1229.2 Moist Heat Sterilization of Aqueous Liquids

Printed on: Tue Mar 15 2022, 06:48:18 AM(EST) Official Status: Currently Official on 15-Mar-2022 DocId: 1_GUID-D516FF11-E0D7-4288-9979-2074A9B283FD_2_en-US
Printed by: Mujtaba Ali Official Date: Official as of 01-Nov-2019 Document Type: GENERAL CHAPTER @2022 USPC
DOI Ref: aw6ef DOI: https://doi.org/10.31003/USPNF_M7411_02_01
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á1229.2ñ MOIST HEAT STERILIZATION OF AQUEOUS LIQUIDS

INTRODUCTION
Steam sterilization of aqueous liquids (including both suspensions and emulsions with mixing), also known as sterilization of
nonporous loads, is the method of choice for aqueous parenteral products, in-process aqueous liquids, laboratory media, and
biological waste materials. This type of sterilization is accomplished primarily in closed containers. During steam sterilization by
direct contact (also called steam sterilization of parts, hard goods, or porous items) the steam in the chamber directly contacts
the surface of load items to effect sterilization (see Steam Sterilization by Direct Contact á1229.1ñ). In contrast, sterilization of
liquids in containers is accomplished by application of heat to the container, heating of the container wall, and ultimately
heating of the internal liquid volume. This can be accomplished using steam, superheated water, and air in various
combinations. Some aqueous liquids are susceptible to over-processing that could render them unfit for their intended use.
Manufacturers should consider the influence of these differences when they establish a suitable process.
During the sterilization of liquid-filled containers, differential pressures between the interior of the containers and the
sterilization chamber may potentially impact container integrity. Air over-pressure is used to minimize the pressure differential
between the container and the sterilizer to protect the integrity of the container, especially prefilled syringes and plastic
containers. Before sterilization of product containers, manufacturers should consider the potential adverse consequences of
excess heat on the materials. In order to ensure sterility as well as functionality, the process definition and validation method
used must incorporate both lower and upper temperature and time limits on the process conditions.
When the overkill method can be used for sterilization of sealed liquid containers, it is the preferred method and is described
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in Steam Sterilization by Direct Contact á1229.1ñ. When product quality attributes can be impaired by excessive heat, the
sterilization process should use less time or a lower temperature to minimize the adverse effect on the materials. Sterilization
time–temperature or F0 conditions (F0 is defined as the equivalent sterilization time relative to a base temperature of 121°)
include both lower (sterility-related) and upper (stability-related) limits.1 Manufacturers commonly employ the bioburden/
biological indicator (BB/BI) or bioburden methods when constraints on the material’s ability to withstand the process require
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the use of less aggressive conditions. This approach requires appropriate controls on presterilization bioburden and/or
product-related D-values in conjunction with bioindicators of lower spore count or resistance to ensure sterilization.
Terminal Sterilization of Products

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The maintenance of product attributes may require the use of sterilizing conditions that are less aggressive and sterilization
equipment, cycles, and validation methods adapted to these more restricted circumstances. The substantial variations in
equipment designs and methods for terminal sterilization preclude a singular description of a typical cycle. All terminal sterilizers
heat the load, but they accomplish this in varying ways: saturated steam; steam–air mixtures, steam–air–water mixtures, and
superheated water. Air over-pressure for maintenance of container integrity and cooling containers and water for heating/
cooling of the load may be present depending upon the autoclave size, throughput expectations, and container.
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In-Process Fluids
In-process fluids are used for pH adjustment, dilution to a specified volume, lubrication, and other purposes. In many instances
these liquids are sterilized in conjunction with items that must be sterilized by direct steam contact, and the sterilization process
must ensure that all items are adequately sterilized.
Laboratory Media
Laboratory media often are sterilized in standard steam autoclaves with minor adaptations. Provision for slow exhaust (to
reduce stress on container integrity and minimize boil over) and jacket cooling can help improve the basic steam sterilizer design
and operation to better accommodate the materials. The sterilization process may be specific for media containers or a
combination of both liquid-filled containers and hard goods. This process may resemble the methods used for terminally
sterilized products (see above). The sterilization of laboratory media may entail the processing of a number of different containers
that contain different materials. Manufacturers should be aware of the potential for under- and over-processing across the load
and must consider container size, container contents, and position. When liquid-filled containers are combined in the same
load with hard goods, manufacturers must consider the unique concerns of each to ensure all items are properly sterilized.
Because laboratory media are considered self-indicating with respect to sterility, the use of internal biological indicators during
validation is not required.
Biowaste Sterilization
The sterilization of biowaste in sealed containers from laboratory or production use is similar to parts sterilization. The process
is defined to ensure a minimum time–temperature exposure or attainment of a specified F0 value throughout all items of the
load. Depending on the potential contaminants present, the autoclave design may incorporate condensate collection/
sterilization or sterilizable exhaust filters to ensure that pathogens are adequately contained. Because the objective of biowaste
1 Degradation kinetics may differ from those of microbial kill, and F0 values may not be sufficient to fully evaluate “worst case” effects.
https://online.uspnf.com/uspnf/document/1_GUID-D516FF11-E0D7-4288-9979-2074A9B283FD_2_en-US 1/5
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sterilization is to render the materials safe for contact and disposal, the overkill method described in Steam Sterilization by Direct
Contact á1229.1ñ is employed.
Change to read:
BIOBURDEN/BIOLOGICAL INDICATOR METHOD
Application of the BB/BI method requires a thorough understanding of the bioburden type, population, and resistance
typically present in the presterilized product-filled container. The method relies on substantial differences between moist heat
resistance and the population of the bioburden present and the biological indicator used during validation (Figure 1).
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Figure 1. Relative resistance and population of typical bioburden and biological indicator microorganisms.
BB/BI is a method 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 process to reliably destroy any bioburden. This is accomplished using
detailed knowledge of the BB and BI populations and their relative resistance.
Typical BB microorganisms have only minimal resistance in comparison to BIs, and this can be confirmed by heat screening
of BB isolates. The BB population is controlled by filtration steps for the fluid, process time limits, environmental controls,
gowning systems, and other means. The conventional BIs for terminal sterilization using the BB/BI method are Clostridium
sporogenes ATCC 7955 and Bacillus subtilis ATCC 5230, although other strains can be used. The use of Geobacillus
stearothermophilus for terminal sterilization is uncommon with the BB/BI method because the organism’s strong resistance to
moist heat makes it poorly suited for this application.
Confirmation of an acceptable probability of a nonsterile unit (PNSU) can be accomplished using physical measurements
and BI response (which define the lethality of the process) in conjunction with processing limits for the BB population and
resistance (which define the N0 and D-value). D-value is the time (customarily in minutes) 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
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at which it was determined. For example, D121 is the D-value at 121°. Articles intended to be sterile must attain a ≤10−6 PNSU,
i.e., less than or equal to 1 chance in 1 million that viable bioburden microorganisms are present. The PNSU can be determined
from Equation 1.
log Nu = −F/D + log N0 [1]
Nu = PNSU
D = D-value of the natural bioburden
F0 = F0-value of the process (lethality)
N0 = bioburden population per container
The following example indicates the resulting PNSU under the defined conditions of validation and routine operation (Table
1).
Table 1. Examples of PNSU Calculation

Validation Routine Usage
F0 = 8.0 min F0 = 8.0 min
D121 of BI = 0.5 min D121 of bioburden = 0.005 min
N0 of BI = 106 N0 of bioburden = 100 (or 102 )
PNSU for BI = 10–10 PNSU for BB = 10–1598
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Determining the resistance of the bioburden is accomplished using a heat-screening process during which a pure culture (a
laboratory culture containing a single species of organism spores with minimal vegetative cells) is boiled at 100° for various
periods. If the bioburden microorganism is viable after exposure, its resistance at 121° can be estimated for use in the PNSU
calculation (Table 2). ci
Table 2. D121 Estimation from Boil Test Results
Exposure Time Approximate D121-value
1 min 0.01 min

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10 min 0.1 min
20 min 0.2 min
100 min 1.0 min
Bioburden Method
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In most respects the BB method is similar to the BB/BI indicator method. The difference lies in the isolation and
characterization of the most-resistant bioburden microorganism. The worst-case isolate is used as the biological indicator in the
evaluation of the process. For use in this manner, it must be cultured to produce a suitable challenge population. When this
method is used, the bioburden of each process cycle must be closely controlled with respect to population and must be
monitored for resistance.
Sterilization Cycle Control

Process equipment for terminal sterilization typically is controlled by calibrated and pressure sensors in/on the chamber/
equipment. During the exposure portions of the cycle, attainment of a minimum dwell time at a predefined temperature is
used to support process lethality. Cycle lethality for terminal sterilization customarily is measured using F0, which is defined as
an actual exposure time at a variable process temperature that is equivalent to an exposure at 121° based on an ideal
microorganism with a z-value of 10°. This can include lethality delivered during the heat-up and cooling phases of the
sterilization process. A z-value is defined as the number of degrees of temperature change necessary to change the D-value
by a factor of 10. The F0 approach is used to evaluate to a single standard sterilization processes that are operated at varying
temperature conditions. The process lethality at temperatures other than 121° can be calculated to determine lethality
equivalent to that provided at 121°. Sterilizer control systems for terminal sterilization deliver conditions within a predefined
time–temperature or F0 range to avoid over-processing.
Simple mathematics can be used to calculate the total lethality over the course of the process. For the specific reference
temperature of 121° and a z-value of 10.0°, the F0 calculation can be determined by Equation 2:
t = time
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T = temperature
Summing the instantaneous lethality contributions over the entire sterilization process allows the calculation of the overall
process lethality or F0 delivered over the course of the entire process at varying conditions.
Validation of Liquid-Filled Container Sterilization

As previously noted, the preferred method for steam sterilization is the overkill method as defined in Sterilization of Compendial
Articles á1229ñ and Steam Sterilization by Direct Contact á1229.1ñ. However, when the processed materials are susceptible to
damage by moist heat at the overkill conditions, the BB/BI method is better suited because it results in reduced heat input while
affording the same degree of process efficacy but with different controls. As noted above, terminal sterilization processes require
greater consideration of the effects of the treatment on material properties. This has implications for many of the elements of
the qualification and validation exercises as indicated below. The validation requirements for the BB/BI and BB methods are
more rigorous than those associated with the overkill method. Although the overkill method can be confidently used without
detailed consideration of the presterilization bioburden, application of the BB/BI and BB methods require continued monitoring
and control of the bioburden, specifically the population and resistance. This is accomplished by testing of filled containers just
before sterilization and measuring the number of colony-forming units per container and confirming the absence of resistant
BB isolates. When resistant isolates are found, their thermal resistance in the fluid should be determined.
EFFECT OF THE STERILIZATION PROCESS

A preliminary determination of the liquid and the container–closure system’s ability to withstand the expected sterilizing
conditions should be made during product development. This can be accomplished by sterilization at conditions slightly in
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excess of the maximum expected and evaluating the effect on the material. The evaluation should encompass the essential
quality attributes with attention focused on known and potential new impurities. Appearance and other physical properties also
should be evaluated as a part of this effort.
EQUIPMENT QUALIFICATION
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Equipment qualification is a predefined program that confirms the equipment has been properly installed and that it operates
as intended. Qualification of the sterilizing equipment provides a baseline for preventive maintenance and change control for
the sterilizer. The sterilization equipment may require qualification of air, water, utility, and other systems that impact the
sterilization equipment’s performance.
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EMPTY CHAMBER TEMPERATURE DISTRIBUTION
The dual considerations of sterility and stability commonly associated with sterilization of liquids require that equal attention
be paid to potential under- and over-processing of the load. For this reason the temperature gradient across the sterilizer may
require substantially tighter control than that expected in sterilization by direct contact. The objective is to minimize the time–
temperature or F0 differences across the load throughout the process. Biological indicators are not required in the evaluation
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of empty chamber temperature distribution.
BIOLOGICAL INDICATORS
The selection of a BI must be considered carefully because of the balance that must be maintained between attaining
sterilization and maintaining the sterilized material’s essential quality attributes. The biological challenge is either directly
inoculated into a liquid-filled container or is introduced via self-contained units provided there is adequate correlation between
their resistance and the resistance that would occur in the process fluid. The liquid can be either the product or a surrogate
fluid. The resistance of the indicator in the product (and surrogate fluid, where used) must be known. The surrogate’s physical
properties should approximate those of the product. If there are surfaces within the container that are not presterilized,
biological challenge of those surfaces may be required.
LIQUID D-VALUE DETERMINATION

Determination of the thermal resistance (D-value and z-value) for the biological indicator in the liquid is required. This must
be performed in a Biological Indicator Evaluation Resistometer (BIER) in replicate. The thermal resistance of each BI lot in the
liquid should be determined. When a surrogate liquid is used for convenience (e.g., a master solution approach) or because of
microbial inhibition of the BI by the liquid, the thermal resistance in the surrogate must be determined.
CONTAINER MAPPING
Liquid-fill containers with volumes greater than or equal to 100 mL should be mapped to determine internal cold spots. The
mapping should be performed with product containers oriented as they would be within the load. The temperature probes
should be introduced into the containers using methods that maintain container integrity. Internal supports (of minimally
heat-conductive materials) may be required to ensure proper positioning of the probe within the container. After these locations
are determined, they are used as either monitoring locations or are correlated to external conditions on the container during
validation and routine processing. Smaller containers (less than 100 mL) have fewer discernable cold spots, the importance of
which is reduced as container size decreases. Smaller containers (less than 100 mL) can be monitored with temperature probes
secured to their exterior. The “cold spot” should be considered a “region” and not a single point in the container.
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LOAD POSITIONING AND MAPPING

A fixed loading position within the sterilizer may be necessary for proper sterilization to ensure uniformity of heating and
cooling in routine use. Once the load is positioned properly, its size can vary within a defined range. Load-mapping studies
should be performed to determine the coldest and hottest locations within the load. These locations may not be specific
individual containers but rather regions. This ensures that the containers are neither under- nor over-processed in routine
operation of the sterilizer. Validation of variable-size load patterns is accomplished using a bracketing approach for which success
with maximum and minimum loads (avoiding both under- and over-processing) establishes the acceptability of
intermediate-size loads. However, evaluation of intermediate load sizes may be beneficial. In product sterilization, only a
single-size container with a single product lot is processed concurrently.
HEAT PENETRATION AND MICROBIOLOGICAL CHALLENGE

The core of the validation activity is confirmation of acceptable heat penetration using temperature measurements and
microbial challenge inactivation. Temperature probes and biological indicators are placed within the load at worst-case locations
(e.g., the coldest portions of the loaded chamber). Introduction of the thermocouples must not alter the integrity of the
container. Biological challenges are placed in containers adjacent to those that contain heat penetration probes (or the same
unit with external temperature measurement).
Proof of cycle efficacy is provided by replicate studies in which the BIs perform as expected and the physical measurements
correspond to the expected values of time and temperature or F0. If the microbial and physical measurements do not correlate,
an investigation is in order, and corrective action must be taken to rectify the discrepancy. Samples from the hottest regions of
the load are used for evaluation of material stability and quality.
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PRODUCT QUALITY AND STABILITY EVALUATION
Manufacturers must conduct ongoing evaluation of the product’s ability to withstand the routine sterilizing conditions. The
evaluation should encompass the essential quality attributes with attention focused on known and potential new impurities and
those materials that receive the most heat input. Manufacturers also should evaluate appearance, other physical properties, and
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container–closure integrity as a part of this effort. For microbiological media, the ability of the media to meet growth promotion
and other requirements is required as indicated in the appropriate test chapter(s) (e.g., Microbiological Examination of Nonsterile
Products: Microbial Enumeration Tests á61ñ, Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
á62ñ, and Sterility Tests á71ñ).
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Routine Process Control
All sterilization processes should be subject to formalized practices that maintain them in a controlled state. The practices
outlined in Sterilization of Compendial Articles á1229ñ include the general requirements appropriate for all sterilization systems.
This is accomplished by a number of related practices that are essential for continued use of the process over an extended
period of time. The practices include: ▲calibration,▲ (ERR 1-Nov-2019) physical measurements, physicochemical integrators,
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indicators for sterilization, monitoring of bioburden, ongoing process control, change control, preventive maintenance, periodic
reassessment, and training.
The use of parametric release is common in the terminal sterilization of finished product containers. This subject is
addressed in Terminally Sterilized Pharmaceutical Products—Parametric Release á1222ñ.
REFERENCES
Berger, T., & Trupp, K., Validation of Terminal Sterilization, chapter in Validation of Pharmaceutical Processes: 3rd edition,
edited by J. Agalloco & F. J. Carleton, InformaUSA, New York, 2007.
Owens, J., Sterilization of LVPs and SVP’s, chapter in Morrissey, R., & Phillips, G.B., Sterilization Technology—A Practical
Guide for Manufacturers and Users of Health Care Products, Van Nostrand Reinhold, New York, 1993.
Young, J., Sterilization with Steam under Pressure, chapter in Morrissey, R., & Phillips, G.B., Sterilization Technology—A
Practical Guide for Manufacturers and Users of Health Care Products, Van Nostrand Reinhold, New York, 1993.

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Printed on: Tue Mar 15 2022, 06:48:18 AM(EST) Official Status: Currently Official on 15-Mar-2022 DocId: 1_GUID-D516FF11-E0D7-4288-9979-2074A9B283FD_2_en-US

á1229.2ñ MOIST HEAT STERILIZATION OF AQUEOUS LIQUIDS

Terminal Sterilization of Products

log Nu = −F/D + log N0 [1]

Table 1. Examples of PNSU Calculation

D121 of BI = 0.5 min D121 of bioburden = 0.005 min

N0 of BI = 106 N0 of bioburden = 100 (or 102 )

PNSU for BI = 10–10 PNSU for BB = 10–1598

1 min 0.01 min

20 min 0.2 min

100 min 1.0 min

Sterilization Cycle Control

Validation of Liquid-Filled Container Sterilization

EFFECT OF THE STERILIZATION PROCESS

of empty chamber temperature distribution.

LIQUID D-VALUE DETERMINATION

LOAD POSITIONING AND MAPPING

HEAT PENETRATION AND MICROBIOLOGICAL CHALLENGE

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