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

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Table 20.5 Effect of membrane

disc filter diameter on filtration

volumes

fixed number of resterilizations; sintered filters may be resterilized many times. Filtration

sterilization is an aseptic process and careful monitoring of filter integrity is necessary

as well as final product sterility testing (Chapter 23).

Membrane filters, in the form of discs, can be assembled into pressure-operated

filter holders for syringe mounting and in-line use or vacuum filtration tower devices.

Filtration under pressure is generally considered most suitable since filling at high

flow rates directly into the final containers is possible without problems of foaming,

solvent evaporation or air leaks. The filtration capacity of a range of membrane filter

discs is given in Table 20.5. To increase the filtration area, and hence process volumes,

several discs can be used in parallel in multiple-plate filtration systems or, alternatively,

membrane filters can be fabricated into plain or pleated cylinders and installed in

cartridges. Membrane filters are often used in combination with a coarse-grade fibreglass

depth prefilter to improve their dirt-handling capacity.

7.2 Filtration sterilization of gases

The principal application for filtration sterilization of gases is in the provision of sterile

air to aseptic manufacturing suites, hospital isolation units and some operating theatres.

Filters employed generally consist of pleated sheets of glass microfibres separated and

supported by corrugated sheets of Kraft paper or aluminium; these are employed in

ducts, wall or ceiling panels, overhead canopies, or laminar airflow cabinets (Chapter

22). These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of

particles greater than 0.3 fim in diameter and thus are acting as depth filters. In practice

their microorganism removal efficiency is rather better since the majority of bacteria

are found associated with dust particles and only the larger fungal spores are found in

the free state. Air is forced through HEPA filters by blower fans, and prefilters are used

to remove larger particles to extend the lifetime of the HEPA filter. The operational

efficiency and integrity of a HEPA filter can be monitored by pressure differential and

airflow rate measurements, and dioctylphthalate smoke particle penetration tests.

Other applications of filters include sterilization of venting or displacement air in

tissue and microbiological culture (carbon filters and hydrophobic membrane filters);

decontamination of air in mechanical ventilators (glass fibre filters); treatment of

exhausted air from microbiological safety cabinets (HEPA filters); and the clarification

and sterilization of medical gases (glass wool depth filters and hydrophobic membrane

filters).

Principles and practice of sterilization 407

Filter

(mm)

142

293

diameter

Effective filtration

area (cm

)

0.8

3.9

11.3

530

Typical batch

volume (litres)

<0.01

0.05-0.1

0.1-0.3

0.3-5

5-20

>20

Conclusions

A sterilization process should always be considered a compromise between achieving

good antimicrobial activity and maintaining product stability. It must, therefore, be

validated against a suitable test organism and its efficacy continually monitored during

use. Even so, a limit will exist as to the type and size of microbial challenge which can

be handled by the process without significant loss of sterility assurance. Thus,

sterilization must not be seen as a 'catch-all' or as an alternative to good manufacturing

practices but must be considered as only the final stage in a programme of

microbiological control.

Acknowledgements

The assistance of the following is gratefully acknowledged: F.J. Ley, Isotron

pic, Swindon (for discussions and permission to reproduce Fig. 20.9); M.S. Copson,

Albert Browne Ltd, Leicester (for discussions and permission to reproduce Fig.

20.6).

Appendix

Examples of typical conditions employed in the sterilization of pharmaceutical and

medical products

Sterilization method

Moist heat (autoclaving)

Dry heat

Ethylene oxide

Low-temperature steam and formaldehyde

Irradiation

Gamma-rays or accelerated

electrons

Filtration

Conditions

121°Cfor 15min

134°Cfor3min

160°Cfor 120min

170°Cfor60min

180°Cfor30min

Gas concentration:

800-1200 mgl"

45-63°C

30-70% relative humidity

1-4 hours sterilizing time

Gas concentration:

15-100 mgl"

Steam admission to 73°C

40-180min sterilizing time

depending on type of process

25kGy (2.5 Mrad) dose

=s0.22,um pore size, sterile

membrane filter

10 Further reading

Baird R.M. & Bloomfield S.F. (eds) (1996) Microbial Quality Assurance in Cosmetics, Toiletries and

Non-sterile Pharmaceuticals. London: Taylor & Francis.

British Pharmacopoeia (1993) London: HMSO.

British Standards Institution (1991) Specification for Steam Sterilizers for Aqueous Fluids in Rigid

Sealed Containers: BS 3970. London: BSI.

British Standards Institution (1990) Sterilizing and Disinfecting Equipment for Medical Products. BS

3970, Parts, 1, 3,4, 5. London: BSI.

Denyer S.P. & Baird R.M. (eds) (1990) Guide to Microbiological Control in Pharmaceuticals. Chichester:

Ellis Horwood. (Chapters 7, 8 and 9 provide additional information.)

European Pharmacopoeia, 3rd edn. (1997) Maisonneure: SA.

Gardner J.F. & Peel M.M. (1991) Introduction to Sterilisation, Disinfection and Infection Control.

Melbourne: Churchill Livingstone.

Gilbert P. & Allison D. (1996) Redefining the 'sterility' of sterile products. Eur J Parenteral Sci, 1,

19-23.

Health Technical Memorandum (1994) Sterilisers. HTM 2010. London: Department of Health.

Russell A.D. (1982) The Destruction of Bacterial Spores. London: Academic Press.

Russell A.D., Hugo W.B. & Ayliffe G.AJ. (eds) (1998) Principles and Practice of Disinfection,

Preservation and Sterilization, 3rd edn. Oxford: Blackwell Scientific Publications.

Stumbo CR. (1973) Thermobacteriology in Food Processing, 2nd edn. London: Academic Press.

United States Pharmacopeia (1995) 23rd revision. Rockville MD: US Pharmacopeial Convention.

Principles and practice of sterilization 409

Sterile pharmaceutical products

2.1

2.2

2.2.1

2.2.2

2.3

2.3.1

2.4

3.1

3.2

3.3

3.4

Introduction

Injections

Design philosophy

Intravenous infusions

Intravenous additives

Total parenteral nutrition (TPN)

Small-volume aqueous injections

Problems of drug stability

Small-volume oily injections

Non-injectable sterile fluids

Non-injectable water

Urological (bladder) irrigation solutions

Peritoneal dialysis and haemodialysis

solutions

Inhaler solutions

4 Ophthalmic preparations

4.1 Design philosophy

4.2 Eye-drops

4.3 Eye lotions

4.4 Eye ointments

4.5 Contact-lens solutions

4.5.1 Wetting solutions

4.5.2 Cleaning solutions

4.5.3 Soaking solutions

5 Dressings

6 Implants

7 Absorbable haemostats

7.1 Oxidized cellulose

7.2 Absorbable gelatin foam

7.3 Human fibrin foam

7.4 Calcium alginate

8 Surgical ligatures and sutures

8.1 Sterilized surgical catgut

8.2 Non-absorbable types

9 Instruments and equipment

10 Further reading

Introduction

Certain forms of drug administration and other pharmaceutical products, such as

dressings and sutures, must be sterile in order to avoid the possibility of nosocomial

(hospital-induced) infection arising from their usage. This applies particularly to

medicines which are administered parenterally but also to any material or instrument

likely to contact broken skin or internal organs. While inoculation of human pathogenic

bacteria, fungi or viruses poses the most obvious danger to the patient, it should also be

realized that microorganisms usually regarded as non-pathogenic which inadvertently

gain access to body cavities in sufficiently large numbers can also result in a severe,

often fatal, infection. Consequently, injections, ophthalmic preparations, irrigation fluids,

dialysis solutions, sutures and ligatures, implants, certain surgical dressings, as well as

instruments necessary for their use or administration, must be presented for use in a

sterile condition and in such a way that they remain sterile throughout the period of

use.

Principles of the methods employed to sterilize pharmaceutical products are

described in Chapter 20. The British Pharmacopoeia (1993) recommends autoclaving

and filtration as suitable methods applicable to aqueous liquids, and dry heat for non-

aqueous and dry solid preparations. The choice is determined largely by the ability of

the formulation and container to withstand the physical stresses applied by moist heat

treatment. The use of ionizing radiation or ethylene oxide is also appropriate in specific

instances. The primary considerations relate to the ability of active ingredients to

withstand the applied stress and of the container to maintain the product in a sterile

condition until use. It should be realized that all products intended to be sterilized must

be rendered and kept thoroughly clean and therefore of low microbial content prior to

sterilization. Thus, the process itself is not overtaxed and is generally well within safety

limits to guarantee sterility with minimal stress applied to the product. Because of the

clinical consequences (such as granuloma in the lung) of injecting solid particles into

the bloodstream, the numbers of particles present in injections and in other solutions

used in body cavities must be restricted. The British Pharmacopoeia (1993) set

limits for injections based on operation of a particle-detecting apparatus. The European

Pharmacopoeia (1997) describes a microscope method for particulate contamination

of injections and intravenous infusions, i.e. extraneous, mobile, undissolved particles,

other than bubbles, unintentionally present in the solutions. The test method provides

a qualitative method for identifying and detecting the characteristics of such particles

together with an indication of their possible origin. It might then be possible to

develop means of avoiding such contamination. Limits are given in the United States

Pharmacopoeia (1995) for large-volume injections, using this method.

Injections

Design philosophy

Any injectable product must be designed and produced to the highest possible

pharmaceutical standards. Not only must the product have the minimum possible levels

of particles and pyrogenic substances, but also the formulation and packaging must

maintain product integrity throughout the production processes, the shelf-life and during

administration. The formulation must be such as to ensure that the product remains

physically and chemically stable over the designated shelf-life. To achieve this,

excipients such as buffers and antioxidants may be required to ensure chemical stability,

and solubilizers, such as propylene glycol or polysorbates, may be necessary for drugs

with poor aqueous solubility to maintain the drug in solution. The packaging must

prevent water, excipient or drug loss during sterilization and storage and, in addition,

retain microbiological integrity. Axiomatically, ingress of microorganisms must be

prevented. The packaging must not contribute any significant amounts of extractable

chemicals to the contents, for example vulcanizing agents from rubber or plasticizers

from polyvinyl chloride (PVC) infusion containers.

Most injections are formulated as aqueous solutions, with Water for Injections BP

as the vehicle. The formulation of injections depends upon several factors, namely the

aqueous solubility of the active ingredient, the dose to be employed, thermal stability

of the solution, the route of injection and whether the product is to be prepared as a

multidose one (i.e. with a dose or doses removed on different occasions) or in a single-

dose form (as the term suggests, only one dose is contained in the injection). Nowadays,

most injections are prepared as single-dose forms and this is mandatory for certain

routes, e.g. spinal injections such as the intrathecal route and large-volume intravenous

infusions (section 2.2). Multidose injections may require the inclusion of a suitable

Sterile pharmaceutical products 411

preservative to prevent contamination following the removal of a dose on different

occasions. Single-dose injections are usually packed in glass ampoules containing 1, 2

or 5 ml of product; to ensure removal of the correct volume by syringe, it is necessary

to add an appropriate overage to an ampoule. Thus, a 1-ml ampoule will actually contain

1.1 ml of product, with 2.15 ml in a 2-ml ampoule. Full details are to be found in the

British Pharmacopoeia (1993).

Some types of injections must be made iso-osmotic with blood serum. This applies

particularly to large-volume intravenous infusions if at all possible; hypotonic solutions

cause lysis of red blood corpuscles and thus must not be used for this purpose.

Conversely, hypertonic solutions can be employed: these induce shrinkage, but not

lysis, of red cells which recover their shape later. Intraspinal injections must also be

isotonic, and to reduce pain at the site of injection so should intramuscular and

subcutaneous injections. Adjustment to isotonicity can be determined by the following

methods.

1 Depression of freezing-point, which depends on the number of dissolved particles

present in solution. A useful equation is given by:

in which W is the percentage (w/v) of adjusting substance, a the freezing-point of

unadjusted solution and b the depression of the freezing-point of water by 1 % w/v of

adjusting substance.

2 Sodium chloride equivalent, which is produced by dividing the value for the

depression of freezing-point produced by a solution of the substance by the cor-

responding value of a solution of sodium chloride of the same strength.

For details of these and other methods, the Pharmaceutical Codex (1993) should

be consulted.

2.2 Intravenous infusions

These consist of large-volume injections or drips (500 ml or more) that are infused at

various rates (e.g. 50-500 ml h

) into the venous system; they are sterilized in an

autoclave (see Chapter 20). The most commonly used infusions are isotonic sodium

chloride and glucose. These are used to maintain fluid and electrolyte balance, for

replacement of extracellular body fluids (e.g. after surgery or during prolonged periods

of fluid loss), as a supplementary energy source (1 litre of 5% w/v glucose = 714kJ)

and as a vehicle for drugs. Such solutions are prepared using freshly distilled water as

a vehicle under rigidly controlled conditions to minimize pyrogen (see Chapters 1 and

18) and particle content, and filtered to remove remaining particles immediately before

distribution to the final clean container.

Other important examples are blood and blood products, which are collected and

processed in sterile containers, and plasma substitutes, for example dextrans and

degraded gelatin. Dextrans, glucose polymers consisting essentially of (1 -^6) a-links,

are produced as a result of the biochemical activities of certain bacteria of the genus

Leuconostoc, e.g. L. mesenteroides (see Chapter 25).

A small range of intravenous infusions, e.g. those containing amino acids or

412 Chapter 21

chlormethiazole, are prepared in glass containers. These are sealed with a rubber closure

held on by an aluminium screw cap or crimp-on ring. The rubber should be non-

fragmenting, not release soluble extractives, and be sufficiently soft and pliable to

seal around the giving set needle inserted immediately prior to use. Although bottles

are sterilized by autoclaving, it is still possible for the infusion in glass bottles to become

contaminated with microorganisms before use. For instance, during the final part of

the autoclave process, bottles may be spray-cooled with water to hasten the cooling

process and therefore reduce the total autoclaving time. However, due to the poor fit

between bottle lip and rubber plug (a skirted insert type is used) it is possible for the

spray-cooling water to spread by capillary movement between bottle thread and screw

cap and even enter the bottle contents. This process is encouraged if the bottle contains

a vacuum as a consequence of rubber seal failure during heating-up. It should also be

remembered that autoclaving leads to considerable heat and pressure stresses on the

container. Failure may result from any imperfection in the bottle or plug. Microbes

may also gain access to the contents of bottles during storage if hair-line cracks (a

result of bad handling and rough treatment) are present, through which fluid may seep

outwards and microorganisms inwards to contaminate the fluid. Finally, contamination

may occur during use due to poor aseptic techniques when setting up the infusion, via

an ineffective air inlet (allowing replacement of infused fluid with air) or when changing

the giving set or bottle.

Most infusions are now packed in plastic containers. The plastic material should be

pliable, thermoresistant, transparent and non-toxic. Plasticized PVC and polyethylene

are commonly used. The former is transparent and very pliable, allowing the pack to

collapse as the contents are withdrawn (consequently no air inlet is required). These

packs are also amenable to the inclusion of ports into the bag, allowing greater safety

during use. Such ports can be protected by sterile overseals. Two problems arise: (i) the

possibility of toxic extractives, e.g. diethyl phthalate, from the plastic entering the

fluid if poor quality PVC is used; and (ii) moisture permeability leading to loss of

water if the packs are not protected by a water-impermeable outer wrap. Bags of high-

quality polyethylene are readily moulded (although separate ports cannot be included),

translucent and free from potential toxic extractives. As stated, these packs normally

collapse readily during infusion. An important advantage of all plastic packs is that the

containers are hermetically sealed prior to autoclaving and, therefore, spray-cooling

water cannot enter the pack unless there is seal failure, an easily detected occurrence.

However, the autoclaving of plastic bags is more complex than that of bottled fluids

because a steam-air mixture is necessary to prevent bursting of the bags when heated

(air-ballasting); adequate mixing of the steam and air is therefore required to prevent

layering of gases inside the chamber.

2.2.1 Intravenous additives

A common practice in hospitals is to add drugs to infusions immediately prior to, or

during, administration. The most common additives are potassium chloride, lignocaine,

heparin, certain vitamins and antibiotics.

Potentially, this can be a hazardous practice. For instance, the drug may precipitate

in the infusion fluid because of the pH (e.g. amphotericin) or the presence of calcium

Sterile pharmaceutical products 413

salts (e.g. thiopentone). The drug may degrade rapidly (e.g. ampicillin in 5% w/v

glucose). Multiple additions may lead to precipitation of one or both of the drugs or to

accelerated degradation. Finally, drug loss may occur because of absorption by the

container. For instance, insulin is absorbed by glass or PVC, glyceryl trinitrate and

diazepam are absorbed by PVC. Apart from these problems, if the addition is not

carried out under strict aseptic conditions the fluid can become contaminated with

microorganisms during the procedure. Thus, any addition should be made in a laminar-

flow work station or isolator, preferably in the pharmacy, and the fluid administered

within 24 hours, unless prepared under strict aseptic conditions.

Another approach to the problem of providing an intravenous drug additive service

is to add the drug to a small-volume (50-100 ml) infusion in a collapsible plastic

container and store the preparation at -20°C in a freezer. The infusion can be removed

when required and thawed rapidly by microwave. Many antibiotics are stable for several

months when stored in minibags at -20°C and are unaffected by the thawing process in

a suitable microwave oven. Other antibiotics, e.g. ampicillin, are degraded when frozen.

2.2.2 Total parenteral nutrition (TPN)

Total parenteral nutrition is the use of concentrated mixtures of amino acids, vitamins,

inorganic salts and an energy source (carbohydrate or fat emulsion, e.g. soyabean oil

with lecithin as emulsifying agent) for the long-term feeding of patients who are

unconscious or unable to take food. Many hospital pharmacies operate a TPN service.

All or most of the ingredients to feed a patient for 1 day are combined in one large (3-

litre capacity) collapsible plastic bag. The contents are infused over a 12-24 hour period.

Transfer of amino acid, glucose and electrolyte infusions, and the addition of vitamins

and trace elements, must be carried out with great care under aseptic conditions to

avoid microbial contamination. These solutions often provide good growth conditions

for bacteria and moulds. Fats are administered as oil-in-water emulsions, comprising

small droplets of a suitable vegetable oil (e.g. soyabean) emulsified with egg lecithin

and sterilized by autoclaving. In many cases, the fat emulsion may be added to the 3-

litre bag.

2.3 Small-volume aqueous injections

This category comprises single-dose injections, usually of 1-2 ml but as high as 50 ml,

dispensed in borosilicate glass ampoules, plastic (polyethylene or polypropylene)

ampoules or, rarely, multidose glass vials of 5-15 ml capacity stoppered with a rubber

closure through which a hypodermic needle can be inserted, e.g. insulins, vaccines.

The closure is designed to reseal after withdrawal of the needle. It is unwise to include

too many doses in a multidose container because of the risk of microbial contamination

during repeated use. Bactericides must be added to injections in multidose containers

to prevent contamination during withdrawal of successive doses, except as detailed

below. Bactericides may not be used in injections in which the total volume to be

injected at one time exceeds 15 ml. This may occur if the solubility of a drug is such

that a therapeutic dose is only soluble in this order of volume (e.g. Bemegride Injection).

There is also an absolute prohibition on the inclusion of bactericides in injections of

414 Chapter 21

the following categories: intra-arterial, intracardiac, intrathecal or subarachnoid,

intracisternal and peridural.

Small-volume injections may be sterilized by the following methods.

1 Heating in an autoclave for injections packed in glass ampoules.

2 Filtration followed by aseptic sealing (plastic containers). Since the product is not

sterilized in its final container, a bactericide may be included to reduce the risks of

contamination.

2.3.1 Problems of drug stability

1 Thermostability. The choice of sterilization method depends on the thermostability

of the active ingredient, autoclaving being applied only to drugs that are heat stable in

aqueous solution.

2 Chemical stability. Some medicaments undergo chemical change in aqueous

solutions. If the change is due to oxidation, a reducing agent such as sodium

metabisulphite is included (e.g. Adrenaline Injection BP).

Aqueous solutions of some drugs are so unstable that chemical stabilization is

impossible. In this case the drug itself, not its aqueous solution, is sterilized by dry heat

(160°C for 2 hours or its equivalent at higher temperatures) in its final container and

dissolved immediately before use by the addition of sterile water (Water for Injections

BP). For drugs which are both thermolabile and unstable in aqueous solution, a sterile

solution of the drug is freeze-dried in the final container and is reconstituted as above

just before use (e.g. many antibiotics, Hyaluronidase BP).

Details of time-temperature regimens as dictated by injection volume and heat

transfer to the whole of the product (section 2.2) and of possible interactions between

active ingredients and containers must be considered (see also Chapter 20).

2.4 Small-volume oily injections

Certain small-volume injections are available where the drug is dissolved in a viscous

oil because it is insoluble in water; non-aqueous solvent must be used. In addition,

drags in non-aqueous solvents provide a depot effect, for example for hormonal

compounds. The intramuscular route of injection must be used. The vehicle may be a

metabolizable fixed oil such as arachis or sesame oil (but not a mineral oil) or an ester

such as ethyl oleate which is also metabolizable. The latter is less viscous and therefore

easier to administer but the depot effect is of shorter duration. The drug is normally

dissolved in the oil, filtered under pressure and distributed into ampoules. After sealing,

the ampoules are sterilized by dry heat, for example, at 160°C for 2 hours. A bactericide

is probably ineffective in such a medium and therefore offers very little protection

against contamination in a multidose oily injection.

3 Non-injectable sterile fluids

There are many other types of solution required in a sterile form for use particularly in

hospitals.

Sterile pharmaceutical products 415

3.1 Non-injectable water

This is sterile water, not necessarily of injectable water standards, which is used widely

during surgical procedures for wound irrigation, moistening of tissues, washing of

surgeons' gloves and instruments during use and, when wanned, as a haemostat. Isotonic

saline may also be used. Topical water (as it is often called) is prepared in 500-ml and

1-litre polyethylene or polypropylene containers with a wide neck, to allow for ease

for pouring, and tear-off cap. Hospitals in the UK probably use larger quantities of

topical fluids than of intravenous infusions.

3.2 Urological (bladder) irrigation solutions

These are used for the rinsing of the urinary tract to aid tissue integrity and cleanliness

during or after surgery. Either water or glycine solution is used, the latter eliminating

the risk of intravascular haemolysis when electrosurgical instruments are used. These

are sterile solutions produced in collapsible or semi-rigid plastic containers of up to 3-

litre capacity.

3.3 Peritoneal dialysis and haemodialysis solutions

Peritoneal dialysis solutions are admitted into the peritoneal cavity as a means of

removing accumulated waste or toxic products following renal failure or poisoning.

They contain electrolytes and glucose (1.4-7% w/v) to provide a solution equivalent to

potassium-free extracellular fluid. Lactate or acetate is added as a source of bicarbonate

ions. Slightly hypertonic solutions are usually employed to avoid increasing the water

content of the intravascular compartment. A more hypertonic solution, containing a

higher glucose concentration, is used to achieve a more rapid removal of water. In fact,

the peritoneal cavity behaves as if it were separated from the body organs by a semi-

permeable membrane. Warm peritoneal solution (up to 5 litres) is perfused into the

cavity for 30-90 minutes and then drained out completely. This procedure can then be

repeated as often as required. Since the procedure requires large volumes, these fluids

are commonly packed in 2.5-litre containers. It is not uncommon to add drugs (for

instance potassium chloride or heparin) to the fluid prior to use.

Haemodialysis is the process of circulating the patient's blood through a machine

via tubing composed of a semi-permeable material such that waste products permeate

into the dialysing fluid and the blood then returns to the patient. Haemodialysis solutions

need not be sterile but must be free from heavy bacterial contamination.

3.4 Inhaler solutions

In cases of severe acute asthmatic attacks, bronchodilators and steroids for direct delivery

to the lungs may be needed in large doses. This is achieved by direct inhalation via a

nebulizer device; this converts a liquid into a mist or fine spray. The drug is diluted in

small volumes of Water for Injections BP before loading into the reservoir of the machine.

This vehicle must be sterile and preservative-free and is therefore prepared as a terminally

sterilized unit dose in polyethylene nebules.

416 Chapter 21