Contamination Control and Sterile Manufacturing

SOM2 – Semester 2

Microorganisms• Microorganisms pose a threat because they are small, grow quickly and are abundant

in the atmosphere and environment. Their main types are; viruses, fungi, bacteria and spores.

• They can cause disease (pathogenic) and some elements of some pathogens are enough to cause an immune response. This means the killing of some pathogens is not enough to prevent the patient developing symptoms of infection.

• Spores are produced by some bacteria, and these are resistant to heat treating, chemical, radiation and drying eradication methods.

• Bacteria are only 0.7 - 4µm in size, are unicellular, can be a variety of shapes, can be gram positive or gram negative and can be anaerobic or aerobic.

• Viruses are much smaller at 20 – 250nm in size, allowing them to pass through bacterial filters. They are destroyed by hear and most are inactivated at 60°C for 30 minutes. Chemical disinfectants generally have poor viricidal activity.

Cleanrooms • Sterile medicinal products should be prepared in specially designed and constructed

manufacturing areas, separate from others. The different types of operation should be separated; solution preparation, filling, component preparation and sterilization are four different stages of the operational process.

• Negative air pressures can be used in isolation rooms to prevent the escape of air from a cleanroom environment. This means that when doors are opened, air flows into the room and not out of it.

• Positive air pressures are used in grade A/B environments and prevent the entry of new air into the room via the door. The pressure gradient forces air outwards if the door is opened. The air is cycled in the room via filtration systems and air enters this way.

• Air is important to control as though not a nutrient store in its own right, airborne organisms are found on airborne matter, attached to particles that are not visible to the naked eye. Particles sediment in the air and are redistributed when agitated, so air flows can cause pathogens trapped on sinking particles to become dispersed in the air and settle on the bench.

• High Efficiency Particulate Air (HEPA) filters are used to filter air to an appropriate efficiency.

Cleanroom Specifications• Specifications for the microbial contamination levels of cleanrooms depends on their

grade and the system observed. There are two important classification systems; the EU and the US systems.

• Grade A and B areas are the cleanest, most sterile areas typically used in aseptic preparation of parenterals and high-risk medications.

• Many products are manufactured at one site for global distribution and so will have to be compliant with both specifications.

• Above, the undefined areas refer to limits that vary depending on the nature of the operations being carried out.

EU Grade

US Grade

International Society of Pharm. Eng. Description

Maximum number of particles ≥ 0.5µm/m³ at rest

Maximum number of particles ≥ 5µm/m³ at rest

Maximum number of particles ≥ 5µm/m³ in operation

Maximum number of particles ≥ 5µm/m³ in operation

A 100 Critical 3500 0 3500 0B 1000 Clean 3500 0 350000 2000C 10000 Controlled 350000 2000 350000 20000

D 100000

Pharmaceutical 3500000 20000 undefined undefined

Measuring Aerial Contamination

• Settle plates can be used to test aerial contamination. They are a nutrient plate (i.e. agar), which monitors the settling of particles larger than 100µm via gravity.

• Small particles are not represented by settle plates and we are unaware of the volume of air introduced.

• Slit samplers are perhaps better measures of aerial contamination, as they are able to provide a measure of the number of organisms associated with a volume of air. In these devices, air is drawn in through a series of slits onto a rotating nutrient agar disc.

• The deposition of particles in slit sampling depends on the slit size, the rate of air flow and distance between holes. These machines have a 99% efficiency with particles even as small as 1μm in size.

Personnel• Operators should operate to high standards of personal cleanliness, reporting any

medical conditions such as colds and skin infections, so that the situation can be evaluated.

• Within clean room environments, there should be minimal staff and activities are limited to avoid excess shedding of microorganisms.

• Personnel should be trained on how to gown appropriately, which will vary depending on the grade of room being used and the product being prepared.

Sterilisation• Microorganisms are expressed as either viable or dead according to their ability to

grow. After exposure to a lethal agent, the viable cells begin to die, but not usually all at once.

• The reduction in the number of viable cells following exposure to a lethal agent is plotted as the log number of viable cells or log percentage of viable cells against time. This therefore provides a constant proportion of viable cells and not a constant number of organisms killed per unit time.

• Plotting this graph gives a log-linear representation of death rate of viable microorganisms. This can give a constant proportion of viable organisms killed per minute, which is good but does not indicate sterility when a batch contains 1000 units; after 10 minutes of 90% of viable death per minute, 100 of the batches would contain a microorganism.

• Although the count would be just one, rapid reproduction that batches can quickly become further contaminated and are not sage for administration.

• Allowance for these factors, and the unknown distribution of resistance to sterilisation is made in the sterility assurance level (SAL) which is 10-6. This means that sterility is accepted if there is a probability of not more than one in one million units being affected.

Measures of Sterilisation• The D value (decimal reduction value) is a measure of a sterilisation parameter

(duration of sterilisation or absorbed dose of agent) required to reduce the number of viable organisms by 90% (i.e. to 10% of the original number).

• The inactivation factor (IF) is a measure of the reduction in the number of viable microorganisms produced by the sterilising process. It is calculated using the sterilisation time and the D value in the following manner: 10t/D.

• Products that are sterilised in their final containers are referred to as terminally sterilised, which is the method of choice whenever possible. Products such as this are manufactured in clean rooms to produce pyrogen-free products with low microbial and particulate counts even before sterilisation has occurred.

• Those products that are not sterilised in their final container are referred to as non-terminally sterilised. They have been prepared using previously sterilised materials, under aseptic conditions and so are assumed to be sterile despite the entire finished product being sterilised alone. They cannot be assembled or filled in their final container anywhere other than an aseptic clean room.

Approaches to Sterilisation• The bioburden is an expression of the number of viable microorganisms a batch

contains. • The overkill method of sterilisation uses sterilisation cycles that inactivate a greater

number of microorganisms than the actual bioburden of the batch. A minimum sterility assurance level is defined for the process.

• Traditional BP methods of sterilisation adopt this overkill approach to sterilisation with recommended combinations of time and temperature.

• An alternative approach to sterility is called the bioburden method. This requires the operator to know the D value of the batch, the bioburden and the most resistant organism in the batch. This is all done prior to defining the sterilisation cycle. This approach allows cycles to be run with acceptable sterility assurance levels with minimal product degradation.

• Many methods of sterilisation are based on heat, such as dry heat sterilisation, steam sterilisation and autoclaving, though some do not involve heat in any capacity; ionising radiation and filtration are examples of these. Filtration is used where products are non-terminally sterilised.

Heat Sterilisation• Heat sterilisation methods are the most reliable, cheap and safe methods to use as

they do not require the use of harmful chemical gases such as ethylene oxide, or harmful radiation. Heating bacteria increases the rate of chemical reactions in cells and causes them to grow, but above a certain temperature will destabilise cellular components and result in death of viable cells. The efficiency of this process will depend on the balance between these forces.

• Increasing temperature will cause bacterial colonies to reach a point where population growth and death occurs at the same rate; this is known as bacteriostasis. Above the temperature at which this occurs, a destructive effect dominates leading to a bactericidal effect.

• Sterilisation depends on the time-temperature relationship, and there is an obvious difference in the sterilising effect of moist and dry heat, with dry heat requiring greater temperatures to achieve the same effect.

Heat Sterilisation• Some microorganisms are heat resistant, but most are not and are killed in hot water

at temperature of 60 to 100°C. Resistance depends on the species of microorganisms present and the most resistant microorganism is assumed to be Bacillus stearothermophilus; if a sterilisation method kills this bacteria, then the process is assumed to be very efficient.

• Moist heat causes microorganism death via the influence of H2O, which causes protein denaturation through distortion of H bonds, causing secondary and tertiary structures to unravel. Changes also occur in the cell membrane as phospholipids become more mobile and cause leakage of cell contents and death. This method of sterilisation requires relatively lower temperatures than dry heat and so is preferred where possible.

• On the other hand, dry heat causes death by oxidation of components, which requires high temperatures and is not as effective in causing protein denaturation.

Steam Sterilisation• Technically a method of heat sterilisation, sterilising products with steam occurs in an

autoclave. According to the BP, sterilisation with saturated steam under pressure is preferred whenever possible.

• Aqueous preparations should be heated to a minimum of 121°C for 15 minutes.• Saturated steam is defined as water vapour at a temperature corresponding to the

boiling point of water at the appropriate pressure. Steam is an effective means of sterilisation because it contains more heat than water or air at the same temperature, which is a combination of sensible and latent heat.

• Sensible heat is the heat required to boil water. Latent heat is additional heat that is absorbed when liquid is converted to steam at the same temperature. Out of the two, the latent heat of steam is the more effective in sterilisation.

Dry Heat Sterilisation• Preparations that are to be sterilised by dry heat are distributed in their final

containers and then sealed to exclude microorganisms.• Heating is often at very high temperatures in excess of 250°C, with shorter sterilising

times requiring higher temperatures than longer exposures. • This method can only be used for thermostable products and containers, but offers a

solution to sterilising products that are sensitive to moisture or impermeable to steam.

• This method can also be used in the sterilisation of anhydrous materials like oils, fats, suspensions as well as glass and metal materials, powders, and some rubbers.

Moist Heat vs. Dry Heat• The advantages of moist heat are that it can be used to terminally sterilise products

of any dose volume, with a good safety margin. It also requires lower operating temperatures, kills viruses and is compatible with many containers, dressings and rubber closures. It can also be used for some surgical materials, solutions and suspensions.

• The disadvantages are that it is not compatible with thermolabile drugs, it cannot be used for anhydrous materials like powders, it requires skilled operators and is a batch process. Furthermore, it kills microorganisms but it does not remove them, which makes it unsuitable for endotoxins/bacteria presenting antigenic material when dead, as administration of these products will elicit an immune response.

• The advantages of dry heat sterilisation include the fact that it can be used for anhydrous preparations and containers. This method can also be used for products which are degraded by water/steam, and can also cause less damage to glass and metal than moist sterilisation. It can be used as a terminal sterilisation method, for any dose volume and also kills viruses.

• The disadvantages of dry heat sterilisation are that it requires excessive heat, it does not remove dead organisms, is unsuitable for dressings, rubber and some plastics and also may lead to changes to suspensions on cooling. This is via Ostwald ripening and recrystallisation changes.

Filtration• Filtration is a non-terminal sterilisation method that does not involve the use of dry

heat, moist heat or radiation to achieve sterility. • Sterilisation is achieved through passing liquids through a sterilised filter of nominal

pore size, usually 0.22μm. Therefore, the microorganisms are not killed but removed from the liquid as it passes through the filter.

• Due to the nature of filtration and the small pore size, this method of sterilisation is not available to all preparations. Suspensions, products highly adsorbed to filters, dressings, easily oxidized products and medicaments not stable in solution cannot be sterilised via these means.

• The main advantages to filtration are that it allows some sterility for products which are thermolabile or hydrolytic, and it also removes bacteria whole, meaning endotoxins are less of a concern.

• However, the main disadvantages are that it does not remove viruses, it cannot be used with suspensions, it is not suitable for easily oxidised materials, defects in the filter cannot be detected, sterility must be checked (causing a delay) and it is a difficult technique to perform.

Ionisation• Ionisation radiation can be used as a terminal sterilisation method, where the final

product in its container is exposed to a dose of ionising radiation.• The minimum absorbed does of radiation is 25kGy, where a Gy (Gray) is the SI unit of

dose and is the absorption of 1 Joule of energy from 1Kg matter.• Ionising radiation is of two types; gamma and beta. Gamma radiation is much more

penetrating than beta radiation, and therefore more dangerous. Beta radiation is poorly penetrating and samples must be irradiated from various angles or turned in the beam to receive the full benefit.

• The radiation sterilises samples by damaging the DNA of microorganisms, resulting in death.

• The applications of ionising radiation include the sterilisation of heat-sensitive materials like syringes, needles, blades and gowns, as well as antibiotics, hormones and vitamins.

• The main advantages of ionisation as a means of sterilisation are that it can be achieved at ambient temperatures, can be used for thermolabile products, is very quick and can be used as a continuous process, controlled reliably and accurately.

• The disadvantages are that it is expensive can damage materials like some glass, plastics and PVC (causes covalent bond formation) and protective precautions must be taken.

Reasons for Sterilisation• Sterilisation is compulsory for the preparation of parenteral, ophthalmic and select topical

preparations, where products will bypass the body’s natural defenses. Under these conditions, preparations must be completely free from microorganisms so that they are not introduced directly into the body. This would lead to sepsis and severe infection, potentially embolisms too.

• Sterilisation is a process that kills viable microorganisms, including spores.• Disinfection is a process of reducing the number of viable microorganisms to low levels,

but does not kill spores.• Antisepsis is the process of applying antiseptic agents to tissues. Antiseptics are less

effective and toxic than disinfectants. • Viable cells are capable of division to form a colony on a solid nutrient plate, like agar, or

visible turbidity in a liquid medium. Dead cells are those incapable of division on a nutrient plate.

• Endotoxins are pyrogens that are high MW polysaccharides (i.e. lipopolysaccharide; LPS) found on some bacterial cell walls. They are water soluble, heat stable fragments that are only inactivated by dry heat temperatures of 170 - 350°C.

• Some examples of sterile preparations include: IV injections, TPN bags, small volume injections (bolus) and non-injectable sterile fluids as used in peritoneal dialysis and haemodialysis.

• Ophthalmic preparations like eye drops, lotions and ointments must also be sterile.• Dressings, implants, absorbable haemostats, surgical materials and all surgical

instruments must also be sterile.

Containers• Containers for sterile preparations should be chemically compatible with the product,

should allow safe withdrawal of the medicine, should allow sterilisation of the product and should preserve the sterility of the product once sterile.

• Large volume parenteral containers can be rigid or flexible, glass or plastic, whilst small volume parenteral containers are usually ampoules, vials, prefilled syringes and novel devices (glass or plastic). Irrigations and eye drops are usually contained in glass or plastic, whereas eye ointments are contained in plastic.

• Single-dose containers contain a quantity of product that it intended to be fully or partially used, only once, then discarded. Intraspinal injections and IV injections are always single use, and those IV injections greater than 15mL must not contain bactericides/preservatives and so are also single-use preparations.

• Multi-dose containers contain several doses of a product but not so many as to introduce an excessively long period of opening that could introduce contaminants into the product. These preparations require a bactericide to remain sterile.

Sterile Fluid Containers• Sterile fluid containers can be made from glass, which has disadvantages and advantages.• The min advantages to using glass for sterile fluid containers are that it has a good

chemical resistance and neither absorbs nor elutes organic ingredients, like plastic can. Glass is also rigid and strong, resistant to puncture and tampering, allows inspection of the contents and is easily cleaned. It can also be autoclaved at 121°C and is impermeable to gasses and liquids.

• The disadvantages to using glass as a container material for sterile fluids are that it can break, is attacked by alkaline solutions and can develop small cracks during transportation, which can allow the entry of moulds. Glass is also much heavier than plastic, requires venting when drawing out liquid due to the rigidity of the container and the formation of a vacuum and in all cases except for ampoules, requires another material to be used as the closure, which introduces issues with compatibility. All vessels require inspection and washing prior to use.

• Plastic is an alternative to glass, the main advantages of which are that it is lighter, cheaper and easier to manufacture, is single use, is less prone to breakage through cracking, can be sealed entirely by fusion and has smaller ports, reducing the risk of contamination. This is now the preferred alternative to glass in the packaging of LVPs.

• The disadvantages are mainly that it does not have the same oxygen and moisture barrier properties as glass, it can have inferior clarity, can contain additives that leach into the product and also must be protected from deforming and bursting during sterilisation.

Sterile Fluid Containers• There are many plastics which can be used in the manufacture of plastic sterile fluid

containers, including polyvinyl chloride (PVC) and polyethylene (polythene).• PVC is widely used in the medical industry, is flexible and so can be used for IV

tubing, catheters, blood bags and LVP containers.• Polythene can be used in parenteral containers, providing that a high density is used.

It can withstand sterilisation as it has a high melting temperature, but can be less flexible and more opaque.

Closures for Sterile Containers

• Both glass and plastic products can be closed by fusion, i.e. glass ampoules and sealed plastic containers, however most sterile containers incorporate some elastomeric component in a closure, along with a screw device.

• Elastomeric closures are desirable as they are able to be compressed and resealed.• Choice of rubber closure will be affected by; the active substance and any

interactions with the closure; the vehicle and excipients for the same reason; preservatives (if present – multi dose); the pH of the product and possible buffer systems; the colour and appearance and also the moisture/gas protection required for the final preparation.

• Closures exist to protect the preparation from microbiological, pyrogenic, particulate and chemical contamination, as well as degradation of the product. This is in the interests of patient safety and efficacy of therapy.

• Parenteral containers should be completely sealed and most leaks are not due to failure of closure but due to thermal or mechanical cracks and faulty manufacture.

Labeling of Sterile Products• The BP states that all preparations (sterile or not) must include; the name of the

product or approved synonym; the names and fixed proportions of all medicaments (except for in the case of a fixed recipe); the names and proportions of any added preservatives and; the batch number.

• For injectable preparations, the label must also contain; the amount of API per suitable dose volume (i.e. mg/mL); the name and proportion of any added preservative; name of any buffering agent added; conditions for storage and; the date after which product should be discarded.

• Injections with a capacity above 10mL and parenteral boxes are labeled with; strength, expiry, batch number, dose and RoA and name and concentration of bactericide.

• Injections with a capacity of less than 10mL enclosed in a package (i.e. ampoule) must be labeled with; strength, RoA, expiry and batch number.

• Parenteral preparations containing a CD must be labeled with; total volume, strength, expiry, batch number, name/synonym and list of medicaments and quantities (unless fixed recipe).

• Intravenous infusion labels must include; the volume, the name, the quantities and strengths of components per litre, additional directions (for IV infusion as directed by the physician, do not use if solid particles are present, store below 25°C, to be used on one occasion and remaining solution discarded), expiry and batch number.