STERILISATION

Sterilization

            Among the many facets of patient care is that of the sterilization of a range of materials. These include bacteriologically contaminated glassware and Petri dishes, dressings, sutures, ligatures, surgical instruments, etc., as well as certain raw materials and forms of pharmaceutical dosage. It is considered necessary to sterilise all of these as they could constitute a potential health hazard to patients.

            Sterilization is the process of killing or removing microorganisms. A sterile material is one that contains no living organisms at all and the term sterile is therefore an absolute one. However, the killing or removing of microorganisms follows a probability function, there always being a finite chance of leaving some live microorganisms intact in the system so that sterilization process are designed to minimize the probability of leaving survivors.

            However, with all articles to be sterilized there is the chance that the sterilizing treatment will have a detrimental effect. This is particularly true of pharmaceutical dosage forms where it is important that the chosen process should not cause changes in the formulation, which would reduce its therapeutic efficacy or patient acceptability. For this reason, with the design of all sterilization processes a balance has to be achieved between the maximum acceptable risk of failing to achieve sterility and the maximum permissible concomitant damage caused to the treated articles.

            The methods currently used to kill microorganisms involve exposing the preparation to an inimical physical agent or condition for a known period of time. The agents used are of three different types, namely elevated temperature, ionizing or UV radiation, and toxic gases. The fourth way of achieving sterility is restricted to solutions and gases and involves passing the solution through a filter that will retain the microorganisms.

DEFINITION OF STERILISATION

            Sterilization is a process whereby all the viable life forms are either killed or removed (by filtration) from a product.

‘Sterilisation’ and ‘Sterility’ are two absolute terms i.e. a sterile product should be absolutely free from viable (vegetative + spores) microorganisms.

DRY HEAT STERILIZATION

Principle:

The killing of microorganisms by heat is a function of the time-temperature combination used. If the temperature is increased then the time required for killing all the bacteria will be decreased.

Condition: Cycles recommended as per BP 1988 are:

                        A minimum of 180⁰C for not less than 30 minutes.

                        A minimum of 170⁰C for not less than 1 hour.

                        A minimum of 160⁰C for not less than 2 minutes.

Mechanism of killing the bacteria:

The vital constituents of cells such as proteins (enzymes) and nucleic acids are denatured by oxidation.

Biological indicator:                 Spores of Bacillus subtilis var. niger. or,

                                                Spores of Bacillus globigii or,

                                                Spores of Clostridium tetani (atoxigenic)

 Application of dry heat sterilization:

Dry heat is used to sterilize

1.      Glass ware e.g. test tubes, Pasteur-pippettes, petridishes, flasks, glass syringes etc. The glass wares should be prewashed with apyrogenic water.

2.      Porcelain and metal equipment such as forceps, scalpels, scissors etc.

3.      Dry materials in sealed container like powders.

4.      Fats, oils and greasy materials (like petroleum jelly) those are impermeable to moisture.

Dry heat sterilization is not used in the following cases:

1.      Aqueous solution cannot be sterilized by dry heat beacause the water will evaporate if the container is kept open. If the container is kept close then it may burst due to pressure developed by the steam.

2.      Surgical dressings cannot be sterilized by dry heat because cotton or other cellulosic material will get chared at the high temperature.

3.      Powders that cannot withstand the high temperature are not sterilised by dry heat method.

HEATING IN A HOT AIR OVEN

·         A hot air oven is a double walled chamber made up of mild steel or aluminium. The door is insulated with asbestos gasket to minimize the heat loss. To reduce heat loss by conduction insulating material (glass fibre or puff) is used in between two walls.

·         The inner surface is made reflecting to reduce heat loss by radiation.

·         It consists of one or more shelves.

·         It is thermostatically controlled. Heaters are fitted to heat the air inside. The heat is circulated by normal convection. To spread the heat uniformly forced convection is arranged by fitting fans in strategic places.

·         For effective utilization of the oven and to obtain correct results it should be uniformly packed or loaded so that all the objects are exposed to sterilization temperature for the required period of time.

·         Care should be taken so that once in operation the oven is not opened in the middle of the cycle.

FLAMING

            This is an emergency method, the forceps-tips, the surfaces of the scalpels and the needles may be sterilized by holding the items directly in the flame of a Bunsen burner. This method is generally done in microbiology.

INFRARED RADIATION

·         Infrared radiation (IR) is a thermal radiation, i.e. when absorbed by some article its energy is converted to heat and therefore it is often known as radiant energy.

·         A tunnel having an IR source is used for this purpose. The instruments and glass wares are kept in trays are passed through this tunnel keeping on the conveyor belt, at a controlled speed exposing them to a temperature of 180⁰C for 17 minutes, thereby achieving the sterility. This is a continuous process and is used in hospitals for regular supply of sterile syringes and other apparatus.

·         Heating at or above 200⁰C by IR in vacuum is employed as a means of sterilizing surgical instruments.

·         Cooling is hastened, (after the heating cycle) during the cooling period, by admitting filtered N₂ to the chamber.

MOIST HEAT STERILIZATION

Principle:

Mechanism of killing of microorganisms:

            Bacterial death by moist heat is due to denaturation and coagulation of essential protein molecules (enzymes) and cell constituents.

Conditions:

The USP XXI and BP 1988 recommended the following condition:

·         Pressure:                15 lb / square inch (psi)

·         Temperature:         121⁰C

·         Time:                     15 minutes

·         The following combinations of temperature and holding time are normally employed for sterilizing by heat in autoclave:

Holding temperature (0C)	Holding time (min)
115 to 118 121 to 124 126 to 129 134 to 138	30 15 10 3

Biological indicator:                 Spores of Bacillus stearothermophilus or

                                                Spores of Clostridium sporogenes.

Principles of sterilization by steam under pressure:

            Pressure itself has no sterilizing power. Steam is used under pressure as a means of achieving an elevated temperature.

Steam production: This may be achieved in two ways:

1.      In a small scale, steam may be generated from water within the sterilizer and since water is present this steam is known as ‘wet saturated steam’.

2.      For large scale sterilizers dry saturated steam may be piped from a separate boiler.

Penetration of steam:

            Steam flows quickly into every article in the load ( and into porous articles). This is due to its condensation creating a low-pressure region into which more steam flows.

Rapid heating: The saturated steam heats the load rapidly due to the release of its considerable amount of latent heat.

Moist heat: The condensate produced on cooling hydrates the microorganisms and thus helps in coagulating microbial proteins.

No residual toxicity: The product is free from toxic contamination.

The design and operation of autoclave:

            A portable autoclave is an elaborate pressure cooker. It is a hollow cylindrical vessel fitted with a lid that can be tightly secured in a position by nut-bolts or screws. The body is made up of aluminium or steel or gun metal.

            The lid is provided with a steam vent, a safety valve and a pressure or temperature gauge. It is heated electrically or gas operated. The electrical element is fitted at the bottom of the autoclave. First water is added so that the heating element is fully immersed in water. Then the materials to be sterilized are placed over a perforated platform.

            Heater is switched on. Initially the steam-vent (outlet) is opened. The displaced air is first removed, then the vent is closed. The pressure will then rise to 15 lb/sq inch (psi) and as a result the temperature inside will rise to 121⁰C. At this condition the autoclave is kept for 15 minutes, then the heater is switched off. When the pressure inside and outside the autoclave equals, the steam-vent is opened and the lid is removed to take out the sterilized articles.

Condition:

            Relative humidity (rh)              33%

            Temperature                            25⁰C [generally 40 - 50⁰C, maximum limit 60⁰C

Concentration of ethylene oxide	Time (Temp. 540C)
900 mg / litre 450 mg / litre	3 hour 5 hour

Precautions

·         It is important to ensure that the articles for gaseous sterilization are scrupulously clean. Organic material reduces the efficiency of the process.

·         After sterilization ethylene-oxide gets strongly adsorbed by a wide variety of substances. Desorption of the gas is done by airing the materials in a well-ventilated room for sometime or a powerful vacuum is applied.

·         Ethylene oxide sterilizers:

The features of a suitable equipment include:

1.      An exposure chamber that is gas tight.

2.      A means of heating the chamber: e.g. a steam or hot water jacket or, heating element clipped to the outside of the chamber.

3.      A system for adding water to provide the right humidity.

4.      A means of extracting air before and after the sterilization.

Use:

Ethylene oxide possesses the ability to penetrate paper, a number of plastics and rubber. So disposable syringes, hypodermic needles, prepackaged materials etc. are sterilized by this method.

Advantages:

1.      It is suitable for thermolabile substances, because it can be carried out at room temperature or only slightly above.

2.      It does not damage moisture-sensitive substances and equipment because only a low humidity is required.

3.      It can be used for prepackaged articles, because of the great penetrating power of ethylene oxide.

4.      Though ethylene oxide is highly reactive compound comparatively few materials are damaged by this process.

Disadvantages:

1.      It is slow. Long exposures and desorption periods are necessary and therefore cannot be used in emergency.

2.      The running costs are high.

3.      Ethylene oxide is inflammable. To overcome it ethylene oxide is mixed with inert gases such as carbon dioxide, fluorinated hydrocarbons.

Some marketed mixtures are:

Cryoxide :        11%w/w ethylene oxide

                        79% w/w trichlorofluoromethane

                        10% w/w dichlorodifluoromethane

Sterethox         12% ethylene oxide

                        8% dichlorodifluoromethane

4.      Toxic substances, such as ethylene chlorohydrin are produced in some materials.

FORMALDEHYDE

Like ethylene oxide this is an alkylating agent but it is generally inferior for use as a sterilizing agent. Because

·         formaldehyde has poor penetrating power and is readily inactivated by organic matter.

·         High concentrations are difficult to maintain in the atmosphere because it tends to deposit in the form of solid polymers on contact with cool surfaces.

Condition:

Input concentration:                 2 g/L of HCHO in subatmospheric steam

Temperature:                           90⁰C

Exposure time:             3 hours

Formaldehyde can be obtained as :

1.      Formaldehyde solution (Formalin) B.P.

            Approximately 37%w/w containing stabilizers to prevent deposition of solid polymers

2.      Tablets of paraformaldehyde

* In case of formalin addition of KMnO₄ produces heat by oxidation and the gas will be vaporized.

Use:     Dressing packs.

Disadvantages:

1.      Low penetrability. It cannot penetrate polymeric packaging.

2.      Polymerize to inactive forms on the surface of low exposure temperature.

3.      Can fall in the active concentration

4.      Cannot sterilize narrow lumen.

Solution of the above problems:

1.      Reducing the size of the load.

2.      Increasing exposure temperature.

STERILIZATION BY RADIATION

Radiation can be divided into two groups:

1.         Electromagnetic waves:                       (i)         infra-red radiation (IR)                        (iii)       X-rays

                                                (ii)        ultraviolet radiation (UV)         (iv)       gamma rays

                                                                                                                       

2.         Streams of particulate matter   (i)         alpha radiation             (ii)        beta radiation

For sterilization infrared, ultraviolet, gamma radiation and high velocity electrons (a type of beta radiation) are used for sterilization.

Radiation	Wave length	Energy
UV-radiation Gamma radiation High velocity electrons	190 to 370 nm 1 to 10-4 nm	5 eV 1.3 MeV* 4MeV

* MeV = Million electron volt

ULTRAVIOLET RADIATION

Source: Low current of high voltage is passed through mercury vapor in an evacuated tube made of borosilicate glass.

Dose of sterilizing radiation: 10 to 60 microwatts / cm² reduce the populations of vegetative cells by 90% in a short period.

Mode of action: Only a narrow range of wavelength (220 to 280 nm) is effective in killing micro-organisms, and wavelengths close to 253.7 nm are the most effective. It has been found that a maximum biological efficiency exists at 253.7 nm, which is also the absorption peak of isolated DNA, this suggests strongly therefore that DNA or the nucleic acid is the target for UV-induced lethal events.

 

Use:     (i)         Surface sterilization

            (ii)        Sterilization of clean air and water in thin layers.

Disadvantages:

1.      It has very poor penetration power.

         [N.B. UV radiation does not penetrate normal packaging material, such as glass or plastic and hence, it is not used to sterilize pharmaceutical dosage forms.]

2.      UV-light sterilization is not absolutely reliable because DNA may get repaired in some favorable condition.

IONISING RADIATION

Mode of action of ionizing radiation:

            Ionizing radiation can cause excitations, ionization and where water is present free radical formation. Free radicals are powerful oxidizing ( OH, HO₂) and reducing (H) agents, which are capable of damaging essential molecules (enzymes and DNAs) in living cells. This results in cell death.

Biological indicator: Bacillus pumilis.

HIGH SPEED RADIATION

Source: This type of sterilizing radiation is most widely used in Denmark and the USA. In a machine known as a van de Graph accelerator electrons are generated from a suitable source and then accelerated along a highly evacuated tube by a tremendous potential difference between the ends.

Dose: 5 MeV to 10 MeV (Million electron Volt).

Use: The beam, which is narrow and intense, is used to irradiate articles on a conveyor-belt.

GAMMA RAYS

Source: Radiation from the radioactive isotope of Cobalt ⁶⁰ Co, is used as a source of gamma emission.

Dose: 1.25 MeV,

            Some users take the adequate dose as 2.5 Mrad (Mega radiation unit)

Procedure: Articles for sterilization by radiation are packed in boxes of standard size, which are suspended from a monorail and sterilized by slow-passage around the gamma-ray source.

Uses:

·         Articles regularly sterilized on a commercial scale include plastic syringes, catheters, hypodermic needles and scalpel blades, adhesive dressings, single-application capsules of eye-ointment and catgut

·         Containers made of polyethylene and packaging materials using aluminum foil and plastic films.

Disadvantages:

·         The damage of the cells is mediated through radiation-induced free radicals in water, hence the extent of degradation is found to be maximum in pharmaceuticals in aqueous solutions. Practically the amount of degradation in aqueous solution is so great that this sterilization method is only confined to sterilize surgical sutures, instruments etc.

·         The necessary apparatus is much expensive for installation in hospital. It is employed commercially for the sterilization of large amount prepackaged disposable items such as plastic syringes and catheters, which are unable to withstand heat.

STERILIZATION BY FILTRATION

            This method is used for sterilizing thermolabile solutions, which will otherwise be degraded by other conventional heating methods.

            The drug solutions are passed through the sterile bacteria proof filter unit and subsequently transferring the product aseptically into the sterile containers which are then sealed.

            The process involves considerable hazards. Hence IP and BP require that the tests for sterility be carried out on the filtered product.

Biological indicator: Micromonospora  diminuta.

Procedure:

            The solutions to be sterilized is passed through the filter and collected in the sterile receiver by the application of positive pressure to the nonsterile compartment or negative pressure to the sterile side.

Advantages of sterilization by filtration:

1.   Thermolabile solutions can be sterilized.

2.   It removes all the living microorganisms.

Disadvantages of sterilization by filtration:

1.   Filters may break down suddenly or gradually on use.

2.   Sterility testing is obligatory on the filtered solution.

3.   Filter media may be absorbed on the filter surface.

4.   Viruses are not removed by filtration.

5.   Suspensions and oils cannot be sterilized by this method due to their heavy load of particulate matters  and viscosity.

Mode of action:

The filters are thought to function by one or usually a combination of the following:

1.   Sieving or screening,

2.   Entrapment,

3.   Electrostatic attraction.

            When a particle is larger than the pore size of the filter the particle is retained on the filter - this known as sieving or screening.

            Entrapment occurs when a particle smaller than the size of the pore enters into the pore channel and lodges onto the curves of the channel while passing through it.

            Electrostatic attraction causes particles, opposite in charge to that of the surface of the filter pore, to be held or adsorbed onto the surface.

MEMBRANE FILTERS

·         Membrane filters are made of cellulose-derivative (acetate or nitrate). They are very fine. They are fixed in some suitable holders.

·         Nominal pore size is 0. 22 ± 0. 02 mm or less is required.

·         The membranes are brittle when dry. In this condition they can be stored for years together. They become very tough when dipped in water.

·         They are sterilized by autoclaving or by ethylene oxide gas. They cannot be sterilized by dry heat as they decompose above 120⁰ C.

·         They are suitable for sterilizing aqueous and oily solutions but not for organic solvents such as alcohol, chloroform etc.

·         Membrane filters are generally blocked by dirt particles and organisms. Pre-filtration (through glass-fibre paper prefilter) reduces the risks of blockage of the final filter.

Examples of membrane filters:

      MF-Millipore – it is a mixture of cellulose esters

      Sartorius Regular ­­– it is made of cellulose nitrate

      Gelmen Triacetate Metricel – cellulose triacetate

SINTERED (or FRITTED) GLASS FILTERS

            Borosilicate glass is finely powdered in a ball-mill and the particles of required size are separated. This is packed into disc mounted and heated till the particles get fused. The disc thus made have pore size of 2 mm and are used for filtration.

They are cleaned with the help of sulfuric acid.

SIETZ FILTER:            They are made of asbestos pad.

                                    [For further details see Cooper & Gunn Dispensing pp. 582]

CERAMIC FILTERS: They are made of either porcelain or keiselghur. These are supplied in the shape of candles mounted to metallic joint.

TESTING OF FILTERS:

The BP requires that the integrity of an assembled sterilizing filter be verified before use and confirmed after use by means of a suitable test.

Bacteriological test:

            A diluted solution of broth culture of Serratia marcescens is passed though the filter and the filtrate is collected aseptically and incubated at 25⁰C for 5 days. The filter passes this test if no growth is found after 5 days of incubation.

Bubble point test:

            The bubble point of a test filter is the pressure at which the largest pore of a wetted filter is able to pass air.

Objectives:

1.   Filtration should normally be performed at pressures lower than the bubble point of a membrane. This prevents gas from passing through the filter at the end of a filtration cycle and thereby prevents excessive foaming.

2.   For testing membrane efficiency and integrity.

Procedure:

1.   First the membrane is wetted and usually has a liquid above and a gas below.

2.   Since the pore is full of liquid there is no passage of gas at zero pressure. The pressure is increased gradually. At bubble point pressure a small bubble will form at the largest opening.

3.   As the pressure is further increased, rapid bubbling begins to occur.

Inference:

            Bubble point pressure for a given set of membrane and liquid is constant. If the bubble point pressure got reduced it can be concluded that the membrane integrity is lost or efficiency is reduced.

MANUFACTURE OF STERILE BULK POWDER

            Some drugs are difficult to sterilize terminally (i.e. when the total product is ready within the container and package), the raw materials (i.e. bulk drugs) of those products are required to be sterile.

Generally, the manufacture of a sterile bulk substance usually includes the following steps:

1.      Conversion of non-sterile drug substance to the sterile form by dissolving in an (organic or aqueous)  solvent, sterilization of the solution by filtration and collection in a sterilized reactor (crystallizer).

2.      Aseptic precipitation or crystallization of the sterile drug substance in the sterile reactor.

3.      Aseptic isolation of the sterile substance by centrifugation or filtration.

4.      Aseptic drying (spray drying,  lyophilization), milling and blending of the sterile substance.

All the above mentioned operations should be performed in closed systems, with minimal operator handling.

Sterilization of the processing equipment

            Equipment used in the processing of sterile bulk substances should be sterile and capable of being sterilized. This includes the crystallizer, centrifuge and dryer.

A. Steam sterilization: The method of choice for the sterilization of equipment and transfer lines (pipings) is saturated, clean steam under pressure.

B. Formaldehyde: Formaldehyde is used rarely due to its residue present in the environment and the equipment after the sterilization process. This residue is very difficult to remove.