ISO/TS 22456:2021

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 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved

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
Foreword
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iv © ISO 2021 – All rights reserved

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.
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 products
ISO 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
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 agent
3.2
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 donor
3.4
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 product
3.6
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
constituents
Note 1 to entry: This can include tissues from tissue banks or material of animal origin.
2 © ISO 2021 – All rights reserved

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 sterility
5.1 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.
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

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.
b) Some tissues can include complex surface characteristics. The location and adherence of
microorganisms on
...