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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〉〉
•

1.

2.

3.

4.

5.
6.

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.

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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 bioburden-based 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 bioburden-based 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.

relative resistance.

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.

D-Value and Microbial Resistance

The D-value 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 D-value is a function of temperature. In gas sterilization (ethylene oxide, ClO2,
or O3), D-values are a function of the chemical concentration, relative humidity, and temperature. Similarly, for liquid chemical
sterilization the D-value is a function of the temperature and sterilant concentration. [NOTE—Determining the D-value 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.]

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Figure 2. Graphical representation of D-value.

The D-value 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 D-value 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.
Figure 3):
1. Survivor curve region— the death curve.

approximately 10 CFU.
2. slope. This can

3. Estimated region— negative method is

Figure 3 by the

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.

be considered.

sterilization processes.

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 Physicochemical Integrators and Indicators for Sterilization 〈 1229 .9〉〉.

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

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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 half-cycle 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 small-volume parenterals, in-process 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 in-line.

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

Physical Integrators/Indicators immediate indication of

Parametric Release— Sterilized Pharmaceutical

Products—Parametric Release
Additional Considerations— requirements

process in a validated state.

Change Control— the sterilization process should

Preventive Maintenance— of sterilizing

change control.
Periodic Reassessment—In of sterilization

Training—
engineers well-grounded 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

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.

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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.)

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< 1229 > STERILIZATION OF Radhakrishna S Tirumalai GCM2015 General Chapters-Microbiology

COMPENDIAL ARTICLES Principal Scientific Liaison 2015

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