Relevance of Microorganisms in Pharmaceutical Processing 47

• Investigation of reports from pharmacists and users on undesirable characteristics likely to

be initiated by microbiological activity in the finished product and incidents where a batch
has failed to meet specifications before marketing.
• Participation in validation and in-process control efforts with respect to microbiological
elements such as: determination of bio-burdens for water and other raw materials,
processing environment through environmental monitoring, containers and closures,
finished products before filling, personnel, processing equipment etc. This also includes the
efficiency of environmental parameters and product sterilization systems, notably the
efficiency of filters and of other sterilizing equipment and determination of parameters
such as F and D values for common microbiological contaminants at the processing sites.
For terminal sterilization often the performance of each sterilizer load is tested for delivery
of the parametric energy required for sterilization during the sterilization cycle being the
summation of the bio-burden and the sterilizing effectiveness for the process.
• Conducting evaluation tests for disinfectants, antiseptics, sterilants, sanitizers and
preservatives. Some such tests are conducted periodically (such as efficiency of
disinfectants) while others are conducted basically upon initiation of a new formula for a
product (such as in the case of preservative).
• Conducting quality control evaluation of the potency of active ingredients using
microbiological methods as is the case in the assay of antibiotics and accessory factors.
• Participation in the microbiological elements in the auditing and certification of
manufacturing facilities. A major role is played in auditing aseptic filling, the
microbiological quality of the processing environment, microbiological compliance of
various areas with specifications “at rest” and during operation throughout the
manufacturing period and the overall preparation/sterilization process. Similar
environmental in-process control procedures are applied in non-sterile products
production departments.
• Most pharmacopoeias currently require that each sterilizer load be validated by using,
among others, biological indicators which are tested for viability after sterilization. This is
usually among the responsibilities of the microbiology laboratory.
• Validation of aseptic filling systems require occasional or periodical “media fill” or “dry fill”
testing where sterile material is filled in a small batch (usually multiples of 3,000 or more)
containers and subsequent testing of the sterility of all filled containers which is performed
by the microbiology laboratory.

2.4.5 Current Pharmacopeial Guidance on Non-Sterile Dosage Forms

Current pharmacopeial guidance on sterilization, sterility testing and microbial quality of non-
sterile dosage forms

As mentioned before, while special compendial monographs were introduced on sterilization,

sterility testing and microbiological attributes of non-sterile products, the pace of expanding
compendial monographs accelerated significantly in the last 40 years or so, when more
knowledge of the role played by microorganisms in drug quality and safety gained higher
recognition. This is evidenced by the size and level of detail which microbiological elements
48 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

presently occupy in recent compendial editions. The following examples are representative of a
world trend:

• In reviewing GMP, Title 21 of the United States Code of Federal Regulations (CFR) (18)
devoted Part 210 to cGMP in manufacturing practice in manufacturing, processing,
packing, or holding of drugs and Part 211 to cGMP for finished pharmaceuticals. Section
211.113 discussed control of microbiological contamination which required “Appropriate
written procedures, designed to prevent objectionable microorganisms in drug products
not required to be sterile, shall be established and followed” and that “Appropriate written
procedures, designed to prevent microbiological contamination of drug products
purporting to be sterile, shall be established and followed. Such procedures shall include
validation of any sterilization process”. Both Canadian Good Manufacturing Practices and
European Union Good Manufacturing Practices regarding medicinal products for human
and veterinary use have similar requirements. Part 820 of the CFR, directed at the medical
device industry, have similar requirements.
• The United States Pharmacopeia 32/National Formulary 27 (19), in force on May 2008 the
entire Section 10 of the General Chapters to “Microbiology”. This chapter comprised:

o Chapter <61>: Microbiological examination of non-sterile products: microbiological

enumeration tests.
o Chapter <62>: Microbiological examination of non-sterile products: tests for specified
microorganisms.
o Chapter <1111>: Microbiological examination of non-sterile products: acceptance
criteria for pharmaceutical preparations and substances for pharmaceutical use.
o Chapter <2>: Biotechnology-derived drug substances.
o Chapter <5>: Biotechnology-derived drug products.

Under the General tests and determinations, the following chapters on “Microbiological Tests”
are described:

o Chapter <51>: Antimicrobial effectiveness test.

o Chapter <55>: Biological indicators – resistance performance tests.
o Chapter <61>: Microbial limit tests.
o Chapter <62>: Microbiological examination of non-sterile products tests.
o Chapter <71>: Sterility tests. Here it is interesting to note a new deviation from older
traditions: a repeat test on the same sample size is allowed only “if the first test is
invalid” based on four defined criteria, reflecting greater confidence in the test
methods which guards against contamination during testing. If the second test yields
growth again, the article “does not comply with the test”.
o Chapter <151>: Pyrogen test

Under the Biological tests and assays, the following chapters are included:

o Chapter <81>: Antibiotics –microbial assays.

o Chapter <85>: Bacterial endotoxin test.
 Relevance of Microorganisms in Pharmaceutical Processing 49

In addition, Chapter <1211> is devoted to "Sterilization and sterility assurance of compendial

articles.

• Similarly, the BP (20), which now incorporates a sizable part of the European
Pharmacopeia, which became official on first January 2009, in Part II devotes the sizable
Appendix XVIII to “Methods of sterilization” and Appendix XVIC to “Antimicrobial
preservatives”. In Volume III (General Monographs) the following new requirements are
described:

o While eye drops remain sterile with a maximum volume of 10 ml, ear drops and nasal
preparations (Chapter 2.6.1) too may have to be sterile for certain uses.
o Any preparation labeled “sterile” must pass the sterility test.
o Chapter 2.6.8 describes the rabbit test for Pyrogens.
o Chapter 2.6.14 describes the test for bacterial endotoxins.
o Chapter 5.1.1 is devoted to “Methods of preparation of sterile products” while
Chapter 5.1.4 is devoted to the “Microbiological quality of pharmaceutical
preparations”.

The Control Methods described in Volume IV, includes reference to six methods for the
“microbial test to assure microbial quality”.

This is contrasted against earlier editions of the BP, most notably:

 Introduction of “Tyndalization” as a method of sterilization in the 1934 edition, whereby

the preparation is heated to 98 for 20 to 45 minutes on 3 successive days with incubation
at 37 for 24 hours in the intervening period. This was removed in the 1941 edition when it
was found not to be sufficiently sporocidal and to result in pyrogenic products.
 Introduction of “heating with bactericide” in 1934 for “materials which would not
withstand temperatures over 100 and as an alternative method for sterilization of
containers made of thermo-labile plastics”. This method was later eliminated being not
sufficiently sporocidal.

2.5 THE FUTURE

Will all pharmaceuticals be aseptically processed?

Of course, pharmaceutical practice has moved from essentially a chemical specialty to a

patient-oriented field and has been significantly influenced by advances in physical and
engineering sciences as well. However, looking slightly less than a century back, the rate of
growth of microbiology in pharmacy practice has indeed been phenomenal moving from a zero
point to a pivotal role. It is safe to conclude than no other discipline has made such deep
inroads into pharmacy. The question arises: what next? Currently pharmacy is linked to
microbiology essentially from a safety aspect and this is not likely to change. The current major
emphasis on pharmaceutical biotechnology products – with this field’s close inter-linkage with
microbiology is most likely to expand dramatically rather than be a passing mode, but this is a
50 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

matter of development of novel pharmaceuticals not currently available which will require
further interaction with microbiology (and other contemporary biological sciences) with
respect to quality control and quality assurance of such novel products. Many such elements
will also relate to safety, not in the sense of microbiological quality but more in terms of
efficacy and potency.

The future of the microbiological quality control tests

Elements of microbiological quality however will remain important safety considerations not
only in terms of protection against infections but also to reduce the chances of un-necessary
immune response of patients to microbial antigens. With further decline in relative cost of
reducing bio-burden and simplification of technologies which target such reduction, one may
expect an expansion in this direction and a consequent reduced reliance on de-contamination
procedures. This is evidenced by the trend to establish lower in-house total count limits for
most non-sterile products from the common compendial 100 cfu/g or ml to 5 and often to 2, by
many producers and the strong trend of reliance on low-bioburden approaches to sterilization
at mild conditions rather than the more expensive and damaging over-kill approaches.

Microbiological quality control tests on finished products are and will continue to be essential
elements for the release of pharmaceutical products since it is a basic concept in
pharmaceutical practice and guards against any gross failure in a process. It is also basically a
legal requirement. The more recent approval of “parametric release” for sterile products
resolved an economic dilemma without sacrificing the safety of the product and is likely to be
applied on a wider scale. Similar innovative procedures may be devised for non-sterile products
too. However, methodologies for simpler, more reliable and reproducible microbiological
quality control tests has so far defied innovation. Considerable efforts have been put into a
simplified test for sterility which did not depend on “visible growth” as an end point but none
has met with much support and, with present day scientific knowledge none is expected to
command universal approval. The same seems to apply to microbiological quality control tests
for non-sterile products. It is possible, however, that more reproducible tests in this direction
could be devised based on possible recognition of unique identifiers for specified un-desirable
“species” based on nucleic acid contents and/or their expression products. The total counts too
may be replaced by similar fast tests if an when reliable and reproducible systems are designed
to correlate a given microbiological activity to numbers of cells – which are currently not
available.

2.6 REFERENCES

1. Sydney D. Rubbo and Joan F. Gardner (1965) A review of Sterilization and disinfection as
Applied to Medical, industrial and Laboratory Practice, Lloyd-Luke Medical Books Ltd,
London)
2. The United States Code of Federal Regulations (CFR) 1965. Chapter I, Part 133. Office of
the Federal Register, Washington, D.C.
3. The United States Code of Federal Regulations (CFR) 1965. Section 133.8. Office of the
Federal Register, Washington, D.C.
 Relevance of Microorganisms in Pharmaceutical Processing 51

4. The United States Code of Federal Regulations (CFR) 1965. Section 141.2. Office of the
Federal Register, Washington, D.C.
5. British Pharmaceutical Codex (BPC) 1968. pages 1418-1429. The Pharmaceutical Press,
London.
6. United States Pharmacopeia (USP) X, pages 837-840 and 719-723. United States
Pharmacopeial Convention, Washington, D.C.
7. United States Pharmacopeia (USP) XVII, pages 810-814 United States Pharmacopeial
Convention, Washington, D.C.
8. United States Pharmacopeia (USP) XVII, pages 829-832 United States Pharmacopeial
Convention, Washington, D.C.
9. British Pharmaceutical Codex (BPC) 1968, pages 1103-1107. The Pharmaceutical Press,
London.
10. British Pharmaceutical Codex (BPC) 1963, pages 1148-1149. The Pharmaceutical Press,
London.
11. British Pharmacopeia (BP) 1968, pages 1357-1358. The Pharmaceutical Press, London.
12. British Pharmaceutical Codex (BPC) 1968, pages 1076-1090. The Pharmaceutical Press,
London.
13. British Pharmaceutical Codex (BPC) 1968, pages 1091-1093. The Pharmaceutical Press,
London.
14. British Pharmaceutical Codex (BPC) 1968, pages 1262-1263. The Pharmaceutical Press,
London.
15. British Pharmaceutical Codex (BPC) 1968, pages 1032-1033. The Pharmaceutical Press,
London.
16. British Pharmaceutical Codex (BPC) 1968, page 1042. The Pharmaceutical Press, London.
17. British Pharmaceutical Codex (BPC) 1968, pages 1417-1418. The Pharmaceutical Press,
London.
18. The United States Code of Federal Regulations (CFR). 2008. Title 21. Office of the Federal
Register, Washington, D.C.
19. United States Pharmacopeia 32/National Formulary 27. 2009 United States Pharmacopeial
Convention, Washington, D.C.
20. British Pharmacopeia, 2007. The Pharmaceutical Press, London.
52 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

“I have been trying to point out that in our lives chance may have an
astonishing influence and, if I may offer advice to the young laboratory worker,
it would be this—never neglect an extraordinary appearance or happening. It
may be — usually is in fact — a false alarm that leads to nothing, but may on
the other hand be the clue provided by fate to lead you to some important
advance.”

Sir Alexander Fleming

 Microbial Contamination and Spoilage 53

Chapter 3

Microbial Contamination and Spoilage

David G Allison, Ph.D., School of Pharmacy and Pharmaceutical Sciences, University of

Manchester, Oxford Road, Manchester, M13 9PT, UK

Contents

3.0 Introduction
3.1 Sources of Microbial Contaminants
3.2 Raw Materials
3.3 Materials of Natural Origin
3.3.1 Microorganisms from Plant Material
3.3.2 Microorganisms from Animal Sources
3.3.3 Microorganisms from Mineral-Derived Materials
3.4 Synthetic Raw Materials
3.5 Water
3.5.1 Types of Water
3.5.2 Disinfection of Water
3.6 Microbial Contamination from the Manufacturing Environment
3.6.1 Air supply
3.6.2 Equipment and facilities
3.6.3 Personnel
3.6.4 Users / consumers
3.7 Factors affecting Microbial Spoilage of Pharmaceutical Products
3.7.1 Preparation and Storage
3.7.2 Nature of the Contaminant Inoculum
3.7.3 Moisture Content
54 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

3.7.4 Nutritional Factors

3.7.5 pH and Redox
3.8 Consequences of Microbial Growth
3.9 Microbiological Control of Raw Materials
3.10 Summary
3.11 References
 Microbial Contamination and Spoilage 55

3.0 Introduction

The risks to society of spoiled or otherwise adulterated pharmaceuticals has been widely
appreciated for many centuries. It is only in the last century however that the particular
role played by microorganisms in this process has been appreciated. Microbial
contamination can be defined as the presence of any microorganism, vegetative or
otherwise, including by-products and toxins, at levels that exceed acceptable limits in the
end product. In this respect, microbial contamination and spoilage of pharmaceuticals
will not only alter the aesthetic qualities of a product (colour, smell, texture etc) but
may also render the product dangerous to the user by constituting an infection hazard,
and / or nullify any intended therapeutic value of the product. The consequence s of
pharmaceutical preparations containing microrganisms will vary in severity according to
the purpose of the preparation, its route of administration, the health status of the user and
the nature of the contaminating microorganism. Thus, those products which are injected
(parenteral products) directly into blood vessels or tissues and those that are applied directly
to the eyes and ears represent a greater infection risk than products that are taken orally or
applied to intact healthy skin. While parenteral products are required to be free from all viable
microorganisms (sterile), oral and topical products are not required to be sterile but are
subject to strict guidelines limiting the number and types of acceptable microorganisms
(microbial limits). Indeed, products made in the Pharmaceutical Industry today must meet
high microbiological specifications and have no more than a minimal microbiological
population at the time of product release. With the exception, therefore, of preparations
which are terminally sterilised in their final container, the microbiological quality of the
final product will be determined by the formulation components used, the environment in
which they are manufactured and the manufacturing process itself. Irrespective of whether
manufacturing takes place in industry or on a smaller scale in e.g. a hospital pharmacy, control
and quality must be built into the product at all stages of the manufacturing process. Perhaps
the most demanding in terms of microbiological control of the three areas mentioned above is
to produce, and continually maintain, a high microbiological standard in raw materials.

3.1 Sources of Microbial Contaminants

Microorganisms contaminate pharmaceutical products due to their presence in raw

materials used in their manufacture, including that used for packaging. Such
microorganisms might originate from the manufacturing environment (industrial plant, air,
surfaces, personnel) or they might enter during storage of the product if the packaging is
inadequate or faulty. The final and arguably most severe microbial challenge to a
pharmaceutical product is administered at the hands of the user / consumer.
Microorganisms are ubiquitous, and extremely varied in their ability to survive and grow
under different conditions, as well as cause harm to either the patient or to the pharmaceutical
product itself. Any moist natural environment will support microbial growth and virtually all
natural environments will harbour bacteria. Microorganisms that contaminate pharmaceutical
products and cause disease in patients may be classified as true pathogens or opportunistic
pathogens. Pathogenic organisms such as Clostridium tetani and Salmonella spp. rarely occur in
56 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

products, but when present cause serious problems. On the other hand, opportunistic
pathogens such as Pseudomonas aeruginosa, Klebsiella spp. and Serratia spp. are more
frequently isolated from pharmaceutical products, and as their name suggests, may be
pathogenic if presented with the right set of circumstances. The main concern with these
microorganisms is that their simple nutritional requirements enable them to survive in a wide
6 7
range of pharmaceuticals. Whilst such organisms may be present in high numbers (eg 10 – 10
CFU/g or ml), the product may not show any visible sign of deterioration. Compromised patients
such as the elderly, immunosuppressed and burn victims are considered to be at particular risk
from infection by these organisms. (1)

In addition to whole cell contamination, some microorganisms that are destroyed by in-process
heat treatment may still leave cell residues which may contain either lipopolysaccharide (LPS)
which is considered an endotoxin (pyrogen), or secreted extracellular toxins.
Lipopolysaccharide is a component of the outer membrane of the cell envelope of viable
and non-viable Gram-negative bacteria (Figure 1) that is released by the cell during either
growth or processing (Figure 2). The lipid A component is the endotoxin moiety that can
survive moist heat sterilisation. Although inactive by the oral route, it can induce a number of
physiological effects such as fever, endothelial cell damage and fatal febrile shock if it enters
the bloodstream, even in nanogram quantities. By comparison, acute bacterial toxins
normally associated with food poisoning are not commonly reported in pharmaceutical
products.

Lipid A Core O-Antigen

Figure 1: Schematic diagram of the lipopolysaccharide (LPS) component of the Gram-

negative bacterial cell envelope. The lipid A component is an endotoxin.

Figure 2: Examples of LPS shedding from a Gram-negative bacterial cell by (A) blebbing and (B)
cell lysis.
 Microbial Contamination and Spoilage 57

3.2 Raw materials

Raw materials are defined as those substances which can be brought into a manufacturing
unit either for further processing or to aid in such processing. In the microbiological control of
pharmaceutical raw materials there is one primary aim – to exclude any microorganism which
may subsequently result in deterioration of the product or may harm the patient.
Contaminants in raw materials are important not only because they may contaminate the
product directly, but also because they may contaminate the manufacturing plant, possibly
giving rise to problems in the future. Experience has shown that where free-living reservoirs of
microorganisms become established within the manufacturing plant, they may be hard to
eradicate and can often result in continuous or intermittent contamination of the product.

Mechanisms of control involve the sampling of raw material and the testing of part or the
entire sample for the presence of microorganisms, with special reference to pathogenic
species. This is known as bioburden testing. However, the range of raw materials used in
pharmaceutical products is extremely large and any attempt to sample and test every one
would prove to be too onerous. A logical approach must, therefore, be used. By examining
data collected by the Pharmaceutical Industry over the years, it is apparent that some raw
materials are almost invariably contaminated whereas some never contain any detectable
contamination. Common sense would suggest that those in the latter category need only be
examined on occasion whereas those in the former category may need regular
examination. The criteria which may be used to identify materials to be examined are: (2)

 Is the product of natural origin?

 Is it synthetic?
 Is it produced in a manner likely to reduce or increase the level of contamination?
 Are microorganisms likely to multiply in it?

3.3 Materials of Natural Origin

Ideally, pharmaceutical preparations should be formulated with raw materials that are unlikely
to be sources of contamination. To do so would mean avoiding raw materials originating from
plants, animals or mineral material (e.g. gums, sugars, gelatin talc etc). Although
specifications for raw materials are generally in the order of 103 CFU per gram or ml, higher
microbial limits for certain raw materials used in critical applications may occur. Quality
specifications for raw materials should include tests for the absence of specific pathogens (eg
Salmonella spp., Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa). Tests
for these organisms are given in various pharmacopoeias. With the above exceptions, at
present there are no official limits for total viable counts (TVCs) for raw materials in the UK.
Although the value of TVCs for in-house process control is recognised, the general consensus in
the UK is against the inclusion of TVCs in official standards because of factors such as inter-
laboratory variation, problems associated with sampling, quantification of viable but non-
culturable bacteria and the dynamics of microbial growth. However, in recent years the
accuracy of TVCs has improved due to the use of validated pharmacopoeial methods,
58 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

calibrated equipment, proper sampling plans and provision of appropriate training and
interpretation for analysts. (3)

Due to its varied and widespread use, water is perhaps the most significant source of
contamination in any pharmaceutical preparation. Water for pharmaceutical manufacture
requires further treatment, usually ion exchange, reverse osmosis or distillation. Such
processes require careful monitoring, as does the microbiological quality of the water after
treatment. The many grades of water used in pharmaceutical manufacturing, and their
treatments, are discussed below.

3.3.1 Microorganisms from Plant Material

As with any natural material, the microbial count from a plant origin will reflect the natural
flora. Generally, this will comprise a mixture of yeasts, moulds, Gram-positive bacteria and
bacterial spore-formers. Hence, the microflora of plant materials such as gum acacia and
tragacanth, agar, powdered rhubarb and starches will be indigenous to the respective plants
and may include bacteria such as Erwinia spp., Pseudomonas spp., Lactobacillus spp., Bacillus
spp. and streptococci, moulds such as Cladosporium spp., Alternaria spp., and Fusarium spp.
The numbers of microorganisms present on the plant material may also reflect storage and
harvest conditions and should be taken into account when performing any bioburden
estimation. In addition, some microbial species have inherent anti-microbial properties
which must be taken into account when testing. If fungal growth on the material is suspected,
then the presence and significance of fungal toxins must be evaluated. It is not
unreasonable to expect bacterial numbers of anywhere between 100 per gram to 106 per
gram of plant material. Whilst the material from roots, tubers, bulbs etc is sterile in the
healthy plant, they are likely to be contaminated with soil organisms, predominantly Gram-
positive spore forming bacteria. There, numbers may vary from 100 per gram to 10,000 per
gram. (4)

3.3.2 Microorganisms from Animal Sources

Historically, animal derived products include vaccines, hormones and growth factors, sutures
and organ explants and implants. However, with concerns over TSE / BSE infections, the
number of animal products used these days as raw materials is vastly reduced. Those that are
still obtained from animals are required to be sourced from approved suppliers who have given
sound (documented and audited) consideration to hygiene and microbiological control in the
design of their production (eg farming, cultivation, extraction etc) and distribution practices.
In order to trace the source of any possible contaminant, each organ / extract effectively
becomes its' own batch number. Products from animal sources such as gelatin, dessicated
thyroid, pancreas and cochineal may be contaminated with animal-borne pathogens. For
this reason, statutory bodies such as the British Pharmacopoeia and European
Pharmacopoeia require freedom of such materials from E. coli and Salmonella spp. at a stated
level before they can be used in the preparation of pharmaceutical products. Such raw
materials must also comply with a total viable count limit in some cases. (4)
 Microbial Contamination and Spoilage 59

3.3.3 Microorganisms from Mineral-Derived Materials

Mi ned sour c es of m i ner al s suc h as tal c um and gypsum m ay al so c ontai n

microorganisms. Often they reflect the microflora of the surrounding soil , or the method
of extraction, but can harbour potentially harmful microorganisms. C. tetanii rarely
occurs in pharmaceutical products, but when present in, for example, contaminated talcum
powder, has been know to cause serious wound infections and several cases of neonatal death.
Generally speaking, mineral-derived materials present only minor sources of microbiological
contamination. However, it is essential that they be sourced from suppliers who have given
sound consideration to hygiene and microbiological control in the design of their
manufacturing and distribution practices. (2)

3.4 Synthetic Raw Materials

Materials of synthetic or semi-synthetic origin are, if stored correctly, usually free from all
but incidental microbial contamination. Although microorganisms may contaminate all types
of raw material, the potential for growth in synthetic materials is very low. In general, these
incidental organisms are considered to be of little practical significance. Contamination by
pathogens is extremely unlikely. Exceptions to this rule can occur, for example, where one of
the preparation stages involves washing with or crystallizing from water of poor
microbiological quality. (4)

3.5 Water

A working knowledge of the microbial ecology of water is of vital importance as it is the

most widely used raw material in the pharmaceutical industry. It may be used as a constituent
of many products, cleansing agent, temporary suspending agent or for cooling processes.
Water may also act as a vector for transmitting contamination and encourage the
proliferation of contaminants. The quality of the raw water, its treatment, storage and
distribution all have an influence on its microbial quality. All of these factors must be taken
into account when assessing its quality.

Natural inhabitants of freshwater include Pseudomanas spp., Alcaligenes spp.,

Flavobacterium spp., Chromobacterium spp., and Serratia spp. Indeed, an examination
of stored water supplies showed that 98% of the contaminants were Gram-negative
bacteria, with obvious implications for pyrogen presence (1, 2). Other organisms isolated
were Micrococcus spp., and Cytophaga spp. Many of these bacteria may be considered
as opportunistic pathogens, and as the name suggests, may be pathogenic if provided with
the right opportunity. The main concern with these organisms is that they have simple
nutritional requirements and often have a relatively low optimum growth temperature. This
enables them to not only survive but also multiply using traces of organic matter present in
treated water, such that in some situations they might be present in high numbers, perhaps
in excess of 106 CFU per ml at room temperature. This level of contamination cannot be
seen with the naked eye, but is clearly a high-risk raw material. Water should, therefore,
be stored at temperatures in excess of 65°C (usually 80°C) and circulated in the
60 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

distribution system at a flow rate of 1-2 M/s to prevent build up of bacterial biofilm in the
piping. (1, 2)

Bacteria may also be introduced to water by indirect means, for example soil erosion resulting
from heavy rainfall, and decaying plant matter might lead to to an increase in the number of
Bacillus subtilus, B. megaterium, Enterobacter aerogenes and E. cloacae. Similarly,
contamination by sewage results in the presence of Proteus spp., E. coli and other
enterobacteria. However, in many cases microorganisms that are derived from animal or
plant debris do not survive for long due to unfavourable growth (nutrient) conditions. (4)

The most heavily contaminated water in pharmaceutical manufacturing facilities is in the

drains, the main habitat for Gram-negative microorganisms. Since these may be transferred
throughout the facility on the feet and garments of operators who work close to the drains,
the locations should be minimised and the drains designed to cope with the expected
volumes of water without fear of backflow. Any unused drain should be capped. Water of
poor microbiological quality may also be used as a coolant on, for example, stirrers. As such,
great care should be taken to ensure that all seals are in place and intact, and that no leaks occur.

3.5.1 Types of Water

There are many grades of water used in the pharmaceutical industry. Water for
manufacturing may be potable mains water, water purified by ion-exchange, reverse osmosis
or distillation or Water for Injection. Water used for parenteral products must be apyrogenic
and is usually produced in a specially designed still. Water prepared in this way can be stored
at a temperature of 80°C to prevent bacterial growth and consequent pyrogen production.
The microbiological limit applying to purified water is normally in the region of no more than
100 CFU per ml. (4)

Potable Water

Potable water may be used for some pharmaceuticals, but perhaps more so for cosmetics
and toiletries. In the Pharmaceutical Industry potable water is deemed good enough for
cleaning purposes (eg walls and floors in non-sterile units). The degree to which cleaning water
must be microbiologically controlled is a function of where it is to be used, what products and
equipment it is being used in association with, and of the volumes to be used. In general,
potable water has a microbiological specification of no more than 500 CFU per ml and the
absence of Enterobacteriaceae. However, the quality of water may vary both from time to
time and from place to place. In the UK water authorities will not allow water to be used
directly from the mains but insist on break tanks. This represents a significant source of
potential contamination because on prolonged storage microorganisms either settle out or
attach themselves to the storage vessel surfaces and grow as living biofilms. This results in the
so called ‘bottle effect’, whereby bacterial growth and activity are substantially enhanced
through growth as a biofilm ( due primarily to increased nutrient trapping and concentration)
as opposed to a free floating (planktonic) lifestyle. The intermittent throughput of the storage
tank ensures that, unless treated, the contents serve as a source of infection. (1)
 Microbial Contamination and Spoilage 61

Deionised Water

Deionised water is used extensively in the manufacture of tablets, syrups, suspensions,

creams, lotions and for washing of all manufacturing equipment. It is prepared by passing
potable water through anion and cation exchange resin beds to remove the ions. Any bacteria
present in the mains water will therefore be present in the deionised water. Deionisation beds
are prone to contamination because they must be protected from the corrosive potential of
chlorine which acts as a bacteriostat in potable water. Those beds that are not regenerated
frequently with strong acids or alkali are often heavily contaminated. Consequently, there is a
lot of emphasis on the development of new resins that are able to resist microbial
contamination.

Water Produced by Reverse Osmosis

The process of producing water by reverse osmosis involves forcing water by an osmotic
pressure through a semi-permeable membrane which acts as a molecular filter. Solubles
dissolved in the water arc impeded and those with a molecular weight in excess of 250 do not
diffuse at all. In this manner microorganisms, and pyrogens, are removed, resulting in sterile
water being produced. Contamination may, however, occur in the storage vessel on the
distribution system if they are not kept free from microorganisms. Care must also be taken to
disinfect the membrane at regular intervals. This interval will be determined by the results of
regular sampling but will probably be of the order of once per month, depending on use. (2, 4)

Distilled Water

Distilled water is very high quality water, similar in standard to reverse-osmosis water, if
produced by a still designed to prevent the entrainment of water droplets. As it leaves the
still, distilled water is sterile, but its microbiological quality can deteriorate quickly as a
result of a fault in the cooling system, the distribution system or incorrect storage conditions.
The flora of contaminated distilled water is usually Gram-negative bacteria (commonly
Pseudomonas spp.), often as a pure culture. Owing to its high cost, distilled water is usually
used only for parenteral manufacture either as an ingredient or as a pyrogen-free rinsing agent
for product contact surfaces. It can on occasion be used in the formulation of oral and
topical pharmaceutical products where a low bacterial count is acceptable. (2, 4)

3.5.2 Disinfection of Water

Many options are available for treating and improving water quality. The use of any particular
method depends on what is causing the microbial deterioration, the source of the problem,
the quality of the water required and the volume to be treated and the type of distribution
system. The design of a system is influential on the size of the microbial populations and the
ability of the user to remove them. Dead-legs, long pipework runs to taps, un-drainable pipes
and U-bends all create microbiological problems once installed. (2)
62 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

Three methods are routinely used for treating water, namely chemicals, filtration or UV light.
Chemical treatment (eg sodium hypochlorite, chlorine gas) is applicable to raw, mains water,
but can also be used to treat distribution systems of water produced by distillation,
deionisation and reverse osmosis. The concentration of the chemical used will vary depending
upon the location of the water in the distribution system. Membrane filtration, using a 0.22 µm
porosity-filter, is useful where the usage is moderate and a continuous circulation of water can
be maintained. Thus, with the exception of that drawn off for use, water is continually being
returned to the storage tank and re-filtered. In principle, filtration works well but is relatively
expensive for high throughputs because the filters may need regular changing to prevent
blockages and ‘grow through’. For this reason, use of 0.22 µm filters as a means of controlling
contamination in waters used directly for product manufacture is frowned upon. In essence,
filters should only be used prior to the distribution process. Ultraviolet radiation (254nm) is
used for the disinfection of water of good optical clarity, and works particularly well in a re-
circulating system where water flows over a multiple lamp system. Caveats are that
penetration of UV light into water is small, and any dead bacteria present in the system will
further hinder penetration. (4)

3.6 Microbial Contamination from the Manufacturing Environment

Regardless of whether manufacture takes place in industry or on a smaller scale in hospital

pharmacy, the microbiological quality of the finished product will be determined by the
formulation components used, the environment in which they are manufactured and the
manufacturing process itself. Quality must therefore be built into the product at all stages
of the manufacturing process. As such, manufacture should take place in suitable
premises, supplied with filtered air, for which the environmental requirements vary
according to the type of product being made; all processing equipment should be subject
to planned preventative maintenance and should contribute the minimal amount of
contamination and / or cross-contamination; personnel involved in manufacture should
not only have good health, but also a sound knowledge of the importance of good personal
and production hygiene. Generally, quality control procedures will determine the numbers
and types of microorganisms that contaminate each and every batch. A satisfactory batch
will be free from specified pathogenic bacteria, whilst the total microbial population will
be below a pre-determined value appropriate to a particular product. (5)

3.6.1 Air Supply

Small numbers of microorganisms present within the air of manufacturing environments may
ingress into the products where these are open and exposed. Some of these will die during
storage, with the rest probably remaining at a static level of 102 – 103 CFU per gram or ml.
Microorganisms can be associated with dust particles drawn in from outside atmospheres or
they may be generated from the shedding of skin cells by operators and personnel entering the
manufacturing facility. These become airborne when air currents are created (e.g. opening of
doors and hatches, moving personnel) or when the dust layer is physically disturbed.
Microorganisms isolated from air samples will clearly reflect the environment from which they
were taken. (4)
 Microbial Contamination and Spoilage 63

Aerosols created by turbulent flow of liquids may also give rise to significant numbers of
airborne organisms. Thus, the operation of sinks and drains, the running of water taps, and the
vigorous cleaning of the manufacturing plant will generate airborne microorganisms. Ingress of
airborne contaminants from the exterior is generally controlled by containing the production
environment and providing it with a separate supply of filtered air. Given that the air is
sufficiently rapidly recycled within the room, and that separate supplies of air protect filling and
mixing equipment, then bacteria introduced along with the clothing and bodies of personnel
require control.

3.6.2 Equipment and Facilities

Clearly, if equipment is inadequately cleaned between uses, or if it is allowed to collect

moisture in ports and recesses which directly come into contact with the product, then they
can represent potent and chronic sources of contamination. Contamination levels in the
manufacturing environment may, however, be minimised by observing good manufacturing
practices. For example, equipment and pipelines should be regularly cleaned and stored in a
dry state, heating traps could be installed in sink U-bends thereby destroying the main
reservoir of contaminants and cleaning of production units by contractors should be carried
out to pharmaceutical specification.

3.6.3 Personnel

The biggest single sources of microorganisms in an enclosed environment are the people
within it. (4, 6) Individuals shed many thousands of skin cells to the atmosphere per day. Many
of these will be contaminated with skin microorganisms such as yeasts, Staphylococci spp. and
Micrococci spp. Numbers of microorganisms will rise with increasing numbers of people in a
room and also with the degree of physical exertion and movement that they are undergoing.
The wearing of protective clothing will do much to contain the shedding of skin cells and
bacteria to the air. Gowning procedures and hair coverings are intended to minimise such
shedding to the environment and the wearing of face masks contain aerosols generated by
breathing and sneezing. The axillae of the body and the hair covered surfaces (armpits, crotch,
head, neck) are especially prone to shedding (Figure 3).

Figure 3: Thermal imagery of the human body showing thermal body currents and ‘hot-spots’
from where skin cells are more readily shed.
64 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

Particular attention is therefore paid to these areas in the design of specialised clothing for
use in clean rooms and controlled areas. Overshoes prevent the movement of dust borne
microorganisms from one area to another. Strict entry procedures must be adhered to,
involving hand wash routines, and removal of cosmetics, jewelry, and outside clothing.
Personnel must be trained in the use of controlled environments in order to minimise their
contribution to airborne contamination. A major objective is to minimise the body movements
made by personnel since it is well documented that rapid movements and exertion increases
the rate of skin cell shedding and reduces the effectiveness of the protective clothing (see
Table 1 and (6)).

Particles (≥ 0.3μm)
TYPE OF MOVEMENT OR ACTIVITY
dispersed per minute
Sitting quite still 100,000
Gentle movement of arms, hands or head 500,000
Quite active movement of arms, head, rapid turning of head 1,000,000
Change from sitting to standing or reverse 2,500,000
Rapid mobility such as climbing stairs, walking or similar action 10,000,000

Table 1: Relationship between activity and particle shedding from the human body

3.6.4 Users / Consumers

The final abuse that a product has to withstand is that given by the user / consumer. During
normal usage, patients may contaminate their medicine with their own microbial flora;
subsequent use of such products may or may not result in self-infection. If the product is
multi-use (i.e. opened, used several times and stored between uses) then microorganisms
may enter it at each use. Such organisms may grow during subsequent storage of the opened
product. Whilst storage conditions prior to sale can be adequately controlled, those at the
point of use cannot. Products might therefore be stored in hot humid environments (eg
bathrooms) where microbial growth will be favoured. (5)

Contamination by the user of is particularly problematic for topical products (eg creams and
ointments) which are applied by the fingers and hands, thus introducing contaminants from
the resident skin flora but also perhaps transient contaminants such as Pseudomonas spp.
which would normally be removed by effective hand-washing. Even with products such as ear
and eye drops it is possible for the user to contaminate the applicator and return organisms to
the bottle. In some instances the design of the container is intended to minimise consumer
contamination. In other instances this is not possible.
 Microbial Contamination and Spoilage 65

3.7 Factors Affecting Microbial Spoilage of Pharmaceutical Products

Microbial spoilage of a pharmaceutical product may be deemed to have occurred if (i) low
levels of acutely pathogenic bacteria or higher levels of non-pathogenic bacteria are
present that may provide an infection risk; (ii) toxic microbial metabolites are present,
even in the absence of live microorganisms and (iii) detectable microbial-mediated physical
or chemical changes have occurred in the product. Spoilage of a product can depend on
the intended mode of usage and on a number of other factors including the preparation
and storage of the product, moisture content, available nutrients, active ingredients, pH
and redox values as well as product packaging. (5)

3.7.1 Preparation and Storage

Raw materials account for a high proportion of the microorganisms introduced during the
manufacture of pharmaceuticals, and the selection of materials of good microbiological
quality aids in the control of contamination in both products and the environment. However, it
is generally accepted that raw materials will be used which have some non-pathogenic
microorganisms present. An equally important consideration is that the method of
preparation of the raw materials may in itself lead to increased levels of contamination.
Hence, a knowledge of the method of preparation of raw materials is essential in order
to determine the full extent of microbiological testing for any material. For example, some
refining processes modify the microflora of raw materials. Drying may concentrate the level of
spore-forming bacteria, whilst some solubilisation processes may introduce waterborne
bacteria such as E. coli.

The careful storage of raw materials, particularly hygroscopic substances, is important in order
to prevent growth of the organisms and spoilage of the material. If stable, natural products
with a high microbial count may be sterilised. Staff handling raw materials must be given
adequate training to prevent cross-contamination. However, for the majority of raw
materials pre-product sterilisation is not an option.

Packaging can also have a major influence on microbial stability of some formulations in
controlling the entry of contaminants during both storage and use. Considerable thought
and energies have gone into the design and re-design of containers to prevent the ingress
of contaminants and include the use of self-sealing rubber stoppers or wads on multi-dose
injection containers, widemouthed cream jars replaced by narrow nozzles and flexible
screw-capped tubes, thereby reducing the likelihood of operator or consumer-introduced
contamination during use of the product. Where pharmaceutical products rely on their low
water content to prevent spoilage, packaging such as strip foils must be of water vapour-
proof materials with fully efficient seals. Cardboard outer packaging and even labels
themselves can become substrates for microbial attack under humid conditions, and
preservatives are often included to reduce the risk of damage. (5)
66 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

3.7.2 Nature of the Contaminant Inoculum

Once they have ingressed into a pharmaceutical product the contaminating microorganism
will either die immediately (especially if the product is then subjected to a termina l
sterilisation process), survive for some time but unable to grow and divide (static
contamination) or, given conditions favourable for growth, multiply (dynamic
contamination). So long as there are viable microorganisms remaining within a product
then such organisms have the potential to cause infection in the consumer during product
use. An important measure is the number of bacterial cells present in a non-sterile product
that are required to initiate an infection. These are termed the Minimum Infectious
Number (MIN) and are defined as the minimum number of cells that are required to be
introduced to particular sites in the body in order to initiate infection. These vary from
organism to organism, and will also be affected by the health status of the host and site of
infection.

If the physico-chemical conditions within the product change then static contaminants may
be able to recommence growth. When an aggressive microorganism contaminates a
product there may be an appreciable lag period before significant spoilage begins, the
duration of which decreases disproportionately with increasing contaminant loading.
Sometimes growth of species may be sequential, one organism paving the way for another.
For instance, growth of an aerobic organism such as a pseudomonad may be followed by
that of an anaerobe. This sequence can be quite common in oils that eventually become
blackened due to the proliferation of sulphate reducing bacteria that are the secondary
colonisers.

Risks to the consumer may also occur even in the absence of any viable microorganisms.
Before removal or destruction, bacteria and fungal cells may release extracellular
metabolites which can either lead to the degradation of the product or be toxic to the
user. A significant risk is retention in the product of the lipid A component of the LPS
released by Gram-negative bacteria. This endotoxin is pyrogenic and in sufficient
concentrations can lead to death. This hazard in parenteral products is well understood; in
non-sterile products the significance of pyrogens is doubtful.

3.7.3 Moisture Content

An important parameter for storage is the moisture content of the environment. (5,7)
Microorganisms require readily accessible water in sufficient quantities for growth to
occur. By measuring a product’s water activity, A w, it is possible to obtain an estimate of
the proportion of uncomplexed water that is available in the formulation to support
microbial growth. Water activity is calculated by the formula: A w = vapour pressure of the
formulation / vapour pressure of pure water under similar conditions. Greater the solute
concentration, lower is the water activity. With a few exceptions, most bacteria grow best
in dilute solutions (high A w) and, as solute concentration rises (thereby lowering A w),
growth rates decline until a minimal growth-inhibitory Aw value is reached. Conversely, any
increase in water activity (Aw) above the minimum required for growth (Table 2) will encourage
 Microbial Contamination and Spoilage 67

population growth especially of moulds and yeasts, as will insect or rodent infestation during
storage.

ORGANISM TYPE Aw Value Representative example

Xerophilic Fungi 0.70 Xeromyces spp.
Spoilage Yeasts & Moulds 0.80 Penicillium spp., Saccharomyces spp.
Gram-positive Bacteria 0.90 Staphylococci spp.
Gram-negative Bacteria 0.95 Pseudomonas spp., Escherichia spp.

Table 2: Water Activity (Aw) for various groups of spoilage microorganisms. (7)

Precautions should be taken to ensure that dry materials are held below these levels.
Hence, it is important to hold raw materials at a constant temperature in order to prevent
evaporation and condensation occurring. It is worth noting that some materials such as
oils and sugar solutions may contain local pockets of free water where microbial
proliferation may take place. Care should also be taken with packaging as some types (eg
unlined paper sacks) may absorb moisture and may itself be subject to microbial
deterioration and so contaminate the contents.

3.7.4 Nutritional Factors

Growth of microorganisms will only occur in a product if appropriate nutrients are provided. All
microorganisms require sources of carbon and nitrogen. Moreover, some organisms such as
obligate pathogens require additional, specific growth factors that are not found in
pharmaceutical formulations. However, microorganisms are highly metabolically versatile.
Indeed, many common spoilage microorganisms have simple nutritional requirements and,
coupled with their metabolic adaptability, are able to utilise many formulation components as
substrates for biosynthesis and growth (Table 3). Almost every compound can be
microbiologically altered and degraded. The use of crude vegetable or animal products in a
formulation provides an additional nutritious environment. Even demineralised water can
support the growth of many waterborne bacteria such as P. aeruginosa.

COMMONLY DEGRADED INGREDIENTS

Surfactants
Organic Polymers
Humectants
Fats and Oils
Sweeteners, Flavours and Colourings
Therapeutic Agents
Preservatives and Disinfectants
Table 3: Examples of common microbiologically degraded ingredients found in pharmaceutical
products. (5)
68 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

Anionic surfactants such as the amine soaps of fatty acids are generally stable due to the
slightly alkaline pH of the formulation. By comparison, non-ionic surfactants such as
alkylpolyoxyethylene alcohol emulsifiers are readily metabolised by a wide range of
microorganisms. Increasing chain lengths and branching decrease ease of attack. The cationic
surfactants used as antiseptics and preservatives in pharmaceutical applications are usually
only slowly degraded at high dilution in sewage. Pseudomonads have been found growing
readily in quaternary ammonium antiseptic solutions, largely at the expense of other
ingredients such as buffering materials. (5)

Organic polymers are often used as thickening and suspending agents in pharmaceutical
preparations and are subject to microbial depolymerisation by specific classes of extracellular
enzymes, such as amylases, pectinases, cellulases and dextranases. Similarly, low molecular
weight humectants such as glycerol and sorbitol are included in some products to reduce water
loss and may be readily metabolised unless present in high concentrations. Hydrophobic fats
and oils are usually attacked fairly extensively when dispersed in aqueous formulations such as
oil-in-water emulsions, aided by the high solubility of oxygen in many oils. As might be
expected, many of the sugars and other sweetening agents used in pharmaceutical
preparations are ready substrates for microbial growth. Some are used at very high
concentrations to reduce water activity in aqueous products and inhibit microbial attack.
Historically, colouring agents such as tartrazine and flavourings such as peppermint water were
kept as stock solutions for extemporaneous dispensing purposes, but were prone to
contamination by Pseudomonas spp. These days such solutions are either made up as required,
or kept as concentrated alcoholic solutions that are much less susceptible to microbial attack.
(5)

Microorganisms have the potential to metabolise active drug constituents to less, or indeed
non-active forms. A diverse range of materials including alakaloids (eg morphine, atropine),
analagesics (eg aspirin, paracetamol), barbiturates and steroid esters can be used as substrates
for growth by a wide range of microorganisms. Examples include the metabolism of atropine in
eye drops, steroid metabolism in damp tablets and creams by fungi and hydrolysis of aspirin in
suspension by esterase producing bacteria. Similar problems can be encountered with
preservatives and disinfectants where a wide range of Gram-negative bacteria have the ability
to metabolise such agents. Although problems of this nature are more common at
concentrations below their effective ‘use’ levels, growth of organisms such as Pseudomonas
spp. in stock solutions of e.g. quaternary ammonium antiseptics or chlorhexidine has resulted
in serious infections in patients.

3.7.5 pH and Redox

Extremes of pH prevent microbial attack. Around neutrality bacterial spoilage is more likely,
with reports of pseudomonads and related Gram-negative bacteria growing in antacid
mixtures, flavoured mouthwashes and even in distilled or demineralised water. Above pH 8
spoilage is rare. In products with low pH levels (eg pH 3 - 4) yeast or mould attack is more likely.
Yeasts can metabolise organic acids and raise the pH to levels where secondary bacterial
growth can occur.
 Microbial Contamination and Spoilage 69

Since the growth of many bacteria, particularly anaerobic, fermentative organisms leads to the
generation of organic acids, then a product of neutral pH showing dynamic contamination with
bacteria will become progressively more acidic. Eventually acid production will inhibit the
growth of the bacteria, and facilitate the germination and growth of fungal spores. Dynamic
contamination with one type of organism can therefore lead, at a later stage, to dynamic
contamination by a previously static organism. (5)

The oxidation – reduction balance (redox potential) of a product can also influence bacterial
growth, as they will require compatible terminal electron acceptors to permit their respiratory
pathways to function. The redox potential in even fairly viscous emulsions may be quite high
due to the appreciable solubility of oxygen in most fats and oils.

3.8 Consequences of Microbial Growth

Pharmaceutical products are highly processed materials and contain an enormous range of
natural and synthetic materials. Their susceptibility to microbial attack may result in
extensive growth of contaminants and lead to devastating chemical and physical changes
in the product. Moreover, since the infection risk is related to the numbers of
microorganisms (MIN) a user is exposed to, then microbial multiplication will increase
numbers and hence the degree of risk. This is also associated with the route of
administration / use of the product. Spoilage organisms are unlikely to be primary
pathogens but, in high number, environmental contaminants might be able to initiate
infection in a compromised patient.

If the source of nutrients to the contaminating organisms consists of active ingredients of

the product then the therapeutic efficacy of the product will be compromised as the
ingredient is metabolised. In this fashion certain antibiotics such as the ß-lactams, some
steroid agents and many antimicrobial preservatives (e.g. parabens, phenolics etc) can act
as nutrients for microbial growth. Consequently, modification and / or consumption of the
active ingredient of a product may alter its pharmacological profile and render it safe but
functionless.

Homogeneous or colonial growth in, or on, a product probably constitutes the most
striking and frequent manifestations. Contaminants may be seen as a sediment, turbidity
or pellicle in liquid products. On solid preparations, coloured colonies may form. Colour
changes due to alterations in product components themselves may result as a direct
consequence of metabolism of, or indirectly, because of alteration of parameters such as
pH or redox. Growth is not so readily detected in suspensions, except at the surface,
because of inherent opacity but preparations of this type can thin, separate, decolourise or
change colour as a result of microbial contamination. The same sort of change can occur in
emulsions which may become visibly heterogeneous owing to hydrolysis (‘cracking’ –
separation of oil and water phase) of the oil phase or changes in pH of the aqueous phase.
Mould growth is one of the most common visible manifestations of spoilage of creams and
may also be visible on the surface of powders and tablets if they have been stored under
damp conditions (Figure 4).
70 Microbiology and Sterility Assurance in Pharmaceuticals and Medical Devices

Fungal growth

Figure 4: Fungal growth on a tablet which has become damp (adapted from Baird, 2004).
(5)

Microbial growth in or on products may not be directly visible. However, spoilage can also
occur through production of secondary metabolites by contaminating organisms with
equally disastrous effects. Early indications of spoilage are often organoleptic, with the
release of unpleasant smelling and tasting metabolites, often organic acids, produced
through fermentative growth of microorganisms. The production of gases such as
hydrogen sulphide and methane from fermentative metabolism can also cause the
creation of gas pockets. On occasion gas production can be heard as bubbles trapped in
viscous formulations burst. Changes in the pH of a formulation brought about by bacterial
growth and fermentation can affect the stability of ingredients, causing cracking to occur
in emulsions and can bring about colour changes through production of bacterial pigments.
Thickening and suspending agents such as acacia or carboxymethylcellulose can be
depolymerised resulting in a loss of viscosity, and sedimentation of suspended ingredients.
Alternatively, microbial polymerisation of sugars and surfactant molecules can produce
slimy, viscous masses in syrups, shampoos and creams, whilst fungal growth in creams can
produce ‘gritty’ textures, causing serious modification to the mechanical properties of the
product. The texture of topical products is vital to their acceptability. (5)

3.9 Microbiological Control of Raw Materials

The aim of microbiological control is to give the manufacturer assurance that the raw materials
used will not directly or indirectly represent a hazard to the product and ultimately to the
consumer. (1) It is very difficult to make a microbiologically clean product from
microbiologically dirty raw materials. In deciding suitable microbiological methods
for raw materials, it is necessary to consider at the outset the use to which the raw materials
 Microbial Contamination and Spoilage 71

are to be put. The standard adopted must reflect the type of raw material, the type of
product, its method of manufacture and intended use. Mechanisms of control involve the
sampling of raw materials and the testing of part of or the entire sample for the presence of
microorganisms, with special reference to pathogenic species. Accordingly, improvements over
the years in both manufacturing technology and Good Manufacturing Practices have helped
minimise microbial contamination. In addition, Hazard Analysis of Critical Control Points
(HACCP; Table 4) (8) is a tool that is becoming more commonly used in the Pharmaceutical
Industry, providing a structured thought process for GMP. The objective of HACCP is to
improve the microbiological safety of the product in a cost-effective manner, assisted by
the development of rapid methods for the detection of microorganisms. Documentation is a
vital part of any quality assurance process and HACCP is no exception. (8)

 Conduct a hazard analysis for the process

 Determine the critical control points (CCPs)
 Establishing target levels and critical limits
 Establish a system to monitor the CCPs
 Establish the corrective action to be taken when monitoring indicates that a particular
CCP is not under control
 Establishing procedures to verify that the HACCP regime is functioning as intended
 Establishing documentation concerning all procedures and keep records appropriate to
these principles and their application.

Table 4: The principles of HACCP Analysis. (8)

3.10 Summary

The aim of this chapter has been to examine the role played by microorganisms in spoilage of
pharmaceutical products, and the consequences for not only the manufacturing company but
also the end user. In essence, this chapter has covered potential sources of contaminating
microorganisms in raw materials and the manufacturing environment, microbial products and
metabolites that cause spoilage, factors affecting spoilage of pharmaceutical products, the
consequences of microbial growth and mechanisms of control.

In covering these topics the chapter has undoubtedly emphasised that prevention is better
than cure in minimising the risk of product-borne infections. Without doubt good
manufacturing practices must be employed and control measures built in at all stages of
manufacture. This not only applies to the manufacturing plant itself, but also to all suppliers
(and transporters) of raw materials such that they too comply with in-house specifications. All
aspects of the manufacturing process should be subject to regular microbiological monitoring
as should the end product. It is important to ensure that standards (in-house and international)
are maintained so that the product is (microbiologically) suitable for its intended use.