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

Status

Published

Publication Date

18-Mar-2021

ICS

07.100.10 - Medical microbiology

11.080.01 - Sterilization and disinfection in general

Technical Committee

ISO/TC 198 - Sterilization of health care products

Drafting Committee

ISO/TC 198/WG 8 - Microbiological methods

Current Stage

5060 - Close of voting Proof returned by Secretariat

Start Date

10-Feb-2021

Completion Date

10-Feb-2021

Ref Project

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ISO/TS 22456:2021 - Sterilization of healthcare products -- Microbiological methods— Guidance on conducting bioburden determinations and tests of sterility for biologics and tissue-based products

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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

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 (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
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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
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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

© ISO 2021 – All rights reserved 1
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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 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
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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 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.
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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
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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.

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 fluid

Generally, 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.

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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

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