200 g/day.
Oral challenge trials to elicit nickel dermatitis have
utilized higher doses of 500-5,600 g (Ricciardi 2001).
Therefore, using safety limits based upon 1-5% of the
NAAQS (30-150 g/day) should provide an adequate
safety margin against allergic manifestations.
Slide 373
Ricciardi L et al, Allergy 201; 56 (Suppl 67) : 109-112
Other Safety Considerations

For foreign particles where the threshold data for
clinical disease are lacking, manufacturers may
need to rely upon toxicology studies or other
safety data
It should also be recognized there are
subpopulations of extremely sensitive individuals
who may react adversely to very low levels of
certain types of foreign particulate
Slide 374
Conclusions
Safety limits can be developed once foreign
particulates have been characterized in terms of
number and identity
Safety limits for foreign particulates 10 m are
most appropriate since these can penetrate into
the lungs
Establishing safety limits based on 1-5% of the
EPA NAAQS PM10 (30-150 g/day) should
provide adequate protection for most types of
foreign particulates, but not necessarily all.
Slide 375
Acknowledgements
IPAC-RS Foreign Particulate Working Group
Courtney Crim, MD, GSK for clinical safety
assessments
Ron Wolff, PhD, Novartis for development of the
NAAQS safety assessment
Slide 376
References
Blanchard, J., J. Coleman, C. DAbreu Hayling, R. Ghaderi, B.

Haeberlin, S. Jensen, R. Malcolmson, S. Mittelman, L.M. Nagao, I.
Saracovan, C. Snodgrass-Pilla, M. Sundahl, and R. Wolff.
Commentary: Foreign particles testing in orally inhaled and nasal
drug products. Pharm. Res. 21: 2137-2147, 2004.
Blanchard, J., J. Coleman, C. Crim, C. DAbreu Hayling, L. Fries, R.

Ghaderi, B. Haeberlin, R. Malcolmson, S. Mittelman, L.M. Nagao, I.
Saracovan, L. Shtohyrn, C. Snodgrass-Pilla, M. Sundahl, and R.
Wolff. Commentary: Best practices for managing quality and
safety of foreign particles in orally inhaled and nasal drug products,
and an evaluation of clinical relevance. Pharm. Res. 24: 471-479,
2007.
Slide 377

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Welcome to the

USP Workshop on Particle Size:

USP Workshop on Particle Size:

Welcome and Opening Remarks

USP Workshop on Particle Size:

USP Workshop on Particle Size:

Session I: Particle and Product Size

Particle Size Fundamentals

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

Particle size detection and

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Company Confidential Copyright 2000 Eli Lilly and Company

Potential effects of API and excipient

Company Confidential Copyright 2000 Eli Lilly and Company

Parenteral and Ophthalmic solutions

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

A particle may be:

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

What is particle size

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Variability of Size Measurements of a

Powder Technology, 57, 143.

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Equivalent Spherical Diam.

08 Dec 2010 USP Workshop

Not adequately described by a single number

Variety of ways to describe and display distribution

Need to define the parameter or statistic used to

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

Size without distribution information

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

Actual size distribution:

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

Actual size distribution

Volume distribution density

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

Cumulative size distribution plot

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop

Company Confidential Copyright 2000 Eli Lilly and Company

08 Dec 2010 USP Workshop