INTERNATIONAL
`STANDARD
`
`ISO
`14161
`
`First edition
`2000-10-01
`
`Sterilization of health care products -
`Biological indicators - Guidance for the
`selection, use and interpretation of results
`
`lndicateurs biologiques - Directives
`Sterilisation des produits de sante -
`generates pour la selection, /'utilisation et /'interpretation des resultats
`
`~ -
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`~1S01
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`Reference number
`ISO 14161:2000(E)
`
`© ISO 2000
`
`Novartis Exhibit 2188.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`ISO 14161:2000(E)
`
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`

`ISO 14161 :2000(E)
`
`Contents
`
`Page
`
`Foreword ...................................................................................................................................................................... v
`
`Introduction ................................................................................................................................................................ vi
`
`1
`
`2
`
`3
`
`4
`
`5
`5.1
`5.2
`5.3
`5.4
`
`6
`6.1
`6.2
`
`7
`7.1
`7.2
`7.3
`7.4
`
`8
`8.1
`8.2
`8.3
`8.4
`8.5
`8.6
`
`9
`9.1
`9.2
`9.3
`
`10
`10.1
`10.2
`
`11
`11.1
`11.2
`11.3
`11.4
`
`Scope .............................................................................................................................................................. 1
`
`Normative references .................................................................................................................................... 1
`
`Terms and definitions ................................................................................................................................... 2
`
`General ............................................................................................................................................................ 4
`
`Characteristics of biological indicators ...................................................................................................... 6
`General ............................................................................................................................................................ 6
`Test organism suspension for direct inoculation of products ................................................................. 6
`Inoculated carriers ......................................................................................................................................... 7
`Biological indicators ..................................................................................................................................... 7
`
`Selection of supplier ..................................................................................................................................... 8
`General ............................................................................................................................................................ 8
`Documentation ............................................................................................................................................... 9
`
`Biological indicators in process development ......................................................................................... 10
`General .......................................................................................................................................................... 10
`Reference microorganism method ............................................................................................................ 11
`Combined biological indicator and bioburden method ........................................................................... 11
`Bioburden method ....................................................................................................................................... 12
`
`Biological indicators in sterilization validation ........................................................................................ 13
`General .......................................................................................................................................................... 13
`Placement and handling of biological indicators ..................................................................................... 13
`Sterilizer qualification ................................................................................................................................. 13
`Performance qualification ........................................................................................................................... 13
`Review and approval of validation ............................................................................................................. 14
`Requalifications ........................................................................................................................................... 14
`
`Biological indicators in routine monitoring .............................................................................................. 14
`General .......................................................................................................................................................... 14
`Placement and handling of biological indicators ..................................................................................... 14
`Process challenge device (PCD) ................................................................................................................ 15
`
`Results .......................................................................................................................................................... 15
`General .......................................................................................................................................................... 15
`Interpretation of results .............................................................................................................................. 15
`
`Application of biological indicator standards .......................................................................................... 16
`General assessment of biological indicator performance by the user .................................................. 16
`Nominal population of test organism ........................................................................................................ 17
`D-value determination ................................................................................................................................. 17
`Testing equipment ....................................................................................................................................... 19
`
`Culture conditions ....................................................................................................................................... 20
`12
`General .......................................................................................................................................................... 20
`12.1
`12.2
`Incubation temperature ............................................................................................................................... 20
`Incubation period ......................................................................................................................................... 20
`12.3
`12.4 Choice of growth medium ........................................................................................................................... 21
`
`13
`13.1
`
`Third-party requirements ............................................................................................................................ 21
`General .......................................................................................................................................................... 21
`
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`ISO 14161:2000(E)
`
`13.2 Minimum requirements for replicates and total number of biological indicators ................................. 22
`13.3
`Test equipment ............................................................................................................................................ 22
`
`14
`15
`
`Personnel training ....................................................................................................................................... 22
`Storage and handling .................................................................................................................................. 23
`
`Disposal of biological indicators ............................................................................................................... 23
`16
`Annex A (informative) Microbiological inactivation kinetics and enumeration techniques .............................. 24
`
`Annex B (informative) Process challenge devices ................................................................................................ 28
`Annex C (informative) Formulae for fraction negative methods for D-value calculations ................................ 29
`
`Annex D (informative) Examples of documentation of biological indicators collected by the user ................ 35
`
`Bibliography .............................................................................................................................................................. 39
`
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`ISO 14161 :2000(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.
`
`International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
`
`Draft International Standards adopted by the technical committees are circulated to the member bodies for voting .
`Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote .
`
`Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
`patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
`
`International Standard ISO 14161 was prepared by Technical Committee ISO/TC 198, Sterilization of health care
`products .
`
`Annexes A, B, C and D of this International Standard are for information only.
`
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`V
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`

`

`ISO 14161:2000(E)
`
`Introduction
`
`This International Standard provides guidance regarding the selection, use and interpretation of results of biological
`indicators when used to develop, validate and monitor sterilization processes . The procedures described in this
`document are of a general nature and do not, of themselves, constitute a comprehensive development, validation ,
`or monitoring programme with regard to the sterilization of health care products. The intent of this International
`Standard is not to mandate the use of biological indicators in a process, but, if they are used, to provide guidance
`for their proper selection and use, to avoid misleading results .
`
`Biological indicators are not intended for use in any process other than that specified by the manufacturer on the
`product labelling . The use of an inappropriate biological indicator can give misleading results. In this International
`Standard , the user will find guidance on selection of the correct biological indicator for their particular sterilization
`process and critical parameters as well as guidance on its appropriate use .
`
`The user should select a biological indicator that is appropriate for the particular process to be employed . There are
`wide variations in sterilization processes and biological indicator manufacturers are not able to foresee all possible
`uses of their product. Manufacturers, therefore, label biological indicators according to their intended use . It is the
`responsibility of the users of biological indicators to select, use, recover and interpret the results as appropriate for
`the particular sterilization process used .
`
`Biological indicators should always be used in combination with physical and/or chemical measurements in
`demonstrating the efficacy of a sterilizing process . When a physical and/or chemical variable of a sterilization
`is outside
`its specified
`limits, cycle parameters should be evaluated . It should be noted
`that
`process
`measurements, which need to be evaluated, may be made during the cycle in the context of the overall cycle .
`Systems and/or procedures should be established to evaluate any deviations from the cycle process limits, and
`reasons for accepting any deviation should be fully documented .
`
`The performance of a biological indicator can be adversely affected by the conditions of storage and transport prior
`to its use, the use of the biological indicator, the sterilizer operating parameters , or the techniques employed after
`exposure to the process . For these reasons , the recommendations of the biological indicator manufacturer for
`storage and use should be followed . After exposure, biological indicators should be aseptically transferred and
`subjected to the validated recovery conditions as specified by the biological indicator manufacturer.
`
`It should be noted that biological indicators are not intended to indicate that the products, nor any other load being
`sterilized , are sterile . Biological indicators are utilized to test the effectiveness of a given sterilization process and
`employed equipment by assessing microbial lethality according to the concept of sterility assurance level. Suitably
`trained personnel should conduct these studies .
`
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`

`

`INTERNATIONAL STANDARD
`
`ISO 14161 :2000(E)
`
`Sterilization of health care products - Biological indicators -
`Guidance for the selection, use and interpretation of results
`
`1 Scope
`
`This International Standard provides guidance for the selection, use and interpretation of results from application of
`biological indicators when used in the development, validation and routine monitoring of sterilization processes .
`This International Standard applies to biological indicators for which International Standards exist.
`
`NOTE 1
`
`See for example the ISO 11138 series.
`
`The general information provided in this International Standard may have useful application for processes and
`NOTE 2
`biological indicators not currently addressed by existing International Standards, e.g. new and developing sterilization
`processes.
`
`International Standard does not consider those processes that rely solely on physical removal of
`This
`microorganisms, e.g. filtration .
`
`This International Standard is not intended to apply to combination processes, using for example washer
`disinfectors or flushing and steaming of pipelines .
`
`This International Standard is not intended to apply to liquid sterilization processes.
`
`2 Normative references
`
`The following normative documents contain provisions which , through reference in this text, constitute provisions of
`this International Standard . For dated references , subsequent amendments to, or revisions of, any of these
`publications do not apply. However, parties to agreements based on this International Standard are encouraged to
`investigate the possibility of applying the most recent editions of the normative documents indicated below. For
`undated references, the latest edition of the normative document referred to apply. Members of ISO and IEC
`maintain registers of currently valid International Standards.
`
`ISO 11134:1994, Sterilization of health care products - Requirements for validation and routine control -
`Industrial moist heat sterilization.
`
`ISO 11135:1994, Medical devices - Validation and routine control of ethylene oxide sterilization.
`
`ISO 11138-1 :1994, Sterilization of health care products - Biological indicators - Part 1: General.
`
`ISO 11138-2:1994, Sterilization of health care products - Biological indicators - Part 2: Biological indicators for
`ethylene oxide sterilization.
`
`ISO 11138-3:1995, Sterilization of health care products - Biological indicators - Part 3: Biological indicators for
`moist heat sterilization.
`
`ISO 11737-1 : 1995, Sterilization of medical devices - Microbiological methods - Part 1: Estimation of population
`of microorganisms on product.
`
`© ISO 2000 - All rights reserved
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`1
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`

`

`ISO 14161:2000(E)
`
`ISO 13683:1997, Sterilization of health care products - Requirements for validation and routine control of moist
`heat sterilization in health care facilities.
`
`ISO 14937, Sterilization of health care products - General criteria for characterization of a sterilizing agent and
`development, validation and routine control of a sterilization process.
`
`3 Terms and definitions
`
`For the purposes of this International Standard , the following terms and definitions apply.
`
`3.1
`accreditation
`procedure by which an authoritative body gives formal recognition that a body or person is competent to carry out
`specific tasks
`
`NOTE 1
`
`See reference [3].
`
`Accreditation does not itself qualify the laboratory to approve any particular product. However, accreditation may be
`NOTE 2
`relevant to approval and certification authorities when they decide whether or not to accept data produced by a given laboratory
`in connection with their own activities.
`
`3.2
`aseptic technique
`conditions and procedures used to exclude the introduction of microbial contamination
`
`3.3
`bioburden
`population of viable microorganisms on or in a product and/or package
`
`3.4
`biological indicator
`Bl
`inoculated carrier contained within its primary pack ready for use and providing a defined resistance to the
`specified sterilization process
`
`NOTE
`
`See ISO 11138-1.
`
`3.5
`D-value
`D10 value
`time or radiation dose required to achieve inactivation of 90 % of a population of the test microorganism under
`stated exposure conditions
`
`NOTE
`
`See ISO 11138-1 .
`
`3.6
`inoculated carrier
`carrier on which a defined number of test organisms have been deposited
`
`NOTE 1
`
`See ISO 11138-1 .
`
`NOTE 2
`
`The carrier is the supporting material on which test organisms are deposited .
`
`NOTE 3
`
`The test organism is a microorganism used for the manufacture of inoculated carriers.
`
`3.7
`inoculation
`transferral of a defined microbial entity into or on an item
`
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`

`ISO 14161:2000(E)
`
`3.8
`log reduction
`LR
`reduction in number of viable microorganisms, expressed in log10 units, after fractional exposure to a sterilization
`cycle
`
`3.9
`process challenge device
`PCD
`item which is deemed to present one of the greatest challenges to the effective performance of the sterilizing
`agent(s) in the collection of items to be sterilized
`
`The item is so constituted that a biological indicator can be placed in the position that is most difficult for the
`NOTE 1
`sterilizing agent to reach .
`
`NOTE 2
`procedure.
`
`The design of the process challenge device depends on the type of goods to be sterilized and the sterilization
`
`NOTE 3
`
`The biological indicator should not interfere with the function of the process challenge device.
`
`NOTE 4
`
`In some process challenge devices an inoculated carrier may be used in place of a biological indicator.
`
`3.10
`process challenge location
`PCL
`site that simulates "worst case" conditions as they are given for sterilizing agent(s) in the goods to be sterilized
`
`The site is so constituted that a biological indicator can be placed in the position that represents a rigorous
`NOTE 1
`challenge for the sterilizing agent to reach.
`
`NOTE 2
`
`The site depends on the type of goods to be sterilized and the sterilization process parameters.
`
`NOTE 3
`
`The biological indicator should not interfere with the function of the goods.
`
`NOTE 4
`
`In some sites an inoculated carrier may be used in place of a biological indicator.
`
`3.11
`process parameter
`specified value for a process variable
`
`NOTE
`
`Specifications for a sterilization process include the process parameters and their tolerances .
`
`3.12
`resistometer
`equipment designed to create defined combinations of the physical and/or chemical variables of a sterilization
`process within defined limits
`
`NOTE 1
`
`See ISO 11138-1 .
`
`NOTE 2
`
`Also referred to as Biological Indicator Evaluator Resistometer (BIER).
`
`3.13
`sterilization cycle development
`procedure for determination of the appropriate processing parameters and conditions which are consistent with
`attaining the desired specifications and label claims for a given product or group of products
`
`3.14
`sterilization cycle validation
`documented procedure for obtaining, recording and interpreting the results required to establish that a process
`would consistently yield product complying with predetermined specifications
`
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`

`ISO 14161:2000(E)
`
`3.15
`sterile
`free from viable microorganisms
`
`3.16
`sterilization
`validated process used to render a product free from viable microorganisms
`
`In a sterilization process, the nature of microbial inactivation is described by an exponential function . Therefore , the
`NOTE
`presence of viable microorganisms on any individual item can be expressed in terms of probability. While this probability may be
`reduced to a very low number, it can never be reduced to zero.
`
`3.17
`supplier
`organization that provides a product to the customer
`
`NOTE 1
`
`In a contractual situation , the manufacturer may be called the "contractor''.
`
`The supplier may be, for example, the manufacturer, distributor, vendor, importer, assembler or service
`NOTE 2
`organization. The supplier can be either external or internal to the organization. The supplier is a person or business concern
`that manufactures goods or owns a factory and represents the "first party" (see reference [41).
`
`3.18
`third party
`person or body that is recognized as being independent of the parties involved , as concerns the issue in question
`
`NOTE 1
`
`See reference [1].
`
`NOTE 2
`
`Parties involved are usually supplier ("first party") and purchaser ("second party") interests.
`
`3.19
`user
`person or body employing biological indicators for a given purpose
`
`NOTE 1
`
`See reference [4] .
`
`The user is the customer who is the recipient of a product provided by the supplier (see reference [41). In a
`NOTE 2
`contractual situation, the user is called "purchaser''. The user may be the customer, beneficiary or purchaser. The user can be
`either external or internal to the organization and represents the "second party".
`
`3.20
`z-value
`<thermal sterilization process> the change in exposure temperature which corresponds to a 10-fold change in
`D-value
`
`NOTE
`
`See ISO 11138-3.
`
`4 General
`
`This guidance International Standard provides information on biological indicators that may apply generally for any
`sterilization process, including new sterilization processes not yet covered by International Standards.
`
`The use of biological indicators is normally documented in procedures and/or instructions.
`
`NOTE
`[121).
`
`4
`
`Employing quality systems complying with ISO 13485 or ISO 13488 satisfies this provision (see references [11] and
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`

`

`ISO 14161:2000(E)
`
`Biological indicators that are defined in ISO 11138-1 , ISO 11138-2 and ISO 11138-3 give requirements for the
`manufacture of biological indicator systems where the biological component is a microorganism , such as a bacterial
`endospore or other microbiological form . The ISO 11138 series gives requirements for biological indicators for use
`in sterilization processes . These International Standards require that suitably trained personnel carry out the
`procedures and methods described .
`
`A suitable biological indicator consists of carrier material and packaging , and has a microbiological component that
`is known to be suitable for handling without special containment facilities . The growth conditions should be well
`documented and the use of the indicator should be as simple and well described to the user as possible to avoid
`misinterpretation .
`
`No formal approval system exists internationally for biological indicators that are marketed and used for stated
`purposes or under stated conditions. Some national regulatory authorities, however, have particular requirements
`for biological indicators and for the choice and use of biological indicators for the validation and control of products
`marketed as sterile or sterilized .
`
`A biological indicator represents a microbiological challenge to a sterilization process, and is used to verify that a
`sterilization process has the ability to inactivate microorganisms that have a known resistance to a referenced
`sterilization process . Test organisms employed in biological indicators typically have resistance to sterilization
`which exceeds that of common bioburden microorganisms, although some organisms may exhibit a resistance to
`sterilization in excess of that of the test organisms. The appropriate biological indicator has a combination of
`population and resistance that exceeds that of the bioburden . If there is reason to believe that the goods to be
`processed may be contaminated with particularly resistant organisms, extended sterilization processing , based on
`the bioburden, may be required .
`
`The user should ensure that the biological indicator has been validated for use with the particular range of
`sterilization conditions that are used. This may require additional information than that given in the labelling . When
`biological indicators are used outside reference conditions , the user may require information on the reaction to be
`expected from the indicator, e.g. the effect of sub-optimal moisture conditions on the biological indicators used in
`an ethylene oxide process . Users who employ biological indicators for non-standard sterilization techniques should
`thoroughly characterize the resistance of the biological indicators to the particular sterilization process as compared
`to a wide range of microorganisms, including any hazardous microorganisms or infectious agents that may
`constitute a part of the bioburden of the product. The relationship of the response of the biological indicator to
`process parameters should be clearly demonstrated .
`
`It is incumbent upon those responsible for the sterilization of product to ensure that the type of biological indicator
`employed to validate and/or routinely monitor a given sterilization process is appropriate for that use.
`
`The manufacturer's recommendations for the use and storage of the biological indicators should always be
`followed . Failure to do so may compromise the integrity of the biological indicator. If the user removes the
`inoculated carrier from the biological indicator's primary packaging, changes in the resistance characteristics may
`occur. Guidance should be sought from the manufacturer on the extent of this change or the user may evaluate
`changes in the resistance characteristics. The user should document that the performance characteristics of the
`inoculated carrier are appropriate for their use .
`
`Biological indicators should not be used beyond the expiration date stated by the manufacturer.
`
`Those who employ biological indicators for validation and/or routine monitoring of sterilization should be properly
`trained in their use. Post-sterilization handling of inoculated carriers and inoculated products should be performed
`according to validated guidelines or in compliance with the directions provided by the manufacturer of the
`inoculated carriers . The transfer of microorganisms exposed to the sterilization process to the appropriate recovery
`medium should employ aseptic technique.
`
`The ISO 11138 series gives requirements for the information that the manufacturer should provide for biological
`indicators. The information may be provided on the label , as a packet insert, or as a general specification
`accompanying the biological indicators. These International Standards also include minimum requirements for
`resistance characteristics . Testing conditions and methods are given as reference methods .
`
`Users of biological indicators come from a wide variety of industries, private enterprises and health care facilities .
`Users generally are not required to perform resistance assays on biological indicators, but may have differing
`requirements for their quality assuranc