ASTM E3231-19

Standard Guide for Cell Culture Growth Assessment of Single-Use Material

SIGNIFICANCE AND USE
5.1 A risk-based approach must be used to determine the cell lines, test articles, and materials used for testing. An evaluation of relevant factors should be made to determine if a test article is representative of the intended use.  
5.2 Cell culture compatibility should be assessed if the material is in direct contact with cell culture medium regardless of duration of contact. Test articles can be of a single material or assembled from a multitude of materials.  
5.3 Two perspectives to single-use material cell culture compatibility assessments are the supplier and the end user perspectives. It is understood that the supplier may have better access to single-use materials and material manufacturing processes, while having limited access to representative cell lines. Supplier assessment of materials are best tested using cell lines available that have shown known material sensitivity. The end users may have more limited access to materials but access to more representative cell lines and processes. Therefore assessment of compatibility of material with a specific cell line in a process is best evaluated by the end user.  
5.4 This guide outlines best practices to establish test procedures. Appendix X1 outlines an example test procedure for a commercially available CHO cell line.
SCOPE
1.1 This guide outlines best practices to consider when setting up a representative leachable test method to detect if a material is compatible with cell culture media or manufacturing processes. This guide does not replace or supersede cell growth tests like USP , USP  (plastic/elastomeric materials), or ISO 10993 (medical device materials), that are used in assessing biological reactivity in humans. Polymeric materials that have passed these tests have been found to leach compounds under normal process conditions that can inhibit cell culture growth for some cell lines. See Refs (1-5).2 Test methods that are representative of the manufacturing conditions will help identify materials that are appropriate for use during manufacturing.  
1.2 This guide may be relevant to biopharmaceutical manufacturing, cell-based therapeutics, vaccines, cell-based diagnostics, and other areas.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 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.  
1.5 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.

General Information

• Status: Published
• Publication Date: 30-Sep-2019
• ICS: 11.100.01 - Laboratory medicine in general
• Technical Committee: E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
• Drafting Committee: E55.07 - Single Use Systems

Overview

ASTM E3231-19: Standard Guide for Cell Culture Growth Assessment of Single-Use Material provides best practices for evaluating the compatibility of single-use materials in cell culture processes. Developed by ASTM International, this guide focuses on assessing whether the materials used in bioprocessing – such as disposable bags, tubing, and other polymeric components – are suitable for their intended cell-based applications. The standard addresses the importance of risk-based approaches, proper study design, representative sample preparation, and test execution to ensure the safety and performance of single-use systems in environments where direct contact with cell culture occurs.

Significance

• Ensures materials do not release leachables that inhibit cell growth.
• Supports biopharmaceutical quality, process safety, and product integrity.
• Complements, but does not supersede, biological reactivity tests like USP <87>, USP <88>, and ISO 10993.

Key Topics

Risk-Based Assessment

• Selection of cell lines and test articles should be based on risk analysis relevant to the application.
• Representative cell lines (sensitive to known leachables) and materials closely matching manufacturing processes are critical for valid results.

Testing Perspectives

• Supplier Perspective: Typically has better access to materials and manufacturing knowledge but may have limited access to relevant cell lines.
• End User Perspective: Often can utilize more representative cell lines and processes most applicable to their unique applications.

Study Design & Controls

• Use of negative and positive controls to benchmark cell growth and assay sensitivity.
• Inclusion of multiple replicates and proper reference standards to ensure data reliability.

Material and Test Preparation

• Materials should mimic real-world manufacturing scenarios in terms of form, sterilization, and exposure conditions.
• Test articles must be appropriately prepared, sized, sterilized, and extracted to simulate worst-case usage.

Extraction and Culture Methods

• Extraction conditions, such as media type, duration, and temperature, must reflect the intended application.
• The duration and agitation during extraction should be optimized for worst-case scenarios based on industry experience.

Data Analysis

• Growth rates are compared between test articles and controls.
• Statistical evaluation determines if significant inhibition or promotion of cell growth is associated with the test material.

Applications

ASTM E3231-19 is particularly relevant in:

• Biopharmaceutical Manufacturing: Assessment of single-use components for media preparation, cell culture, and downstream processing.
• Cell-Based Therapeutics: Ensuring polymeric devices and consumables do not adversely affect cell growth or function.
• Vaccine Production: Validates that materials in direct contact with cell culture maintain process consistency and safety.
• Cell-Based Diagnostics: Supports development and qualification of diagnostic systems reliant on cell culture compatibility.
• Quality and Risk Management: Facilitates proactive evaluation and mitigation of risks posed by unknown leachables from disposable systems.

Key benefits include reduced risk of process failures, improved reproducibility, and enhanced regulatory compliance for manufacturers and end users.

Related Standards

ASTM E3231-19 should be considered in conjunction with established cell culture and biocompatibility standards, including:

• USP <87>: Biological Reactivity Tests, In Vitro
• USP <88>: Biological Reactivity Tests, In Vivo
• ISO 10993: Biological Evaluation of Medical Devices
• Other relevant ASTM guides for bioprocessing and single-use systems

Keywords: cell culture growth assessment, single-use material compatibility, leachable testing, biopharmaceutical manufacturing, cell-based assays, ASTM E3231-19, polymeric material evaluation, risk-based testing, disposable bioprocessing systems, media extraction.

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: E3231 − 19
Standard Guide for
Cell Culture Growth Assessment of Single-Use Material
This standard is issued under the fixed designation E3231; 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. Referenced Documents
1.1 This guide outlines best practices to consider when 2.1 USP Documents:
USP <87> Biological Reactivity Tests, In Vitro
setting up a representative leachable test method to detect if a
material is compatible with cell culture media or manufactur- USP <88> Biological Reactivity Tests, In Vivo
ing processes. This guide does not replace or supersede cell
2.2 ISO Documents:
growth tests like USP <87>, USP <88> (plastic/elastomeric
ISO 10993 Biological Evaluation of Medical Devices
materials), or ISO 10993 (medical device materials), that are
used in assessing biological reactivity in humans. Polymeric 3. Terminology
materials that have passed these tests have been found to leach
3.1 Definitions:
compounds under normal process conditions that can inhibit
3.1.1 bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP),
cell culture growth for some cell lines. See Refs (1-5). Test
n—leachable compound known to inhibit cell growth (CAS #
methods that are representative of the manufacturing condi-
69284-93-1).
tions will help identify materials that are appropriate for use
3.1.2 dimethyl sulfoxide (DMSO), n—(CH3)2SO (CAS #
during manufacturing.
67-68-5).
1.2 This guide may be relevant to biopharmaceutical
3.1.3 test articles, n—the material being tested.
manufacturing, cell-based therapeutics, vaccines, cell-based
3.1.4 test media, n—cell culture media that has been used to
diagnostics, and other areas.
extract potential leachables from the test articles.
1.3 The values stated in SI units are to be regarded as
3.1.5 the gray (Gy), n—the SI unit for absorbed radiation
standard. No other units of measurement are included in this
dose, and defined as the absorption of one joule of energy, in
standard.
the form of ionizing radiation, per kilogram of matter, that is
one gray = 1 J/kg.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
4. Summary of Guide
responsibility of the user of this standard to establish appro-
4.1 This guide outlines best practices to assess compatibility
priate safety, health, and environmental practices and deter-
of polymeric materials with animal cell cultures used in the
mine the applicability of regulatory limitations prior to use.
manufacture and processing of vaccine, gene, cell and protein
1.5 This international standard was developed in accor-
therapies reliant on cell-based processes. The best practices
dance with internationally recognized principles on standard-
may be used to reveal compatibility issues in a cell culture
ization established in the Decision on Principles for the
system that includes the cell culture medium, the cell line and
Development of International Standards, Guides and Recom-
the polymeric test articles that are under evaluation.
mendations issued by the World Trade Organization Technical
4.2 The guide starts with an overview of typical cell culture
Barriers to Trade (TBT) Committee.
compatibility studies and then outlines best practices for each
aspect of the study.
This guide is under the jurisdiction of ASTM Committee E55 on Manufacture
of Pharmaceutical and Biopharmaceutical Products and is the direct responsibility of
Subcommittee E55.07 on Single Use Systems. Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
Current edition approved Oct. 1, 2019. Published October 2019. DOI: 10.1520/ Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
E3231-19. Available from International Organization for Standardization (ISO), ISO
The boldface numbers in parentheses refer to a list of references at the end of Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier,
this standard. Geneva, Switzerland, http://www.iso.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3231 − 19
5. Significance and Use cell lines available that have shown known material sensitivity.
The end users may have more limited access to materials but
5.1 A risk-based approach must be used to determine the
access to more representative cell lines and processes. There-
cell lines, test articles, and materials used for testing. An
fore assessment of compatibility of material with a specific cell
evaluation of relevant factors should be made to determine if a
line in a process is best evaluated by the end user.
test article is representative of the intended use.
5.4 This guide outlines best practices to establish test
5.2 Cell culture compatibility should be assessed if the
procedures. Appendix X1 outlines an example test procedure
material is in direct contact with cell culture medium regardless
for a commercially available CHO cell line.
of duration of contact. Test articles can be of a single material
or assembled from a multitude of materials.
6. Cell Culture Compatibility Testing Overview
5.3 Two perspectives to single-use material cell culture 6.1 Fig. 1 highlights the main stages of organizing cell
compatibility assessments are the supplier and the end user culture compatibility testing. Key aspects are highlighted in the
perspectives. It is understood that the supplier may have better figure and detailed best practices for each stage are covered in
access to single-use materials and material manufacturing the subsequent sections. Appendix X1 shows an example of
processes, while having limited access to representative cell
what a test could look like using an industrially available media
lines. Supplier assessment of materials are best tested using and cell line.
FIG. 1 Cell Culture Compatibility Testing Design: Key Considerations for Success
E3231 − 19
7. General Study Design and Controls 8. Cell Line and Test Medium
7.1 Fig. 2 shows a typical process flow for cell culture 8.1 Cell Lines:
compatibility testing. The test article is first prepared to a size
8.1.1 The cell lines should be representative of the final
that is scaled to be representative of the contacted surface area
application. In order to detect leachables better it is recom-
during use. Then the test article is sterilized in a representative
mended to use a cell line that demonstrates a known sensitivity
way. Following sterilization the test article is extracted in the
to known leachables that effect cell growth.
cell culture medium (test medium) in parallel to control
8.2 Extraction Solution (Test Medium): Cell Culture Me-
conditions. Then these test medium are inoculated with cell
dium:
culture and cell growth is monitored. The growth rate is used
8.2.1 Media that is representative of the intended process
to analyze the material’s effect on cell growth.
should be used. When possible serum-free, chemically defined
7.2 Controls:
cell culture medium will help ensure test reproducibility.
7.2.1 Several replicates of negative and positive controls
Serum-containing media are known to give false negative test
should be used during each test. These flasks filled with media
results and should not be used if possible. See Ref (4). If your
must be incubated under the same conditions as the extraction
intended process and the representative test cell line requires
containers of the test articles. The number of replicates should
serum containing medium, then serum containing medium can
be chosen based on expected cell growth variability. Typically
be used to best represent the final intended use case.
a minimum of three replicates are recommended.
8.2.2 The medium must be temperature stable for the
7.2.2 Multiple positive and negative controls could be used.
duration of the testing.
For example, each test could include multiple standard growth
inhibitors to demonstrate growth inhibition. Multiple vessel
9. Material Preparation and Sterilization
types could be used to demonstrate representative cell growth
9.1 A representative test article should be used. The material
(glass and plastic flasks).
should be manufactured using methods as similar as possible to
7.3 Negative Controls (Growth Benchmark):
the envisioned manufacturing process of the component. It is
7.3.1 The media from the negative controls should exhibit
important to use representative chemical grades, multiple
normal growth performance following the extraction period.
production lots, anticipated process (machining or molding),
This controls for any problem with the extraction process
type of irradiation process, use of cleaning agents or process
affecting cell growth. This extraction container could have no
aids, etc.
test article or a material known to not inhibit cell growth in the
9.2 Select the test article size based on the worst case use
media during the extraction duration.
condition and contact area during extraction. Using consistent
7.4 Positive Control (Extracted or Spiked with Growth
surface areas during extraction helps when comparing materi-
Inhibitor or Growth Promoter):
als. It is recommended, as a worst case test condition, to test a
7.4.1 The media from the positive controls should demon-
surface area of 3 cm of material per ml of extraction medium
strate reduced or enhanced growth performance following
for materials >0.5-mm thick (ISO 10993). Smaller test articles
extraction. The positive controls show that the assay was
can be used as multiples to achieve a surface area of 3 cm per
executed properly to demonstrate sensitivity to a known
millilitre of extraction medium. If the material floats, this
leachable that has proven to effect the cell line used. It does not
should be accounted for in the surface area calculation. For
indicate that if the test article media also demonstrates an effect
some materials with a low surface area, an extraction ratio of
that the same leachable compound is present. For this reason,
3 cm /mL cannot be achieved and a lower ratio is required.
positive controls are not an absolute requirement.
9.3 If cutting is required to achieve the target sample size,
7.4.2 One example of a positive control would be to spike
cutting tools need to be clean to prevent introduction of foreign
the media from the flask with bDtBPP or similar growth
matter. Test articles should be dusted to remove small particles
inhibitor of interest.
that could be generated during cutting, sawing, or similar
7.4.3 The sensitivity of the cell line to the positive control
procedures. Avoid cleaning fluids, if possible, as they can
should be demonstrated with a dose-response curve test.
extract leachables and reduce reactivity prior to the test.
Consider that cutting the material might expose material layers
that would normally not contact the cell culture.
FIG. 2 Cell Culture Compatibility Study Process Flow
E3231 − 19
9.4 Extraction containers and test article packaging need to 11. Culture Preparation
be tested with separate cell culture experiments to ensure that
11.1 Expand culture from vial thaw until adequate cell
they don’t exhibit a growth inhibition effect. For example:
density is reached to inoculate all test flasks simultaneously.
shake flasks and sterilization pouches.
11.2 Allow adequate number of cell passages for cell
9.5 Non-sterilized test articles should be used to prevent
growth recovery. A minimum of two passages after thaw is
repeatedly sterilizing the test article if multiple sterilization
recommended. Material test flasks should be inoculated after a
does not represent the final use case.
consistent number of passages following vial thaw and a
9.6 The test articles should be sealed and then sterilized
consistent range of cell culture generation number should be
using a representative method. Target dose should be represen-
targeted.
tative of the worst case dose or treatment: kGy for e-beam or
gamma, or temperature for autoclaving.
12. Cell Culture Compatibility Study
9.7 Special Geometries: Tubing and Components with Small
12.1 It is critical to achieve consistent seed density and to
Surfaces:
dilute the test medium as little as possible. It is recommended
9.7.1 Calculate the necessary length of tubing to be used,
that the final cell culture volume contains greater than 90 % test
accounting for the inner and outer surface area. If the inner or
medium if possible. It is also critical to keep this ratio of
outer surface is not wetted during extraction, the necessary
extracted to fresh medium consistent from tests to test
...