Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology

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Fig. 20.1 Typical survivor

curves for bacterial spores

exposed to moist heat or

gamma-radiation.

Expressions of resistance

D-value

The resistance of an organism to a sterilizing agent can be described by means of the

D-value. For heat and radiation treatments, respectively, this is defined as the time

taken at a fixed temperature or the radiation dose required to achieve a 90% reduction

in viable cells (i.e. a 1 log cycle reduction in survivors; Fig. 20.2A). The calculation of

the D-value assumes a linear type A survivor curve (Fig. 20.1), and must be corrected

to allow for any deviation from linearity with type B or C curves. Some typical D-

values for resistant bacterial spores are given in Table 23.2 (Chapter 23).

z-value

For heat treatment, a D-value only refers to the resistance of a microorganism at a

particular temperature. In order to assess the influence of temperature changes on thermal

resistance a relationship between temperature and log D-value can be developed leading

to the expression of a z-value, which represents the increase in temperature needed to

reduce the D-value of an organism by 90% (i.e. 1 log cycle reduction; Fig. 20.2B). For

bacterial spores used as biological indicators for moist heat (B. stearothermophilus)

Principles and practice of sterilization 387

and dry heat (B. subtilis) sterilization processes, mean z-values are given as 10°C and

22°C, respectively. The z-value is not truly independent of temperature but may be

considered essentially constant over the temperature ranges used in heat sterilization

processes.

2.3

Sterility assurance

The term 'sterile', in a microbiological context, means no surviving organisms

whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not,

and so there are no levels of contamination which may be considered negligible or

insignificant and therefore acceptable.

From the survivor curves presented, it can be seen that the elimination of viable

microorganisms from a product is a time-dependent process, and will be influenced by

the rate and duration of biocidal action and the initial microbial contamination level. It

is also evident from Fig. 20.2A that true sterility, represented by zero survivors, can

only be achieved after an infinite exposure period or radiation dose. Clearly, then, it is

illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus,

the likelihood of a product being produced free of microorganisms is best expressed in

terms of the probability of an organism surviving the treatment process, a possibility

not entertained in the absolute term 'sterile'. From this approach has arisen the concept

of sterility assurance or a microbial safety index which gives a numerical value to the

probability of a single surviving organism remaining to contaminate a processed product.

For pharmaceutical products, the most frequently applied standard is that the probability,

post-sterilization, of a non-sterile unit is ^1 in 1 million units processed (i.e. =sl0

-6

The sterilization protocol necessary to achieve this with any given organism of known

D-value can be established from the inactivation factor (IF) which may be defined as:

IF = 10*°

388 Chapter 20

Fig. 20.3 Sterility assurance. At Y, there is (literally) 10"

bacterium in one bottle, i.e. in 10 loads of

single containers, there would be one chance in 10 that one load would be positive. Likewise, at Z,

there is (literally) 10

-6

bacterium in one bottle, i.e. in 1 million (10

) loads of single containers, there

is one chance in 1 million that one load would be positive.

where t is the contact time (for a heat or gaseous sterilization process) or dose (for

ionizing radiation) and D is the D-value appropriate to the process employed.

Thus, for an initial burden of 10

spores an inactivation factor of 10

will be needed

to give the required sterility assurance of 10

-6

(Fig. 20.3). The sterilization process will

therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in

viable organisms; this will require exposure of the product to eight times the D-value

of the reference organism (8D). In practice, it is generally assumed that the contaminant

will have the same resistance as the test spores unless full microbiological data are

available to indicate otherwise. The inactivation factors associated with certain

sterilization protocols and their biological indicator organisms (Chapter 23) are given

in Table 20.1.

3 Sterilization methods

The British Pharmacopoeia (1993) recognizes five methods for the sterilization of

pharmaceutical products. These are: (i) dry heat; (ii) heating in an autoclave (steam

sterilization); (iii) filtration; (iv) ethylene oxide gas; and (v) gamma or electron radiation.

In addition, other approaches involving steam and formaldehyde and ultraviolet (UV)

light have evolved for use in certain situations. For each method, the possible

permutations of exposure conditions are numerous, but experience and product stability

Principles and practice of sterilization 389

requirements have generally served to limit this choice. Nevertheless, it should be

remembered that even the recommended methods and regimens do not necessarily

demonstrate equivalent biocidal potential, but simply offer alternative strategies for

application to a wide variety of product types. Thus, each should be validated in their

application to demonstrate that the minimum required level of sterility assurance can

be achieved (section 2.3 and Chapter 23).

In the following sections, factors governing the successful use of these sterilizing

methods will be covered and their application to pharmaceutical and medical products

considered. Methods for monitoring the efficacy of these processes are discussed in

Chapter 23.

4 Heat sterilization

Heat is the most reliable and widely used means of sterilization, affording its

antimicrobial activity through destruction of enzymes and other essential cell

constituents. These lethal events proceed at their most rapid in a fully hydrated state,

thus requiring a lower heat input (temperature and time) under conditions of high

humidity where denaturation and hydrolysis reactions predominate, rather than in the

dry state where oxidative changes take place. This method of sterilization is limited to

thermostable products, but can be applied to both moisture-sensitive and moisture-

resistant products for which the British Pharmacopoeia (1993) recommends dry (160-

180°C) and moist (121-134°C) heat sterilization, respectively. Where thermal

degradation of a product might possibly occur, it can usually be minimized by selecting

the higher temperature range since the shorter exposure times employed generally result

in a lower fractional degradation.

4.1 Sterilization process

In any heat sterilization process, the articles to be treated must first be raised to

sterilization temperature and this involves a heating-up stage. In the traditional approach,

timing for the process (the holding time) then begins. It has been recognized, however,

that during both the heating-up and cooling-down stages of a sterilization cycle (Fig.

20.4), the product is held at an elevated temperature and these stages may thus contribute

to the overall biocidal potential of the process.

390 Chapter 20

Table 20.1 Inactivation factors (IF) for selected sterilization protocols and their correspond

biological indicator (BI)

Sterilization protocol

Moist heat

<121°Cfor 15 minutes)

Dry heat

(160°Cfor2 hours)

Irradiation

(25kGy;2.5Mrad)

organisms

BI organism D-value

B. stearothermophilus 1.5min

B. subtilis Max. 10min

var. niger

B. pumilus 3 kGy (0.3 Mrad)

ing

Min. 12

8.3

Fig. 20.4 Typical temperature profile of a heat sterilization process.

A method has been devised to convert all the temperature-time combinations

occurring during the heating, sterilizing and cooling stages of a moist heat (steam)

sterilization cycle to the equivalent time at 121 °C. This involves following the

temperature profile of a load, integrating the heat input (as a measure of lethality), and

converting it to the equivalent time at the standard temperature of 121°C. Using this

approach the overall lethality of any process can be deduced and is defined as the F-

value, which expresses heat treatment at any temperature as equal to that of a certain

number of minutes at 121 °C. In other words, if a moist heat sterilization process has an

F-value of x, then it has the same lethal effect on a given organism as heating at 121 °C

for x minutes, irrespective of the actual temperature employed or of any fluctuations in

the heating process due to heating and cooling stages. The F-value of a process will

vary according to the moist heat resistance of the reference organism; when the reference

spore is that of B. stearothermophilus with a z-value of 10°C, then the F-value is known

as the F

-value.

A relationship between F- and D-values, leading to an assessment of the probable

number of survivors in a load following heat treatment, can be established from the

following equation:

F = F>(log;V

-logAO

in which D is the D-value at 121 °C, and iV

and N represent, respectively, the initial and

final number of viable cells per unit volume.

The F-concept has evolved from the food industry and principally relates to the

sterilization of articles by moist heat. Because it permits calculation of the extent to

which the heating and cooling phases contribute to the overall killing effect of the

autoclaving cycle, the F-concept enables a sterilization process to be individually

developed for a particular product. This means that adequate sterility assurance can be

achieved in autoclaving cycles in which the traditional pharmacopoeial recommendation

of 15 min at 121

C is not achieved. The holding time may be reduced below ,15 min if

there is a substantial killing effect during the heating and cooling phases, and an adequate

cycle can be achieved even if the 'target' temperature of 121 °C is not reached. Thus, F-

values offer both a means by which alternative sterilizing cycles can be compared in

terms of their microbial killing efficiency, and a mechanism by which over-processing

of marginally thermolabile products can be reduced without compromising sterility

Principles and practice of sterilization 391

assurance. They have found application in the sterilization of medical and pharmaceutical

products by moist heat where, for aqueous preparations, the British Pharmacopoeia

(1993) generally requires a minimum F

-value of 8 from a steam sterilization process.

There is an apparent anomaly in that it also states that the 'preferred' combination

of temperature and time is a minimum of 121 °C maintained for 15 minutes, which, by

definition, equates to an F

value of 15. The latter, however, is applicable where the

material to be sterilized may contain relatively large numbers of thermophilic bacterial

spores, and an F

of 8 is appropriate for a 'microbiologically validated' process where

the bioburden is low and the spores likely to be present are those of (the generally more

heat sensitive) mesophilic species.

values may be calculated either from the 'area under the curve' of a plot of

autoclave temperature against time constructed using special chart paper on which the

temperature scale is modified to take into account the progressively greater lethality of

higher temperatures, or by use of the equation below:

= AtZW

where At = time interval between temperature measurements; T = product temperature

at time t; z is (assumed to be) 10°C.

Thus, if temperatures were being recorded from a thermocouple at 1.00 minute

intervals then At= 1.00, and a temperature of, for example, 115°C maintained for

1 minute would give an F

value of 1 minute x 10

(115

121)/10

which is equal to 0.251

minutes. In practice, such calculations could easily be performed on the data from

several thermocouples within an autoclave using PC-driven software, and, in a

manufacturing situation, these would be part of the batch records. Such a calculation

facility is offered as an optional extra by most autoclave manufacturers.

Application of the F- value concept has been largely restricted to steam sterilization

processes although there is a less frequently employed, but direct parallel in dry heat

sterilization (see section 4.3).

4.2 Moist heat sterilization

Moist heat has been recognized as an efficient biocidal agent from the early days of

bacteriology, when it was principally developed for the sterilization of culture media.

It now finds widespread application in the processing of many thermostable products

and devices. In the pharmaceutical and medical sphere it is used in the sterilization of

dressings, sheets, surgical and diagnostic equipment, containers and closures, and

aqueous injections, ophthalmic preparations and irrigation fluids, in addition to the

processing of soiled and contaminated items (Chapter 21).

Sterilization by moist heat usually involves the use of steam at temperatures in the

range 121-134°C, and while alternative strategies are available for the processing of

products unstable at these high temperatures, they rarely offer the same degree of sterility

assurance and should be avoided if at all possible. The elevated temperatures generally

associated with moist heat sterilization methods can only be achieved by the generation

of steam under pressure.

By far the most commonly employed standard temperature/time cycles for bottled

fluids and porous loads (e.g. surgical dressings) are 121 °C for 15 minutes and 134°C

392 Chapter 20

Table 20.2 Pressure-temperature relationships and antimicrobial efficacies of alternative steam

sterilization cycles

Temperature

(°C)

115

121

126

134

Holding time

(minutes)

Steam

(kPa)

103

138

207

pressure

(psi)

Inactivation factor*

(decimal reductions)

5.2

* Calculated for a spore suspension having a D

l2l

of 1.5 minutes and a Z value of 10°C.

for 3 minutes, respectively. Not only do high temperature-short time cycles often result

in lower fractional degradation (see section 4), they also afford the advantage of

achieving higher levels of sterility assurance due to greater inactivation factors (Table

20.2). The 115°C for 30 minute cycle was considered an acceptable alternative to 121 °C

for 15 minutes prior to the publication of the 1988 British Pharmacopoeia, but it is no

longer considered sufficient to give the desired sterility assurance levels for products

which may contain significant concentrations of thermophilic spores.

4.2.1 Steam as a sterilizing agent

To act as an efficient sterilizing agent, steam should be able to provide moisture and

heat efficiently to the article to be sterilized. This is most effectively done using saturated

steam, which is steam in thermal equilibrium with the water from which it is derived,

i.e. steam on the phase boundary (Fig. 20.5). Under these circumstances, contact with

a cooler surface causes condensation and contraction drawing in fresh steam and leading

to the immediate release of the latent heat, which represents approximately 80% of the

heat energy. In this way heat and moisture are imparted rapidly to articles being sterilized

and dry porous loads are quickly penetrated by the steam.

Steam for sterilization can either be generated within the sterilizer, as with portable

bench or 'instrument and utensil' sterilizers, in which case it is constantly in contact

with water and is known as 'wet' steam, or can be supplied under pressure (350-400kPa)

from a separate boiler as 'dry' saturated steam with no entrained water droplets. The

killing potential of 'wet' steam is the same as that of 'dry' saturated steam at the same

temperature, but it is more likely to soak a porous load creating physical difficulties for

further steam penetration. Thus, major industrial and hospital sterilizers are usually

supplied with 'dry' saturated steam and attention is paid to the removal of entrained

water droplets within the supply line to prevent introduction of a water 'fog' into the

sterilizer.

If the temperature of 'dry' saturated steam is increased, then, in the absence of

entrained moisture, the relative humidity or degree of saturation is reduced and the

steam becomes superheated (Fig. 20.5). During sterilization this can arise in a number

of ways, for example by overheating the steam jacket (see section 4.2.2), by using too

dry a steam supply, by excessive pressure reduction during passage of steam from the

boiler to the sterilizer chamber, and by evolution of heat of hydration when steaming

Principles and practice of sterilization 393

over-dried cotton fabrics. Superheated steam behaves in the same manner as hot air

since condensation and release of latent heat will not occur unless the steam is cooled

to the phase boundary temperature. Thus, it proves to be an inefficient sterilizing agent,

and although a small degree of transient superheating can be tolerated, a maximum

acceptable level of 5°C superheat is set, i.e. the temperature of the steam is never

greater than 5°C above the phase boundary temperature at that pressure.

The relationship between temperature and pressure holds true only in the presence

of pure steam; adulteration with air contributes to a partial pressure but not to the

temperature of the steam. Thus, in the presence of air the temperature achieved

will reflect the contribution made by the steam and will be lower than that normally

attributed to the total pressure recorded. Addition of further steam will raise the

temperature but residual air surrounding articles may delay heat penetration or, if a

large amount of air is present, it may collect at the bottom of the sterilizer, completely

altering the temperature profile of the sterilizer chamber. It is for these reasons that

efficient air removal is a major aim in the design and operation of a boiler-fed steam

sterilizer.

4.2.2 Sterilizer design and operation

Steam sterilizers, or autoclaves as they are sometimes known, are stainless steel vessels

designed to withstand the steam pressures employed in sterilization. They can be: (i)

394 Chapter 20

'portable' sterilizers, where they generally have internal electric heaters to produce

steam and are used for small pilot or laboratory-scale sterilization and for the treatment

of instruments and utensils; or (ii) large-scale sterilizers for routine hospital or industrial

use, operating on 'dry' saturated steam from a separate boiler (Fig. 20.6). Because of

their widespread use within pharmacy this latter type will be considered in greatest

detail.

There are two main types of large sterilizers, those designed for use with porous

loads (i.e. dressings) and generally operated at a minimum temperature of 134°C, and

those designed as bottled-fluid sterilizers employing a minimum temperature of 121 °C.

The stages of operation are common to both and can be summarized as air removal and

steam admission, heating-up and exposure, and drying or cooling. Many modifications

of design exist and in this section only general features will be considered. Fuller

treatments of sterilizer design and operation can be found in Health Technical

Memorandum 2010 (1994).

General design features. Steam sterilizers are constructed with either cylindrical or

oblong chambers, with preferred capacities ranging from 400 to 800 litres. They can be

sealed by either a single door or by doors at both ends (to allow through-passage of

processed materials; see Chapter 22, section 3.2.3). During sterilization the doors are

held closed by a locking mechanism which prevents opening when the chamber is

under pressure and until the chamber has cooled to a pre-set temperature, typically

80°C.

In the larger sterilizers the chamber may be surrounded by a steam-jacket which

can be used to heat the autoclave chamber and promote a more uniform temperature

throughout the load. The same jacket can also be filled with water at the end of the

cycle to facilitate cooling and thus reduce the overall cycle time. The chamber floor

slopes towards a discharge channel through which air and condensate can be removed.

Temperature is monitored within the opening of the discharge channel and by

thermocouples in dummy packages; jacket and chamber pressures are followed using

pressure gauges. In hospitals and industry, it is common practice to operate sterilizers

on an automatic cycle, each stage of operation being controlled by a timer responding

to temperature- or pressure-sensing devices.

Operation

1 Air removal and steam admission. Air can be removed from steam sterilizers either

by downward displacement with steam, evacuation or a combination of the two. In the

downward displacement sterilizer, the heavier cool air is forced out of the discharge

channel by incoming hot steam. This has the benefit of warming the load during air

removal which aids the heating-up process. It finds widest application in the sterilization

of bottled fluids where bottle breakage may occur under the combined stresses of

evacuation and high temperature. For more air-retentive loads (i.e. dressings), however,

this technique of air removal is unsatisfactory and mechanical evacuation of the air is

essential before admission of the steam. This can either be to an extremely high level

(e.g. 2.5 kPa) or can involve a period of pulsed evacuation and steam admission, the

latter approach improving air extraction from dressings packs. After evacuation, steam

penetration into the load is very rapid and heating-up is almost instantaneous. It is

Principles and practice of sterilization 395

axiomatic that packaging and loading of articles within a sterilizer be so organized as

to facilitate air removal.

During the sterilization process, small pockets of entrained air may still be released,

especially from packages, and this air must be removed. This is achieved with a near-

to-steam thermostatic valve incorporated in the discharge channel. The value operates

on the principle of an expandable bellows containing a volatile liquid which vaporizes

at the temperature of saturated steam thereby closing the valve, and condenses on the

passage of a cooler air-steam mixture, thus reopening the valve and discharging the

air. Condensate generated during the sterilization process can also be removed by this

device. Small quantities of air will not, however, lower the temperature sufficiently to

operate the valve and so a continual slight flow of steam is maintained through a bypass

around the device in order to flush away residual air.

It is common practice to package sterile fluids, especially intravenous fluids, in

flexible plastic containers. During sterilization these can develop a considerable internal

pressure in the airspace above the fluid and it is therefore necessary to maintain a

proportion of air within the sterilizing chamber to produce sufficient overpressure to

prevent these containers from bursting (air ballasting). In sterilizers modified or designed

to process this type of product, air removal is therefore unnecessary but special attention

must be paid to the prevention of air 'layering' within the chamber. This is overcome

by the inclusion of a fan or through a continuous spray of hot water within the chamber