Principles of sterilization

R.M.E. Richards
Introduction
Pharmaceutical products are generally required to be free from contamination
with micro-organisms (bacteria, viruses, yeasts, moulds, etc.). Such organism
may cause spoilage by adversely affecting the appearance or composition of a
product and may cause serious adverse effects in the patient. Adverse effects
are particularly likely if the preparation is introduced into the body via a route
which bypasses some of the bodys normal defence mechanisms, especially in a
seriously ill or immunocompromised patient.
Dosage forms designed for parenteral, ophthalmic or surgical use, as well
as irrigation solutions and topical preparations for application to large open
wounds, must be free from microbial and particulate contamination. These
products are requaired to be prepared and maintained in a sterile state until
used. Some of the terms used in connection with sterile pharmaceutical
products are defined below.
Sterility. Sterility my be defined as the total absence of viable microorganisms and is an absolute state. The production of sterile pharmaceutical
products may be achieved by aseptic technique (see Ch. 22) or by means of a
terminal sterilization process (see Chs 20, 21).
Sterilization. This is the subjection of products to a process whereby all
viable life forms are either killed or removed. The sterilization process is ussually
the final stage in the preparation of the product. The fundamental properties of
micro-organisms and the actions of chemical and physical agents upon them are
discussed in the companion volume (Aulton 1998), which also discusses the
principles and practice of sterilization. The methods of sterilization in reguler use
include exposure to : saturated steam under pressure, dry heat, ionizing
radiation, ethylene oxide or passage through a bacteriaretaining filter. When
possible, exposure to saturated steam under pressure is the sterilization method
of choice.
Aseptic technique. This is the preparation of pharmaceutical products
from sterile ingridients by procedures that exclude the access of viable microorganisms into the products. It is used for those products that would be
adversely affected by being subjected to a sterilization process.
Sterility testing. Tests for sterility of pharmaceutical products attempt to
reveal the presence or absence of viable micro-organism in a sample number of
containers taken from a production batch. From the results of such tests an
inference is made as to the sterility of the batch.

Disinfection. Disinfection is a process which aims to reduce the number

of harmful (phatogenic) microorganisms in a particular situation. Disinfection will
destroy infectivevvegetative organisms but not necessarily resistant spores, i.e.
it is not an absolute process. It often involves the use of chemicals althought
other means of disinfection may be employed, e.g. the pasteurization of milk is a
disinfection process that uses heat.
Disinfectant. This is a chemical agent used to destroy harmul microorganisms ususally in inanimate objects.
Antiseptic. This is a chemical agent usually applied to living tissues in
humans or animals in order to destroy harmful micro-organisms.
STERILIZATION Cor RITERIA
The bioburden
In order to select the appropriate parameters for any method intended to kill
micro-organisms in a given product or associated with a given material, it is
necessary to know the initial number of organisms, the bioburden or bioload,
and their resistance to the cosen process. For example, it has been the practice
to choose the time and temperature relationship for steam sterilization to ensure
that a large number of the known most resistant pathogen would be killed. This
treatment would not necessarily be sufficient to kill a large number of the known
most resistant non-pathogenic organism which, however, are extremely unlikely
to be present and is greatly in excess of the treatment necessary to kill the small
number of heat-sensitive contaminants likely to be present in pharmaceutical
solutions.
Establishing microbial death
Death in a microbial population is determined by assessing the reduction in the
number of viable micro-organisms resulting from contact with a given destructive
force. Viable organisms are those which when transferred to a medium can form
a colony. This places the onus on the investigator to provide suitable culture
conditions for recovery and growth of any surviving micro-organisms.
Death rates
The kinetics of microbial cell inactivation are discussed in detail in the
companion volume (Chapter 25, Aulton 1988). When a population of microorganism is subjected to a destructive sterilization procedure the order of death
is generally logarithmic. That is a constant proportion of the microbial population
is inactivated in any given time interval, approximating to first-order kinetics.
A typical survivor curve is shown in Figure 19.1. If N0 is the initial number of
organisms and Nt surviving after time t, the death rate constant (inactivation
constant) can be calculated.
Nt =Noe-t

In Nt = In N0 t

Log10 Nt = log10 N0 - 2.303

The death rate constant can be calculated as follows from Figure 19.1
Let N0 = 106 Nt = 102 t =10
Then, = 1/10 (log10 106/102 )
=1/10 (6 2) = 0.4
The dertiminationof death rate providers the facility to compare the
resntanceof the same organism at different temperatures or to compare the
resistance of different organisms to the same lethal agent, e.g. temperature,
ionizing radiation, chemical agent, etc. Death rates my also be used to give a
quantitative measure of the effect of enviromental factors such as pH, os
molarity and the presence of various chemicals on the sterilization process. Since
the same percentage of bacteria die each minute, it is impossible in theory to
reach a point of zero survivors. From figure 19.1 an extension of the process to
30 minutes would gave the assurance that the probability of survival of one
member of the original population would be 1 in 10. In terms of containers of
solution this would mean a probability of one container in a million being
contaminated.
The concept of percentage reduction of a bacterial population reinforces
the need for as small a bioburden as possible when a product is to be sterilized.
The methods that are used to prepare sterile fluids ensure that the
biorbuden is very low (see Chs 24, 29). If containers are filled througa bacteriaproof filter prior to sterilization the biorbuden is effectively zero.

De value or decimal reduction time

The death rate can also be expressed as the decimal reduction time or D
value. This is the time in minutes atany defined temperature to destroy 90% of
viable organisms. In Figure 19.1 this is 2.5 minutes. Numerically it is also the
reciprocal of the death rate constant. D values are often given a subscript to
indicate the temperature at which they were measured, e.g. D1210C. From the D
value for a particular combination of an organisms/time/temperature an
innactivatio factor (IF) can be calculated, e.g.e if the D value of an organism
exposed to a temperature of 1210 C for 15 minutes was 2 minutes the IF would
be 1015/2, i.e 107.5.
Z value or thermal destruction value
This relates the heat of a micro-organism to changes in temperature. The Z value
is the number of degrees of temperature change to produce a ten-fold change in

D value. Bacterial spores have a Z value in the range 10-15 0C while most nonsporing organisms have Z values of 4-60C.
Q or temperature coefficient
The F value is a measure of the lethality of the total process of sterilization and
equates heat treatment at any particular temperature with the time in minutes
at a designated reference temperature that would be required to produce the
same lethality in a organism of stated Z value. For a temperature of 121 0C and
organism with a Z value of 10 0C F becomes Fo. Annex 2 of Appendix of F0: The F0
value of a sturated steam process is the lethality expressed in terms of the
equivalent time in minutes at 1210C delivered by that process to the product in
its final containers with refernce to micro-organisms possesing a Z value of 10 0C.
One F unit is equivalent to heating the load for 1 minute at 121 0C.
Mathematically F is defined as :
Fo = 10(Tc-121/Z) dt
Where Tc = load temperature at time dt and Z = 100C.
The total lethality (Fo) for the cycle is equal to the sum the F o values for
each individual time/temperature segment. The accuary of the estimated F O is
related to the time interval represented by each segment of the profile. Small
intervals are conventionalty handled by computer, the BP 1998 suggest the use
of Fo calculation for the overall lethalty delivered to aqueos preparations for a
microbiologically validated steam sterilizations process and states that a total F o
value of not less than 8 applied to every container in load would be considered
satisfactory.
Where the product is especially heat sensitive an F o of less than 8 is
deemed justifiable so long as great care is tajen to ensure that an adequate
assurance of sterility is consistently achieved. That is : it is necessary not only to
validate the process microbiologically, but also to perform continuous, rigorous,
microbiological monitoring during routine production to demonstrate that micro
biological parameters are within the estabilshd tolerances so as to give a
theorretical level of not more than one living organism in 10 6 containers of the
final product (1988).
Kirk et al (1985) investigated the degradiation under autoclaving
conditions of 2,4-dihyroxybenzoic acid in autoclave cycles delivering a standart
process lethality (Fo=8) by means of different degress of degradation of the
chemical. That is hight temperature, short time cycles (112 oC/Fo=8) caused
approximately 12% degradation but prolonged exposure at a relatively low
temperature (112oC/Fo=8) caused approximately 30% degredation. Thus in the in
terest of stability these works suggested that, where other factores permit, the
optimum sterilization cycle would consist of the load being subjected to a
temperature which continuously increased, no holding phase, until a prefixed F 0
had been achieved. The load would then be cooled under controlled conditions

and this cooling phase would provide the remaining F

the predetermined level of sterility assurance.
There is further discussion on the use of the F
the companion volume ( Aulton 1988 ).

units required to achieve

concept in Chapter 26 of

STERILIZATION VALIDATION AND MONITORING

Tests for sterility of the products subjected to a sterilization procedure are
discussed in Chapter 30. Whenever possible addtional validation and monitoring
of the sterilization process is carried out using indicatores other then the product.
Biologicval, chemical and physical indicators have all been used. ( see also
companion volume, Aulton 1988, Chapter 43 ).
Biological indicators
Biological indicators are supplied in one of two main forms, each of which
incoporates a viable culture of a stated species of micro-organism. One form
consists of spores added to a carrier such as disc or strip of filter paper, glass or
plastic, so packaged as to protect the contents before use but to allow the
sterilizing agent to reach the spores an exert its effect during use. In the other
form the spores are added to representative units of the product to be sterilized
or to similar units if it is not practicable to add the spores to selected units of a
particular product.
Choice of the biological indicator is critical if the indication given is to be a
valid reflection of the efficacy of sterilization cycle. The viability of the organisms,
the storage conditions before use and the incubation and culture conditions after
sterilization must be standarized for the result to be meaningful, especially for
the less chalenging protocols ( e.g. F 0 values of 8 or less ).
The reader is referred to the monographs in the British Pharmacopoeia and
United States Pharmacopeia for current recommendations.
The organisms used s biological indicators include:
Bacillus subtilis var. Niger dry heat
Bacillus stearothermophilus steam
Clostridium sporogenes steam
Bacillus subtilis var. Niger ethylene oxide
Bacillus pumulis ionizing radiation
In order to use biological indicators effectively in the monitoring of a
sterilization process a knowledge of the product bioburden in terms of numbers
and resistance to the sterilization methode is required. The biological indicator

must be so chosen as to provide a greater challenge to the sterilization process

than the natural bioburden of the product.
Sterilzation by filtration
For the biological validation of sterilization by filtration se Chapter 22.
Chemical indicators
These are used to indicate whether a particular batch of product has been
through a sterilization process, they do not generally indicate whether the
process was successful. The indicator chosen undergoes some change in physical
or chemical nature when exposed to the conditions of the sterilization process.
Brownes tubes
These are sealed glass tubes (manufactured by A. Browne, Ltd Leicester
containing a red fluid which change colour through yellow and brown to green on
heating at the specified temperature for the appropriate length of time. Various
types are available for different sterilization processes, e.g. moist heat and try
heat processes. They should not be used as quatitative indicators.
Heat-sensitive tape
The Bowie-Dick test is valuable for confirming that steam has displaced all
the air from a porous load in a high vacuum autoclave (Bowie et al 1963). It
consists of using autoclave tape which has heat-sensitive bars, at intervals of
about 15 mm, which change colour after contact with steam. The tape is placed
suitably wrapped at the centre of a test pack. All the bars on the tape should
change colour to demonstrate full penetration of the steam. Duration of exposure
or temperature attained is not indicated by this type of indicator.
Chemical degradation tests
Hoskins (1981) has described a test which follows the degradation kinetics of
2,4-dihydroxybenzoic acid by u.v. spectrophotometry. Bunn & Sykes (1981) were
able to calibrate Thermalog S indicator strips in terms of F 0 unit in the
temperature range 115-123oC. Thermalog S (produced by Bio Medical Sciences,
Fairfield, USA) would appear to be a useful monitor for moist heat sterilization.
Ethylene oxide sterilization
The royce sachet contains an indicator which changes colour on exposure to a
given time/concentration of ethylene oxyde.
Chemical dosimeters
There are used to monitor the quantity of the radiation sterilization. Qualitative
indicators of exposure to radiation are also available.
Phisycal validation and monitoring

In the UK, guidelines on the acceptability of a sterilization cycle are given in the
Health Technical Memorandum 10 on sterilizers (DHSS). A Master Temperatured
Record (MTR) is prepared as part of the validation procedure for a particular
autoclave and for each specified product and load configuration. This is then
used as a reference for the process record obtained from a single thermocouple
placed in a strategic part of each load (Temperature Recording Chart, TRC).
In scotland the MTR is established on the evidence of three succesful sterilization
cycles as recorded on a 12-point recorder on a situations. For examples, ten
thermocouples could be the load, one thermocouple could be left free in the
centre of the autoclave and one thermocouple should be placed sparately in
containers distributed throughout the load, one thermocouple could be left free
in the centre of the autoclave and one thermocouple should be represent the
coolest spot. The MTR should be checked at annual intervals and whenever
significant changes occur in the TRC when compared with the MTR.
It is likely that microprocessor controlled sterilization cycles will replace the MTR
approach to controlling sterilization cycles. This is because the microprocessor
gives the possibility of a much tighter control and description of sterilization
cycles than comparison of the TRC with the MTR as used at present in the UK.
Conclusion
From the foregoing it can be seen that the factors affecting sterilization include:
Factors relating to the biorburden.
These are concerned with the Initial number of organisms present, their heat
resistance and in growth requirements.
Factors relating to the sterilization process.
These are concerned with the method chosen, the probability of sterilization
which is related to the overall lethality or efficiency of the process and the
methods of validating and monitoring the sterilization processes.
Factors relating to the product to be sterilized.
These are concerned with the physical and chemical properties of the product.
That is its solubility, hydrogen ion concentration and nutritive properties and in
particular its stability to heat and moisture.
In addition, it should be mentioned that the properties of the container are
Interrelated with the factors relating to the sterilization process and those
relating to the product.