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1229 STERILIZATION OF COMPENDIAL ARTICLES
BACKGROUND AND SCOPE
This general information chapter provides an overview of the concepts and principles involved in sterilization (by
various modes) of compendial articles that must be sterile. It includes information about supportive sterilization
processes utilized in their preparation.1 More detailed recommendations are presented in specific information
chapters for each sterilization mode:
Steam Sterilization by Direct Contact 1229.1
Moist Heat Sterilization of Aqueous Liquids 1229.2
Monitoring of Bioburden 1229.3
Sterilizing Filtration of Liquids 1229.4
Biological Indicators for Sterilization 1229.5
Liquid Phase Sterilization 1229.6
Gaseous Sterilization 1229.7
Dry Heat Sterilization 1229.8
Physicochemical Integrators and Indicators for Sterilization 1229.9
Radiation Sterilization 1229.10
Vapor Phase Sterilization 1229.11
In the strictest definition of sterility, a specimen is deemed sterile only when there is a complete absence of viable
microorganisms (bacteria, yeasts, and molds), but sterility cannot be demonstrated with respect to compendial
articles and other items because of the inherent limitations of the current test (see Sterility Tests 71 ).
Sterility, therefore, is defined in probabilistic terms that establish an acceptable level of risk. Sterility can be
accomplished only by the use of a validated sterilization process under appropriate current good manufacturing
practices and cannot be demonstrated by reliance on sterility testing alone. The basic principles for control of
sterilization processes, including method development, validation, and ongoing assurance, are as follows:
1. Sterilization process development that includes evaluation of the stability and compatibility of materials,
container integrity, expected presterilization bioburden, equipment method control parameters, etc.
2. Identification of sterilization process parameters that preserve the inherent properties of the materials yet
inactivate or remove microorganisms.
3. Demonstration that the sterilization process and equipment are capable of operating within the prescribed
parameters and corresponding to independent measurements of the critical parameters.
4. Performance of replicate studies that represent the operational range of the equipment and employ actual
or simulated product. The use of biological indicators for correlation between the measured physical
parameters and the expected lethality is recommended wherever possible.
5. Maintenance and monitoring of the validated process during routine operation.
6. Assurance that the bioburden (number and type) of the materials is acceptable and is maintained within
predetermined limits during routine operation.
VALIDATION OF STERILIZATION PROCESSES
Validation of sterilization processes requires knowledge of sterilization technology and use of the appropriate
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instrumentation and equipment to control and verify critical sterilization process parameters. An important aspect
of the sterilization validation program involves the use of biological indicators when appropriate. All sterilization
processes should be maintained in a state of validation that includes periodic requalification. The validation
program for each type of sterilization comprises several formally documented stages.
The general principles of validation programs are applicable to all sterilization procedures. Individual details are
presented in the specific USP informational chapters for each sterilization mode.
The process development stage investigates and establishes the operating parameters that define the controls that
will be used for the sterilization process. Portions of the cycle development can be performed in a laboratory
setting. The installation qualification stage establishes that equipment controls and other instrumentation are
installed as specified and calibrated. Documentation should demonstrate the acceptability of the required utilities
such as steam, water, and air. The operational qualification stage confirms that the equipment functions within the
defined sterilization parameters. The performance qualification stage of the validation program directly evaluates
the sterilization of materials or articles. This determination requires independent parameter measurement during
the sterilization process, as well as biological challenges in operational configurations. Correlation between the
physical measurements and the demonstrated microbiological lethality or removal capability for sterilizing filtration
methods supports the effectiveness of the sterilization process. The routine process control stage of the
sterilization process requires a number of supportive practices and is outlined in detail below.
ESTABLISHING AND JUSTIFYING STERILIZATION PROCESSES THAT RELY ON MICROBIAL INACTIVATION
Articles intended to be sterile must attain a 10 6 probability of a nonsterile unit, i.e., less than or equal to one
chance in one million that viable bioburden microorganisms are present. [NOTE—This
is also called the
Sterility Assurance Level. The term Probability of a Nonsterile Unit (PNSU) is used
throughout this chapter because it is descriptive and substantially easier to understand. ] This
PNSU can be accomplished by balancing the method effect on the materials and the destruction of the bioburden
(see Figure 1). Three methods are currently in use: overkill, bioburden/biological indicator, and bioburdenbased
methods. These methods are described in greater detail below. Regardless of the method chosen, the objective is
a maximum PNSU of 10 6 for the bioburden. An overkill method is the simplest method to establish but has
the greatest impact on materials. The bioburdenbased method requires the most method control but subjects the
materials to the least stressful conditions. Confidence in the process's lethality is the same, regardless of the
method utilized.
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Figure 1. A basic comparison of various validation methods.
For items that are essentially unaffected by the sterilization process, the overkill method is preferred because of its
simplicity. Overkill sterilization can be defined as a method in which the destruction of a high concentration of a
resistant microorganism supports the destruction of reasonably anticipated bioburden present in routine
processing. That objective can be demonstrated by attaining any of the following: a defined minimum lethality; a
defined set of method conditions; or confirmation of minimum log reduction of a resistant biological indicator.
If articles could be damaged by extended exposure to the sterilization process, it may not be feasible to employ an
overkill method. In these instances, demonstrating the effectiveness of the sterilization cycle requires not only
information about the delivered method conditions but also knowledge about resistance and control of the
population of the materials' bioburden. This method is widely used for the terminal sterilization of heatlabile
solutions and laboratory media. This sterilization strategy is variously called the bioburden/biological indicator or
combination based method and is defined thus:
Bioburden/biological indicator based sterilization is an approach in which the incomplete destruction (or
destruction of a modest population) of a resistant biological indicator can be used to demonstrate the capability of
the method to reliably destroy the bioburden present. This is accomplished using detailed knowledge of the
bioburden and biological indicator populations and their relative resistance.
The bioburdenbased method is used when material stability is limited or when there are no suitable biological
indicator microorganisms available to use with the sterilizing process. Customarily, radiation sterilization is
validated using the bioburden based method. The bioburdenbased method can be defined as:
A method in which bioburden samples from the material are routinely evaluated for resistance to the sterilization
process and may be utilized to demonstrate the effectiveness of the process. Routine monitoring of the bioburden
population and its resistance to the sterilization process is mandatory.
Filter sterilization of liquids and gases differs from other sterilization modes because filtration relies on removal of
microorganisms from the fluid rather than inactivation by chemical or physical means.
DValue and Microbial Resistance
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The Dvalue is the time (customarily in minutes) or radiation dose (customarily in kGy) required to reduce the
microbial population by 90% or 1 log10 cycle (i.e., to a surviving fraction of 1/10) and must be associated with the
specific lethal conditions at which it was determined (see Figure 2). For steam and dry heat, the Dvalue is a
function of temperature. In gas sterilization (ethylene oxide, ClO2, or O3), Dvalues are a function of the chemical
concentration, relative humidity, and temperature. Similarly, for liquid chemical sterilization the Dvalue is a
function of the temperature and sterilant concentration. [NOTE—Determining
the Dvalue for vapor
(condensing) systems such as H2O2, H2O2 plasma, and peracetic acid is complex because of
the biphasic nature of the materials. Radiation and filtration sterilization are validated using
unique methods. These processes are validated by methods that differ from those in this
introductory chapter. ]
Figure 2. Graphical representation of Dvalue.
The Dvalue is not an inherent attribute of the microbe only, so the influence of other factors such as substrate,
matrix, recovery media, and test methodology must be considered in Dvalue determination. The resistance of a
biological indicator is defined for the indicator as a system. Accurate assessment and comparison between D
values requires standardization of test methods. To properly evaluate the effectiveness of a sterilization process,
analysts must evaluate the resistance of the bioburden experimentally or via a literature search.
The death curve for microorganisms subjected to a sterilization process is comprised of three distinct regions (see
Figure 3):
1. Survivor curve region—Where viable microorganisms can be recovered and counted to determine the slope
of the death curve. Using survivor counts in short exposure periods, the first section of the death curve can
be drawn to where the population is approximately 10 CFU.
2. Fraction negative region—Where replicate studies with multiple biological indicators are used to estimate
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the slope. This can extend the demonstrable portion of the death curve to approximately 10 2 to 10 3.
3. Estimated region—Where the death rate curve established by either the survivor curve method or fraction
negative method is extrapolated to the desired PNSU. Below 10 3 the death curve is assumed to be linear
and is depicted in Figure 3 by the dashed line beyond the point assuming that the death of microorganisms
continues at the same rate.
Figure 3. Death curve showing the various regions.
As stated earlier, the goal of the sterilization process is inactivation of the bioburden without adversely affecting
product quality attributes. Demonstration of the lethality of a sterilization process under routine operation relies on
differences in the relative resistance and population of the bioburden relative to the biological indicator (see Figure
4). Where the overkill method is used, bioburden controls can be less rigorous.
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Figure 4. Relative resistance and population of typical biological indicator and bioburden microorganisms.
Validation of sterilization processes links physical measurements with biological indicator performance to establish
method lethality. [NOTE—In
radiation sterilization bioburden response is linked to physical
irradiation dosage measurements. ] Knowledge of the method's effectiveness coupled with bioburden
controls on the materials under processing and information on bioburden resistance allow determination of the
probability of a nonsterile unit.
Analysts must know the resistance of the biological indicator to the process in order to ensure that the organism's
response to the process is properly understood. Equally important is an understanding of the bioburden present
during routine use of the sterilization process and its possible resistance to the chosen process.
Biological and Physical Data
The biological indicator, when used, is a convenient means to simplify the sterilization validation effort. Biological
indicators customarily are bacterial spores that have established resistance to the sterilization process under
evaluation. When supplied as spores (on a substrate or as a suspension) with known initial population and
resistance, their response to the method can be correlated to the measured physical conditions. Spores are
preferable as biological indicators because their resistance and population are predictable and stable when they
are handled, stored, and used as recommended.
The spores can be placed in the sterilization load in locations where physical parameter measurements such as
temperature or gas concentration cannot be easily obtained (e.g., within needle lumens, syringes, and ampuls) or
where measurement may alter the delivered conditions (e.g., sampling of the lethal gas). The biological indicator
provides a means to directly assess the sterilizing effect of the method in a manner not possible by physical
measurements. The lethalitybased physical measurement and biological inactivation data from a sterilization
process should be in reasonable agreement. When this is not the case, an investigation should be considered.
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STERILIZATION INDICATORS AND INTEGRATORS
The execution of sterilization processes can be supported by physical and chemical indicators and integrators that
provide an indication that processing is completed. These are available in many different forms for use in
conjunction with many common sterilization processes.
Sterilization indicators respond to sterilization process parameters in a nonquantitative fashion; i.e., they show
passing or failing results. They are useful in an operating environment as a means to identify whether an item has
been exposed to a sterilization process. They are of minimal use in directly establishing process efficacy.
Sterilization integrators are more sophisticated devices that react quantitatively in response to one or more of the
critical sterilization parameters and yield a result that can be correlated to lethality. The most sophisticated
integrators are radiation dosimeters that are so accurate and robust that their use has displaced the use of
biological indicators for the validation of radiation sterilization. Additional detail about integrators and indicators
can be found in Sterilization—Chemical and Physicochemical Indicators and Integrators 1209 .
STERILIZATION PROCESS DEVELOPMENT
An important consideration in any sterilization activity is the selection of an appropriate process from the many
possible alternatives: steam, dry heat, gas, radiation, vapor, chemical, or filtration. The choice of the appropriate
method for a given item requires knowledge of the sterilization process and information concerning effects of the
process on the material being sterilized. The selection of a particular sterilizing treatment and the details of its
execution often represent a compromise between the conditions required to destroy or remove the bioburden to
the desired level and the impact of the sterilization process on the materials being processed. Sterilization
processes should be sufficiently robust for certainty of microbial inactivation while avoiding adverse
consequences to material quality attributes.
The overkill method employs conditions that are capable of destroying a high concentration of a resistant biological
indicator and thus are a greater challenge to material integrity and stability. Overkill is employed only where the
items being sterilized can withstand extended exposure to the sterilizing process and is used most commonly for
metal, glass, and other items that are unaffected by process exposure. Its use is always preferable where
materials can tolerate the more aggressive conditions utilized.
The halfcycle validation method is a special case of the overkill method in which a lethal cycle to the biological
indicator is arbitrarily doubled. Its use unnecessarily exposes materials to harsh conditions, and it should be
avoided.
Bioburden/biological indicator (or combination) methods are appropriate when the product has some sensitivity to
the sterilizing conditions. Analysts commonly use it for large and smallvolume parenterals, inprocess solutions,
and laboratory media for which the material properties would be impaired by a lengthy exposure to the sterilizing
conditions. The proper use of the method requires control over the presterilization bioburden levels and confidence
that the bioburden's resistance is such that it will be destroyed during processing. The complete destruction of or
the use of a high population of the bioburden/biological indicator is not necessary for use of this method because
it relies on differences in the relative resistance and population of the biological indicator and bioburden
microorganisms.
The bioburden method bases the sterilization duration solely on the expected population and resistance of the
bioburden on the materials. This is the method of choice for all radiation sterilization. It relies on periodic
bioburden monitoring and resistance screening to establish confidence in the method. The bioburden method does
not require use of a biological indicator.
Filtration is used for liquids and gases that either will not withstand heat, radiation, or chemical sterilization
processes or are more conveniently sterilized inline.
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ROUTINE PROCESS CONTROL
After a sterilization process has been initially validated, it must be maintained in that state to ensure continued
acceptable operation. This is accomplished by a number of related activities that are essential for continued use
of the method.
Calibration—Equipment instrumentation must be periodically calibrated against a traceable standard. This
includes recording as well as controlling instruments that regulate the operation of the equipment.
Physical Measurements—Data reported by the equipment sensors and recorders must be verified after the
completion of each sterilization cycle. The records from the sterilization equipment are an essential part of the
documentation.
Physical Integrators/Indicators—When they are used, integrators, and to a lesser extent indicators, provide an
immediate indication of method execution and differentiate between processed and unprocessed materials. When
these integrators provide a direct indication of method lethality (e.g., radiation dosimeters), they can be used for
material release.
Parametric Release—The release of finished products without sterility testing is addressed in Terminally Sterilized
Pharmaceutical Products—Parametric Release 1222 .
Additional Considerations—Depending on the specifics of the particular sterilization process, there may be
additional requirements for confirming method efficacy. These can include bioburden sampling, bioburden
resistance determination, biological indicator resistance determination, and supplier audits. As applicable, these
activities should be carried out to maintain the sterilization process in a validated state.
Change Control—To maintain a validated state, materials, procedures, and equipment that influence the
sterilization process should be monitored to ensure that all changes are recorded and evaluated in terms of their
potential impact. To accomplish this, analysts must establish a formalized system for change control.
Preventive Maintenance—The user should establish a defined preventive maintenance program for each piece of
sterilizing equipment in accordance with the equipment manufacturer's written recommendation. Preventive
maintenance represents a special class of predefined changes that have no adverse effects on the operation of
the system and thus do not require evaluation under change control.
Periodic Reassessment—In the absence of change to the materials, method, or equipment, the effectiveness of
sterilization processes should be reconfirmed on a periodic basis. This system should be formalized and should
address the potential impact of a number of de minimus changes or undetected changes to the validated system.
In the absence of change, the amount of information required to support a sterilization process is less than that
required for initial acceptance of the sterilization process.
Training—Sterilization processes rely heavily on scientific principles for the effective destruction of
microorganisms. Scientists and engineers wellgrounded in the principles of microbial death and removal develop
processes to ensure effective sterilization. Individuals involved in the development of sterilization processes
require a background in microbiology, physics, chemistry, and engineering, and they must be familiar with good
manufactoring principles and regulations. Sterilization is an interdisciplinary activity where the combined
knowledge of a group of individuals is generally required for the establishment of a reliable process. In addition to
the sterilization process development team, individuals responsible for maintenance and operation of sterilization
processes must also be trained appropriately to ensure that their actions contribute to success. These individuals
are often the first to identify upsets and shifts in process performance because of their intimate involvement with
it. Effective training programs should be established and documented. Training programs should emphasize
sterilization principles, adherence to established processes and procedures, and the importance of documenting
deviations from normal operations.
REFERENCES
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Agalloco, J., & Carelton, F., Validation of Pharmaceutical Processes, InformaUSA, New York, 2007.
Block, S., Disinfection, Sterilization, and Preservation, 5th edition, Lippincott Williams & Wilkins, Philadelphia,
2001.
Morrissey, R., & Phillips, G.B., Sterilization Technology—A Practical Guide for Manufacturers and Users of
Health Care Products, Van Nostrand Reinhold, New York, 1993.
Perkins, J., Principles and Methods of Sterilization in Health Sciences, 2nd edition, Charles C. Thomas,
Springfield, IL, 1969.
Pflug, l., Microbiology and Engineering of Sterilization Processes, 14th edition, Environmental Sterilization
Laboratory, Otterbein, IN, 2010.
Russell, A., Hugo, W., & Ayliffe, G., Principles and Practices of Disinfection, Preservation and Sterilisation,
Blackwell Scientific, Oxford, U.K., 1982.
1 These processes may also provide depyrogenation, the extent of which depends on the actual sterilization conditions. (Depyrogenation by
various means will be addressed in a chapter under development.)
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph
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