ASTM E3230-20

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3230 − 20
Standard Practice for
Extraction of Particulate Matter from the Surfaces of Single-
Use Components and Assemblies Designed for Use in
Biopharmaceutical Manufacturing
This standard is issued under the fixed designation E3230; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 USP Documents:
USP <788> Particulate Matter in Injections, 2012
1.1 This practice describes the requirements for
USP <790> Visible Particulates in Injections, 2017
development, qualification, and routine application of a proce-
USP <1788> Methods for the Determination of Particulate
dure for the effective liquid extraction of particulate matter
Matter in Injections and Ophthalmic Solutions, 2012
from the surfaces of single-use components and assemblies
USP <1790> Visual Inspection of Injections, 2018
designed for use in biopharmaceutical manufacturing pro-
2.3 ISO Documents:
cesses. The extraction generates a suspension of particulate
ISO 16232:2018 Road Vehicles – Cleanliness of Compo-
matter in liquid which makes the particulate matter readily
nents and Systems
available for analytical characterization.
2.4 Other Documents:
BPSA Recommendations for Testing, Evaluation, and Con-
1.2 The values stated in SI units are to be regarded as
trol of Particulates from Single-Use Process Equipment,
standard. No other units of measurement are included in this
standard.
JP 6.07 Insoluble Particulate Matter Test for Injections
1.3 This standard does not purport to address all of the
Ph. Eur. 2.9.19 Particulate Contamination: Sub-Visible Par-
safety concerns, if any, associated with its use. It is the 7
ticles
responsibility of the user of this standard to establish appro-
VDA 19 Part 1 Inspection of Technical Cleanliness, March
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3. Terminology
1.4 This international standard was developed in accor-
dance with internationally recognized principles on standard-
3.1 Definitions:
ization established in the Decision on Principles for the
3.1.1 agitation, n—an extraction method by which a test
Development of International Standards, Guides and Recom- article partially filled with test liquid is moved to create liquid
mendations issued by the World Trade Organization Technical motion relative to the internal surfaces of the test article.
Barriers to Trade (TBT) Committee.
3.1.2 bioprocess container, n—a container (bag, bottle, tank,
etc.) used primarily for liquid, frozen liquid, or powder storage
2. Referenced Documents
during various stages of biopharmaceutical manufacturing
processing.
2.1 ASTM Standards:
E3060 Guide for Subvisible Particle Measurement in Bio- 3.1.3 background particle count, n—average or range, or
pharmaceutical Manufacturing Using Dynamic (Flow) both, of particle counts obtained upon executing the extraction
Imaging Microscopy procedure without the test article present.
Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
This practice is under the jurisdiction of ASTM Committee E55 on Manufac- Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
ture of Pharmaceutical and Biopharmaceutical Products and is the direct responsi- Available from International Organization for Standardization (ISO), ISO
bility of Subcommittee E55.07 on Single Use Systems. Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
Current edition approved March 1, 2020. Published May 2020. DOI: 10.1520/ Geneva, Switzerland, http://www.iso.org.
E3230-20. Available from BioProcess Systems Alliance, 1400 Crystal Drive, Suite 630
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Arlington, VA 22202, https://bpsalliance.org.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Available from Japanese Pharmacopoeia.
Standards volume information, refer to the standard’s Document Summary page on Available from European Pharmacopoeia.
the ASTM website. Available from Verband der Automobilindustrie, https://www.vda.de/en.html.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3230 − 20
3.1.4 background test, n—application of the same extraction 3.1.22 particle count total (PTn), n—total particle count
conditions required for the extraction procedure, but without obtained for all extraction steps up to and including extraction
the test article present. step n.
3.1.5 collection equipment, n—any device (conical flask,
3.1.23 particle count specification, n—maximum particle
beaker, tray, funnel, etc.) suited to the collection of all the count allowed in a single-use component or assembly.
extraction liquid obtained from the test article.
3.1.24 particle load, n—(1) particulate matter load; (2)
3.1.6 external rinsing, n—an extraction method based upon
totality of all particles present on the surfaces of a test article.
flow of test liquid on the outside surfaces a test article.
3.1.25 particle measurement, n—implementation of a par-
3.1.7 extraction, n—operation required to transfer particu-
ticle measurement method to obtain a particle count.
late matter present on a surface into a test liquid, thus
3.1.26 particle measurement method, n—a qualified method
collecting the particulate matter for subsequent analysis.
for counting and sizing particles for a defined particle size
3.1.8 extraction apparatus, n—all equipment applied in
range.
delivering clean test liquid to a test article, and all collection
3.1.27 particle size, n—chosen measure of the size of a
equipment applied in collecting the extraction liquid obtained
particle (for example, Feret diameter, equivalent circular
from a test article
diameter, fiber length, etc.).
3.1.9 extraction curve, n—trend of the extraction effective-
3.1.28 qualification criterion, n—criterion required for
ness index (EIn) as a function of the number of extraction steps
qualification of an extraction procedure (EIn ≤ 0.10 for any of
(n).
the extraction steps n, where n = 6).
3.1.10 extraction conditions, n—parameters under which an
3.1.29 qualification test, n—process by which a chosen
extraction procedure is carried out (for example, test liquid
extraction method is verified to meet the criteria for effective
volume and flow rate, time, temperature, etc.).
extraction of particulate matter.
3.1.11 extraction effectiveness index (EIn for extraction step
3.1.30 reference particles, n—standardized test particles of
n), n—the ratio of the particle count (Pn) extracted in an
known type, size and concentration.
extraction step n, divided by the particle count total (PTn),
3.1.31 routine test, n—a test performed regularly on samples
which is the sum of Pn up to and including step n.
of components/assemblies from a manufacturing process in-
3.1.12 extraction liquid, n—test liquid loaded with particu-
volving a qualified particle extraction procedure combined
late matter extracted from the test article.
with a qualified particle measurement method.
3.1.13 extraction method, n—technique (agitation or rins-
3.1.32 single-use assembly, n—parts assembled together to
ing) applied to detach particulate matter from the test article.
produce a stand-alone single-use system.
3.1.14 extraction procedure, n—complete sequence of ac-
3.1.32.1 Discussion—An assembly is comprised of multiple
tions under controlled extraction conditions applying one or
single-use components.
more extraction methods to the test article.
3.1.33 single-use component, n—parts assembled together
3.1.15 extraction step (n), n—one application of the extrac-
to produce single-use assemblies.
tion procedure which creates one batch of extraction liquid.
3.1.33.1 Discussion—The most common components are
bioprocess containers, tubing, connectors, clamps, valves,
3.1.16 extraction volume, n—volume of test liquid used to
extract particulate matter from a test article. sensors and filters.
3.1.17 extraction time factor, n—for a method based upon 3.1.34 single-use system (SUS), n—stand-alone process
equipment used in biopharmaceutical manufacturing.
rinsing, the time factor is the total volume of test liquid applied
for a method based upon agitation, the time factor is the total 3.1.34.1 Discussion—A system is comprised of multiple
single-use assemblies.
agitation time applied.
3.1.34.2 Discussion—Often composed of plastic compo-
3.1.18 final rinsing, n—application of test liquid to remove
nents which are used once and then disposed or recycled.
any residual particulate matter from the surfaces of the
3.1.35 soaking time period, n—a time period over which the
collection equipment.
surfaces of the test article are wetted by the test liquid, but no
3.1.19 internal rinsing, n—an extraction method based upon
energy input (agitation or rinsing) is applied.
flow of test liquid on the internal surfaces of a test article.
3.1.36 test article, n—a single component or assembly from
3.1.20 particulate matter; particle, n—particulate matter
which particulate matter is extracted.
and particle are equivalent terms (USP <1790>), a portion,
piece or fragment of potentially loose mobile and non-soluble 3.1.37 test liquid, n—liquid used to extract particulate mat-
matter present on the surfaces of a single-use component or ter from a test article.
assembly, which may contact or end up in the process fluid or 3.1.37.1 Discussion—This liquid is terminally filtered to
drug product during biopharmaceutical processing. ensure it has minimal background particle count.
3.1.21 particle count (Pn), n—number of particles in a 3.1.38 test liquid delivery system, n—equipment to deliver a
defined particle size range (determined by a particle measure- controlled flow of filtered test liquid (equipment to pressurize
ment method) found in the extraction liquid obtained from the the test liquid, a filtration system, tubing, adapters, nozzle,
extraction step n. etc.).
E3230 − 20
4. Significance and Use VDA 19 Part 1). The standard practice described here for the
extraction of particulate matter from the surfaces of single-use
4.1 Conventional stainless-steel process equipment for bio-
components and assemblies is closely based upon the prin-
pharmaceutical manufacturing require cleaning and steriliza-
ciples described in the ISO 16232:2018 standard for automo-
tion prior to implementation. Single-use systems (SUS), stand-
tive components. This “multiple extractions” approach to
alone equipment typically composed of plastic components and
qualification of an extraction procedure significantly increases
assemblies, are usually assembled in cleanrooms and are
the probability that particulate matter adhering to surfaces is
usually not cleaned or rinsed prior to implementation (with the
removed upon extraction, and that the extraction procedure so
exception of filters, which are often rinsed prior to use). SUS
qualified is effective. The qualification criterion described in
cleanliness with respect to particulate matter depends upon the
this practice is essentially the same as the “declining criterion”
quality of the SUS manufacturing process, and also upon the
described in ISO 16232:2018. In essence, this criterion requires
care and handling of the SUS upon implementation by the
that during qualification the chosen extraction procedure must
end-user.
achieve an effectiveness of greater than 90% particle removal
4.2 In the process of manufacturing single-use components
on a relative basis.
or assemblies, particulate matter may adhere to the interior
4.7 Note that this practice does not specify the particle
(fluid contacting) or exterior surfaces of SUS (BPSA). Visual
measurement method required to count and size the particles.
inspection of SUS components and assemblies for particulate
The qualification of the extraction procedure described in this
matter is often limited by translucent or opaque materials
practice will be compatible with particle measurement methods
which inhibit visualization, especially of interior fluid-
typically used for both so-called “visible” (≥ 100 micron) or
contacting surfaces. Also in some cases, the large size of
“sub-visible” (10 to 100 micron) particle size ranges (USP
single-use assemblies significantly reduces the effectiveness of
<788>, USP <1788>, USP <790>), which includes methods
visual inspections. A more complete assessment of particulate
such as light obscuration, membrane microscopy or dynamic
matter load requires a method to extract particulate matter from
flow imaging (Guide E3060). In order for this practice to be
the surfaces of single-use components or assemblies using a
effective, the chosen particle measurement method shall have
test liquid, which makes the particles readily available for
been qualified for reliable determination of the particle count
analytical characterization using counting, sizing and
(number of particles in the particle size range of interest).
chemical/physical identification methods.
4.8 The overall goal of a chosen extraction procedure for
4.3 Pharmaceutical manufacturers use a wide variety of
particulate matter on the surfaces of single-use components and
configurations and sizes of single-use components and
assemblies is to maximize the probability that particles are
assemblies, such as bioreactors, bioprocess containers, tubing,
extracted in an effective, practical, consistent and controlled
connectors, clamps, valves, sensors and filters. Extraction of
way.
particulate matter may be relatively easy from small compo-
nents with readily accessible surfaces, however, extraction of
5. Requirements for Effective Extraction of Particulate
particulate matter from large and complex assemblies with less
Matter
readily accessible interior surfaces may require significantly
5.1 General Principles:
more effort.
5.1.1 In order to determine the particle load of single-use
components and assemblies, the first procedure required is an
4.4 The wide variety of single-use components and assem-
blies inhibits specification of a narrowly defined extraction extraction procedure, whereby the particulate matter is re-
moved from the surfaces of components/assemblies (test ar-
procedure with a universally prescribed volume of test liquid
and energy input (rinsing/agitation) conditions. The approach ticle) by means of a liquid extraction method, which is
essentially a surface cleaning process. A test liquid is applied to
described in this practice allows for flexibility and innovative
the surfaces of a test article using rinsing or agitation extraction
approaches to maximize particle extraction which are specifi-
methods, or both, and the extraction liquid (test liquid contain-
cally tailored to the component or assembly of interest.
ing the extracted particles) is collected for further analysis
4.5 In most cases, relatively small amounts of particulate
using a particle measurement method. The chosen particle
matter are non-uniformly dispersed over large surface areas,
measurement method (not specified in this practice) shall have
and the particulate matter also is often inhomogeneous in
been qualified for determination of the particle count in the
chemical composition and morphology. Standardized single-
particle size range of interest.
use components and assemblies with controlled amounts of
5.1.2 Since reference test articles with a known amount of
known standardized particulate matter which simulate real
standardized particulate matter are not readily available to
systems are challenging to prepare. Thus in the development of
allow absolute quantitative determination of extraction
a particle extraction procedure, a practical and expedient
effectiveness, development of an extraction procedure with a
methodology is required to assess whether the chosen extrac-
high degree of effectiveness (measured on a relative basis)
tion procedure is effective and extracts as many particles as
requires the following steps:
practically possible
5.1.3 Qualification Test:
4.6 A well-established standardized methodology for dem- 5.1.3.1 The appropriate extraction procedure for effective
onstrating effective extraction of particles from the surfaces of extraction of particles from the surfaces of a component or
automotive components provides guidance (ISO 16232:2018, assembly is determined by performing the qualification test.
E3230 − 20
The qualification approach and qualification criteria stated in equipment used to receive the extraction liquid, particles shed
this document ensure that as many as possible of the particles by the test operator (for example, clothing fibers), and particles
adhering to the surfaces of the test article are removed upon present in the general environment. It is recommended to
extraction. Once a specific test article has undergone a quali- terminally filter the test liquid prior to use through a non fiber
fication test, it cannot be retested. The requirements, shedding filter having a nominal pore size smaller than the
parameters, and results of the chosen extraction procedure shall smallest particle size to be counted by the chosen particle
be documented. measurement method. Carefully clean the extraction apparatus,
properly gown the test operator, and perform all procedures in
5.1.4 Routine Test:
a clean environment.
5.1.4.1 Depending upon requirements, the routine test will
5.4.2 The cleanliness of the environment under which the
consist of one or more extractions routinely performed on a
extraction procedure occurs must be adequate to meet the
sampling of components or assemblies, typically for process
required background particle count. In most cases in order to
monitoring and control, or component/assembly product re-
meet the required background particle count, a controlled
lease based upon particle count specifications, or both.
laboratory environment will be required: a laminar-flow
5.1.5 Background Test:
cabinet, proper operator gowning (garments and gloves), and
5.1.5.1 During qualification and routine testing, a back-
decontaminated tools and sample handling equipment. The
ground test is required. In the background test, the extraction
suitability of the environment is confirmed by meeting the
procedure for the test article of interest is applied, but without
required background particle count.
the test article present. The result of the background test is the
background particle count, a measure of the amount of par-
5.5 Test Liquid:
ticulate matter introduced from sources other than the test 5.5.1 Particulate matter adheres to the fluid-contacting sur-
article, such as the extraction apparatus and the surrounding
faces via a variety of forces, and the role of the test liquid is to
environment. overcome these forces and remove (wash off) particulate
matter from the surfaces. The test liquid chosen combined
5.2 Selection of the Extraction Method:
along with the extraction conditions shall meet the require-
5.2.1 Particulate matter is extracted from the surfaces of a
ments for effective extraction as described in the qualification
component or assembly in a surface cleaning process, whereby
test. The test liquid chosen shall not chemically attack or etch
the surfaces are flushed with a test liquid. The ease with which
the surfaces of the test components. If agitation is applied, the
particulate matter is removed from surfaces by liquid extrac-
test liquid shall be non-foaming since foam may not com-
tion depends upon the strength of adhesion between particle
pletely drain from the component and could potentially trap
and surface, and the ability to deliver test liquid with the
particles. Care shall be taken that the test liquid is also
required force to flush the desired surfaces of the test article.
compatible with the chosen particle measurement method. In
The extraction method is determined by the features of the test
most cases, purified water or water plus salt(s) (for example,
article, such as size, shape, and the accessibility of the surfaces.
buffers) meet the criteria noted above.
The purpose of selecting an extraction method, determining
5.6 Extraction Apparatus:
extraction conditions, and qualifying the procedure, is to
5.6.1 The extraction apparatus generally consists of the
optimize the removal of particulate matter from the test article.
following items:
The main parameters which influence the extraction result are
5.6.1.1 A system adequate to prepare clean test liquid with
the wetting and suspension properties of the test liquid,
low particle count which meets the required background
temperature, energy input (shear forces imparted by the test
particle count.
liquid upon rinsing or agitating with the test liquid, or both)
5.6.1.2 A system to deliver the test liquid to the test article.
and the amount of time the surfaces of the test article are
This system provides test liquid at flow rates adequate to rinse
exposed to the test liquid (ISO 16232:2018, VDA 19 Part 1).
particulate matter from test article surfaces, and also to rinse
For this practice, the extraction procedure shall have rinsing or
surfaces of collection equipment (funnels, collection
agitation steps, or both.
containers, etc.) applied in the extraction procedure.
5.3 Disassembly of Single-Use Assemblies:
5.6.1.3 If needed, adapters to connect the test liquid delivery
5.3.1 Extraction of large or complex assemblies, or both,
system directly to components or assemblies, or plugs, or both,
may require careful disassembly (for example, cutting of
to close component or assembly openings.
tubing lines) in order to allow practical handling, or to allow
5.6.1.4 If needed, equipment to impart controlled agitation
access to flow-restricted interior regions of the assembly. Care
(at specified amplitude and frequency).
should be taken to minimize the generation of particulate
5.6.1.5 Collection equipment to receive the extraction liquid
matter during disassembly (for example, cut tubing lines using
obtained after extraction of the test article (for example, flasks,
a sharp knife).
beakers, trays, funnels, etc.).
5.4 Environmental Conditions:
6. Qualification Test
5.4.1 In the implementation of this practice, care must be
taken to minimize particulate matter originating from foreign 6.1 General Principles:
sources other than the surfaces of the single-use components or 6.1.1 In order to properly assess the particle load of a
assemblies of interest. Potential foreign sources include the test single-use component or assembly, the chosen extraction
liquid, the delivery system for the test liquid, the collection procedure shall be determined to be effective. Effective means
E3230 − 20
that it shall be proved that the chosen extraction procedure 6.1.4 The qualification test procedure comprises the follow-
extracts the maximum amount of detachable particulate matter ing steps:
from the test article surfaces of interest.
6.1.4.1 Determination and procuring the number of test
6.1.2 Absolute determination of the particle load of a
articles required for qualification.
single-use component or assembly is challenging, since cre-
6.1.4.2 Cleaning the extraction apparatus.
ation of standard test articles with a defined load of standard-
6.1.4.3 Executing a background test to determine back-
ized reference particles is difficult. In many cases, creation of
ground particle count.
a test article with a standardized load of particles is impractical,
6.1.4.4 Determination if background particle count require-
considering the broad range of materials and geometries of
ments are satisfied.
single-use components and assemblies, the possible range of
6.1.4.5 Preparation of the test article.
particulate matter types and concentrations, and potential
variations in adhesion strength between particles and material
6.1.4.6 Extraction of the test article.
surfaces. Consequently, this practice describes a multiple
6.1.4.7 Final rinsing of the collection equipment (optional).
extractions approach for the qualification of an effective
6.1.4.8 Determination of the particle count in the extraction
extraction procedure, which avoids the need for reference
liquid.
particles.
6.1.4.9 Determination of the extraction effectiveness index
6.1.3 Qualification tests are carried out to define an effective
(EIn).
extraction procedure for routine testing. The qualification test
6.1.4.10 Repeat the above steps up to 5 more times until the
determines the extraction curve, the trend of the extraction
qualification criterion is met.
effectiveness index (EIn) as a function of the number of
6.1.5 If the background particle counts meet requirements,
extraction steps (n) applied. Each extraction step (n) is a repeat
of the same extraction procedure (same conditions and and the qualification criterion for effective extraction is met,
the extraction procedure is qualified, and the extraction proce-
method). After each extraction step, the particle count (Pn) in
the extraction liquid is determined, and the EIn for each step n dure (extraction conditions and methods, along with back-
ground particle count) shall be documented in detail.
calculated by dividing the particle count from step n (Pn) by
PTn, which is the particle count total for all extraction steps
6.1.6 A qualification test procedure shall never be repeated
executed up to and including the extraction step n. Thus, EIn =
on a test article which has already been put through the
Pn/PTn and should generally decrease as n increases, since
qualification test. The qualification test shall be carried out for
after each extraction step, the test article should be cleaner and
a component/assembly or a representative member of a closely
release less particles (Pn decreases), and the cumulative total of
related family of compone
...