ISO/TS 22456:2021
(Main)Sterilization of healthcare products -- Microbiological methods— Guidance on conducting bioburden determinations and tests of sterility for biologics and tissue-based products
Sterilization of healthcare products -- Microbiological methods— Guidance on conducting bioburden determinations and tests of sterility for biologics and tissue-based products
1.1.1 This document provides guidance for bioburden testing and tests of sterility for biologics and tissue-based products, where this testing is in relation to product sterilization. NOTE This document is intended to be used in conjunction with ISO 11737-1 and ISO 11737-2. 1.1.2 Guidance in this document can be applicable to biologics and tissue-based products that are not sterile but are microbiologically controlled.
Stérilisation des produits de santé -- Méthodes microbiologiques -- Recommandations pour la réalisation de déterminations de la charge biologique et de contrôles de stérilité des produits biologiques et des produits à base de tissus
General Information
Buy Standard
Standards Content (sample)
TECHNICAL ISO/TS
SPECIFICATION 22456
First edition
2021-03
Sterilization of healthcare products —
Microbiological methods—
Guidance on conducting bioburden
determinations and tests of sterility
for biologics and tissue-based
products
Reference number
ISO/TS 22456:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 22456:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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 the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 22456:2021(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ..................................................................................................................................................................................................................................v
1 Scope ................................................................................................................................................................................................................................. 1
1.1 Inclusions ..................................................................................................................................................................................................... 1
1.2 Exclusions .................................................................................................................................................................................................... 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 2
4 Definition and maintenance of product families ................................................................................................................ 3
5 Selection and testing of product for bioburden and tests of sterility ............................................................3
5.1 General ........................................................................................................................................................................................................... 3
5.2 Nature of product ................................................................................................................................................................................. 4
5.3 Sample Item Portion (SIP) ............................................................................................................................................................ 4
5.4 Sampling conditions ........................................................................................................................................................................... 4
5.4.1 General...................................................................................................................................................................................... 4
5.4.2 Considerations for human tissue donor batches in sterilization ........................................... 4
5.4.3 Use of multiple batches .............................................................................................................................................. 5
5.4.4 Considerations for packaging ............................................................................................................................... 5
5.5 Microbiological testing ..................................................................................................................................................................... 5
5.5.1 Bioburden test considerations for biologics/tissues........................................................................ 5
5.5.2 Test of sterility considerations for biologics/tissues ....................................................................... 8
5.5.3 Verification of microbiological methods ..................................................................................................10
5.5.4 Rapid microbiology tests .......................................................................................................................................11
Bibliography .............................................................................................................................................................................................................................12
© ISO 2021 – All rights reserved iii---------------------- Page: 3 ----------------------
ISO/TS 22456:2021(E)
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
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
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 documents 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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.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 198, Sterilization of health care products.
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.iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 22456:2021(E)
Introduction
The sources and types of some microorganisms, as well as the test methods used to evaluate biologics
and tissue-based products, can be unique relative to other health care products, such as plastic and
metal medical devices. This document provides guidance to address issues that are applicable to the
microbiological testing of biologics and tissue-based products, where this testing constitutes bioburden
testing or a test of sterility performed in relation to product sterilization. Except where otherwise
indicated in this document, the requirements in ISO 11737-1:2018 and ISO 11737-2:2019 apply.
© ISO 2021 – All rights reserved v---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 22456:2021(E)
Sterilization of healthcare products — Microbiological
methods— Guidance on conducting bioburden
determinations and tests of sterility for biologics and
tissue-based products
1 Scope
1.1 Inclusions
1.1.1 This document provides guidance for bioburden testing and tests of sterility for biologics and
tissue-based products, where this testing is in relation to product sterilization.
NOTE This document is intended to be used in conjunction with ISO 11737-1 and ISO 11737-2.
1.1.2 Guidance in this document can be applicable to biologics and tissue-based products that are not
sterile but are microbiologically controlled.1.2 Exclusions
1.2.1 This document does not include guidance for validation requirements for testing, eliminating
and/or inactivating viruses and prions or sterilization of tissue-based products.
NOTE Guidance on inactivating viruses and prions can be found in ISO 22442-3.1.2.2 This document does not include guidance for containment or biosafety issues for biologics and
tissue-based products.1.2.3 This document does not include guidance for testing biologics and tissue-based products for
specific infectious agents as listed in relevant national or international guidance (e.g. viruses/protozoa/
parasites, intracellular microorganisms or mycoplasma screening).1.2.4 This document does not include guidance for the acceptance criteria for biologics and tissue-
based products during procurement or tissue to be processed and/or released for use.
1.2.5 This document does not include guidance for the testing associated with procurement and
screening of biologics and tissue-based products.2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 11737-1:2018, Sterilization of health care products — Microbiological methods — Part 1: Determination
of a population of microorganisms on productsISO 11737-2:2019, Sterilization of health care products — Microbiological methods — Part 2: Tests of
sterility performed in the definition, validation and maintenance of a sterilization process
© ISO 2021 – All rights reserved 1---------------------- Page: 6 ----------------------
ISO/TS 22456:2021(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 11737-1:2018, ISO 11737-2:2019
and the following apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp— IEC Electropedia: available at http:// www .electropedia .org/
3.1
biologics
product that is synthesized from living organisms, or their products, and used as a diagnostic,
preventive or therapeutic agent3.2
companion tissue
tissue from the same donor(s) that is not intended to be used for transplantation
Note 1 to entry: For the purposes of this document, companion tissue is expected to be processed in the same
manner as tissue that is used for transplantation. Companion tissue is representative of tissue intended for
transplantation but is only used for evaluation and/or testing purposes.3.3
donor identification
unique identifier assigned to all biologics (3.1)/tissue and companion tissue (3.2) that originates from
the same donor3.4
method suitability test
bacteriostasis/fungistasis (B/F) test
technical operation performed to detect the presence of substances that inhibit microbial multiplication
Note 1 to entry: This testing is also referred to as “method verification.”[SOURCE: ISO 11139:2018, 3.20, modified — Note 1 to entry has been added]
3.5
processing
any activity performed in the preparation, manipulation,
preservation for storage and packaging of a biological or tissue-based product3.6
product family
group or subgroup of product characterized by similar attributes determined to be equivalent for
evaluation and processing purposes[SOURCE: ISO 11139:2018, 3.218]
3.7
tissue-based product
product consisting of organization of cells, cells and extra-cellular constituents, or extra- cellular
constituentsNote 1 to entry: This can include tissues from tissue banks or material of animal origin.
2 © ISO 2021 – All rights reserved---------------------- Page: 7 ----------------------
ISO/TS 22456:2021(E)
4 Definition and maintenance of product families
4.1 Product families can be organized for a multitude of purposes. The purpose for which the family
is being organized will dictate the criteria for that family. Care should be taken in organizing families to
ensure that appropriate and relevant criteria are in place.4.2 In cases where a product family representative is tested instead of each type of health care product,
an appropriate rationale should be written to ensure that results for the product family representative
are representative of the whole family. The applicable standards, i.e. ISO 11137-2, ISO 11135, etc. provide
guidance specific to establishment of a product family. Some of that guidance can be applicable to
establishment of a product family for other testing purposes. For other sterilization modalities, refer to
the applicable standard for additional guidance. Additionally, the following items can be considered:
a) the purpose for which the family is being established (e.g. for sterilization purposes, or for
bioburden testing purposes);b) processing procedures that are applied to the biologic/tissue;
c) storage conditions that are applied to the various tissue types or sizes;
d) tissue collection methods.
4.3 For biologics or tissue-based product that is sterilized, the final determination of the product
family representative is based on recommendations per the relevant sterilization process standard (e.g.
ISO 11137-2 for radiation, ISO 11135 for EO, etc.).4.4 In considering the relative size of biologic/tissue products, a larger size might not necessarily
correspond to a higher product bioburden if processing of the product is the same. If equivalence can
be demonstrated within a family of products, a rationale should be provided. If equivalence cannot be
demonstrated, either a larger size should be tested, or an SIP employed (see 5.3).
5 Selection and testing of product for bioburden and tests of sterility5.1 General
The results of raw material pre-disinfection cultures of a biologic/tissue are typically utilized to
determine the suitability of that biologic/tissue for further processing (e.g. collection, cleaning
processes, disinfection processes, washing, soaking, etc.), or its supplier, or for the defined monitoring
program and can also be used for trending/monitoring purposes.In performing bioburden applicable to a sterilization process, such as dose- establishment or for routine
bioburden monitoring, the relevant bioburden is that present on the product immediately prior to
sterilization. If product is subjected to cleaning, rinsing and disinfection steps, the bioburden is usually
low and comparable to the bioburden (numbers and types) of other healthcare products. This is due to:
a) the reduction of the bioburden through the cleaning processes, disinfection processes, washing,
soaking, ultrasonication and/or centrifugation or other processes;NOTE 1 If these processes are not appropriately controlled and monitored, they could introduce
additional bioburden to the product.b) the contribution of microorganisms from the environment, contact surfaces and handling during
processing or manufacturing steps.NOTE 2 Refer to ISO 11737-1:2018 for guidance on bioburden characterization. ISO 14160 provides
guidance on characterization and evaluation for pre-sterilization bioburden for liquid chemical sterilization
used on animal tissues.© ISO 2021 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO/TS 22456:2021(E)
NOTE 3 As described in ISO 11737-1:2018, it is not necessary in all cases to fully characterize product
bioburden. The extent of characterization performed is expected to be based on the purpose for which the
testing is being carried out.NOTE 4 When evaluating bioburden to support a microbicidal process validation (i.e. disinfection,
sterilization), the resistance of the species to the relevant microbicidal process is more relevant than its
pathogenicity. A pathogenic microorganism could have a low resistance to the disinfectant/sterilant, while a
non-pathogenic microorganism could be highly resistant.5.2 Nature of product
Processing of biologics/tissues should be controlled to maintain low or consistent levels of bioburden.
The unique source of these biologics/tissues, compared to other health care products made of synthetic
materials, creates the potential for a microflora different than that found on plastics or metal devices.
However, due to the processing, antimicrobial treatment and chemicals often applied to biologics/
tissues, the bioburden after controlled and proven processing tends to be relatively consistent and
low. When a biologic/tissue is pooled during processing it typically results in a relatively consistent
bioburden within the pool. See also ISO 11737-1:2018.5.3 Sample Item Portion (SIP)
In many cases a limited amount of biologic/tissue product is available for testing, therefore, it is
reasonable to utilize product that is not clinically suitable or is non- conforming product, but which
is handled in the same manner and undergoes the entire manufacturing process. For example, to
maintain an SIP of 1,0, a biologic/tissue can be used that is not considered clinically suitable for use but
is microbiologically representative of the biologic/tissue and process.In general, if an SIP < 1,0 is used, a rationale applying the principles of ISO 11737-1:2018 should be used.
If an SIP <1,0 is used, qualification of the SIP could be required. Use of a product that is smaller than
the largest product produced can also be addressed through establishment of product families and
substantiation of an equivalent product approach. For radiation sterilization, guidance is provided in
ISO 11137-2.NOTE If it has been demonstrated that product within a product family is considered equivalent because of
the disinfection process and rationale, and if smaller size pieces or quantities of a biologic/tissue in that family
are used for dose establishment, all sizes are considered an SIP=1.5.4 Sampling conditions
5.4.1 General
The conditions the test sample has been exposed to should represent typical shipping, storage and
processing conditions, including any refrigeration, frozen conditions or lyophilization. Shipping, storage
and sampling conditions should be designed to minimize conditions which are conducive to microbial
growth on the biologic/tissue.5.4.2 Considerations for human tissue donor batches in sterilization
For medical devices, the term batch means a quantity of identical devices manufactured under similar
conditions. Some sterilization validations (e.g. radiation) require that a specific number of products be
tested from each batch. Human tissue-based products and some biologics are typically processed in
batches based on a single donor ("processing batch"). Processing batch sizes often vary in the number
of products per batch. Products in a processing batch might not be multiples of the same product type.
For example, a human tissue batch is often a mixture of tissues from multiple sites within the donor
that are further processed together, and the number of products per batch can depend on the donor and
[11]might be small. Refer to Kowalski. Due to this distinction in definition and constituents of a batch for
biologics/tissues, some allowance, with documented rationale, should be made regarding sample sizes
and batches when following standards initially written for other types of health care products.
4 © ISO 2021 – All rights reserved---------------------- Page: 9 ----------------------
ISO/TS 22456:2021(E)
5.4.3 Use of multiple batches
Biologics/tissues from different processing batches and/or donor identifications may be combined for
testing purposes in order to achieve the required number of samples for the particular bioburden or
sterilization method, based on the rationale for the sampling. For example, it might be necessary to
use five test samples from each of six batches rather than 10 from each of the three batches in order
to obtain a sample size of 30. It is the number of test samples that is critical, not the number of batches
from which they originate as long as a minimum of 3 batches are represented. A batch may be given a
unique identifier apart from the processing batch and/or donor identification, as long as traceability to
the original donor samples comprising the batch is maintained.NOTE For qualification of biologics/tissues in radiation dose setting, the batch sampling concept from
ISO 11137-2 can be modified.5.4.4 Considerations for packaging
Typically, a bioburden determination or a test of sterility is performed on product after its removal
from its packaging system. It is common to omit the packaging system from these determinations.
Depending on the intent for sterility, internal packaging components, such as a tray or product insert,
might need to be tested based upon factors such as whether:a) the component is intended to be sterile;
b) the package is an integral part of the product; or
c) specific evaluation is required.
When packaging is tested, it might be preferred that it be tested separately from product, depending on
the circumstances.5.5 Microbiological testing
5.5.1 Bioburden test considerations for biologics/tissues
5.5.1.1 General
Establishing and maintaining a sterilization dose or bioburden-based sterilization process is dependent
upon an estimated determination of bioburden. The requirements, guidelines, and processes that are
in place for biologic/tissue products usually result in finished products that are relatively consistent
and low in bioburden if requirements and guidelines for microbial control are appropriately followed.
However, there are some characteristics that are unique to biologic/tissue types compared to other
health care products. For sterilization validations that utilize an overkill approach (e.g. typically using
a biological indicator and 12-log reduction), the sterilization process is not directly dependent on the
product bioburden count. In some cases, the bioburden count is used for trending and demonstration of
process control.Characteristics of biologics/tissues that can affect the determination of bioburden include:
a) Each human tissue donor or animal tissue batch is a separate case, therefore bioburden diversity can
vary. The bioburden might contain microbial species that are not commonly associated with typical
medical device materials, such as synthetic materials or metal alloys. Based on an understanding
of the biologic/tissue type and processes involved, an assessment should be performed of the
predominant types of microorganisms present at a stage in the process that is appropriate to
the purpose for the assessment. For example, in the bioburden assessment for a bioburden-based
sterilization process, the appropriate stage is usually immediately prior to sterilization rather
than upon receipt (which is prior to cleaning and disinfection). The information obtained in this
assessment should be used to develop appropriate test methods. For example, with some tissue
types it could be appropriate to test for anaerobic microorganisms, increase incubation time or use
specialized media, where this practice might not be common for other health care products.
© ISO 2021 – All rights reserved 5---------------------- Page: 10 ----------------------
ISO/TS 22456:2021(E)
b) Some tissues can include complex surface characteristics. The location and adherence of
microorganisms on and/or in certain complex tissues could make them difficult to remove. Thus,
the bioburden recovery efficiency should be evaluated and used as appropriate for bioburden
determination.ISO 11737-1:2018 is primarily written in the context of conventional medical devices, but includes
methods that are also applicable to most biologics/tissues, including shaking, ultrasonication,
stomaching, blending, culturing, membrane filtration with rinsing for neutralization, and recovery
evaluation. The aspects in 5.5.1.2 to 5.5.1.10 should be considered when performing bioburden
tests on biologics/tissues.If bioburden samples are pooled for testing, reference ISO 11737-1:2018 for additional information.
5.5.1.2 Extraction fluidGenerally, the extraction fluids used with conventional medical devices are also appropriate to use with
biologic/tissue products. Residual processing chemicals and antibiotics can be difficult to completely
remove from some biologics/tissues and could leach into extraction fluids during testing. Thus, if
inhibitory substances are present, it might be necessary to use a neutralizer in the extraction fluid,
rinsing, or a neutralizing step to the testing process to prevent inhibition of microbial growth. Most
extraction fluids can be filtered, which can help to eliminate inhibitory factors from the tests.
Guidance on general bioburden testing eluents and diluents is provided in ISO 11737-1:2018. Guidance
on method suitability to demonstrate the absence/presence of inhibitory substances can be found in
5.5.3.1. Additional guidance can be found in Annex B of ISO 11737-1:2018, which describes methods to
screen for release of substances affecting bioburden determinations.5.5.1.3 Extraction methods
Bioburden on biologics/tissues tends to be influenced by the environment the biologic/tissue exterior
is exposed to, and thus the bioburden is primarily found externally. Based on an understanding
of the biologic/tissue processing, if the bioburden is expected to reside externally, traditional
extraction methods are appropriate. These methods can include mechanical shaking, manual shaking,
ultrasonication or stomaching. If it is expected that bioburden might also be located inside the biologic/
tissue, other extraction methods could be indicated. These methods can include blending, stomaching,
grinding/milling, maceration or enzymatic digestion. The effect of such treatment on product bioburden
should be evaluated.5.5.1.4 Membrane filter selection
Membrane filtration may be used to facilitate removal of inhibitory substances in the extract or rinse
solution(s). If inhibition is observed, cellulose-based filters commonly used for bioburden testing
are prone to trap antibiotics and/or chemicals inside the filter, even after multiple rinses. Therefore,
certain filter types that are less apt to retain residual antibiotics and/or chemicals during filtration
should be used for biologics/tissues. Polycarbonate, nylon and hydrophobic-edge filters could be better
suited for filtration of fluids that contain inhibitory substances. Wetting the filter with sterile fluid
prior to filtration and adequately rinsing the filter with sterile fluid after filtration can also be helpful
in reducing inhibitory substances on membrane filters. Even with the use of these filters and methods,
additional neutralization steps might still be necessary. Guidance on method suitability to demonstrate
the absence/presence of inhibitory substances can be found in 5.5.3.1. Additional guidance can be found
in Annex B of ISO 11737-1:2018, which describes methods to screen for release of substances affecting
bioburden determinations.There can be cases where a membrane filter with a nominal pore size less than 0,45 µm is required, for
example, to minimize loss of contaminants that can pass through a 0,45 µm filter.
6 © ISO 2021 – All rights reserved---------------------- Page: 11 ----------------------
ISO/TS 22456:2021(E)
5.5.1.5 Filtration considerations
Some biologic/tissue extracts might not filter well due to particulates, lipids and/or fats that sometimes
elute from the biologic/tissue during extraction. In these situations, smaller volumes can be filtered
through multiple filters, or direct culture methods can be used to test the extract solution, such as pour
plates, spread plates and the most probable number (MPN) test. A large pore pre-filter can be used
to reduce the quantity of materials in solution that could obstruct filters. However, use of a pre-filter
can also trap microorganisms. Thus, an evaluation should be performed to account for microorganisms
that could be trapped by the pre-filter.5.5.1.6 Most Probable Number (MPN)
MPN testing is an option provided in ISO 11737-1:2018 and can be a good option for some biologic/
tissue products (see B.3.3 of ISO 11737-1:2018). This is because most biologics/tissues from a donor or
batch are processed together in the same disinfecting/rinsing solutions, assisting in creating a more
evenly distributed bioburden. Also the MPN method can be desirable because bioburden can b
...TECHNICAL ISO/TS
SPECIFICATION 22456
First edition
Sterilization of healthcare products —
Microbiological Methods—
Guidance on conducting bioburden
determinations and tests of sterility
for tissue-based products
PROOF/ÉPREUVE
Reference number
ISO/TS 22456:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 22456:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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 the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 22456:2021(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
Introduction ..................................................................................................................................................................................................................................v
1 Scope ................................................................................................................................................................................................................................. 1
1.1 Inclusions ..................................................................................................................................................................................................... 1
1.2 Exclusions .................................................................................................................................................................................................... 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 2
4 Definition and maintenance of product families ................................................................................................................ 3
5 Selection and testing of product for bioburden and tests of sterility ............................................................3
5.1 General ........................................................................................................................................................................................................... 3
5.2 Nature of product ................................................................................................................................................................................. 4
5.3 Sample Item Portion (SIP) ............................................................................................................................................................ 4
5.4 Sampling conditions ........................................................................................................................................................................... 4
5.4.1 General...................................................................................................................................................................................... 4
5.4.2 Considerations for human tissue donor batches in sterilization ........................................... 4
5.4.3 Use of multiple batches .............................................................................................................................................. 5
5.4.4 Considerations for packaging ............................................................................................................................... 5
5.5 Microbiological testing ..................................................................................................................................................................... 5
5.5.1 Bioburden test considerations for biologics/tissues........................................................................ 5
5.5.2 Test of sterility considerations for biologics/tissues ....................................................................... 8
5.5.3 Verification of microbiological methods ..................................................................................................10
5.5.4 Rapid microbiology tests .......................................................................................................................................11
Bibliography .............................................................................................................................................................................................................................12
© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii---------------------- Page: 3 ----------------------
ISO/TS 22456:2021(E)
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
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
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 documents 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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.For an explanation on 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 the following
URL: www .iso .org/ iso/ foreword .html.This document was prepared by Technical Committee ISO/TC 198, Sterilization of health care products.
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.iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 22456:2021(E)
Introduction
The sources and types of some microorganisms, as well as the test methods used to evaluate biologics
and tissue-based products, can be unique relative to other health care products, such as plastic and
metal medical devices. This document provides guidance to address issues that are applicable to the
microbiological testing of biologics and tissue-based products, where this testing constitutes bioburden
testing or a test of sterility performed in relation to product sterilization. Except where otherwise
indicated in this document, the requirements in ISO 11737-1:2018 and ISO 11737-2:2019 should be
followed.© ISO 2021 – All rights reserved PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 22456:2021(E)
Sterilization of healthcare products — Microbiological
Methods— Guidance on conducting bioburden
determinations and tests of sterility for tissue-based
products
1 Scope
1.1 Inclusions
1.1.1 This document provides guidance for bioburden testing and tests of sterility for biologics and
tissue-based products, where this testing is in relation to product sterilization.
NOTE This document is intended to be used in conjunction with ISO 11737-1 and ISO 11737-2.
1.1.2 Guidance in this document can be applicable to biologics and tissue-based products that are not
sterile but are microbiologically controlled.1.2 Exclusions
1.2.1 This document does not include guidance for validation requirements for testing, eliminating
and/or inactivating viruses and prions or sterilization of tissue-based products.
NOTE Guidance on inactivating viruses and prions can be found in ISO 22442-3.1.2.2 This document does not include guidance for containment or biosafety issues for biologics and
tissue-based products.1.2.3 This document does not include guidance for testing biologics and tissue-based products for
specific infectious agents as listed in relevant national or international guidance (e.g. viruses/protozoa/
parasites, intracellular microorganisms or mycoplasma screening).1.2.4 This document does not include guidance for the acceptance criteria for biologics and tissue-
based products during procurement or tissue to be processed and/or released for use.
1.2.5 This document does not include guidance for the testing associated with procurement and
screening of biologics and tissue-based products.2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 11737-1:2018, Sterilization of health care products — Microbiological methods — Part 1: Determination
of a population of microorganisms on productsISO 11737-2:2019, Sterilization of health care products — Microbiological methods — Part 2: Tests of
sterility performed in the definition, validation and maintenance of a sterilization process
© ISO 2021 – All rights reserved PROOF/ÉPREUVE 1---------------------- Page: 6 ----------------------
ISO/TS 22456:2021(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 11737-1:2018, ISO 11737-2:2019
and the following apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp— IEC Electropedia: available at http:// www .electropedia .org/
3.1
biologics
product that is synthesized from living organisms, or their products, and used as a diagnostic,
preventive or therapeutic agent3.2
companion tissue
tissue from the same donor(s) that is not intended to be used for transplantation
Note 1 to entry: For the purposes of this document, companion tissue is expected to be processed in the same
manner as tissue that is used for transplantation. Companion tissue is representative of tissue intended for
transplantation but is only used for evaluation and/or testing purposes.3.3
donor identification
unique identifier assigned to all biologics (3.1)/tissue and companion tissue that originates from the
same donor3.4
method suitability test
bacteriostasis/fungistasis (B/F) test
technical operation performed to detect the presence of substances that inhibit microbial multiplication
Note 1 to entry: This testing is also referred to as “method verification.”[SOURCE: ISO 11139:2018, 3.20, modified — Note 1 to entry has been added]
3.5
processing
any activity performed in the preparation, manipulation,
preservation for storage and packaging of a biological or tissue-based product3.6
product family
group or subgroup of product characterized by similar attributes determined to be equivalent for
evaluation and processing purposes[SOURCE: ISO 11139:2018, 3.218]
3.7
tissue-based product
product consisting of organization of cells, cells and extra-cellular constituents, or extra- cellular
constituentsNote 1 to entry: This can include tissues from tissue banks or material of animal origin.
2 PROOF/ÉPREUVE © ISO 2021 – All rights reserved---------------------- Page: 7 ----------------------
ISO/TS 22456:2021(E)
4 Definition and maintenance of product families
4.1 Product families can be organized for a multitude of purposes. The purpose for which the family
is being organized will dictate the criteria for that family. Care should be taken in organizing families to
ensure that appropriate and relevant criteria are in place.4.2 In cases where a product family representative is tested instead of each type of health care product,
an appropriate rationale should be written to ensure that results for the product family representative
are representative of the whole family. The applicable standards, i.e. ISO 11137-2, ISO 11135, etc. provide
guidance specific to establishment of a product family. Some of that guidance can be applicable to
establishment of a product family for other testing purposes. For other sterilization modalities, refer to
the applicable standard for additional guidance. Additionally, the following items can be considered:
a) the purpose for which the family is being established (e.g. for sterilization purposes, or for
bioburden testing purposes);b) processing procedures that are applied to the biologic/tissue;
c) storage conditions that are applied to the various tissue types or sizes;
d) tissue collection methods.
4.3 For biologics or tissue-based product that is sterilized, the final determination of the product
family representative is based on recommendations per the relevant sterilization process standard (e.g.
ISO 11137-2 for radiation, ISO 11135 for EO, etc.).4.4 In considering the relative size of biologic/tissue products, a larger size might not necessarily
correspond to a higher product bioburden if processing of the product is the same. If equivalence can
be demonstrated within a family of products, a rationale should be provided. If equivalence cannot be
demonstrated, either a larger size should be tested, or an SIP employed (see 5.3).
5 Selection and testing of product for bioburden and tests of sterility5.1 General
The results of raw material pre-disinfection cultures of a biologic/tissue are typically utilized to
determine the suitability of that biologic/tissue for further processing (e.g. collection, cleaning
processes, disinfection processes, washing, soaking, etc.), or its supplier, or for the defined monitoring
program and can also be used for trending/monitoring purposes.In performing bioburden applicable to a sterilization process, such as dose- establishment or for routine
bioburden monitoring, the relevant bioburden is that present on the product immediately prior to
sterilization. If product is subjected to cleaning, rinsing, and disinfection steps, the bioburden is usually
low and comparable to the bioburden of other health care products. If product is subjected to cleaning,
rinsing and disinfection steps, the bioburden is usually low and comparable to the bioburden (numbers
and types) of other healthcare products. This is due to:a) the reduction of the bioburden through the cleaning processes, disinfection processes, washing,
soaking, ultrasonication and/or centrifugation or other processes;NOTE 1 If these processes are not appropriately controlled and monitored, they could introduce
additional bioburden to the product.b) the contribution of microorganisms from the environment, contact surfaces and handling during
processing or manufacturing steps.© ISO 2021 – All rights reserved PROOF/ÉPREUVE 3
---------------------- Page: 8 ----------------------
ISO/TS 22456:2021(E)
NOTE 2 Refer to ISO 11737-1:2018 for guidance on bioburden characterization. ISO 14160 provides
guidance on characterization and evaluation for pre-sterilization bioburden for liquid chemical sterilization
used on animal tissues.NOTE 3 As described in ISO 11737-1:2018, it is not necessary in all cases to fully characterize product
bioburden. The extent of characterization performed is expected to be based on the purpose for which the
testing is being carried out.NOTE 4 When evaluating bioburden to support a microbicidal process validation (i.e. disinfection,
sterilization), the resistance of the species to the relevant microbicidal process is more relevant than its
pathogenicity. A pathogenic microorganism could have a low resistance to the disinfectant/sterilant, while a
non-pathogenic microorganism could be highly resistant.5.2 Nature of product
Processing of biologics/tissues should be controlled to maintain low or consistent levels of bioburden.
The unique source of these biologics/tissues, compared to other health care products made of synthetic
materials, creates the potential for a microflora different than that found on plastics or metal devices.
However, due to the processing, antimicrobial treatment and chemicals often applied to biologics/
tissues, the bioburden after controlled and proven processing tends to be relatively consistent and
low. When a biologic/tissue is pooled during processing it typically results in a relatively consistent
bioburden within the pool. See also ISO 11737-1:2018.5.3 Sample Item Portion (SIP)
In many cases a limited amount of biologic/tissue product is available for testing, therefore, it is
reasonable to utilize product that is not clinically suitable or is non- conforming product, but which
is handled in the same manner and undergoes the entire manufacturing process. For example, to
maintain an SIP of 1,0, a biologic/tissue can be used that is not considered clinically suitable for use but
is microbiologically representative of the biologic/tissue and process.In general, if an SIP < 1,0 is used, a rationale applying the principles of ISO 11737-1:2018 should be used.
If an SIP <1,0 is used, qualification of the SIP could be required. Use of a product that is smaller than
the largest product produced can also be addressed through establishment of product families and
substantiation of an equivalent product approach. For radiation sterilization, guidance is provided in
ISO 11137-2.NOTE If it has been demonstrated that product within a product family is considered equivalent because of
the disinfection process and rationale, and if smaller size pieces or quantities of a biologic/tissue in that family
are used for dose establishment, all sizes are considered an SIP=1.5.4 Sampling conditions
5.4.1 General
The conditions the test sample has been exposed to should represent typical shipping, storage and
processing conditions, including any refrigeration, frozen conditions or lyophilization. Shipping, storage
and sampling conditions should be designed to minimize conditions which are conducive to microbial
growth on the biologic/tissue.5.4.2 Considerations for human tissue donor batches in sterilization
For medical devices, the term batch means a quantity of identical devices manufactured under similar
conditions. Some sterilization validations (e.g. radiation) require that a specific number of products be
tested from each batch. Human tissue-based products and some biologics are typically processed in
batches based on a single donor ("processing batch"). Processing batch sizes often vary in the number
of products per batch. Products in a processing batch might not be multiples of the same product type.
For example, a human tissue batch is often a mixture of tissues from multiple sites within the donor
4 PROOF/ÉPREUVE © ISO 2021 – All rights reserved---------------------- Page: 9 ----------------------
ISO/TS 22456:2021(E)
that are further processed together, and the number of products per batch can depend on the donor and
[11]might be small. Refer to Kowalski. Due to this distinction in definition and constituents of a batch for
biologics/tissues, some allowance, with documented rationale, should be made regarding sample sizes
and batches when following standards initially written for other types of health care products.
5.4.3 Use of multiple batchesBiologics/tissues from different processing batches and/or donor identifications may be combined for
testing purposes in order to achieve the required number of samples for the particular bioburden or
sterilization method, based on the rationale for the sampling. For example, it might be necessary to
use five test samples from each of six batches rather than 10 from each of the three batches in order to
obtain a sample size of 30. It is the number of test samples which is critical, not the number of batches
from which they originate as long as a minimum of 3 batches are represented. A batch may be given a
unique identifier apart from the processing batch and/or donor identification, as long as traceability to
the original donor samples comprising the batch is maintained.NOTE For qualification of biologics/tissues in radiation dose setting, the batch sampling concept from
ISO 11137-2 can be modified.5.4.4 Considerations for packaging
Typically, a bioburden determination or a test of sterility is performed on product after its removal
from its packaging system. It is common to omit the packaging system from these determinations.
Depending on the intent for sterility, internal packaging components, such as a tray or product insert,
might need to be tested based upon factors such as whether:a) the component is intended to be sterile;
b) the package is an integral part of the product; or
c) specific evaluation is required.
When packaging is tested, it might be preferred that it be tested separately from product, dependent on
the circumstances.5.5 Microbiological testing
5.5.1 Bioburden test considerations for biologics/tissues
5.5.1.1 General
Establishing and maintaining a sterilization dose or bioburden-based sterilization process is dependent
upon an estimated determination of bioburden. The requirements, guidelines, and processes that are
in place for biologic/tissue products usually result in finished products that are relatively consistent
and low in bioburden if requirements and guidelines for microbial control are appropriately followed.
However, there are some characteristics that are unique to biologic/tissue types compared to other
health care products. For sterilization validations that utilize an overkill approach (e.g. typically using
a biological indicator and 12-log reduction), the sterilization process is not directly dependent on the
product bioburden count. In some cases, the bioburden count is used for trending and demonstration of
process control.Characteristics of biologics/tissues that can affect the determination of bioburden include:
a) Each human tissue donor or animal tissue batch is a separate case, therefore bioburden diversity can
vary. The bioburden might contain microbial species that are not commonly associated with typical
medical device materials, such as synthetic materials or metal alloys. Based on an understanding
of the biologic/tissue type and processes involved, an assessment should be performed of the
predominant types of microorganisms present at a stage in the process that is appropriate to
the purpose for the assessment. For example, in the bioburden assessment for a bioburden-based
© ISO 2021 – All rights reserved PROOF/ÉPREUVE 5---------------------- Page: 10 ----------------------
ISO/TS 22456:2021(E)
sterilization process, the appropriate stage is usually immediately prior to sterilization rather
than upon receipt (which is prior to cleaning and disinfection). The information obtained in this
assessment should be used to develop appropriate test methods. For example, with some tissue
types it could be appropriate to test for anaerobic microorganisms, increase incubation time or use
specialized media, where this practice might not be common for other health care products.
b) Some tissues can include complex surface characteristics. The location and adherence of
microorganisms on and/or in certain complex tissues could make them difficult to remove Thus,
the bioburden recovery efficiency should be evaluated and used as appropriate for bioburden
determination.ISO 11737-1:2018 is primarily written in the context of conventional medical devices, but includes
methods that are also applicable to most biologics/tissues, including shaking, ultrasonication,
stomaching, blending, culturing, membrane filtration with rinsing for neutralization, and recovery
evaluation. The aspects in 5.5.1.2 to 5.5.1.10 should be considered when performing bioburden
tests on biologics/tissues.If bioburden samples are pooled for testing, reference ISO 11737-1:2018 for additional information.
5.5.1.2 Extraction fluidGenerally, the extraction fluids used with conventional medical devices are also appropriate to use with
biologic/tissue products. Residual processing chemicals and antibiotics can be difficult to completely
remove from some biologics/tissues and could leach into extraction fluids during testing. Thus, if
inhibitory substances are present, it might be necessary to use a neutralizer in the extraction fluid,
rinsing, or a neutralizing step to the testing process to prevent inhibition of microbial growth. Most
extraction fluids can be filtered, which can help to eliminate inhibitory factors from the tests.
Guidance on general bioburden testing eluents and diluents is provided in ISO 11737-1:2018. Guidance
on method suitability to demonstrate the absence/presence of inhibitory substances can be found in
5.5.3.1. Additional guidance can be found in Annex B of ISO 11737-1:2018, which describes methods to
screen for release of substances affecting bioburden determinations.5.5.1.3 Extraction methods
Bioburden on biologics/tissues tends to be influenced by the environment the biologic/tissue exterior
is exposed to, and thus the bioburden is primarily found externally. Based on an understanding
of the biologic/tissue processing, if the bioburden is expected to reside externally, traditional
extraction methods are appropriate. These methods can include mechanical shaking, manual shaking,
ultrasonication or stomaching. If it is expected that bioburden might also be located inside the biologic/
tissue, other extraction methods could be indicated. These methods can include blending, stomaching,
grinding/milling, maceration or enzymatic digestion. The effect of such treatment on product bioburden
should be evaluated.5.5.1.4 Membrane filter selection
Membrane filtration may be used to facilitate removal of inhibitory substances in the extract or rinse
solution(s). If inhibition is observed, cellulose-based filters commonly used for bioburden testing
are prone to trap antibiotics and/or chemicals inside the filter, even after multiple rinses. Therefore,
certain filter types that are less apt to retain residual antibiotics and/or chemicals during filtration
should be used for biologics/tissues. Polycarbonate, nylon and hydrophobic-edge filters could be better
suited for filtration of fluids that contain inhibitory substances. Wetting the filter with sterile fluid
prior to filtration and adequately rinsing the filter with sterile fluid after filtration can also be helpful
in reducing inhibitory substances on membrane filters. Even with the use of these filters and methods,
additional neutralization steps might still be necessary. Guidance on method suitability to demonstrate
the absence/presence of inhibitory substances can be found in 5.5.3.1. Additional guidance can be found
in Annex B of ISO 11737-1:2018, which describes methods to screen for release of substances affecting
bioburden determinations.6 PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 11 ----------------------
ISO/TS 22456:2021(E)
There can be cases where a membrane filter with a nominal pore size less than 0.45 µm is required, for
example, to minimize loss of contaminants that can pass through a 0.45 µm filter.
5.5.1.5 Filtration considerationsSome biologic/tissue extracts might not filter well due to particulates, lipids and/or fats that sometimes
elute from the biologic/tissue during extraction. In these situations, smaller volumes can be filtered
through multiple filters, or direct culture methods can be used to test the extract solution, such as pour
plates, spread plates and the most probable number (MPN) test. A large pore pre-filter can be used
to reduce the quantity of materials in solution that could obstruct filters. However, use of a pre-filter
can also trap microorganisms. Thus, an evaluation should be performed to account for microorganisms
that could be trapped by the pre-filter.5.5.1.6 Most Probable Number (MPN)
MPN testing is an option provided in ISO 11737-1:2018 and can be a good option for some biologic/
tiss...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.