450〈1035〉 Biological Indicators for Sterilization / General Information USP 34分手送什么花
A third form of biological indicator is a lf-contained indi-〈1035〉 BIOLOGICAL INDICATORS cator. A lf-contained biological indicator is designed so
that the primary package, intended for incubation following FOR STERILIZATION sterilization processing, contains the growth medium for re-
covery of the process-expod microorganisms. This form of
biological indicator together with the lf-contained growth
medium can be considered a system. In the ca of lf-
A biological indicator is broadly defined as a characterized contained biological indicators, the entire system provides preparation of a specific microorganism that provides a de-resistance to the sterilization process.
fined and stable resistance to a specific sterilization process.If the biological indicator is a paper strip or disk in a lf-Microorganisms widely recognized as suitable for biological contained package that includ
es an available culture me-indicators are spore-forming bacteria, becau, with the ex-dium, the package design should be readily penetrable by ception of ionizing radiation process, the microorgan-the sterilizing agent. To allow for the time lag that may isms are significantly more resistant than ur while the sterilizing agent reaches the contained mi-A biological indicator can be ud to assist in the perfor-croorganisms in the system, the D value, process endpoint mance qualification of the sterilization equipment and in the kill time, and the survival time should be characterized for development and establishment of a validated sterilization the system and not solely for the paper strip in the lf-process for a particular article. Biological indicators are ud contained unit. Following the sterilizing treatment, the spore in process that render a product sterile in its final package strip or disk is immerd in the lf-contained medium by
or container, as well as for the sterilization of equipment,manipulation, which allows contact with the culture materials, and packaging components ud in aptic medium.
processing. Biological indicators may also be ud to moni-Self-contained biological indicators may also consist of a tor established sterilization cycles and in periodic revalida-spore suspension in its own medium, and they often also tion of sterilization process. Biological indicators may also contain a dye, which indicates positive or negative growth be ud to evaluate the capability of proce
ss ud to de-following incubation. Resistance of the lf-contained system contaminate isolators or aptic clean-room environments.is dependent upon penetration of the sterilant into the
The principles and requirements for the applications are package. Penetration may be controlled by the manufac-described under Sterilization and Sterility Assurance of Com-turer through varying designs and composition of the lf-pendial Articles 〈1211〉.contained biological indicator package, ampul, or container.
Self-contained ampul biological indicators may be incubated
directly following exposure to the sterilization process. The TYPES OF BIOLOGICAL INDICATORS
entire system is then incubated under the specified condi-
tions. Growth or no growth of the treated spores is deter-There are at least three types of biological indicators. Each
mined visually (either by obrving a specified color change type of indicator incorporates a known species of a microor-
of an indicator incorporated in the medium or by turbidity) ganism of known sterilization resistance to the sterilization
or by microscopic examination of the inoculated medium. mode. Some biological indicators may also contain two dif-
The lf-contained system resistance characteristics must ferent species and concentrations of microorganisms.
also comply with the labeling of the lf-contained system One form of biological indicator includes spores that are
and the relevant biological indicator monograph. The lf-added to a carrier (a disk or strip of filter paper, glass,
contained biological indicator system should withstand plastic, or other materials) and packaged to maintain the
transport in the condary packaging and handling at the integrity and viability of the inoculated carrier.
point of u without breakage. The design of the lf-con-Carriers and primary packaging shall not contain any con-
tained system should be such to minimize the loss of the tamination (physical, chemical, or microbial) that would ad-
original inoculum of microorganisms during transport and verly affect the performance or the stability characteristics
handling. During or after the sterilization process, the名牌红酒
of the biological indicator. The carrier and primary packag-
materials ud in the lf-contained system shall not retain ing shall not be degraded by the specific sterilization pro-
or relea any substance that can inhibit the growth of low cess, which is ud in a manner that will affect the perfor-
numbers of surviving indicator microorganism under culture mance of the biological indicator. The carrier should
conditions. Adequate steps must be taken to demonstrate withstand transport in the primary and condary packaging
that the recovery medium has retained its growth support and handling at the point of u. The design of the carrier
characteristics after exposure to the sterilization process. and primary packaging should minimize the loss of the orig-
inal inoculum during transport, handling, and shelf life
storage.Preparation
Another form of biological indicator is a spore suspension
that is inoculated on or into reprentative units of the All operations associated with the preparation of biologi-product to be sterilized. This reprents an inoculated prod-cal indicators are controlled by a documented quality sys-uct; however, a simulated inoculated product may be ud tem. Traceability is maintained for all materials and compo-if it is not practical to inoculate the actual product. A simu-nents incorporated in or coming into direct contact with the lated product is a prepar
ation that differs in one or more microorganism suspension, the inoculated carrier, or the bi-ways from the actual product, but performs as the actual ological indicator.
product using test conditions or during actual production The preparation of stock spore suspensions of lected mi-sterilization processing. Spore suspensions with a known D croorganisms ud as biological indicators requires the de-value should be ud to inoculate the actual or simulated velopment of appropriate procedures, including mass cultur-product. If a simulated inoculated product is ud, it must ing, harvesting, purification, and maintenance of the spore be demonstrated that it will not degrade the sterilization suspensions. The stock suspension should contain predomi-resistance of the bioindicator. The physical design of actual nantly dormant (nongerminating) spores that are held in a or simulated product can affect the resistance of spore sus-nonnutritive liquid.
pensions that are inoculated on or into the products. In the The finished product (microbial suspension, inoculated ca of liquid inoculated products, it is often advisable to carriers, or biological indicators) supplied by commercial determine both the D value and z value of the specific manufacturers shall have no microorganisms, other than the biological indicator microorganism in the specific liquid test microorganism, prent in sufficient numbers to ad-product. The population, D value, z value where applicable,verly affect the product. The system to minimize the pres-and end
point kill time of the inoculated actual or simulated ence of microorganisms other than the biological indicator product should be determined.
USP 34General Information / 〈1035〉 Biological Indicators for Sterilization451
microorganism in the product will be validated, monitored,cess will result in a probability of nonsterility at much less and recorded.than 10−6 in actual practice. Overkill process design and
evaluation may differ depending upon the sterilization pro-
cess under test. The u of an overkill design and validation Selection for Specific Sterilization Process approach may minimize or obviate the need for bioburden
enumeration and identification.
The lection of a biological indicator requires a knowl-Moist Heat—For moist heat sterilization process, spores edge of the resistance of the biological indicator system to of suitable strains of Bacillus stearothermophilus are commer-the specific sterilization process. It must be established that cially available as biological indicators and frequently em-the biological indicator system provides a challenge to the ployed. Other heat-resistant spore-forming microorganisms sterilization process that
exceeds the challenge of the natu-such as Clostridium sporogenes, Bacillus subtilis, and Bacillus ral microbial burden in or on agulans have also been ud in the development and vali-The effective u of biological indicators for the cycle de-dation of moist heat sterilization process.
velopment, process, and product validation, and routine
Dry Heat—For dry heat sterilization, spores of Bacillus production monitoring of a sterilization process requires a
subtilis spp. are sometimes ud to validate the process. thorough knowledge of the product being sterilized, along
During the validation of dry heat sterilization process, en-with its component parts (materials and packaging). Only
dotoxin depyrogenation studies are frequently conducted in the widely recognized biological indicators specified in the
果字组词lieu of microbial inactivation studies during the establish-particular biological indicator monograph should be ud in
ment of sterilization cycles becau the inactivation rate of the development or validation of a sterilization process. This
endotoxin is slower than the inactivation rate of Bacillus sub-will ensure that the biological indicator lected provides a
tilis spores. In practice the reduction of endotoxin titer by greater challenge to the sterilization process than the bi-
three or more logs will result in a process that also achieves oburden in or on the product. Some urs may require bio-
a probability of nonsterility substantially lower than 10−6. logical indicators with characteristics that differ from tho
Ionizing Radiation—Spores of Bacillus pumilus have been widely available commercially. In such cas, urs may
ud to monitor sterilization process using ionizing radia-grow their own spore cultures for the express purpo of
tion; however, this is a cedlining practice. Radiation do-preparing in-hou biological indicators for their specific u.
tting methods that do not u biological indicators have In such a ca, the ur is well advid to u organisms
been widely ud to establish radiation process. Further-already described in the scientific literature as indicator or-
more, certain bioburden microorganisms can exhibit greater ganisms, and the ur must have the capability of determin-
大学生犯罪resistance to radiation than Bacillus pumilus.
ing D and z values for in-hou biological indicators. When
biological indicators are prepared in-hou, urs must con-Ethylene Oxide—For ethylene oxide sterilization, spores firm the population, purity, and shelf life of the biological of a subspecies of Bacillus subtilis (Bacillus subtilis var. niger) indicator to ensure the validity of any test conducted using are commonly ud. The same biological indicator systems the in-hou biological indicator.
When a bioburden-bad are generally ud when 100% ethylene oxide or different sterilization process design is ud, data comparing the re-ethylene oxide and carrier gas systems are ud as sterilants. sistance of the biological indicator to that of bioburden are Vapor-Pha Hydrogen Peroxide (VPHP)—This process esntial. Enumeration of the bioburden content of the arti-has been shown to be an effective surface sterilant or
cles being sterilized is also required. The process must result decontaminant. VPHP is capable of achieving sterilization
in a biologically verified lethality sufficient to achieve a(probability of nonsterility of less than one in a million) probability of obtaining a nonsterile unit that is less than when process conditions so dictate and if the target of ster-one in a million.ilization is suitably configured. However, VPHP is also com-Alternatively, the overkill method may be ud in the de-monly ud as a surface decontaminating agent in the treat-sign of a sterilization process. In this ca, specific assump-ment of sterility testing, biological and chemical
tions are made regarding the resistance assumption ud in containment, manufacturing isolators, and clean rooms. establishing sterilization process lethality requirements. In Surface decontaminatio
n is a process that is distinct from general, all overkill process are built upon the assumption sterilization of product contact materials, container-closure that the bioburden is equal to one million organisms and systems, or product. It is a process designed to render an that the organisms are highly resistant. Thus, to achieve the environment free of detectable or recoverable microorgan-required probability of a nonsterile unit that is less than one isms. Biological indicators are widely ud to verify the effi-in a million, a minimum 12 D process is required. A 12 D cacy of the decontamination process. However, in the ca process is defined as a process that provides a lethality suffi-of decontamination, a spore log reduction value of three to cient to result in a 12 log reduction, which is equivalent to four is adequate becau the goal is decontamination rather 12 times a D value for organisms with sufficiently higher than sterilization.
resistance than the mean resistance of bioburden. Becau
the bioburden is assumed to be one million, an overkill pro-
Table 1. Typical Characteristics for Commercially Supplied Biological Indicator Systems
Limits for a Suitable Resistance (depending
Range of D values for Selecting
on the particular D value [minutes]) Example of a Typical D a Suitable Biological Indicator
Sterilization Mode value (minutes)(minutes)
Survival Time Kill Time
Dry heat a 1.9Min. 1.0Min. 4.010.0 160°Max. 3.0Max. 14.032.0 Ethylene oxide b
600 mg per L 3.5Min. 2.5Min. 10.025.0 54°Max. 5.8Max. 27.068.0
60% relative humidity
Moist heat c 1.9Min. 1.5Min. 4.513.5 121°Max. 3.0Max. 14.032.0
a For 1.0 × 106 to 5.0 × 106 spores per carrier.
b For 1.0 × 106 to 5.0 × 107 spores per carrier.
c For 1.0 × 105 to 5.0 × 106 spores per carrier.
452〈1035〉 Biological Indicators for Sterilization / General Information USP 34
Bacillus stearothermophilus is the most prevalently ud bi-zation process. Disposal instructions should also be provided ological indicator for validating VPHP. Other microorganisms by the manufacturer of the biological indicator.
that may be uful as biological indicators in VPHP process
are spores of Bacillus subtilis and Clostridium sporogenes.Ur’s Responsibility
Other microorganisms may be considered if their perfor-
mance respons to VPHP are similar to tho of the micro-
organisms cited above.Commercial Product—When biological indicators are The spores may be inoculated on the surface of various purchad from a commercial source, their suitability for u gas-impervious carrier systems having glass, metal, or plastic in a specific sterilization process should be established surfaces. Highly absorbent surfaces, such as fibrous sub-through developmental sterilization studies unless existing strates, or any other substrate that readily absorbs VPHP or data are available to support their u in the process. The moisture may adverly influence the VPHP concentration ur should establish in-hou acceptance standards for bio-avail
able for inactivation of inoculated microorganisms. Pa-logical indicator lots and consider rejection in the event the per substrates are not ud becau VPHP will degrade cellu-biological indicator lot does not meet the established in-lo-bad materials.hou performance standards. A Certificate of Performance For reprentative characteristics of commercially supplied should be obtained for each lot of indicators, and the ur biological indicators, e Table 1.should routinely perform audits of the manufacturer’s facili-The biological indicator may also be individually packaged ties and procedures. If certificates are not obtained and au-in a suitable primary overwrap package that does not ad-dits have not been performed, or if the biological indicators verly affect the performance of the indicator, and is pene-are to be ud outside of the manufacturer’s label claims, trable by VPHP. Spunbound polyolefin materials have verification and documentation of performance under con-proven to be well suited as an overwrap of biological indica-ditions of u must exist.
tors intended for u in evaluation of VPHP process. The Upon initial receipt of the biological indicator from a overwrap material may facilitate laboratory handling of the commercial supplier, the ur should verify the purity and biological indicators following exposure to VPHP. Also, the morphology of the purchad biological indicator microor-u of an overwrap material to package VPHP biological in-ganisms. Verification of at least the proper genus is desira-dicators must be caref
ully assd to ensure that, following ble. Also, a microbial count to determine the mean count VPHP exposure, residual hydrogen peroxide is not retained per biological indicator unit should be conducted. The man-by the packaging material, possibly inducing bacteriostasis ufacturer’s comments relative to D value range, storage con-during the recovery steps. Microbial D values will be influ-ditions, expiration dating, and stability of the biological indi-enced by the prence of a biological indicator overwrap cator should be obrved and noted. The ur may consider material relative to the rate of inactivation and the potential conducting a D value asssment before acceptance of the prence of residual VPHP. In cas where biological indica-lot. Laboratories that have the capability of performing D tors (inoculated carriers) are being ud without the primary value assays could conduct a D value determination using package, stringent adherence to aptic techniques is one of the three methods cited in the general test chapter required.Biological Indicators—Resistance Performance Tests 〈55〉 and in
the appropriate USP monographs for specific biological in-
dicators. Particularly important is the verification of the D PERFORMANCE EVALUATION value and count stability of the biological indicator system if
long-term storage is employed.
In the event the spore crop is maintained for longer than
12 months under documented storage conditions, both知识测试
Manufacturer’s Responsibility spore count and resistance analysis must be conducted, un-
less performance of an original parent crop has been vali-The initial responsibility for determining and providing to dated for a longer storage period. The result of spore count the urs the performance characteristics of a biological indi-and resistance assays should be within the range of accepta-cator1 lot resides with the manufacturer of biological indica-bility established during initial acceptance of the spore crop tors. The manufacturer should provide with each lot of bio-lot.
logical indicators a certificate of analysis that attests to the
Noncommercial Product—A ur of biological indicator validity of biological indicator performance claims cited on
systems may elect to propagate microorganisms for devel-the biological indicator package label or in the package in-
oping in-hou biological indicators to develop or validate rt of the label package. The manufacturer should define
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sterilization process. In the event a ur becomes a “man-the sterilization process that the biological indicator will be
ufacturer” of biological indicators, biological indicator per-ud to evaluate. The characterization of each type of bio-
formance requirements must be met. If the biological indi-logical indicator, which provides the basis for label claims,
cator system is ud for the development of new sterilization should be performed initially by the manufacturer of the
process or validation of existing process, the same per-biological indicator using specialized and standardized appa-
formance criteria described for commercial manufacturers of ratus under precily defined conditions.1 The manufacturer
biological indicators must be followed.
should also provide information concerning the D value, the
method by which the D value was determined, and micro-
bial count and resistance stability of the biological indicator Spore Crop Preparation
throughout the labeled shelf life of the indicator. Optimum
storage conditions should be provided by the manufacturer,Becau most biological indicators u microbial spores, including temperature, relative humidity, and any other re-accurate records of spore crop identification must be main-quirements for controlled storage. The data obtained from tained by commercial and noncommercial biological indica-the various required performance assays should be cited in a tor manufacturers. The records should include records per-package inrt or on the label of the biological indicator taining to the source of the initial culture, identification, package. The manufacturer should provide directions for traceability to the parent spore crop, subculture frequency, u, including the medium and conditions to be ud for media ud for sporulation, changes in media preparation, the recovery of microorganisms after exposure to the ster
ili-any obrvation of crop contamination, and pre- and post-1See Apparatus under Biological Indicators—Resistance Performance Tests 〈55〉.heat shock data. Records of usage of the spore crop and The apparatus have been designed to provide consistent physical condi-resistance to sterilization (namely, D values and z values tions applicable to the characterization of biological indicators. The required where applicable) should also be maintained.
performance characteristics are also indicated.
USP 34General Information / 〈1041〉 Biologics453
process. It is important that the urs be able to scientifi-Instrumentation
cally justify their lection of a biological indicator.
The instrumentation ud to evaluate the sterilization re-
sistance of spore crops must be consistent with existing
standards2 related to the performance evaluation of biologi-
cal indicator systems.
国外地址Equipment for the determination of D values of microor-
ganisms expod to VPHP should be able to cloly control
equipment operating parameters as described for other bio-〈1041〉 BIOLOGICS
logical indicator systems under Biological Indicators—Resis-
tance Performance Tests 〈55〉. Particularly important is the as-
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surance of a consistently reproducible VPHP concentration,Products such as antitoxins, antivenins, blood, blood de-delivered within a finite time, and maintained within a spec-rivatives, immune rums, immunologic diagnostic aids, tox-ified concentration range or VPHP pressure range for a de-oids, vaccines, and related articles that are produced under fined increment of time. Introduction of biological indicators licen in accordance with the terms of the federal Public into a stabilized concentration of VPHP conditions should be Health Service Act (58 Stat. 682) approved July 1, 1944, as via a system that permits rapid entry and removal of the amended, have long been known as “biologics.” However, test units from the chamber. Also, the design of the test in Table III, Part F, of the Act, the term “biological products”chamber should allow for the attainment of steady-state is a
pplied to the group of licend products as a whole. For VPHP concentrations and pressure, or the u of a defined Pharmacopeial purpos, the term “biologics” refers to amount of cubic feet of free flowing VPHP at a standardized tho products that must be licend under the Act and pressure and temperature. Currently, VPHP concentration comply with Food and Drug Regulations—Code of Federal measurement devices may not be widely ud. Therefore,Regulations, Title 21 Parts 600-680, pertaining to federal exposure conditions may need to be bad on the mainte-control of the products (other than certain diagnostic nance of steady-state VPHP pressures or flow rates resulting aids), as administered by the Center for Biologics Evaluation from a known initial weight of hydrogen peroxide, admitted and Rearch or, in the ca of the relevant diagnostic aids, to the chamber in a defined unit of time. Using this infor-by the Center for Devices and Radiological Health of the mation, together with the known fixed volume of the cham-federal Food and Drug Administration.
ber environment, a calculation of the approximate VPHP Each lot of a licend biologic is approved for distribution concentration can be made. If conditions are maintained when it has been determined that the lot meets the specific constant throughout each D value asssment run, compari-control requirements for that product as t forth by the sons of relative resistance among different biological indica-Office. Licensing includes approval of a specific ries of pro-tor lots may be readily determined.duction steps and in-process control tests as well as end-
product specifications that must be met on a lot-by-lot ba-
sis. The can be altered only upon approval by the Center USE FOR IN-PROCESS VALIDATION
for Biologics Evaluation and Rearch and with the support
of appropriate data demonstrating that the change will yield Regardless of the mode of sterilization, the amount of the
a final product having equal or superior safety, purity, po-initial population of the microorganisms, its resistance to
tency, and efficacy. No lot of any licend biological prod-sterilization, and the site of inoculation on or in the product
uct is to be distributed by the manufacturer prior to the can all influence the rate of biological indicator inactivation.
completion of the specified tests. Provisions generally appli-During product microbial challenges, various areas of the
cable to biologic products include tests for potency, general product should be inoculated with biological indicators. If,
safety, sterility, purity, water (residual moisture), pyrogens, for example, a container with a closure system is sterilized,
identity, and constituent materials (Sections 610.10 to
both the product solution and the closure should be chal-
610.15 and e Safety Tests—Biologicals under Biological Re-lenged to ensure that sterilization equivalent to a 10−6 (one
activity Tests, In Vivo 〈88〉, Sterility Tests 〈71〉, Water Determi-in a million probability of a nonsterile unit) sterilization as-
nation 〈921〉, and Pyrogen Test 〈151〉, as well as Bacterial En-surance level (SAL) will be obtained in the solution as well
dotoxins Test 〈85〉). Constituent materials include ingredients, as at the closure site.
prervatives, diluents and adjuvants (which generally
One may need to determine through laboratory studies
should meet compendial standards), extraneous protein in whether product components are more difficult to sterilize
cell-culture produced vaccines (which, if other than rum-than, for example, a solution or drug within the product.
originating, is excluded) and antibiotics other than penicillin Depending on the locations of the product components
added to the production substrate of viral vaccines (for most difficult to sterilize, different process parameters may
which compendial monographs on antibiotics and antibiotic be involved in assuring microbial inactivation to an SAL of
substances are available). Additional specific safety tests are 10−6. The product performance qualification pha should
also required to be performed on live vaccines and certain identify the most important process parameters for inactiva-
other items. Where standard preparations are made availa-tion of microorganisms at the sites most difficult to sterilize.
ble by the Center for Biologics Evaluation and Rearch Once the critical processing parameters are determined,
(Section 610.20), such preparations are specified for com-during sterilization in-process validation of the product, they
parison in potency or virulence testing. The U.S. Opacity should be operated at conditions less than the conditions
Standard is ud in estimating the bacterial concentration of stated in the sterilization process specifications. Biological in-
certain bacterial vaccines and/or evaluating challenge cul-dicator survival is predicated upon both resistance and pop-
tures ud in tests of them. (See also Units of Potency in the ulation. Therefore, a 106 biological indicator population is
General Notices.)
not always required to demonstrate a 10−6 SAL. The appro-
The Pharmacopeial monographs conform to the Food and priate u for biological indicators is to employ them to
Drug Regulations in covering tho aspects of identity, qual-confirm that the developed process parameters result in the
ity, purity, potency, and packaging and storage that are of desired SAL. In moist heat sterilization, the biological indica-
particular interest to pharmacists and physicians responsible tor is ud to establish that physically measured lethality can
for the purcha, storage, and u of biologics. Revisions of be verified biologically. Biological indicators with substantive
the federal requirements affecting the USP monographs will D values and populations substantially less than 106 are ade-
be made the subjects of USP Supplements as promptly as quate to validate many sterilization and decontamination
practicable.
2BIER/Steam Vesls, American National Standards, ANSI/AAMI ST45:1992.
