ASTM E3251-23

Standard Test Method for Microbial Ingress Testing on Single-Use Systems

SIGNIFICANCE AND USE
4.1 Single-use systems (SUSs) used for biopharmaceutical manufacturing must maintain sterility and product quality of the fluid inside. Such articles or systems should therefore be validated as providing an effective barrier against microbial ingress. The microbial barrier properties of a SUS may be demonstrated using deterministic physical tests that have been correlated to microbial integrity. Such physical test methods are described in Test Method E3336. Two microbial test methods (aerosol exposure and immersion exposure) are described in this test method that can be used to demonstrate microbial integrity of a SUS or determine the MALL, the maximum defect size that does not allow microbial ingress, into a SUS.  
4.2 It is important to note that the results of microbial ingress tests are heavily dependent on the conditions under which the test is performed and are not suitable for routine checking of a SUS due to the test’s destructive nature.  
4.2.1 Any size defect may be forced to fail under sufficiently aggressive conditions (including a large enough sample size, high differential pressure, or high hydrostatic pressure, for example) that would not ordinarily reflect normal use conditions. Thus, it is necessary to clearly define the relevant conditions for a test through a risk assessment of both the actual SUS claims and its final use (Practice E3244). Once that is established, the size of defect that can be detected under those conditions can be determined, if required, using defined defects.  
4.2.2 “Relevant conditions” refers to worse-case actual use conditions but does not mean that a SUS must be tested under theoretically absolute (extreme) “worst-case” conditions.  
4.2.3 Testing may be performed on individual components or entire systems. Considerations for defining “relevant conditions” and testing design should be based on a risk assessment for the SUS intended use and should include:
4.2.3.1 A channel created by a defect or breach through the...
SCOPE
1.1 The microbial test method outlined in this test method applies to microbial ingress risk assessment of a single-use system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the assembly based on a potential risk of a breach to the product or manufacturing process.  
1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold:  
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use.  
1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing methods (see Test Method E3336) can be correlated to this MALL in order to claim microbial integrity. Test articles bearing calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a positive control (defect-based positive control), may be tested to determine the MALL.  
1.3 Both purposes for microbial ingress testing as described in 1.2.1 and 1.2.2 can either be conducted by liquid immersion or aerosol exposure. For the purpose described in 1.2.2, the type of exposure should be determined according to the SUS’s use-case conditions and a risk assessment.  
1.4 The method used to create a breach, hole or defect in single-use film or...

General Information

• Status: Published
• Publication Date: 14-Mar-2023
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
• Drafting Committee: E55.07 - Single Use Systems

Standards Content (Sample)

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: E3251 − 23
Standard Test Method for
1
Microbial Ingress Testing on Single-Use Systems
This standard is issued under the fixed designation E3251; 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 outside of the scope of this test method. The sampling plan for
a given test article should be justified with the rationale of
1.1 The microbial test method outlined in this test method
sampling size to obtain a statistically meaningful effect (Prac-
applies to microbial ingress risk assessment of a single-use
tice E3244). Determining the appropriate number of SUS test
system (SUS) or its individual components that require integ-
articles will depend on a risk assessment of the SUS and the
rity testing either by the assembly supplier or the end user of
conditions of its use and is also outside of this test method’s
the assembly based on a potential risk of a breach to the
scope.
product or manufacturing process.
1.5 Units—The values stated in SI units are to be regarded
1.2 The aim of microbial ingress testing of sterile SUSs
as standard. No other units of measurement are included in this
used in biopharmaceutical manufacturing is two-fold:
standard.
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid
1.6 This standard does not purport to address all of the
path to remain sterile after a SUS has been challenged by
safety concerns, if any, associated with its use. It is the
microbial exposure. Microbial exposure is achieved either by
responsibility of the user of this standard to establish appro-
directly placing a SUS into a container of microbial challenge
priate safety, health, and environmental practices and deter-
solution, or by delivering an aerosolized microbial challenge
mine the applicability of regulatory limitations prior to use.
onto a SUS that is placed inside a test chamber designed to
1.7 This international standard was developed in accor-
generate and deliver the aerosol. The choice of the test
dance with internationally recognized principles on standard-
challenge organism should be justified based on a risk assess-
ization established in the Decision on Principles for the
ment of the SUS and conditions of use.
Development of International Standards, Guides and Recom-
1.2.2 Additionally, microbial ingress testing can be used to
mendations issued by the World Trade Organization Technical
determine the maximum allowable leakage limit (MALL) that
Barriers to Trade (TBT) Committee.
does not allow microbial ingress under specific test conditions.
The defect size that can be detected by specific physical
2. Referenced Documents
integrity testing methods (see Test Method E3336) can be
2
2.1 ASTM Standards:
correlated to this MALL in order to claim microbial integrity.
Test articles bearing calibrated defects over a range of E3244 Practice for Integrity Assurance and Testing of
Single-Use Systems
dimensions, including up to a defect size expected to consis-
tently allow microbial ingress as a positive control (defect- E3336 Test Method for Physical Integrity Testing of Single-
Use Systems
based positive control), may be tested to determine the MALL.
2.2 Other Documents:
1.3 Both purposes for microbial ingress testing as described
USP <1207> Sterile Product Packaging — Integrity
in 1.2.1 and 1.2.2 can either be conducted by liquid immersion
3
Evaluation, 2016
or aerosol exposure. For the purpose described in 1.2.2, the
4
ISO 15747 Plastic Containers for Intravenous Injections
type of exposure should be determined according to the SUS’s
use-case conditions and a risk assessment.
3. Terminology
1.4 The method used to create a breach, hole or defect in
3.1 Definitions:
single-use film or in a SUS test article, as well as the analytical
method used to physically characterize the defect size is
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
1
This test method is under the jurisdiction of ASTM Committee E55 on the ASTM website.
3
Manufacture of Pharmaceutical and Biopharmaceutical Products and is the direct Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
responsibility of Subcommittee E55.07 on Single Use Systems. Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
4
Current edition approved March 15, 2023. Published March 2023
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E3251 − 20 E3251 − 23
Standard Test Method for
1
Microbial Ingress Testing on Single-Use Systems
This standard is issued under the fixed designation E3251; 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
1.1 The microbial test method outlined in this document test method applies to microbial ingress risk assessment of a single-use
system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the
assembly based on a potential risk of a breach to the product or manufacturing process.
1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold:
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial
exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by
delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the
aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use.
1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does
not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing
methods (see Test Method E3336) can be correlated to this MALL in order to claim microbial integrity. Test articles bearing
calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a
positive control (defect-based positive control), may be tested to determine the MALL.
1.3 Both purposes for microbial ingress testing as described in 1.2.1 and 1.2.2 can either be conducted by liquid immersion or
aerosol exposure. For the purpose described in 1.2.2, the type of exposure should be determined according to the SUS’s use-case
conditions and a risk assessment.
1.4 The method used to create a breach, hole or defect in single-use film or in a SUS test article, as well as the analytical method
used to physically characterize the defect size is outside of the scope of this document. test method. The sampling plan for a given
test article should be justified with the rationale of sampling size to obtain a statistically meaningful effect (Practice E3244).
Determining the appropriate number of SUS test articles will depend on a risk assessment of the SUS and the conditions of its use
and is also outside of this document’s test method’s scope.
1.5 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1
This test method is under the jurisdiction of ASTM Committee E55 on Manufacture of Pharmaceutical and Biopharmaceutical Products and is the direct responsibility
of Subcommittee E55.04 on General Biopharmaceutical Standards.
Current edition approved May 1, 2020March 15, 2023. Published May 2020March 2023. Originally approved in 2020. Previous edition approved in 2020 as E3251 – 20.
DOI: 10.1520/E3251-20.10.1520/E3251-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E3251 − 23
1.7 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.
2. Referenced Documents
2
2.1 ASTM Standards:
E3244 Practice for Integrity Assurance and Testing of Single-Use Systems
E3336 Test Method for Physical Integrity Testing of Single-Use Systems
2.2 Other Documents:
3
USP <1207> Sterile Product Packaging — I
...