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

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Medicinal Products in 1989 and are now published in the UK as Rules and Guidance

for Pharmaceutical Manufacturers and Distributors (1997) by the Medicines Control

Agency, Department of Health.

Compliance with the principles of GMP is one of the major factors considered

by the Licensing Authority when examining an application for a licence to manu-

facture under the Medicines Act (1968). Similar codes exist in the USA and other

countries.

5 Conclusions

The sole objective of all hygiene and manufacturing controls is to ensure the quality of

the pharmaceutical product for the safety and protection of the patient. The manufacture

of non-sterile pharmaceutical products requires that certain criteria of cleanliness,

personal hygiene, production methods and storage must be met. Many such products

are for oral and topical use and the question may fairly be posed as to the point of what

are now quite stringent conditions. Nevertheless, some carefully controlled hospital

studies have indeed shown that both types of medicine may be associated with

nosocomial (hospital-acquired) infections and this risk can be minimized by the

application of GMP principles.

A greater degree of stringency is required for the production of terminally sterilized

products. Again, as the final product is subjected to a sterilization process (usually

thermal), it may be asked why so much emphasis is placed upon process and

environmental controls. The single most important reason is to ensure the lowest possible

microbial burden to the sterilizer, thereby ensuring the highest sterility assurance levels

attainable (Chapter 20). It must also be realized (as reiterated in Chapter 23) that it is

far better to control a process from beginning to end, i.e. with frequent checks all along

the line, than to rely solely on tests which can only determine whether a small proportion

of the final products in a batch are satisfactory.

Even further criteria must be satisfied when products are being prepared aseptically

where microbiological quality is entirely dependent upon observance of the highest

possible production standards. It is essential that operatives have a sound working

knowledge of the properties of microorganisms, and that they appreciate the importance

of personal hygiene, of the techniques that will be adopted, and of the possible sources

of contamination and error. In this respect, it is a sobering thought to realize that the

great majority of reported defective medicinal products has resulted from human error

or carelessness, not from technology failure.

6 Further reading

Denyer S.P. (1988) Clinical consequence?, of microbial action on medicines. In: Biodeterioration (eds

D.R. Houghton, R.N. Smith & H.O W. Eggins), vol. 7, pp. 146-151. London: Elsevier Applied

Science.

Denyer S.R (1992) Filtration sterilization. In: Principles and Practice of Disinfection, Preservation

and Sterilization, 2nd edn (eds A.D. Russell, W.B. Hugo & G.A.J. Ayliffe), pp. 573-604. Oxford:

Blackwell Science.

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

Ellis Horwood. (Chapters 4 and 5 provide additional information.)

Factory and hospital hygiene 437

Neiger J. (1997) Life with the UK pharmaceutical isolator guidelines: a manufacturer's viewpoint. Eur

J Parenteral Sci, 2, 13-20.

Ringertz O. & Ringertz S.H. (1982) The clinical significance of microbial contamination in

pharmaceutical and allied products. Adv Pharm Sci, 5, 201-226.

Rules and Guidance for Pharmaceutical Manufacturers and Distributors (1997) London: HMSO.

Spooner D.F. (1996) Hazards associated with the microbiological contamination of cosmetics, toiletries

and non-sterile pharmaceuticals. In: Microbial Quality Assurance in Cosmetics, Toiletries and Non-

sterile Pharmaceuticals, 2nd edn (eds R.M. Baird & S.F. Bloomfield), pp. 9-27. London: Taylor &

Francis.

Underwood E. (1992) Good manufacturing practice. In: Principles and Practice of Disinfection,

Preservation and Sterilization, 2nd edn (eds A.D. Russell, W.B. Hugo & G.A.J. Ayliffe), pp. 274-

291. Oxford: Blackwell Science.

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

(Note the section dealing with microbial limit tests.)

Sterilization control and

sterility assurance

Introduction

Bioburden determinations

Environmental monitoring

Sterilization monitors

Physical indicators

Chemical indicators

Biological indicators

Sterility testing

Methods

Antimicrobial agents

5.2.1

5.2.2

5.2.3

5.3

5.4

5.5

Introduction

A product to be labelled 'sterile' must be free of viable microorganisms. To achieve

this, the product, or its ingredients, must undergo a sterilization process of sufficient

microbiocidal capacity to ensure a minimum level of sterility assurance (Chapter 20).

It is essential that the required conditions for sterilization be achieved and maintained

through every operation of the sterilizer.

Historically, the quality control of sterile products consisted largely, or, in some

cases, even exclusively, of a sterility test, to which the product was subjected at the end

of the manufacturing process. However, a growing awareness of the limitations of

sterility tests in terms of their ability to detect low concentrations of microorganisms,

has resulted in a shift in emphasis from a crucial dependence on end-testing to a situation

in which the conferment of the status 'sterile' results from the attainment of satisfactory

quality standards throughout the whole manufacturing process. In other words, the

quality is 'assured' by a combination of process monitoring and performance criteria;

these may be considered under four headings:

Bioburden determinations (section 2)

Environmental monitoring (section 3)

In-process monitoring of sterilization procedures (section 4)

Sterility testing (section 5).

In well-understood and well-characterized sterilization processes (e.g. heat and

irradiation), where physical measurements may be accurately made, sterility can be

assured by ensuring that the manufacturing process as a whole conforms to the

established protocols for the first three of the above headings. In this case the process

has satisfied the required parameters thereby permitting parametric release of the

product without recourse to a sterility test.

Specific inactivation

Dilution

Membrane filtration

Positive controls

Specific cases

Sampling

Conclusions

Acknowledgements

Further reading

Sterilization control and sterility assurance 439

This chapter will discuss briefly the principles and applications of the various

methods of monitoring and validating sterilization processes.

2 Bioburden determinations

The term 'bioburden' is used to describe the concentration of microorganisms in a

material; this may be either a total number of organisms per millilitre or per gram,

regardless of type, or a breakdown into such categories as aerobic bacteria or yeasts

and moulds. Bioburden determinations are normally undertaken by the supplier of the

raw material, whose responsibility it is to ensure that the material supplied conforms

to the agreed specification, but they may also be checked by the recipient. The

maximum permitted concentrations of contaminants may be those specified in various

pharmacopoeias or the levels established by the manufacturer during product development.

The level of sterility assurance which is achieved in a terminally sterilized

product is dependent upon the design of the sterilization process itself and upon

the bioburden immediately prior to sterilization (see Chapter 19). However, the

adoption of high standards for the quality of the raw materials is not, in itself, a strategy

which will ensure that the product has an acceptably low bioburden immediately prior

to sterilization. It is necessary also to ensure that the opportunities for microbial

contamination during manufacture are restricted (see below), and those organisms that

are present initially do not normally find themselves in conditions conducive to

growth. It is for these reasons that manufacturing processes are designed to utilize

adverse temperatures, extreme pH values and organic solvent exposures in order to

prevent an increase in the microbial load. For example, water is the most common,

and potentially the most significant, source of contamination in the manufactured

product, and maintenance of water at elevated temperatures is commonly employed as

a means of limiting the growth of organisms such as Pseudomonas spp. which can

proliferate during storage, even in distilled or deionized water. Precautions such as

these ensure that chemically synthesized raw materials have bioburdens which are

generally much lower than those found in 'natural' products of animal, vegetable or

mineral origin.

3 Environmental monitoring

The levels of microbial contamination in the manufacturing areas (Chapter 22) are

monitored on a regular basis to confirm that the numbers do not exceed specified limits.

The concentrations of bacteria and of yeasts/moulds in the atmosphere may be

determined either by use of 'settle plates' (Petri dishes of suitable media exposed for

fixed periods, on which the colonies are counted after incubation) or by use of air

samplers which cause a known volume of air to be passed over the agar surface.

Similarly, the contamination on surfaces, including manufacturing equipment, may be

measured using swabs or contact plates (also known as Rodac—replicate organism

detection and counting—plates) which are specially designed Petri dishes slightly

overfilled with agar, which, when set, projects very slightly above the plastic wall of

the dish. This permits the plate to be inverted onto, or against, any solid surface, thereby

allowing transfer of organisms from the surface onto the agar.

440 Chapter 23

Less commonly, environmental monitoring can extend also to the operators in the

manufacturing area whose clothing, e.g. gloves or face masks, may be sampled in

order to estimate the levels and types of organisms which may arise as product

contaminants from those sources.

Sterilization monitors

Monitoring of the sterilization process can be achieved by the use of physical, chemical

or biological indicators of sterilizer performance. Such indicators are frequently

employed in combination.

Physical indicators

In heat-sterilization processes, a temperature record chart is made of each sterilization

cycle with both dry and moist heat (i.e. autoclave) sterilizers; this chart forms part of

the batch documentation and is compared against a master temperature record (MTR).

It is recommended that the temperature be taken at the coolest part of the loaded sterilizer.

Further information on heat distribution and penetration within a sterilizer can be gained

by the use of thermocouples placed at selected sites in the chamber or inserted directly

into test packs or bottles. Since autoclaving depends also upon steam under pressure as

well as temperature, pressure measurements form an essential part of the physical

monitoring of this process. In addition, periodic leak tests are performed on pre vacuum

steam sterilizers to assess the efficiency of air removal prior to the introduction of

steam.

For gaseous sterilization procedures, elevated temperatures are monitored for each

sterilization cycle by temperature probes, and routine leak tests are performed to ensure

gas-tight seals. Pressure and humidity measurements are recorded. Gas concentration

is measured independently of pressure rise, often by reference to weight of gas used.

In radiation sterilization, a plastic (often perspex) dosimeter which gradually darkens

in proportion to the radiation absorbed gives an accurate measure of the radiation dose

and is considered to be the best technique currently available for following the

radiosterilization process.

Sterilizing filters are subject to a bubble point pressure test, which is a technique

employed for determining the pore size of filters, and may also be used to check the

integrity of certain types of filter device (membrane and sintered glass; see Chapter 20)

immediately after use. The principle of the test is that the wetted filter, in its assembled

unit, is subjected to an increasing air or nitrogen gas pressure differential. The pressure

difference recorded when the first bubble of gas breaks away from the filter is related

to the maximum pore size. When the gas pressure is further increased slowly, there is a

general eruption of bubbles over the entire surface. The pressure difference here is

related to the mean pore size. A pressure differential below the expected value would

signify a damaged or faulty filter. A modification to this test for membrane filters involves

measuring the diffusion of gas through a wetted filter at pressures below the bubble

point pressure (diffusion rate test); a faster diffusion rate than expected would again

indicate a loss of filter integrity. In addition, a filter is considered ineffective when an

unusually rapid rate of filtration occurs.

Sterilization control and sterility assurance 441

Efficiency testing of high-efficiency particulate air (HEPA) filters used for the supply

of sterile air to aseptic workplaces (Chapter 22) is normally achieved by the generation

upstream of dioctylphthalate (DOP) or sodium chloride particles of known dimension,

followed by detection in downstream filtered air. Retention efficiency is recorded as

the percentage of particles removed under defined test conditions. Microbiological

tests are not normally performed.

4.2 Chemical indicators

Chemical monitoring of a sterilization process is based on the ability of heat, steam,

sterilant gases and ionizing radiation to alter the chemical and/or physical characteristics

of a variety of chemical substances. Ideally, this change should take place only when

satisfactory conditions for sterilization prevail, thus confirming that a sterilization cycle

has been successfully completed. In practice, however, the ideal indicator response is

Fig. 23.1 Examples of biological and chemical indicators used for monitoring sterilization

processes. (A and B) A spore strip (in a glassine envelope) and a spore disc, respectively; the spores

are dried onto absorbent paper or fabric. (C) Attest™ indicator comprising a plastic vial containing a

spore strip together with a sealed glass ampoule of culture medium; the ampoule is crushed after

exposure and the medium immerses the strip. (D) Chemspor™ indicator in which bacterial spores

are suspended in culture medium; the horizontal band on the ampoule also darkens on autoclaving to

enable steam-exposed and non-exposed ampoules to be distinguished. (E) Plastic carrier with dried

Bacillus stearothermophilus spores designed for monitoring low-temperature steam and

formaldehyde cycles. (F) Browne's tube™; the liquid within the tube changes colour on heat

exposure. (G) Thermalog™ strip in which a blue dye progresses from left to right during heat

exposure. (H) Chemdi™ displays colour change in arrowed section of the strip after heating.

(I) Chemspor™ which is a combined chemical and biological indicator; the ampoule contains a

spore suspension in culture medium together with a second, smaller ampoule which contains a

chemical indicator.

442 Chapter 23

not always achieved and so a necessary distinction is made between (i) those chemical

indicators which integrate several sterilization parameters (i.e. temperature, time and

saturated steam) and closely approach the ideal; and (ii) those which measure only one

parameter and consequently can only be used to distinguish processed from unprocessed

articles. Thus, indicators which rely on the melting of a chemical substance show that

the temperature has been attained but not necessarily maintained.

Chemical indicators generally undergo melting or colour changes (some examples

are given in (Fig. 23.1)), the relationship of this change to the sterilization process

being influenced by the design of the test device (Table 23.1). It must be remembered,

however, that the changes recorded do not necessarily correspond to microbiological

sterility and consequently the devices should never be employed as sole indicators in a

sterilization process. Nevertheless, when included in strategically placed containers or

packages, chemical indicators are valuable monitors of the conditions prevailing at the

coolest or most inaccessible parts of a sterilizer.

4.3 Biological indicators

Biological indicators (Bis) for use in thermal, chemical or radiation sterilization

processes consist of standardized bacterial spore preparations which are usually in the

form either of suspensions in water or culture medium or of spores dried on paper,

aluminium or plastic carriers. As with chemical indicators, they are usually placed in

dummy packs located at strategic sites in the sterilizer. Alternatively, for gaseous

sterilization these may also be placed within a tubular helix (Line-Pickerill) device.

After the sterilization process, the aqueous suspensions or spores on carriers are

aseptically transferred to an appropriate nutrient medium which is then incubated and

periodically examined for signs of growth. Spores of Bacillus stearothermophilus in

sealed ampoules of culture medium are used for steam sterilization monitoring, and

these may be incubated directly at 55°C; this eliminates the need for an aseptic transfer.

Continued on p. 444

Sterilization control and sterility assurance 443

Table 23.1 Examples

Sterilization

method

Heat

Autoclaving or

dry heat

Dry heat only

of chemical indicators for monitoring sterilization processes

Principle

Temperature-sensitive

coloured solution

Temperature-sensitive

chemical

Device

Sealed tubes partly filled with a

solution which changes colour at

elevated temperatures; rate of

colour change is proportional to

temperature, e.g. Browne's tubes

Usually a temperature-sensitive

white wax concealing a black

marked or printed (paper) surface;

at a predetermined temperature the

wax rapidly melts exposing the

background mark(s)

Parameter(s)

monitored

Temperature, time

Temperature

Table 23.1 Continued

Sterilization

method

Heating in an

autoclave only

Gaseous

sterilization

Ethylene oxide

(EO)

Low

temperature

steam and

formaldehyde

Radiation

sterilization

Principle

Steam-sensitive

chemical

Capillary principle

(Thermalog S)

Reactive chemical

Capillary principle

(Thermalog G)

Reactive chemical

Radiochromic

chemical

Dosimeter device

Device

Usually an organic chemical in a

printing ink base impregnated into a

carrier material. A combination of

moisture and heat produces a

darkening of the ink, e.g. autoclave

tape. Devices of this sort can be

used within dressings packs to

confirm adequate removal of air and

penetration of saturated steam

(Bowie-Dick test)

Consists of a blue dye in a waxy

pellet, the melting-point of which is

depressed in the presence of

saturated steam. At autoclaving

temperatures, and in the continued

presence of steam, the pellet melts

and travels along a paper wick

forming a blue band the length of

which is dependent upon both

exposure time and temperature

Indicator paper impregnated with a

reactive chemical which undergoes a

distinct colour change on reaction

with EO in the presence of heat and

moisture. With some devices rate of

colour development varies with

temperature and EO concentration

Based on the same 'migration along

wick' principle as Thermalog S.

Optimum response in a cycle of

600 mgl"

EO, temperature 54°C, rh

40-80%. Lower EO levels and/or

temperature will slow response time,

blue colour of band is fugitive at rh

<30%

Indicator paper impregnated with a

formaldehyde-, steam- and

temperature-sensitive reactive

chemical which changes colour

during the sterilization process

Plastic devices impregnated with

radiosensitive chemicals which

undergo colour changes at relatively

low radiation doses

Acidified ferric ammonium sulphate

or eerie sulphate solutions respond

to irradiation by dose-related

changes in their optical density (see

also section 2.1.3)

Parameter(s)

monitored

Saturated steam

Temperature,

saturated steam, time

Gas concentration,

temperature, time

(selected devices); NB

a minimum relative

humidity (rh) is

required for device to

function

Gas concentration,

temperature, time

(selected cycles)

Gas concentration,

temperature, time

(selected cycles)

Only indicate

exposure to radiation

Accurately measure

radiation doses

Aseptic transfers are also avoided by the use of self-contained units where the spore

strip and nutrient medium are present in the same device ready for mixing after use.

The bacterial species to be used in a BI must be selected carefully, since it must

be non-pathogenic and should possess above-average resistance to the particular

sterilization process. Resistance is adjudged from the spore destruction curve obtained

upon exposure to the sterilization process; recommended BI spores and their decimal

reduction times (D-values; Chapter 20) are shown in Table 23.2. Great care must be

taken in the preparation and storage of Bis to ensure a standardized response to

sterilization processes. Indeed, while certainly offering the most direct method of

monitoring sterilization processes, it should be realized that Bis may be less reliable

monitors than physical methods and are not recommended for routine use, except in

the case of gaseous sterilization.

One of the long-standing criticisms of Bis is that the incubation period required in

order to confirm a satisfactory sterilization process imposes an undesirable delay on

the release of the product. This problem has been overcome, with respect to steam

sterilization at least, by the use of a detection system in which a spore enzyme, a-

glucosidase (reflective of spore viability), converts a non-fluorescent substrate into a

fluorescent product in as little as lhour.

Filtration sterilization requires a different approach from biological monitoring,

the test effectively measuring the ability of a filter to produce a sterile filtrate from a

culture of a suitable organism. For this purpose, Serratia marcescens, a small Gram-

negative rod-shaped bacterium (minimum dimension 0.5 fim), has been recommended

in the Pharmaceutical Codex (1979). The bacterial challenge test is the most severe to

which a filter of any construction can be subjected. In the membrane-filter industry, the

test using Ser. marcescens is usually reserved for filters of 0.45-jUm pore size, and a

more rigorous test involving Brevundimonas diminuta—formerly Pseudomonas

diminuta—having a minimum dimension of 0.3 jjxa is applied to filters of 0.22-jjm

Table 23.2 Biological indicators for monitoring sterilization processes*

Sterilization process

Heating in an

autoclave (121°C)

Dry heat (160°C)

Ethylene oxide (EO)t

(EO600mg|-

temperature 54°C and

60% relative humidity)

Low temperature

steam (73°C) and

formaldehyde

(12mg|-

Ionizing radiation

Species

Bacillus stearothermophilus

Clostridium sporogenes

Bacillus subtilis var. niger

Bacillus stearothermophilus

Bacillus pumilus

Inoculum size

>10

>5x10

—

-10

D-value

1.5min

0.8min

5-10min

2.5 min

5min

3kGy(0.3Mrad)

* British Pharmacopoeia (1993).

f European Pharmacopoeia (1997).

$Soper&Davies(1990).

Sterilization control and sterility assurance 445

pore size. The latter filters are defined as those capable of completely removing Brev.

diminuta from suspension. In this test, using this organism, a realistic inoculum level

must be adopted, since the probability of bacteria appearing in the filtrate rises as the

number of Brev. diminuta cells in the test challenge increases; a standardized inoculum

size of 10

cells cm

-2

is normally employed. The extent of the passage of this organism

through membrane filters is enhanced by increasing the filtration pressure. Thus,

successful sterile filtration depends markedly on the challenge conditions.

Sterility testing

A sterility test is basically a test which assesses whether a sterilized pharmaceutical or

medical product is free from contaminating microorganisms, by incubation of either

the whole or a part of that product with a nutrient medium. It thus becomes a destructive

test and raises the question as to its suitability for testing large, expensive or delicate

products or equipment. Furthermore, by its very nature such a test is a statistical process

in which part of a batch is randomly* sampled and the chance of the batch being passed

for use then depends on the sample passing the sterility test.

A further limitation is that which is inherent in a procedure intended to demonstrate

a negative. A sterility test is intended to demonstrate that no viable organisms are present,

but failure to detect them could simply be a consequence of the use of unsuitable media

or inappropriate cultural conditions. To be certain that no organisms are present it

would be necessary to use a universal culture medium suitable for the growth of any

possible contaminant and to incubate the sample under an infinite variety of conditions.

Clearly, no such medium, or combination of media, are available, and, in practice, only

media capable of supporting non-fastidious bacteria, yeasts and moulds are employed.

Furthermore, in pharmacopoeial tests, no attempt is made to detect viruses, which , on

a size basis, are the organisms most likely to pass through a sterilizing filter. Nevertheless,

the sterility test does have an important application in monitoring the microbiological

quality of filter-sterilized, aseptically filled products and does offer a final check on

terminally sterilized articles. In the UK, test procedures laid down by the European

Pharmacopoeia must be followed; this provides details of the sample sizes to be adopted

in particular cases. The principles of these tests are discussed in brief below.

Methods

There are three alternative methods available when conducting sterility tests.

1 The direct inoculation procedure involves introducing test samples directly into

nutrient media. The British Pharmacopoeia recommends two media: (i) fluid

mercaptoacetate medium, which contains glucose and sodium mercaptoacetate (sodium

thioglycollate) and is particularly suitable for the cultivation of anaerobic organisms

(incubation temperature 30-35°C); and (ii) soyabean casein digest medium, which

will support the growth of both aerobic bacteria (incubation temperature 30-35°C) and

* It has been proposed that random sampling be applied to products which have been processed and

filled aseptically. With products sterilized in their final containers, samples should be taken from the

potentially coolest or least sterilant-accessible part of the load.