Note: Descriptions are shown in the official language in which they were submitted.
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PARTICLE STANDARD AND METHOD OF CALIBRATING OR VALIDATING AN
OPTICAL PARTICLE ANALYZER
TECHNICAL FIELD OF THE INVENTION
The present invention relates to particle standards for calibrating or
validating optical particle
analyzers and to methods of calibrating or validating optical particle
analyzers.
BACKGROUND OF THE INVENTION
In the pharmaceutical industry, an optical particle analyzer is frequently
used to analyze samples
including particles of biological material dispersed in a water-based carrier.
As the optical
properties of the particles in such samples are similar to those of the
carrier, the analysis of such
samples is particularly challenging. Not all of the particles in such samples
may be detectable by
the optical particle analyzer, owing to detection-sensitivity limitations. To
achieve reliable and
repeatable results for the analysis, the optical particle analyzer must be
properly calibrated and
periodically validated by using a particle standard that optically
approximates such samples. In
particular, it is highly desirable that the fraction of particles detected in
such samples is
substantially the same when the samples are analyzed at different times or
locations, with the
same or different optical particle analyzers.
Unfortunately, samples including particles of biological material dispersed in
a water-based
carrier are, generally, unstable and cannot be practically used as particle
standards. Therefore, a
particle standard that may serve as a stable optical surrogate for such
samples and a method of
calibrating or validating an optical particle analyzer by using the particle
standard are required.
However, most conventional methods of calibrating or validating an optical
particle analyzer
involve the use of a particle standard including particles having optical
properties dissimilar to
those of a carrier in which the particles are dispersed. For example, methods
involving the use of
a particle standard including polymer particles having a refractive index
significantly different
from that of a water-based carrier in which the particles are dispersed are
described in U. S.
Patent No. 5,728,582 to Taki, et al., issued on March 17, 1998, in U. S.
Patent No. 4,704,891 to
Recktenwald, et al., issued on November 10, 1987, and in U. S. Patent No.
4,331,862 to Ryan,
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issued on May 25, 1982. For another example, methods involving the use of a
particle standard
including stained particles having fluorescence or transmission properties
significantly different
from those of an unstained carrier in which the particles are dispersed are
described in U. S.
Patent No. 6,542,833 to Nygaard, issued on April 1, 2003, in U. S. Patent No.
5,728,582, and in
U. S. Patent No. 4,704,891.
As the particles in the particle standards used in such methods are easily
detected by the optical
particle analyzer, the particle standards do not challenge the detection
sensitivity of the optical
particle analyzer. Optical particle analyzers having different detection
sensitivities are, typically,
able to detect substantially all of the particles in the particle standards;
however, they may detect
significantly different fractions of particles in samples including particles
of biological material
dispersed in a water-based carrier, leading to unreliable and unrepeatable
results for the analysis
of such samples.
Furthermore, most conventional methods of calibrating or validating an optical
particle analyzer,
such as those mentioned heretofore, and those described in U. S. Patent No.
6,074,879 to
Zelmanovic, et al., issued on June 13, 2000, and in U. S. Patent No. 5,747,667
to Sadar, issued
on May 5, 1998, require that substantially all of the particles in a particle
standard be detected by
the optical particle analyzer. Although, as mentioned heretofore, this
requirement may be easy
to fulfill for a particle standard including particles having optical
properties dissimilar to those of
a carrier in which the particles are dispersed, it may be difficult or
impossible to fulfill for a
2 0 particle standard that optically approximates samples including
particles of biological material
dispersed in a water-based carrier.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a particle standard including
particles having
optical properties similar to those of a carrier in which the particles are
dispersed, as well as a
2 5 method of calibrating or validating a subject optical particle analyzer
with respect to a reference
optical particle analyzer by using the particle standard.
Accordingly, the present invention relates to a particle standard for
calibrating or validating a
subject optical particle analyzer with respect to a reference optical particle
analyzer, comprising:
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a carrier; and particles dispersed in the carrier, wherein the particles have
particle sizes within a
reference particle-size range of the reference optical particle analyzer, and
wherein the particles
have optical properties similar to those of the carrier, such that less than
substantially all of the
particles are detectable by the reference optical particle analyzer.
Another aspect of the present invention relates to a method of calibrating or
validating a subject
optical particle analyzer with respect to a reference optical particle
analyzer, comprising:
providing a particle standard comprising: a carrier; and particles dispersed
in the carrier,
wherein the particles have particle sizes within a reference particle-size
range of the reference
optical particle analyzer, and wherein the particles have optical properties
similar to those of the
carrier, such that less than substantially all of the particles are detectable
by the reference optical
particle analyzer; analyzing the particle standard with the reference optical
particle analyzer to
obtain a reference particle concentration and a reference particle-size
distribution; analyzing the
particle standard with the subject optical particle analyzer to obtain a
subject particle
concentration and a subject particle-size distribution; comparing the subject
particle
concentration to the reference particle concentration and the subject particle-
size distribution to
the reference particle-size distribution to determine a first difference and a
second difference,
respectively; and adjusting the subject optical particle analyzer on the basis
of the first difference
and the second difference.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in greater detail with reference to
the accompanying
drawings, which relate to exemplary, preferred embodiments thereof, wherein:
FIG. 1 is a schematic illustration of an oblique view of a particle standard
according to the
present invention; and
FIG. 2 is a flowchart depicting a method according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
With reference to FIG. 1, the present invention provides a particle standard
100 for calibrating or
validating a subject optical particle analyzer with respect to a reference
optical particle analyzer.
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The subject optical particle analyzer and the reference optical particle
analyzer may be any type
of instrument that employs light having a wavelength between about 200 nm and
1000 nm to
analyze particles, such as a micro-flow imaging (MFI) particle analyzer, a
light-obscuration
particle analyzer, or a light-scattering particle analyzer. Although the
subject optical particle
analyzer and the reference optical particle analyzer may be different types of
instrument, to
achieve maximum reliability and repeatability, it is preferred that the
subject optical particle
analyzer and the reference optical particle analyzer be the same type of
instrument. For example,
in a preferred embodiment, the subject optical particle analyzer and the
reference optical particle
are both MFI particle analyzers. Ideally, the subject optical particle
analyzer and the reference
optical particle analyzer are substantially identical and have the same
instrument specifications.
The reference optical particle analyzer serves as a "gold standard"
instrument, in terms of
detection sensitivity, against which the subject optical particle analyzer is
compared during
calibration or validation of the subject optical particle analyzer. The
particle standard 100
provides an advantageous means for comparing the subject optical particle
analyzer to the
reference optical particle analyzer.
For example, during calibration in manufacture, a particle standard 100
prepared on-site at the
manufacturer's facilities may be used to compare a subject optical particle
analyzer manufactured
on-site to a reference optical particle analyzer housed on-site. For another
example, a particle
standard 100 prepared on-site at the manufacturer's facilities may be used to
compare a subject
2 0 optical particle analyzer located off-site to a reference optical
particle analyzer housed on-site,
during validation or recalibration in the field. Once calibrated or validated
in such a manner, any
optical particle analyzer can itself serve as a reference optical particle
analyzer.
The particle standard 100 includes particles 110 dispersed in a carrier 120.
The particles 110
have particle sizes within a reference particle-size range of the reference
optical particle
analyzer. The particle sizes are also within a subject particle-size range of
the subject optical
particle analyzer, which is, preferably, equivalent to the reference particle-
size range.
The particles 110 are, typically, polydisperse in particle shape, having a
variety of particle
shapes. As the particle shapes are, usually, irregular, they are determined by
measuring the
particle equivalent circular diameter (ECD), the particle Feret diameter, the
particle Feret length,
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the particle Feret width, the particle aspect ratio, the particle circularity,
the particle absorption
intensity, or any other particle dimension measurable by conventional particle-
morphology
algorithms. Preferably, the particles 110 are agglomerates of primary
particles. More
preferably, the particles 110 are porous agglomerates.
The particles 110 are also, typically, polydisperse in particle size, having a
variety of particle
sizes. Preferably, the particle sizes are distributed over a range including
at least a lower part of
the reference particle-size range. More preferably, the particle sizes are
distributed over a range
extending below the reference particle-size range, as well. Depending on the
reference particle-
size range, the particle sizes are, typically, between about 0.1 [an and 1 mm.
For example, for
calibrating or validating a subject optical particle analyzer having a subject
particle-size range of
1 pm to 70 pm with respect to a reference optical particle analyzer having a
reference particle-
size range of 1 p.m to 70 pm, a particle standard 100 including particles 110
having particle sizes
distributed over a range of about 0.5 pm to 10 p.m may be suitable.
The particles 110 are dispersed in the carrier 120 at a particle concentration
within a reference
particle-concentration range of the reference optical particle analyzer. The
particle concentration
is also within a subject particle-concentration range of the subject optical
particle analyzer,
which is, preferably, equivalent to the reference particle-concentration
range. Typically, the
particle concentration is between about 1000 particles/mL and 10 000 000
particles/mL;
however, the particle concentration may also be lower than 1000 particles/mL.
2 0 In most instances, the particle standard 100 has a particle-size
distribution that is substantially
exponential, with particle concentration per particle-size interval increasing
rapidly with
decreasing particle-size interval.
It should be noted that it is not necessary for characteristics such as the
particle concentration and
the particle-size distribution to be known exactly, as the particle standard
100 is intended as a
relative standard for comparing the subject optical particle analyzer against
the reference optical
particle analyzer. Rather, the particle standard 100 is, typically, associated
with a record of a
reference particle concentration and a reference particle-size distribution
obtained by analyzing
the particle standard 100 with the reference optical particle analyzer. This
advantageous feature
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distinguishes the particle standard 100 of the present invention from most
conventional particle
standards, which are absolute standards with defined characteristics.
However, if desired, characteristics such as the particle concentration and
the particle-size
distribution may be determined by analyzing the particle standard 100 with a
non-optical particle
analyzer, such as a Coulter counter, which is able to detect substantially all
of the particles 110 in
the particle standard 100.
The particle concentration and the particle-size distribution
determined in such a manner may then be compared and/or related to the
reference particle
concentration and the reference particle-size distribution.
Another advantageous feature of the particle standard 100 is that the
particles 110 have optical
properties, such as refractive index, transmission properties, and
fluorescence properties, similar
to those of the carrier 120, which allows the particle standard 100 to serve
as a detection-
sensitivity challenge. Preferably, the particles 110 and the carrier 120 are
transparent or near-
transparent.
The particles 110 and the carrier 120 have optical properties sufficiently
similar that less than
substantially all of the particles 110 are detectable by the reference optical
particle analyzer,
owing to detection-sensitivity limitations. Likewise, less than substantially
all of the particles
110 are detectable by the subject optical particle analyzer. Preferably, less
than 95 % of the
particles 110 are detectable by the reference optical particle analyzer.
It is known that less than substantially all of the particles 110 are
detectable by the reference
optical particle analyzer because adjustment of the reference optical particle
analyzer to increase
or decrease its detection sensitivity allows it to detect a larger or smaller
fraction of particles 110.
If desired, the fraction of particles 110 detectable by the reference optical
particle analyzer may
be determined by comparing the reference particle concentration, which is
lower than an actual
particle concentration of the particle standard 100, to the particle
concentration determined by
2 5 using a non-optical particle analyzer, which substantially corresponds
to the actual particle
concentration. Preferably, the reference optical particle analyzer is adjusted
to a detection
sensitivity that is near the highest attainable by the reference optical
particle analyzer, but which
is also attainable by the subject optical particle analyzer upon calibration
or validation.
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As the reference optical particle analyzer and the subject optical particle
analyzer, prior to
calibration or validation, have different detection sensitivities, different
fractions of particles 110
are detectable by each optical particle analyzer. Advantageously, by means of
the particle
standard 100, the detection sensitivity of the subject optical particle
analyzer may be
standardized with that of the reference optical particle analyzer, such that
the fraction of particles
110 detected by each optical particle analyzer is substantially the same.
Ideally, the particles 110 and the carrier 120 have optical properties similar
to those of particles
and carriers, respectively, encountered in samples under study for a
particular application.
The particles 110 and the carrier 120 may be composed of a variety of
materials, which are
selected to ensure that the particle standard 100 has the features and
characteristics detailed
heretofore. Furthermore, the materials are selected to ensure that a
dispersion of the particles
110 in the carrier 120 is sufficiently stable or has a known decay rate for a
time period over
which the particle standard 100 will be used. The material selected for the
particles 110 must be
inert and dispersible in the carrier 120. Likewise, the material selected for
the carrier 120 must
be inert towards the particles 110 and amenable to dispersion of the particles
110 therein. If
necessary, a surfactant, a viscosity modifier, a buffer, a preservative, or
another type of additive
may be included in the material selected for the carrier 120.
Typically, the material selected for the particles 110 is an inorganic oxide,
either crystalline or
amorphous, in solid, glass, or gel form. Alternatively, the material selected
for the particles 110
may be a liquid immiscible with a liquid selected as the material for the
carrier 120, such that the
particles 110 are microdroplets.
A preferred embodiment of the particle standard 100 includes particles 110 of
precipitated silica
dispersed in a water-based carrier 120. Advantageously, the preferred
embodiment of the
particle standard 100 serves as a stable optical surrogate for samples
including particles of
biological material dispersed in a water-based carrier.
In general, precipitated silica, an amorphous form of silica, is produced
commercially by
combining a solution of a metal silicate in water with a mineral acid. In such
a process,
polydisperse particles 110 of precipitated silica are formed, and further
agglomeration into a gel
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is avoided. Similarly to many types of particles of biological material, the
particles 110 of
precipitated silica are porous agglomerates having irregular particle shapes.
Typically, the
particles 110 of precipitated silica have particle sizes between about 0.5
[tin and 20 tm, and pore
sizes between about 10 nm and 50 nm. Furthermore, the particles 110 of
precipitated silica have
optical properties similar to those of many types of particles of biological
material, as well as to
those of the water-based carrier 120.
The preferred embodiment of the particle standard 100 may be produced by
combining the
particles 110 of precipitated silica and the water-based carrier 120 to form a
mixture and by then
agitating the mixture to form a dispersion of the particles 110 in the carrier
120. Typically, the
stability of the dispersion is increased by adjusting the pH of the carrier
120, by passivating
container surfaces, or by other chemical means.
The present invention also provides a method of calibrating or validating the
subject optical
particle analyzer with respect to the reference optical particle analyzer by
using the particle
standard 100. With reference to FIG. 2, in a first step 201 of the method, the
particle standard
100, as described heretofore, is provided. If desired, the particle standard
100 may be analyzed
with a non-optical particle analyzer to determine the particle concentration
and the particle-size
distribution, which may then be compared and/or related to the reference
particle concentration
and the reference particle-size distribution, as mentioned heretofore.
In a second step 202, the particle standard 100 is analyzed with the reference
optical particle
2 0 analyzer, by following standard operating procedures, to obtain the
reference particle
concentration and the reference particle-size distribution. Typically, a
record is made, in print or
electronic format, of the reference particle concentration and the reference
particle-size
distribution, and the record is associated with the particle standard 100. The
record may be
stored at a site of a supplier of the particle standard 100 and/or supplied
together with the particle
standard 100.
Analogously, in a third step 203, the particle standard 100 is analyzed with
the subject optical
particle analyzer, by following standard operating procedures, to obtain a
subject particle
concentration and a subject particle-size distribution. In a fourth step 204,
the subject particle
concentration is compared to the reference particle concentration to determine
a first difference,
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and the subject particle-size distribution is compared to the reference
particle-size distribution to
determine a second difference, typically, by means of the record. The subject
optical particle
analyzer is then adjusted on the basis of the first difference and the second
difference, in a fifth
step 205. For example, software, such as particle-image thresholding
algorithms, and/or
hardware, such as illumination components, magnification components, and
detection
components, of the subject optical particle analyzer may be adjusted, until
the first difference and
the second difference are substantially eliminated.
In some instances, the subject optical particle analyzer is first coarsely
calibrated or validated by
a conventional method and then finely calibrated or validated by the method of
the present
invention. For example, during calibration in manufacture, software and/or
hardware of newly
built optical particle analyzers is, usually, coarsely adjusted by using
optical targets and further
adjusted by using a conventional particle standard including particles having
optical properties
dissimilar to those of a carrier in which the particles are dispersed.
However, different optical
particle analyzers similarly adjusted in such a manner may, nevertheless, have
different detection
sensitivities for samples including particles having optical properties
similar to those of a carrier
in which the particles are dispersed. To standardize the detection
sensitivities at a high level,
software and/or hardware of the optical particle analyzers is finely adjusted
by using the particle
standard 100 according to the method of the present invention.
In other instances, the subject optical particle analyzer is calibrated or
validated by the method of
the present invention alone. For example, during validation or recalibration
in the field, software
and/or hardware of previously calibrated optical particle analyzers is
adjusted by using the
particle standard 100 according to the method of the present invention, to
ensure that the optical
particle analyzers maintain a high level of detection sensitivity.
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