ISO 24190:2023

Biotechnology - Analytical methods - Risk-based approach for method selection and validation for rapid microbial detection in bioprocesses

This document provides guidance, a framework and a risk-based approach for the selection and validation of methods for rapid microbial detection in cellular therapeutic product manufacturing. This document provides a flexible risk-based framework for the detection of microbial contamination in cellular therapeutic products and cellular intermediates. This document provides general requirements and risks associated with cellular therapeutic product manufacturing, with flexibility to address differences in specific manufacturing processes of each unique cellular therapeutic product. This document primarily addresses sterility testing in cellular therapeutic product manufacturing. This document is applicable to other cell-derived therapeutic product manufacturing. This document focuses on rapid microbial test methods (RMTMs) used for both in-process and final product testing. Viral testing in cellular therapeutic product manufacturing is not included in this document.

Biotechnologie — Méthodes d'analyse — Approche basée sur les risques pour la sélection et la validation de méthodes pour la détection microbienne rapide dans les bioprocédés

General Information

• Status: Published
• Publication Date: 30-May-2023
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276/WG 3 - Analytical methods
• Current Stage: 6060 - International Standard published
• Start Date: 31-May-2023
• Due Date: 11-Jun-2023
• Completion Date: 31-May-2023

Overview

ISO 24190:2023 - "Biotechnology - Analytical methods - Risk-based approach for method selection and validation for rapid microbial detection in bioprocesses" - provides a flexible, risk-based framework for selecting and validating rapid microbial test methods (RMTMs) used in cellular therapeutic product manufacturing. The standard focuses on sterility testing for cellular therapeutics and cell-derived products, addressing both in-process and final product testing to support timely patient administration. Viral testing is excluded from this document.

Key technical topics and requirements

• Risk-based approach: Guidance on identifying microbiological contamination risks across unique manufacturing processes and on prioritizing controls and testing strategies.
• Assay and system selection: Criteria for selecting fit-for-purpose assays, kits or systems considering speed, sample volume, specificity and sensitivity.
• User requirement specifications (URS): Defining acceptance criteria and performance expectations for RMTMs (e.g., speed, limit of detection, analytical specificity).
• Validation principles: Quality-by-design for method validation, selection of microorganisms for validation, use of reference materials, and revalidation triggers.
• Validation parameters: Establishing acceptance criteria for precision, detection limit, accuracy, robustness and ruggedness, plus system validation.
• Operational guidance: Recommendations on number/type of samples, testing environment, comparable test data, investigation of positive sterility results, training, documentation and test reporting.
• Informative annexes: Practical tools and examples including risk analyses for input materials and process stages, validation examples, microorganism lists for validation, methods for RMTMs, and environmental control guidance.

Practical applications and intended users

ISO 24190 is intended for organizations involved in development, manufacture and quality control of cell-based and other cell‑derived therapeutics, including:

• Quality assurance / Quality control (QA/QC) teams
• Microbiology and analytical method developers
• Cell and gene therapy manufacturers
• Regulatory affairs and compliance professionals
• Contract development and manufacturing organizations (CDMOs)

Practical uses include establishing RMTM selection criteria, designing validation studies for rapid sterility tests, integrating rapid testing into short-shelf-life workflows, and documenting risk-based testing strategies to support product release and patient safety.

Related standards and context

• Complements pharmacopeial culture-based sterility approaches by enabling rapid microbial detection where time-sensitive decisions are required.
• Builds on definitions and measurement concepts referenced in related ISO documents (e.g., ISO 16140, ISO 18113) and aligns with ISO/TC 276 biotechnology guidance.

Keywords: ISO 24190, rapid microbial detection, RMTM, cellular therapeutics, sterility testing, method validation, risk-based approach, bioprocesses, QA/QC.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 24190
First edition
2023-05
Biotechnology — Analytical methods
— Risk-based approach for method
selection and validation for rapid
microbial detection in bioprocesses
Biotechnologie — Méthodes d'analyse — Approche basée sur
les risques pour la sélection et la validation de méthodes pour la
détection microbienne rapide dans les bioprocédés
Reference number
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
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or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
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Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General considerations .7
5 Risk management for microbiological contamination. 7
5.1 Risk management in manufacturing process . 7
5.2 Risk management in microbial testing . 8
6 Selection of a fit-for-purpose assay .9
6.1 General . 9
6.2 Assay selection . 10
6.3 Kit or system selection . 10
6.4 Considerations for various test types . 11
6.5 User requirement specifications .12
6.5.1 General .12
6.5.2 Speed .12
6.5.3 Sample volume .12
6.5.4 In-process versus final release testing .12
6.5.5 Specificity . 12
6.5.6 Sensitivity . 13
7 Validation . .13
7.1 General concepts . 13
7.2 Selection of microorganisms for validation . 14
7.3 Quality by design of method validation . 15
7.4 Revalidation method . 15
7.5 System validation . 16
7.6 Use of reference material in validation . 16
7.7 Acceptance criteria of targeted validation parameters . 16
7.8 Precision . . . 17
7.9 Detection limit . 17
7.10 Accuracy . 17
7.11 Robustness . 18
7.12 Ruggedness . 18
8 Use and application of rapid microbial tests .18
8.1 Number and type of samples . 18
8.2 Testing environment . 18
8.3 Sensitivity . 19
8.4 Analytical specificity (microorganism detection) . 19
8.5 Comparable test data . 19
9 Investigation of positive sterility results .20
10 Training .20
11 Documentation .21
12 Test report .21
Annex A (informative) Exemplary framework for identifying microbial contamination .22
Annex B (informative) Risk analysis with cellular therapeutic products related to input
materials — Donor selection .23
iii
Annex C (informative) Risk analysis with cellular therapeutic products related to input
materials — Cell transformation and expansion .24
Annex D (informative) Risk analysis with cellular therapeutic products related to input
materials — Packaging storage and administration .26
Annex E (informative) Risk-based classification for monitoring practices for cellular
therapeutic product manufacturing .27
Annex F (informative) Validation of rapid microbial test methods .28
Annex G (informative) Microorganisms for validation of rapid microbial test methods .30
Annex H (informative) Methods for rapid microbial testing .34
Annex I (informative) Environmental control .41
Bibliography .42
iv
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO document should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
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For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
Introduction
Patient safety is essential in providing cell-based therapies. However, novel cell-based therapies
present many challenges with respect to the timely assessment of microbial contamination. Since
many cell-based therapies have short shelf lives, they are administered to patients within hours after
formulation. In addition to final product testing, testing on cell banks and product intermediates is
common. Microbiological testing includes bacteria, fungi, mycoplasma and viral adventitious agents.
Culture-based testing methods (e.g. pharmacopeia methods) have been widely adopted by industry.
However, culture-based testing methods can take days to weeks to obtain a result. More rapid methods
for microbiological testing are needed to ensure patient safety prior to product administration. The
development and use of rapid, validated methods that are sensitive and accurate, and that allow for the
detection of a broad range of microorganisms are therefore desired and supported by this document.
vi
INTERNATIONAL STANDARD ISO 24190:2023(E)
Biotechnology — Analytical methods — Risk-based
approach for method selection and validation for rapid
microbial detection in bioprocesses
1 Scope
This document provides guidance, a framework and a risk-based approach for the selection and
validation of methods for rapid microbial detection in cellular therapeutic product manufacturing.
This document provides a flexible risk-based framework for the detection of microbial contamination
in cellular therapeutic products and cellular intermediates.
This document provides general requirements and risks associated with cellular therapeutic product
manufacturing, with flexibility to address differences in specific manufacturing processes of each
unique cellular therapeutic product.
This document primarily addresses sterility testing in cellular therapeutic product manufacturing.
This document is applicable to other cell-derived therapeutic product manufacturing.
This document focuses on rapid microbial test methods (RMTMs) used for both in-process and final
product testing.
Viral testing in cellular therapeutic product manufacturing is not included in this document.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
acceptance criteria
numerical limits, ranges, or other attributes or variables meeting predefined performance for the
assays described
Note 1 to entry: Acceptance criteria are specified by the user requirement specifications (3.30).
3.2
accuracy
measurement accuracy
closeness of agreement between a measured quantity value and an assigned quantity value of a
measurand
Note 1 to entry: The concept “measurement accuracy” is not a quantity and is not given a numerical quantity
value. A measurement is said to be more accurate when it offers a smaller measurement error.
Note 2 to entry: The term “measurement accuracy” should not be used for measurement trueness and the term
measurement precision should not be used for “measurement accuracy”, which, however, is related to both these
concepts.
Note 3 to entry: “Measurement accuracy” is sometimes understood as closeness of agreement between measured
quantity values that are being attributed to the measurand.
[SOURCE: ISO 16140-1:2016, 2.2]
3.3
analytical sensitivity
quotient of the change in measurement indication and the corresponding change in value of a quantity
being measured
Note 1 to entry: Analytical sensitivity should not be used to mean detection limit (3.8) or quantitation limit and
should not be confused with diagnostic sensitivity (3.9).
[SOURCE: ISO 18113-1:2022, 3.2.4, modified — Admitted term “sensitivity of a measurement procedure”
deleted. Notes 1 to 3 to entry deleted. Note 4 to entry renumbered as Note 1 to entry.]
3.4
analytical specificity
capability of a measuring system, using a specified measurement procedure, to provide measurement
results for one or more measurands which do not depend on each other nor on any other quantity in the
system undergoing measurement
Note 1 to entry: Lack of analytical specificity is called analytical interference.
Note 2 to entry: Analytical specificity should not be confused with diagnostic specificity (3.10).
Note 3 to entry: ISO/IEC Guide 99:2007 uses the term “selectivity” for this concept instead of “specificity”.
[SOURCE: ISO 18113-1:2022, 3.2.5, modified — Admitted term “selectivity of a measurement procedure”
deleted. Notes to entry replaced.]
3.5
aseptic
conditions and procedures used to exclude the introduction of microbial contamination
[SOURCE: ISO 18362:2016, 3.3, modified — “aseptic” replaced “aseptic technique” as the term.]
3.6
cellular therapeutic product
product containing cells as the active substance
EXAMPLE Cell and gene therapy products, tissue engineered products, drug products.
Note 1 to entry: Products produced from cells for gene therapies are included in the definition of cellular
therapeutic product, as cells are not necessarily the active substance for all gene therapies.
Note 2 to entry: Recombinant proteins are not included in this definition of cellular therapeutic product.
[SOURCE: ISO 20399:2022, 3.9, modified — “used for cell therapy or gene therapy” deleted from the
definition.]
3.7
design qualification
DQ
process for verification (3.32) that the proposed specification for the facility, equipment or system of
the assay meets the expectation for the user requirement specifications (URS) (3.30)
[SOURCE: ISO 11139:2018, 3.220.1, modified — Abbreviated term “DQ” and “of the assay” added. “user
requirement specifications (URS)” replaced “intended use”.]
3.8
detection limit
limit of detection
measured quantity value, obtained by a given measurement procedure, for which the probability of
falsely claiming the absence of a component in a material is β, given a probability α of falsely claiming
its presence
Note 1 to entry: IUPAC recommends default values for α and β equal to 0,05.
Note 2 to entry: The abbreviation LOD is sometimes used.
Note 3 to entry: The term “sensitivity” is discouraged for “detection limit”.
[SOURCE: ISO/IEC Guide 99:2007, 4.18]
3.9
diagnostic sensitivity
ability of an in vitro diagnostic examination procedure to identify the presence of a target marker
associated with a particular disease or condition
Note 1 to entry: Also defined as percent positivity in samples where the target marker is known to be present.
Note 2 to entry: Diagnostic sensitivity is expressed as a percentage (number fraction multiplied by 100),
calculated as 100 × the number of true positive values (TP) divided by the sum of the number of true positive
values (TP) plus the number of false negative values (FN), or 100 × TP/(TP + FN). This calculation is based on a
study design where only one sample is taken from each subject.
Note 3 to entry: For microbial detection, diagnostic sensitivity represents the fraction of target organisms that
were detected correctly.
[SOURCE: ISO 18113-1:2022, 3.2.17, modified — “identify the presence of a target marker” replaced
“have positive results”. Second sentence of Note 1 to entry deleted. Note 3 to entry replaced.]
3.10
diagnostic specificity
ability of an in vitro diagnostic examination procedure to recognize the absence of a target marker
associated with a particular disease or condition
Note 1 to entry: Also defined as percent negativity in samples where the target marker is known to be absent.
Note 2 to entry: Diagnostic specificity is expressed as a percentage (number fraction multiplied by 100),
calculated as 100 × the number of true negative values (TN) divided by the sum of the number of true negative
values (TN) plus the number of false positive values (FP), or 100 × TN/(TN+FP). This calculation is based on a
study design where only one sample is taken from each subject.
[SOURCE: ISO 18113-1:2022, 3.2.18, modified — “recognize the absence of a target marker associated
with a” replaced “have negative results associated with an absence of”. Second sentence of Note 1 to
entry deleted. Note 3 to entry deleted.]
3.11
false negative
result indicated by the test method to be negative (3.15) which has subsequently been shown to contain
the target microorganisms
[SOURCE: ISO 13843:2017, 3.14, modified — “microorganisms” replaced “organism”.]
3.12
false positive
result indicated by the test method to be positive (3.19) which was subsequently shown not to contain
the target microorganisms
[SOURCE: ISO 13843:2017, 3.15, modified — “microorganisms” replaced “organism”.]
3.13
fit for purpose
in line with prearranged requirements for an intended use
[SOURCE: ISO 20387:2018, 3.24, modified — Admitted term “fitness for the intended purpose” and Note
1 to entry deleted.]
3.14
installation qualification
IQ
process of establishing by objective evidence that all key aspects of the process equipment and ancillary
system for the assay instrument installation comply with the approved user requirement specifications
(URS) (3.30)
[SOURCE: ISO 11139:2018, 3.220.2, modified — “for the assay instrument” added and “user requirement
specifications (URS)” replaced “specification”.]
3.15
negative
test result indicating the absence of the analyte in a given test portion as defined by the procedure of
the method
[SOURCE: ISO 16140-1:2016, 2.43, modified — “negative” replaced “negative test result” as the term.
“the absence of the analyte” replaced “the analyte was not detected” and “qualitative” deleted before
“method”.]
3.16
nucleic acid amplification techniques
NAT
biochemistry and molecular biology methods that involve the in vitro synthesis of many copies of DNA
or RNA from one original template
Note 1 to entry: NAT is characterized by existence of reverse transcription, amplification method and type of
determination (qualitative or quantitative)
Note 2 to entry: Examples of amplification methods are PCR and iso thermal amplification (NEAR, TMA, LAMP,
HAD, CRISPER, SDA)
3.17
operational qualification
OQ
process of obtaining and documenting evidence that installed equipment operates within predetermined
limits when used in accordance with its operational procedures
[SOURCE: ISO 11139:2018, 3.220.3]
3.18
performance qualification
PQ
process of establishing by objective evidence that the assay process, under anticipated conditions,
consistently produces a result which meets all predetermined user requirement specifications (URS)
(3.30)
[SOURCE: ISO 11139:2018, 3.220.4, modified — “assay” added before “process”, “result” replaced
“product” and “user requirement specifications (URS)” replaced “requirements”.]
3.19
positive
test result indicating the presence of the analyte in a given test portion as defined by the procedure of
the method
Note 1 to entry: When the reference method or alternative method provides a preliminary positive test result
requiring further testing to confirm this result, this test result can be considered as a presumptive positive test
result. If the further testing specified by the method’s procedure confirms that the test result can indeed be
considered as being positive, the test result can be considered as a confirmed positive test result.
[SOURCE: ISO 16140-1:2016, 2.50, modified — “positive” replaced “positive test result” as the term.]
3.20
precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as
standard deviation, variance, or coefficient of variation under the specified conditions of measurement.
Note 2 to entry: The “specified conditions” can be, for example, repeatability conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement (see
ISO 5725-1).
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified — “precision” replaced “measurement precision” as
the term. Notes 3 and 4 to entry deleted.]
3.21
qualification
activities undertaken to demonstrate that utilities, equipment and methods are suitable for their
intended use and perform properly
Note 1 to entry: Qualification of either equipment or processes, or both, generally includes installation qualification
(3.14), operational qualification (3.17) and performance qualification (3.18).
[SOURCE: ISO 11139:2018, 3.220, modified — “or modes” deleted after “methods”.]
3.22
rapid microbial test method
RMTM
analytical method that allows the user to get microbiology test results faster compared with traditional
visual observation methods using direct inoculation and culture-plating
Note 1 to entry: Generally, this means in a significantly reduced time as compared with the traditional method
(e.g. hours or days).
3.23
reference material
material, sufficiently homogeneous and stable with reference to specified properties, which has been
established to be fit for its intended use in measurement or in examination of nominal properties
[SOURCE: ISO/IEC Guide 99:2007, 5.13, modified — Abbreviated term “RM”, notes to entry and examples
deleted.]
3.24
risk assessment
overall process of risk identification, risk analysis and risk evaluation
[SOURCE: ISO Guide 73:2009, 3.4.1]
3.25
risk control
process in which decisions are made and measures implemented by which risks are reduced to, or
maintained within, specified levels
[SOURCE: ISO 14971:2019, 3.21]
3.26
risk-based approach
methodology that allows the prioritization of activities based on a previous analysis of data and
according to the biosafety level
3.27
robustness
measure of a test method’s capacity to remain unaffected by small, but deliberate, variations in method
parameters and to provide an indication of its reliability during normal usage
[11]
[SOURCE: ICH Q2(R1) ]
3.28
shelf life
period of time after production during which a product that is kept under specified conditions retains
its specified properties
[SOURCE: ISO 1382:2020, 3.485, modified — The term “storage life” deleted. “material or” deleted
before “product” and “that is” added.]
3.29
sterility
state of being free from viable microorganisms (3.33)
Note 1 to entry: In practice, no such absolute statement regarding the absence of microorganisms can be proven.
[SOURCE: ISO 11139:2018, 3.274]
3.30
user requirement specifications
URS
requirements specific to a user or requirements that are not covered in general requirements
3.31
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified — Notes to entry deleted.]
3.32
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified — Notes to entry deleted.]
3.33
viable microorganism
microorganism within a sample that has at least one attribute of being alive (e.g. metabolically active,
capable of reproduction, possession of an intact cell membrane, with the capacity to resume these
functions) defined based on the intended measurement purpose
4 General considerations
Prior to patient administration, cellular therapeutic products should be tested for microbial
contamination. Many of these products rely on the activity of viable cells for a therapeutic effect. Viable
cells cannot be terminally sterilized and rely on a combination of aseptic techniques and closed-system
manufacturing to ensure sterility of the final product. These products typically have a relatively
short shelf life and are manufactured as single lots or small lots, presenting challenges for utilizing
[35][36][37][38]
compendial or culture-based methods for detecting microbial contamination.
When selecting a rapid method, the following should be taken into account:
a) the shelf life of the sample;
b) the volume of sample available for testing;
c) the number of samples to be tested;
d) the manufacturing step from which the sample will be collected;
e) the time to result;
f) the microorganisms to be detected;
g) how to distinguish viable from non-viable microorganisms;
h) the ability to speciate microbes that are identified in the sample.
In addition, it is important to take the availability of resources to conduct the tests into account such as
trained personnel and required instrumentation.
NOTE 1 Sampling can introduce microbial contamination into the manufacturing process.
NOTE 2 The amount of material available for testing can be limited, especially for autologous cellular
therapeutic products. In some cases, parallel cellular therapeutic products can be manufactured to assess
microbial contamination.
NOTE 3 Test methods can require specific training and experience to be conducted and analysed.
It is recommended to consider adding cell supernatants (e.g. culture solution, washing solution, frozen
stock solution) instead of cell containing solutions for sterility testing, to solve the problem of small
samples of cell products that cannot be sampled for testing.
5 Risk management for microbiological contamination
5.1 Risk management in manufacturing process
A risk-based approach for determining methods to detect microbial contamination in cellular
therapeutic product manufacturing should be used. It should take into account the source and method
[1][2][3][4]
used for the collection of the cellular starting material.
Potential sources of microbial contamination of cellular therapeutic products include, but are not limited
[5]
to, cellular starting material, raw materials and consumables, and the manufacturing environment.
Apheresis products are the most common source of cellular starting materials. Sources of microbial
contamination can be associated with the incomplete disinfection of the skin, sterility failures in
kits and bags used to collect and store the apheresis products, and technician/operator error. Donor
bacteraemia can also be a source of contamination for the apheresis product.
Microbial contamination and infectious viruses in reagents, ancillary (raw) materials and
recommendations for ancillary materials are described in ISO 20399. Consumables should be pre-
[6]
sterilized and single use to reduce the risk of microbial contamination.
Cell processing/manufacturing should be performed in a closed system or an appropriate clean room
(e.g. ISO 6 to ISO 7) to prevent microbial contamination.
NOTE Open or benchtop processes increase the risk of contamination from the air and surfaces that possibly
have not been adequately cleaned or disinfected.
Some general factors to be taken into account in a risk assessment for RMTMs are outlined in Annex A.
Detailed points to take into account in some critical decisions for the use of RMTMs in cellular
therapeutic product manufacturing that require a risk assessment can be found in Annexes B, C, D
[6][7]
and E.
Environmental controls can minimize the risk of microbial contamination. Examples of environmental
controls are:
— sanitization procedures;
— high efficiency particulate air (HEPA) filtration and air flow;
— gowning procedures;
— aseptic technique;
— clean-room procedures and classifications (in accordance with ISO 14644-1).
Points to take into account when developing risk control can include, but are not limited to:
a) input materials:
1) collection process and donor selection (see Annex B);
2) autologous or allogenic, fresh or frozen;
3) conditions;
b) ancillary materials in accordance with ISO 20399 and consumables (pre-sterilized, single use, etc.);
c) environmental factors;
d) equipment;
e) process steps:
1) closed or open process steps;
2) cell banking;
3) culture or expansion;
4) purification;
5) final product;
f) containment strategy (see Annex C);
g) monitoring (see Annex C);
h) storage, packaging and administration (see Annex D);
i) in-process and final-release testing (see Annex E).
5.2 Risk management in microbial testing
The use of a risk assessment approach to rapid microbial testing in cellular therapeutic product
[8][9][10][11]
manufacturing can limit the risk of validation of a rapid microbial testing system. A risk-
based approach can be used to establish the most appropriate rapid microbial testing mechanism
for intended use. This often focuses on determining the user requirement specifications (URS) as a
foundation.
NOTE The following documents discuss risk assessment and give general guidance on how to implement it
in manufacturing processes:
— ISO 31000;
— ISO 13022;
— ISO Guide 73.
6 Selection of a fit-for-purpose assay
6.1 General
Well- defined user requirements and a clear understanding of an assay’s intended use(s) guide the
design of assays with biological relevance and sufficient performance (e.g. analytical sensitivity,
analytical specificity, precision, accuracy, robustness) to enable subsequent decision-making (fit for the
intended purpose or fit for purpose).
To identify or develop an appropriate assay for detecting microbial contamination in cellular therapeutic
products, the goal of testing should be established and documented. For example, detection versus
quantification. If a test is needed to detect and identify “every” bacterial or fungal contaminant, then a
sequencing approach should be used. If a test is needed to determine taxonomic or quantity resolution
[35][36][37][38]
of only certain reference microorganisms or a limited list of compendial microorganisms,
then multiplexed PCR or a similar targeted approach is most suitable.
Appropriate assay design shall include specifications for the test method and strategies to ensure
measurement quality and reproducibility of results. This can include incorporating replicate
measurements, using sample randomization to reduce biases, and the inclusion of appropriate
measurement controls.
Appropriate assay design shall also include approaches to ensure an appropriate analytical sensitivity
and an established and documented detection limit. The uncertainty of the measurement should also
be established and documented.
The assay shall have a high analytical specificity for the measurement target without significant
interference from other components in the cell preparation.
The intended use of the assay should guide the fit-for-purpose requirements of the measurement. The
uncertainty of measurement should be taken into account.
To determine the appropriate assays, users shall assess the issues of the number and types of
microorganisms required for testing. The extent necessary for identification shall be determined. There
shall be an assessment made as to whether a determination between viable and non-viable microbial
cells is needed.
The assay should be sufficiently robust so that the results are not significantly affected by small changes
in the measurement process (e.g. temperature fluctuations, minor sample handling fluctuations) as
defined by the user for the intended purpose.
The assay should be sufficiently robust for the measurement target so that the results are not
significantly affected by small changes in other components of the cell preparation (e.g. serum
concentration, presence of preservation agents). When using non-compendial methods, the alternative
[35][36][37][38]
method should yield results equivalent or better than compendial methods.
6.2 Assay selection
[5]
Points to take into account when choosing a method include, but are not limited to, whether a method:
[9]
a) is based on sound underlying scientific principles;
b) can detect all the microorganisms which need to be detected;
c) can identify all such microorganisms to the required taxonomic level;
d) can reproducibly quantitate such identified microorganisms, if necessary;
e) can detect analytes in the concentration range of interest;
f) has sufficient analytical specificity and analytical sensitivity for the intended use;
g) can meet specific method performance criteria;
h) has adequate quality assurance (QA) and quality control (QC);
i) can be performed with readily available equipment;
j) uses appropriate resources;
k) has sufficient robustness;
l) addresses the level of expertise required (e.g. technique-sensitive areas, need of specialized
training);
m) contains necessary aspects of QA (e.g. calibrated equipment, media quality, incubation conditions,
amplicon quality, sequence data quality);
n) addresses biosafety concerns (e.g. necessity of specific biosafety practices to handle the test
pathogens);
o) meets specific requirements for the intended use (e.g. appropriate test parameters for materials
that contain bacteria from starting materials that cannot be sterilized through the manufacturing
process);
p) accounts for clinical conditions and natural cell variations within the patient population;
q) is a general microbial testing method or a specific microbial testing method;
r) is fit to detect bacteria, fungi and mycoplasma as appropriate;
s) can distinguish viable from non-viable microorganisms.
6.3 Kit or system selection
A vendor can refer to a contract laboratory that performs the test or does the validation or the
[12][13][14][15]
supplier of the kit or system. In release testing, a kit or system vendor should have proper
quality assessment and should follow guidelines for manufacturers of testing kits, or ISO 13485 when
applicable. The kit or system should be accompanied by applicable validation documents and services.
If a manufacturer is using an external kit or system vendor of RMTMs, the following points for the kit or
system vendor selection should be taken into account:
a) required QA certification of kit or system vendor;
b) QA standards employed by the kit or system vendor (e.g. ISO 9001);
c) audit history and plan (i.e. auditing by regulators, pharmaceutical companies or accreditation
bodies);
d) economic viability of kit or system vendor;
e) references for credibility of the test method (i.e. current user list, scientific publications or
accreditation);
f) technical support services available from the kit or system vendor;
g) documentation supplied by the kit or system vendor (i.e. evaluation and validation documentation).
6.4 Considerations for various test types
The microbial tests are risk-based, so the user can select the preferred technology for their intended
use and balance competing URS including the time to result, specificity, detection limit, sample size and
product attributes.
Recommended approach for microbial contamination detection options includes:
a) after completion of a batch, record the step that includes aseptic manipulations (e.g. manual
replenishment of the cell culture medium), and a sample should be taken to screen for microbial
contamination (i.e. in-process testing);
b) a sample should be taken in advance of the final step with the timing reflecting the screening
method employed, e.g. 48 h to 72 h prior to release for a growth-based contamination check or
shorter for a more rapid microbial test (i.e. surrogate finished product testing);
c) the final product can be subjected to a compendial or growth-based microbial test and released
before the completion of the test with the clinician responsible for the treatment of the patient
being notified if the test becomes positive (i.e. traditional finished product testing);
d) the final product should be tested for microbial contamination using a rapid microbial test and
released at the successful completion of the test (i.e. real-time finished product testing).
Table 1 describes these different release testing strategies with the detection options.
Further information and guidance on specification of various RMTM types can be found in Annex H.
[6]
Table 1 — In-process and release testing strategies
Technology Basis of the test Detection option Viable versus non-viable
distinction
Gram stain Differential staining of Final product testing no
bacterial cells
USP <71> sterility Growth in soybean-casein Final product testing yes
test digest and fluid
thioglycolate medium
Respiration CO production in In-process monitoring, timed yes
proprietary aerobic pre-release testing, final
product testing
Nucleic acid method Nucleic acid amplification Rapid release testing of final no
product
Flow cytometry Vital staining Rapid release testing of final yes
product
Solid phase Vital staining Rapid release testing of final yes
cytometry product
TTabablele 1 1 ((ccoonnttiinnueuedd))
Technology Basis of the test Detection option Viable versus non-viable
distinction
Adenosine ATP production in In-process monitoring, timed yes
triphosphate soybean casein digest pre-release testing, final
(ATP) and fluid thioglycolate product testing
bioluminesce
...