Research Article
ISSN 2250-0480
Vol 2/Issue 4/Oct-Dec2012
STRATEGIES FOR THE VALIDATION OF STEAM STERILIZATION
PROCESS IN AUTOCLAVES FROM THE PHARMACEUTICAL INDUSTRY
LEONARDO TEIXEIRA RODRIGUES1,2, ADERVAL SEVERINO LUNA1 , CRISTIANE
ASSUMPO HENRIQUES1 AND ANTONIO CARLOS AUGUSTO DA COSTA*
1
PPG-EQ/IQ - Universidade do Estado do Rio de Janeiro Rua So Francisco Xavier 524,
Pavilho Haroldo Lisboa da Cunha, Maracan, Rio de Janeiro, RJ CEP: 20550-013,
2
Biomanguinhos Fundao Oswaldo Cruz.
ABSTRACT
The process of sterilization in the asepsis pharmaceutical ensures the materials used in the production of
medicines. The method of saturated steam sterilization is always the first choice in the pharmaceutical
industry because it is the most effective, faster, with better cost / benefit and with less environmental
impact. In order to ensure necessary sterility, the qualification of autoclaves and validation of
requirements when their loads are required by national and international regulatory bodies. The
regulations of these bodies are not always objective and clear, indicating what should be done without
specifying how it should be done. The focus of this work was to create a validation methodology,
change control and revalidation in horizontal autoclaves, highlighting the critical points of the process,
exploring the theoretical concepts of bioengineering. For this, we used statistical techniques for
analyzing data collected in a study of thermal distribution, heat penetration and microbiological
challenges to determine a fast, safe and effective that meets the requirements of regulatory bodies
without affecting the production capacity of industrial plants and quality of the sterilization process.
KEY WORDS : Autoclave; Sterilization; Validation; Microbes; Pharmaceutical Industry.
INTRODUCTION
In the nineteenth century surgeons began to
disinfect hands, tools and environment in order
to prevent infection during surgery, thus
resulting in the surgery asseptic covering
sterilization by physical and chemical
instruments and the surgical environment.
Therefore, one can say that sterilization is the
physical or chemical attempts to destroy or
eliminate all forms of life, especially
microorganisms. Even with the advancement of
science and technology, there are still cases of
infections and hospital products. This is due to
the lack of studies and tests, in some companies
and hospitals, showing that the sterilization
processes are really effective. With this, we
highlight the importance of sterilizing equipment
qualification and validation of sterilization
processes.
1.1 Steam and Sterilization
The heat sterilization is widely used in the
pharmaceutical industry. Table 1 shows the
equivalence of mortality among the different
temperatures with the respective exposure times,
ie mathematically all have the same potential
sterilization. It can be concluded that the
sterilization in the presence of steam is more
efficient since it requires less time and
temperature as the dry heat.
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Table 1. Dry and steam sterilization
Steam sterilization
Temperature (C)
Time (min)
Dry condition
121 126 134 160 170 180
15 10 3
120 60 30
since the boiling point is to be greater than 100
C. This is the case of industrial work with
autoclaves relative pressures around 1.1 kgf/cm
so that the boiling point of water to occur near
121 C. The saturation temperature increases
with pressure, but there is a limit, called the
critical point, above which there is no defined
transition between the two states. Other
important points refer to the dilations that take
place when the water passes into the gaseous
state and back to the contraction when liquid
(Figure 1).
This is due to the fact that as water is heated,
more energy is absorbed to the point the
temperature rises until the boiling state (latent
heat). This energy is used to increase the
temperature, but for transition from liquid to gas.
The spent energy is too high this transition,
causing the reverse process - condensation - the
energy obtained is also very high, confirming
that the sterilization by heat in the presence
steam has a high yield. The yield can be further
increased if the internal pressure of the chamber
is maintained above atmospheric pressure,
Figure 1. Cycle of steam penetration
jacket. The vessel built is the place to
accommodate the load for sterilization and is
called the inner chamber. The normal process of
steam sterilization is basically composed of three
phases. The first stage is known as preconditioning, and constitutes the air is sucked
into the inner chamber through repetitive cycles
of alternating vacuum pulses of injection steam.
At least three vacuum pulses are sufficient, since
the third pulsed vacuum already ensures removal
of 99.994% of the air inside the chamber
(Luqueda, 2004). It is essential to eliminate the
air within the chamber, because the air is
considered the best thermal insulation. The next
step includes the exposition time, where the
maximum temperature is kept at 121C. At this
stage, a temperature sensor installed in the drain
valve controls the steam inlet in about 1.1
kgf/cm, so that the sterilization temperature
The steam to condense and wet the surface of the
instant product undergoes a contraction of
volume (in the order of 1500 times). This causes
the penetration of steam into the object to be
processed to produce an effect of "vacuum
pump" that captures more steam which continues
heating the product and condensing. To
condense the vapor creates a partial vacuum
which tends to be occupied by more steam
which, in turn, provides more energy. Thus, one
cycle is fulfilled. This phenomenon is also called
successive "heat pump" (Luqueda, 2004).
MATERIALS AND METHODS
The horizontal autoclave is basically a
combination of two pressure vessels sealed,
contained in the other. The outer vessel is called
outer chamber, but also is known as a shirt or
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remains close to 121 C. If it was maintained at
134C, the pressure should be adjusted to 2.1
kgf/cm. The drain is the best place to control
temperature, as air and condensate, heavier than
steam, are removed by gravity through the drain
located at the bottom of the camera, turning at
the coldest point of the camera. If the drain is at
121C, it is expected that the other points within
the chamber are greater than or equal to 121C.
The last phase of the sterilization cycle is the
post-conditioning, where the vacuum system is
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switched on while the steam valve is closed. The
presence of vacuum with a heat radiation from
the walls of the chamber takes all moisture from
the chamber (drying). After drying time, the
pressure is equalized to atmospheric pressure,
even outside air through the filter without
aeration. The quality and proper maintenance of
this filter are vital to the success of the
sterilization, since the injection end of the
contaminated air cycle affects the sterilization
process (Figure 2).
Figure 2 Stages of steam sterilization. I) Pr-conditioning; II) Time of exposure; III) Postconditioning.
is found that the microbial death behaves as a
first-order reaction. The decimal reduction time
(D) is defined as the exposure time required to
cause a 90% reduction in the population of a
microorganism. All the values for D are specific
to a temperature, usually cast to 121.1C. This is
calculated by Equation 1 (Russell, 2004):
2.1 Quantification of Steam Sterilization
With the knowledge of the kinetics of microbial
destruction, it is possible to use mathematical
models to predict the behavior microbial steam
sterilization. According Luqueda (2004) the
kinetics of microbial death does not necessarily
follow a linear model and that there is a
mathematical model entirely linear. In practice it
D=
t
log10 N 0 log10 N
(1)
Where:
t = Duration of the thermal treatment
N0 = Initial population of microorganisms
N = Final population of microorganisms
The value z is another way to determine the
microbial resistance to sterilization by moist
heat. Is defined as the number of degrees of
temperature required for switching an output
logarithmic value. The z value is used to
determine the mortality assessment, it is not
suitable above 135C and is not suitable
their extrapolation (Baumer, 2006). For
verification purposes, been standardized in
the pharmaceutical z value of 10. The unit
that quantifies the sterilization is called Lethality
(Fzero or F0), expressed in minutes. According to
the definition Parenteral Drug Association
(PDA, 2007), F0 is the exposure time (min)
equal to 121C with z value of 10C. For
example, when a sterilization cycle accumulated
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value F0 to 15 min, then the product was
exposed theoretically 15 minutes at 121C. In
this case, 121C maintained for 15 min, 115C
maintained for 30 min and maintained at 134C
t2
F0 = 10
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for 3 min, have the same value F0 equal to 15
min. The calculation of the lethality of the
sterilization process by saturated steam is given
by Equation 2.
T 121.1C
Z
dt
(2)
t1
Where:
F0 = Accumulated lethality at 121.1C with a Z value equal to 10C and D equal to 1 min.
t1 = Time when the lower temperature is higher than 100C.
t2 = Time just after t1, when the lower temperature is lower than 100C.
Z = Absolute number of degrees of temperature required for a change in one logarithm in the value of D.
In sterilization processes, this value is equal to 10C.
T = Instant temperature (C).
According to Equation 2, it can be seen that the calculation of mortality has a significant value only above
100 C. For this reason, the data acquisition system automatically performs the calculation of lethality
only when the temperatures are above 100C.
100C 121.1C
10C
F0 = 10
F0 = 0.008min
In this case we can say that sterilization at 100C is 100 times less effective than 121C, for 1 minute to
100C is approximately 0.01 minutes at 121C (Figure 3).
Figure 3 Ramp of accumulated lethality.
The concept of absolute sterilization is being
replaced by the probability of the load to be
sterile. Due to theoretical and practical
limitations, no similar statement regarding the
absence of microorganisms can be proved
(Luqueda, 2004). In the sterilization process, the
killing of microorganisms is described by an
exponential function. Therefore, the presence of
viable microorganisms on any item should be
expressed in probabilities. Although this
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probability can be reduced to a very small
number can never be reduced to zero. This is
why the term sterility assurance level is used to
estimate in the pharmaceutical or evaluate the
efficacy of the sterilization process. He is often
portrayed by its English abbreviation SAL
(Sterile Assurance Level) and expresses the
probability
of
survival
of
viable
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microorganisms. The standardized value of the
SAL, the majority of the International
Pharmacopoeia is 6.10. To achieve a SAL of 106
cycle, must be able to reduce 12 log units of an
initial population of one million, thus expressing
a first probability of survival in 1,000,000
(FDA, 1994) (Figure 4). The probability
calculation is extracted from Equation 3.
F0 = D121C (log10 N0 log10 B)
(3)
Where:
F0 = Minimum required lethality
D121C = Thermal resistance of the bioindicator
N0 = Initial population of the bioindicator
B = Probability of survival of the bioindicator
To estimate the probability of survival should rearrange the formula Equation 4.
log10 B = log10 N 0
F0
D121C
(4)
As an example, a microbial challenge was determined using a biological indicator value D of 121C = 1.3
min with initial population of 1.5 x 106. To obtain a sterilization cycle which is able to provide a SAL of
10-6 to scale a cycle that is capable of accumulating the following minimum required lethality (Equation
5).
F0 = D121C (log10 N 0 log10 B)
F0 = 1.3(log10 1.5x106 log10 106 )
(5)
F0 = 15.8min
Figure 4 Sterile Assurance Level at 10-6.
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Exemplifying an opposite manner, one can estimate the probability of a given sterilization cycle which has
accumulated a mortality rate of 25.3 min and was challenged with the same biological indicator (Equation
6).
log10 B = log10 N 0
F0
D121C
log10 B = log10 1.5x106
25.3
1.3
(6)
B = 1x1013.28
COMPLIMENTARY RESULTS AND DISCUSSION
into hermetically closed and open). Dried
porous loads are intended to put the steam in
contact with microorganisms through their pores
and/or its packaging. There is a need to remove
air, steam penetration, the steam and reevaporation of moisture from the material, for
example packed garments and glassware. Dry
solids loads are intended to bring steam into
direct contact with the surface of the load, for
instance in trays. The processes used to load
porous solid materials or have a common goal to
put the steam in contact with the
microorganism. For liquids, the goal is different
- the water in the product reacts with the
microorganism to kill him, so the steam is used
to evenly heat the liquid. Figure 5 shows the
possible combinations of categories to classify
the matters.
3.1 Classification of the Types of Matters to be
Sterilized
Because they present peculiarities in the preconditioning, exposure time and postconditioning loads of autoclaves were classified
according to their with your final objective
(purpose of the load) and its state of matter. If
the material is used for product handling in a
sterile environment (clean room or cabin
laminar flow) or formulation of product
ingredients, the sterilization process is called. If
the material is classified as Waste Health
Service - RSS, which are usually remnants of
production that had contact with biological
materials and must be discarded, are called and
Decontamination Process (ANVISA, 2006). The
term state of matter refers to solid and liquid
states of the items of charge. They were divided
into dry cargo (which is subdivided into solid
and porous) and liquid cargoes (also subdivided
Figure 5 Classification of autoclavable loads
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control and the ends of the chamber. The control
instrument for sensing the load is dried and
drain liquid loads is the load sensor. For each
study, 12 were spread equidistantly type T
thermocouples into the inner chamber, as shown
in Figure 6. Maximum limits have been defined
(124C) at least (120C) and the limit of
variation between the sensors ( 2C), only
during the exposure time. A variation greater
than 2C has no impact on sterilization, since
the cycle is sized according to the lowest value
of accumulated lethality, but a malfunction of
the autoclave. The presence of air in the
chamber can cause large temperature variation.
3.2 Thermal Distribution
The thermal study with the empty autoclave is
essential to evaluate the performance of the
autoclave. The evaluation of the thermal
uniformity of the load depends on the autoclave
is used, since the positions of the cargo items
can hinder the transit of the steam. The thermal
study of the autoclave provides the thermal
distribution in the inner chamber highlighting
the hot and cold spots. This procedure was
performed with three races to show the
reproducibility of the system. Each type of cycle
(dry, liquid and decontamination), which present
different specificities, was tested separately. The
points to be monitored are instruments of
Figure 6 Distribution of temperature sensors inside the autoclave
The use of a large number of sensors can affect
the nature of the leak-camera, as all the sensors
are in the same hole in the autoclave, called
validation gate. Today this problem is being
solved with data loggers with wireless
technology, but unfortunately it is too
expensive. Since the steam distribution being
evaluated in this study took care that the sensors
do not touch to the inner wall surface of the
chamber, as this would lead to false
measurements.
and loads through the lethality accumulated in
each item. Is typically used as the reference
minimum cumulative mortality and has been
calculated at all times that the sensors were
above 100 C. Twelve were randomly scattered
sensors (one on each item of the load), where
the positions were different in each study,
covering all areas of the autoclave. In order to
evaluate the behavior of the vapor in several
items with different shapes, it was placed more
than one sensor on the same item or identical
items. Were used for most random positions of
sensors and a sensor fixed in the cold spot. For a
satisfactory safety margin, all the cycles were
sized to provide a SAL of 10-6 for a population
of microorganisms with 1x106, a D value of 2
min; all cycles had a mortality of at least 24
minutes (Equation 7).
3.3 Penetration of steam
The materials in thermal study were recorded
via sensors inserted into the load of the items
during the measuring cycle and the heat supplied
to the cargo items. The objective of this
procedure is not to assess the homogeneity
among the items of thermal loads, but to
evaluate the penetration of steam in packaging
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F0 = D121C (log10 N 0 log10 B)
F0 = 2x(log10 1x10 6 log10 106 )
F0 = 24 min
(7)
For microbial challenge was positioned one at each end of the bioindicator thermocouples.
distribution, a study of the penetration of steam
and dry worst case study of penetration of liquid
worst case.
3.4 Determination of the Worst Case
After completing the studies of the thermal
distribution, penetration of steam and
microbiological challenge, split the charges into
three categories: Porous, Solid and Liquid. The
solid cargo, because they are easy sterilization,
were excluded from the process of selecting the
worst case. The porous and liquid loads were
evaluated for a relationship Lethality vs. Time,
one for dry cargo and liquid cargo to other. It
was then possible to determine the dry cargo and
liquid cargo which the highest resistance to
penetration of steam. That it took more time to
accumulate F0 equal to 12 min was considered
the "worst case".
CONCLUSIONS
This validation methodology provided sufficient
data to evaluate the thermal performance of the
autoclave with or without load. She brings more
accurate diagnosis of any problems and can
determine whether the cause is linked to the
wear of the components of the autoclave or in
the assembly and configuration of the loads. It
also covers new concepts for selecting the
"worst case" because contrary to what many
believe, is not always the maximum load has a
lower growth rate of lethality. Another
important aspect was the revalidation. It created
a methodology for revalidation fast, safe and
effective, highlighting the critical points of the
process,
exploring
the
physical
and
microbiological concepts and concentrating
efforts where they really needed. Therefore, it
was possible to meet the requirements of
regulatory bodies without affecting the
production capacity of industrial plants and the
quality of the sterilization process. This fact can
be proven as this methodology has undergone
several national and international audits, and
none made any recommendations.
3.5 Revalidation
Revalidation is the repetition of part or all of the
validation tests intended to reconfirm the
reliability of the process (ANVISA, 2009).
Unfortunately, the regulators do not regulate
which parts of the tests should be repeated,
giving rise to recommendations that propose
different every time the plant is inspected and
must be done every time a repair in the
autoclave can significantly affect the efficiency
of the process. Revalidation should also be
performed at least once every year. Based on the
determination of the worst case, the annual
revalidation process consists in calibrating
instruments critical autoclave conduct a thermal
ACKNOWLEDGEMENTS
Authors are thankful to CNPq, FAPERJ and Bio-Manguinhos for support to conduct this work.
REFERENCES
1. ANVISA Consulta Pblica n 3, de 13 de
janeiro de 2009. D.O.U de 17/02/09
2. ANVISA, Gerenciamento dos Resduos de
Servios de Sade. Braslia: Editora Anvisa,
2006.
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