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

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to mix the air and steam. Air ballasting can also be employed to prevent bottle breakage.

2 Heating-up and exposure. When the sterilizer reaches its operating temperature

and pressure the sterilization stage begins. The duration of exposure may include a

heating-up time in addition to the holding time and this will normally be established

using thermocouples in dummy articles.

3 Drying or cooling. Dressings packs and other porous loads may become dampened

during the sterilization process and must be dried before removal from the chamber.

This is achieved by steam exhaust and application of a vacuum, often assisted by heat

from the steam-filled jacket if fitted. After drying, atmospheric pressure within the

chamber is restored by admission of sterile filtered air.

For bottled fluids the final stage of the sterilization process is cooling, and this needs to

be achieved as rapidly as possible to minimize thermal degradation of the product and

to reduce processing time. In modern sterilizers, this is achieved by circulating water

in the jacket which surrounds the chamber or by spray-cooling with retained condensate

delivered to the surface of the load by nozzles fitted into the roof of the sterilizer

chamber. This is often accompanied by the introduction of filtered, compressed air to

minimize container breakage due to high internal pressures (air ballasting). Containers

must not be removed from the sterilizer until the internal pressure has dropped to a safe

level, usually indicated by a temperature of less than 80°C. Occasionally, spray-cooling

water may be a source of bacterial contamination and its microbiological quality must

be carefully monitored.

4.3 Dry heat sterilization

The lethal effects of dry heat on microorganisms are due largely to oxidative processes

which are less effective than the hydrolytic damage which results from exposure to

steam. Thus, dry heat sterilization usually employs higher temperatures in the range

160-180°C and requires exposure times of up to 2 hours depending upon the temperature

employed (section 10).

Again, bacterial spores are much more resistant than vegetative cells, and their

recorded resistance varies markedly depending upon their degree of dryness. In many

early studies on dry heat resistance of spores their water content was not adequately

controlled, so conflicting data arose regarding the exposure conditions necessary to

achieve effective sterilization. This was partly responsible for variations in recommended

exposure temperatures and times in different pharmacopoeias.

Its application is generally restricted to glassware and metal surgical instruments

(where its good penetrability and non-corrosive nature are of benefit), non-aqueous

thermostable liquids and thermostable powders (see Chapter 21). In practice, the range

of materials which are actually subjected to dry heat sterilization is quite limited, and

consists largely of items used in hospitals. The major industrial application is in the

sterilization of glass bottles which are to be filled aseptically, and here the attraction of

the process is that it not only achieves an adequate sterility assurance level, but that it

also destroys bacterial endotoxins (products of Gram-negative bacteria, also known as

pyrogens, that cause fever when injected into the body). These are difficult to eliminate

by other means. For the purposes of depyrogenation of glass, temperatures of

approximately 250°C are used.

Principles and practice of sterilization 397

The F-value concept which was developed for steam sterilization processes has an

equivalent in dry heat sterilization although its application has been limited. The F

designation describes the lethality of a dry heat process in terms of the equivalent

number of minutes exposure at 170°C, and in this case a z value of 20°C has been

found empirically to be appropriate for calculation purposes; this contrast with the

value of 10°C which is typically employed to describe moist heat resistance.

4.3.1 Sterilizer design

Dry heat sterilization is usually carried out in a hot air oven which comprises an insulated

polished stainless steel chamber, with a usual capacity of up to 250 litres, surrounded

by an outer case containing electric heaters located in positions to prevent cool spots

developing inside the chamber. A fan is fitted to the rear of the oven to provide circulating

air, thus ensuring more rapid equilibration of temperature. Shelves within the chamber

are perforated to allow good air flow. Thermocouples can be used to monitor the

temperature of both the oven air and articles contained within. A fixed temperature

sensor connected to a chart recorder provides a permanent record of the sterilization

cycle. Appropriate door-locking controls should be incorporated to prevent interruption

of a sterilization cycle once begun.

Recent sterilizer developments have led to the use of dry-heat sterilizing

tunnels where heat transfer is achieved by infra-red irradiation or by forced convection

in filtered laminar airflow tunnels. Items to be sterilized are placed on a conveyer belt

and pass through a high-temperature zone (250 - 300 + °C) over a period of several

minutes.

4.3.2 Sterilizer operation

Articles to be sterilized must be wrapped or enclosed in containers of sufficient strength

and integrity to provide good post-sterilization protection against contamination. Suitable

materials are paper, cardboard tubes or aluminium containers. Container shape and

design must be such that heat penetration is encouraged in order to shorten the heating-

up stage; this can be achieved by using narrow containers with dull non-reflecting

surfaces. In a hot-air oven, heat is delivered to articles principally by radiation and

convection; thus, they must be carefully arranged within the chamber to avoid obscuring

centrally placed articles from wall radiation or impending air flow. The temperature

variation within the chamber should not exceed ±5°C of the recorded temperature.

Heating-up times, which may be as long as 4 hours for articles with poor heat-conducting

properties, can be reduced by preheating the oven before loading. Following sterilization,

the chamber temperature is usually allowed to fall to around 40°C before removal of

sterilized articles; this can be accelerated by the use of forced cooling with filtered

air.

5 Gaseous sterilization

The chemically reactive gases ethylene oxide (CH

)

0, and formaldehyde (methanal,

H.CHO) possess broad-spectrum biocidal activity, and have found application in the

398 Chapter 20

sterilization of re-usable surgical instruments, certain medical, diagnostic and electrical

equipment, and the surface sterilization of powders. Sterilization processes using

ethylene oxide sterilization are far more commonly used on an international basis than

those employing formaldehyde.

Ethylene oxide treatment can also be considered as an alternative to radiation

sterilization in the commercial production of disposable medical devices (Chapter 21).

These techniques do not, however, offer the same degree of sterility assurance as heat

methods and are generally reserved for temperature-sensitive items.

The mechanism of antimicrobial action of the two gases is assumed to be through

alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and imino

groups of nucleic acids. At the concentrations employed in sterilization protocols, type

A survivor curves (section 2.1, Fig. 20.1) are produced, the lethality of these gases

increasing in a non-uniform manner with increasing concentration, exposure temperature

and humidity. For this reason, sterilization protocols have generally been established

by an empirical approach using a standard product load containing suitable biological

indicator test strips (Chapter 23). Concentration ranges (given as weight of gas per unit

chamber volume) are usually in the order of 800-1200mgl

for ethylene oxide and

15-100 mg l

-1

for formaldehyde, with operating temperatures in the region of 45-63°C

and 70-75°C, respectively. Even at the higher concentrations and temperatures, the

sterilization processes are lengthy and therefore unsuitable for the resterilization of

high-turnover articles. Further delays occur because of the need to remove toxic residues

of the gases before release of the items for use. In addition, because recovery of survivors

in sterility tests is more protracted with gaseous sterilization methods than with other

processes, an extended quarantine period may also be required.

As alkylating agents, both gases are potentially mutagenic and carcinogenic (as is

the ethylene chlorohydrin which results from ethylene oxide reaction with chlorine),

they also produce symptoms of acute toxicity including irritation of the skin, conjunctiva

and nasal mucosa; consequently, strict control of their atmospheric concentrations is

necessary and safe working protocols are required to protect personnel. Formaldehyde

can normally be detected by smell at concentrations lower than those permitted in the

atmosphere, whereas this is not true for ethylene oxide. Table 20.3 summarizes the

comparative advantages afforded by ethylene oxide and low-temperature steam

formaldehyde (LTSF) processes.

5.1 Ethylene oxide

Ethylene oxide gas is highly explosive in mixtures of >3.6% v/v in air; in order to

reduce this explosion hazard it is usually supplied for sterilization purposes as a 10%

mix with carbon dioxide, or as an 8.6% mixture with HFC 124 (2 chloro-1,1,1,2

tetrafluoroethane) which has replaced fluorinated hydrocarbons (freons). Alternatively,

pure ethylene oxide gas can be used at below atmospheric pressure in sterilizer chambers

from which all air has been removed.

The efficacy of ethylene oxide treatment depends upon achieving a suitable

concentration in each article and this is assisted greatly by the good penetrating powers

of the gas, which diffuses readily into many packaging materials including rubber,

plastics, fabric and paper. This is not without its drawbacks, however, since the level of

Principles and practice of sterilization 399

ethylene oxide in a sterilizer will decrease due to absorption during the process and the

treated articles must undergo a desorption stage to remove toxic residues. Desorption

can be allowed to occur naturally on open shelves, in which case complete desorption

may take many days, e.g. for materials like PVC, or it may be encouraged by special

forced aeration cabinets where flowing, heated air assists gas removal, reducing

desorption times to between 2 and 24 hours.

Organisms are more resistant to ethylene oxide treatment in a dried state, as are

those protected from the gas by inclusion in crystalline or dried organic deposits. Thus,

a further condition to be satisfied in ethylene oxide sterilization is attainment of a

minimum level of moisture in the immediate product environment. This requires a

sterilizer humidity of 30-70% and frequently a preconditioning of the load at relative

humidities of greater than 50%.

5.1.1 Sterilizer design and operation

An ethylene oxide sterilizer consists of a leak-proof and explosion-proof steel chamber,

normally of 100-300 litre capacity, which can be surrounded by a hot-water jacket

to provide a uniform chamber temperature. Successful operation of the sterilizer

requires removal of air from the chamber by evacuation, humidification and con-

ditioning of the load by passage of subatmospheric pressure steam followed by a

further evacuation period and the admission of preheated vaporized ethylene oxide

from external pressurized canisters or single-charge cartridges. Forced gas circulation

is often employed to minimize variations in conditions throughout the sterilizer chamber.

Packaging materials must be air-, steam- and gas-permeable to permit suitable conditions

for sterilization to be achieved within individual articles in the load. Absorption of

ethylene oxide by the load is compensated for by the introduction of excess gas at the

beginning or by the addition of more gas as the pressure drops during the sterilization

400 Chapter 20

Table 20.3 Relative merits of ethylene

processes

Advantages of ethylene

oxide over LTSF

Wider international

regulatory acceptance

Better gas penetration into

plastics and rubber

Relatively slow to form solid

polymers (with the potential to

block pipes etc.)

With long exposure times it

is possible to sterilize at

ambient temperatures

Very low incidence of

product deterioration

oxide and low-temperature steam formaldehyde (LTSF)

Advantage of LTSF

over ethylene oxide

Less hazardous because formaldehyde

is not flammable and is more readily

detected by smell

Cycle times may be shorter

The gas is obtained readily from

aqueous solution (formalin) which is a

more convenient source than gas in

cylinders

process. The ^ame may also be true for moisture absorption, which is compensated for

by supplementary addition of water to maintain appropriate relative humidity.

After treatment, the gases are evacuated either directly to the outside atmosphere

or through a special exhaust system. Filtered, sterile air is then admitted either for a

repeat of the vacuum/air cycle or for air purging until the chamber is opened. In this

way, safe removal of the ethylene oxide is achieved reducing the toxic hazard to the

operator. Sterilized articles are removed directly from the chamber and arranged for

desorption.

The operation of an ethylene oxide sterilizer should be monitored and controlled

automatically. A typical operating cycle for pure ethylene oxide gas is given in Fig.

20.7, and general conditions are summarized in section 10.

5.2 Formaldehyde

Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v

aqueous solution of formaldehyde) to a temperature of 70-75°C with steam, leading to

the process known as LTSF. Formaldehyde has a similar toxicity to ethylene oxide and

although absorption to materials appears to be lower similar desorption routines are

recommended. A major disadvantage of formaldehyde is low penetrating power and

this limits the packaging materials that can be employed to principally paper and cotton

fabric.

5.2.1 Sterilizer design and operation

An LTSF sterilizer is designed to operate with subatmospheric pressure steam. Air is

removed by evacuation and steam admitted to the chamber to allow heating of the load

and to assist in air removal. The sterilization period starts with the release of

formaldehyde by vaporization from formalin (in a vaporizer with a steam jacket) and

continues through either a simple holding stage or through a series of pulsed evacuations

and steam and formaldehyde admission cycles. The chamber temperature is maintained

by a thermostatically controlled water jacket, and steam and condensate are removed

via a drain channel and an evacuated condenser. At the end of the treatment period

formaldehyde vapour is expelled by steam flushing and the load dried by alternating

stages of evacuation and admission of sterile, filtered air. A typical pulsed cycle of

operation is shown in Fig. 20.8 and general conditions are summarized in section 10.

6 Radiation sterilization

Several types of radiation find a sterilizing application in the manufacture of

pharmaceutical and medical products, principal among which are accelerated electrons

(particulate radiation), gamma-rays and ultraviolet (UV) light (both electromagnetic

radiations). The major target for these radiations is believed to be microbial DNA, with

damage occurring as a consequence of ionization and free radical production (gamma-

rays and electrons) or excitation (UV light). This latter process is less damaging and

less lethal than ionization, and so UV irradiation is not as efficient a sterilization method

as electron or gamma-irradiation. As mentioned earlier (section 2), vegetative bacteria

Principles and practice of sterilization 401

generally prove to be the most sensitive to irradiation (with notable exceptions, e.g.

Deinococcus {Micrococcus) radiodurans), followed by moulds and yeasts, with bacterial

spores and viruses as the most resistant (except in the case of UV light where mould

spores prove to be most resistant). The extent of DNA damage required to produce cell

death can vary and this, together with the ability to carry out effective repair, probably

decides the resistance of the organism to radiation. With ionizing radiations (gamma-

ray and accelerated electrons), microbial resistance decreases with the presence of

moisture or dissolved oxygen (as a result of increased free radical production) and also

with elevated temperatures.

Radiation sterilization with high-energy gamma-rays or accelerated electrons has

proved to be a useful method for the industrial sterilization of heat-sensitive products.

However, undesirable changes can occur in irradiated preparations, especially those in

aqueous solution where radiolysis of water contributes to the damaging processes. In

addition, certain glass or plastic (e.g. polypropylene, PTFE) materials used for packaging

or for medical devices can also suffer damage. Thus, radiation sterilization is generally

applied to articles in the dried state; these include surgical instruments, sutures,

prostheses, unit-dose ointments, plastic syringes and dry pharmaceutical products

(Chapter 21). With these radiations, destruction of a microbial population follows the

classic survivor curves (see Fig. 20.1) and a D-value, given as a radiation dose, can be

established for standard bacterial spores (e.g. Bacillus pumilus) permitting a suitable

sterilizing dose to be calculated. In the UK it is usual to apply a dose of 25 kGy (2.5 Mrad)

for pharmaceutical and medical products, although lower doses are employed in the

USA and Canada.

UV light, with its much lower energy, causes less damage to microbial DNA. This,

coupled with its poor penetrability of normal packaging materials, renders UV light

unsuitable for sterilization of pharmaceutical dosage forms. It does find applications,

however, in the sterilization of air, for the surface sterilization of aseptic work areas,

and for the treatment of manufacturing-grade water.

6.1 Sterilizer design and operation

6.1.1 Gamma-ray sterilizers

Gamma-rays for sterilization are usually derived from a cobalt-60 (

Co) source

(caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration

emits radiation at two energy levels of 1.33 and 1.17 MeV. The isotope is held as pellets

packed in metal rods, each rod carefully arranged within the source and containing up

to 20kCi (740 x 10

Bq) of activity; these rods are replaced or rearranged as the activity

of the source either drops or becomes unevenly distributed. A typical

Co installation

may contain up to 1 MCi (3.7 x 10

Bq) of activity. For safety reasons, this source is

housed within a reinforced concrete building with walls some 2 m thick, and it is only

raised from a sunken water-filled tank when required for use. Control devices operate

to ensure that the source is raised only when the chamber is locked and that it is

immediately lowered if a malfunction occurs. Articles being sterilized are passed

through the irradiation chamber on a conveyor belt or monorail system and move

around the raised source, the rate of passage regulating the dose absorbed (Fig. 20.9).

Principles and practice of sterilization 403

Radiation monitors are continually employed to detect any radiation leakage during

operation or source storage, and to confirm a return to satisfactory background levels

within the sterilization chamber following operation. The dose delivered is dependent

upon source strength and exposure period, with dwell times typically up to 20 hours

duration.

The difference in radiation susceptibilities of microbial cells and humans may be

gauged from the fact that a lethal human dose would be delivered by an exposure of

seconds or minutes.

6.1.2 Electron accelerators

Two types of electron accelerator machine exist, the electrostatic accelerator and the

microwave linear accelerator, producing electrons with maximum energies of 5 MeV

and 10 MeV, respectively. Although higher energies would achieve better penetration

into the product, there is a risk of induced radiation, and so they are not used. In the

first, a high-energy electron beam is generated by accelerating electrons from a hot

filament down an evacuated tube under high potential difference, while in the second,

additional energy is imparted to this beam in a pulsed manner by a synchronized

travelling microwave. Articles for treatment are generally limited to small packs and

are arranged on a horizontal conveyor belt, usually for irradiation from one side but

sometimes from both. The sterilizing dose is delivered more rapidly in an electron

accelerator than in a

Co plant, with exposure times for sterilization usually amounting

to only a few seconds or minutes. Varying extents of shielding, depending upon the

size of the accelerator, are necessary to protect operators from X-rays generated by the

bremsstrahlung effect.

6.1.3 Ultraviolet irradiation

The optimum wavelength for UV sterilization is around 260 nm. A suitable source for

UV light in this region is a mercury lamp giving peak emission levels at 254 nm. These

sources are generally wall- or ceiling-mounted for air disinfection, or fixed to vessels

for water treatment. Operators present in an irradiated room should wear appropriate

protective clothing and eye shields.

7 Filtration sterilization

The process of filtration is unique amongst sterilization techniques in that it removes,

rather than destroys, microorganisms. Further, it is capable of preventing the passage

of both viable and non-viable particles and can thus be used for both the clarification

and sterilization of liquids and gases. The principal applications of sterilizing-grade

filters are the treatment of heat-sensitive injections and ophthalmic solutions, biological

products and air and other gases for supply to aseptic areas (see Chapters 21 and 22).

They may also be required in industrial applications where they become part of

venting systems on fermenters, centrifuges, autoclaves and freeze-dryers. Certain types

of filter (membrane filters) also have an important role in sterility testing, where they

can be employed to trap and concentrate contaminating organisms from solutions under

Principles and practice of sterilization 405

test. These filters are then placed on the surface of a solid nutrient medium and incubated

to encourage colony development (Chapter 23).

The major mechanisms of filtration are sieving, adsorption and trapping within the

matrix of the filter material. Of these, only sieving can be regarded as absolute since it

ensures the exclusion of all particles above a defined size. It is generally accepted that

synthetic membrane filters, derived from cellulose esters or other polymeric materials,

approximate most closely to sieve filters, while fibrous pads, sintered glass and sintered

ceramic products can be regarded as depth filters relying principally on mechanisms of

adsorption and entrapment. Some of the characteristics of filter media are summarized

in Table 20.4. The potential hazard of microbial multiplication within a depth filter and

subsequent contamination of the filtrate (microbial grow-through) should be recognized.

7.1 Filtration sterilization of liquids

In order to compare favourably with other methods of sterilization, the microorganism

removal efficiency of filters employed in the processing of liquids must be high. For

this reason, membrane filters of 0.2-0.22 fim nominal pore diameter are chiefly used,

while sintered filters are used only in restricted circumstances, i.e. for the processing

of corrosive liquids, viscous fluids or organic solvents. It may be tempting to assume

that the pore size is the major determinant of filtration efficiency and two filters of

0.2jimi pore diameter from different manufacturers will behave similarly. This is not so

because, in addition to the sieving effect, trapping within the filter matrix, adsorption

and charge effects all contribute significantly towards the removal of particles.

Consequently, the depth of the membrane, its charge and the tortuosity of the channels

are all factors which can make the performance of one filter far superior to that of

another. The major criterion by which filters should be compared, therefore, is their

titre reduction values, i.e. the ratio of the number of organisms challenging a filter

under defined conditions to the number penetrating it. In all cases, the filter medium

employed must be sterilizable, ideally by steam treatment; in the case of membrane

filters this may be for once-only use, or, in the case of larger industrial filters, a small

406 Chapter 20

Table 20.4 Some characteristics of membrane and depth filters

Characteristic

Absolute retention of microorganisms

rated pore size

Rapid rate of filtration

High dirt-handling capacity

Grow-through of microorganisms

Shedding of filter components

Fluid retention

Solute adsorption

Good chemical stability

Good sterilization characteristics

+, applicable; -, not applicable.

greater than

Membrane

Unlikely

Variable (depends

on membrane)

Depth