Hugo W.B., Russel A.D.(ed). Pharmaceutical Microbiology
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6.5
throughout life. DT vaccination is therefore repeated before school entry, at 4-5 years
of age, and once again at puberty.
Haemophilus influenzae Type B (HiB) immunization
Seven different capsular serotypes of Haemophilus influenza Type B are associated
with respiratory infection in young children. The most common presentation of these
infections is as meningitis, frequently associated with bacteraemia. The sequelae
following HiB infection include deafness, convulsions and intellectual impairment.
The fatality rate is ca. 4-5% with 8-11% of survivors having permanent neurological
disorders. The disease, which is rare in children under 3 months, peaks both in its
incidence and severity at 12 months of age. Infection is uncommon after 4 years of
age. Before the introduction of HiB vaccination the incidence of the disease in the UK
was estimated at 34 per 100000. The vaccine utilizes purified preparations of the
polysaccharide capsule of the major serotypes. Polysaccharides are poorly immunogenic
and must be conjugated onto a protein carrier in order to enhance their efficacy. HiB
vaccines are variously conjugated onto diphtheria and tetanus toxoids (above), group
B meningococcal outer membrane protein and a non-toxic derivative of diphtheria toxin
(CRM197) and can now be mixed and co-administered with the DTP vaccine. Three
doses of the vaccine are recommended separated by 1 month. No reinforcement is
recommended at 4 years of age since the risks from infection are negligible at this time.
Juvenile immunization schedule
The timing of the various components of the juvenile vaccination programme is subject
to continual review. In the 1960s, the primary course of DTP vaccination consisted of
three doses given at 3, 6 and 12 months of age, together with OPV. This separation
gave adequate time for the levels of induced antibody to decline between successive
doses of the vaccines. Current recommendations (Table 16.1) accelerate the vaccination
programme with no reductions in its efficacy. Thus, MMR vaccination has replaced
separate measles and rubella prophylaxis and BCG vaccination may now be given at
Table 16.1 Children's immunization schedule for UK (1996)
Vaccine
BCG
DTP and HiB
Poliomyelitis
MMR
Booster DT
Poliomyelitis
MMR booster
BCG
Booster DT
Age
Neonatal (1st month)
1st dose 2 months
2nd dose 3 months
3rd dose 4 months
12-15 months
3-5 years
10-14 years
13-18 years
Notes
If not at 13-14 years
Primary course
Anytime over 12 months
3 years after primary course
If not in infancy
Vaccination and immunization 335
birth. DTP vaccination occurs at 2, 3 and 4 months to coincide with administration of
HiB. It is imperative that as many individuals as possible benefit from the vaccination
programme. Fewer visits to the doctor's surgery translate into improved patient
compliance and less likelihood of epidemic spread of the diseases in question. The
current recommendations minimize the number of separate visits to the clinic whilst
maximizing the protection generated.
8 Immunization of special risk groups
Whilst not recommended for routine administration, vaccines additional to those
represented in the juvenile programme are available for individuals in special risk
categories. These categories relate to occupational risks or risks associated with travel
abroad. Such immunization protocols include those directed against cholera, typhoid,
meningitis (types A, C), anthrax, hepatitis A and B, influenza, Japanese encephalitis,
rabies, tick-borne encephalitis, and yellow fever.
9 Further reading
Salisbury D.M. & Begg N.T. (eds) (1996) Immunisation Against Infectious Disease. HMSO: London.
(Updated every 2-3 years).
Mims C.A. (1987) The Pathogenesis of Infectious Disease, 3rd edn. London: Academic Press.
Salyers A.A. & Whitt D.D. (1994) Bacterial Pathogenesis: A Molecular Approach. Washington:
American Society for Microbiology Press.
336 Chapter 16
Part 3
Microbiological Aspects of
Pharmaceutical Processing
Many failures in pharmaceutical processing have arisen because of the inability of
those responsible for its design to be aware of the distribution and survival potential of
microorganisms in the environment and in the raw materials and equipment used in a
pharmaceutical factory.
The first chapter in this section provides a unique account of the ecology,
i.e. distribution, survival and life-style, of microorganisms in the factory environment,
and should enable process designers, controllers and quality control personnel to
comprehend, trace and eradicate the sources of failure due to extraneous microbial
contaminants in the finished product. Much of the information given here is applicable
to hospital manufacture also, and this is extended in a contribution (Chapter 19) dealing
with contamination in hospital pharmaceutical products and in the home.
The dire consequences of failure to heed the precepts enunciated in Chapter 17
are considered in Chapters 18 and 19 which review the spoilage wreaked upon
pharmaceutical products as a consequence of microbial infestation. The wide, and at
first sight bizarre, range of substrates used by contaminating microorganisms and the
range of biochemical reactions that follow and which, in turn, give rise to the overall
picture of spoilage are well documented; it would be no exaggeration to state that
microbial spoilage and its prevention, through both good working conditions and the
use of preservatives, represent the major problem of pharmaceutical microbiology.
An important group of pharmaceutical products, including those intended for
parenteral administration for instillation into the eye, are required to be free from living
microorganisms, and with parenteral products, from those residues of the bacterial cell
which may give rise to fever. The principles of their preparation and sterilization are
considered in Chapter 21, while the theory of sterilization processes is dealt with in the
preceding chapter. These two chapters in toto cover the most exacting operation in
medicine preparation, and one in which failure has given rise to several disasters ranging
from patient death, for instance as a result of sterilization failure in intravenous drips,
to blindness in the case of contaminated eyedrops.
Chapter 22 deals with general factory and hospital hygiene and the principles of
good manufacturing practice (GMP) which if adhered to go a long way towards
compounding the success of the processes described in Chapters 17 and 20.
The subject of quality control and surveillance is discussed in a chapter on
sterilization control and sterility testing, which deals with aspects Of in-process and
post-process control.
Finally, lest it be thought that microorganisms are always harmful, two chapters
(24 and 25) describe ways in which they can be harnessed for the benefit of mankind.
Ecology of microorganisms as it affects
the pharmaceutical industry
1
2
2.1
2.2
2.3
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4
4.1
4.2
Introduction
Atmosphere
Microbial content
Reduction of microbial count
Compressed air
Water
Raw or mains water
Softened water
Deionized or demineralized water
Distilled water
Water produced by reverse osmosis
Distribution system
Disinfection of water
Skin and respiratory-tract flora
Microbial transfer from operators
Hygiene and protective clothing
5
6
7
7.1
7.2
7.3
8
8.1
8.2
8.3
8.4
9
10
Raw materials
Packaging
Buildings
Walls and ceilings
Floors and drains
Doors, windows and fittings
Equipment
Pipelines
Cleansing
Disinfection and sterilization
Microbial checks
Cleaning equipment and utensils
Further reading
Introduction
The microbiological quality of pharmaceutical products is influenced by the
environment in which they are manufactured and by the materials used in their
formulation. With the exception of preparations which are terminally sterilized in their
final container, the microflora of the final product may represent the contaminants
from the raw materials, from the equipment with which it was made, from the
atmosphere, from the person operating the process or from the final container into
which it was packed. Some of the contaminants may be pathogenic whilst others may
grow even in the presence of preservatives and spoil the product. Any microorganisms
which are destroyed by in-process heat treatment may still leave cell residues
which may be toxic or pyrogenic (Chapter 1), since the pyrogenic fraction, lipid A,
which is present in the cell wall is not destroyed under the same conditions as the
organisms.
In parallel to improvements in manufacturing technology there have been
developments in Good Manufacturing Practices to minimize contamination by a study
of the ecology of microorganisms, the hazards posed by them and any points in the
process which are critical to their control. This approach has been distilled into the
concept of Hazard Analysis of Critical Control Points (HACCP), with the objective
of improving the microbiological safety of the product in a cost-effective manner,
which has been assisted by the development of rapid methods for the detection of
microorganisms.
Microorganism ecology and the pharmaceutical industry 339
17
The type of formulation being prepared determines the microbiological standard of
the air supply required and the hazard it poses. In areas where products for injection
and ophthalmic use which cannot be terminally sterilized by moist heat are being
manufactured, the air count should be very low and regarded as a critical control point
in the process since although these products are required to pass a test for sterility
(Chapter 23), the test itself is destructive, and therefore only relatively few samples are
tested. An unsatisfactory air count may lead to the casual contamination of a few
containers and be undetected by the test for sterility. In addition if the microbiological
air quality is identified as a critical point, it may also give an early warning of potential
contamination and permit timely correction. The manufacture of liquid or semi-solid
preparations for either oral or topical use requires a clean environment for both the
production and filling stages. Whilst many formulations are adequately protected by
chemical preservatives or a pH unfavourable to airborne bacteria that may settle in
them, preservation against mould spores is more difficult to achieve.
2.2 Reduction of microbial count
The microbial count of air may be reduced by filtration, chemical disinfection and to a
limited extent by ultraviolet (UV) light. Filtration is the most commonly used method
and filters may be made of a variety of materials such as cellulose, glass wool, fibreglass
mixtures or polytetrafluorethylene (PTFE) with resin or acrylic binders. For the most
critical aseptic work, it may be necessary to remove all particles in excess of 0.1 /mi in
size, but for many operations a standard of less than 100 particles per 3.5 litres (1.0ft
3
)
of 0.5 ^im or larger (class 100) is adequate. Such fine filtration is usually preceded by
a coarse filter stage, or any suspended matter is removed by passing the air through
an electrostatic field. To maintain efficiency, all air filters must be kept dry, since
microorganisms may be capable of movement along continuous wet films and may be
carried through a damp filter.
Filtered air may be used to purge a complete room, or it may be confined to a
specific area and incorporate the principle of laminar flow, which permits operations to
be carried out in a gentle current of sterile air. The direction of the airflow may be
horizontal or vertical, depending upon the type of equipment being used, the type of
operation and the material being handled. It is important that there is no obstruction
between the air supply and the exposed product, since this may result in the deflection
of microorganisms or particulate matter from a non-sterile surface and cause
contamination. Airflow gauges are essential to monitor that the correct flow rate is
obtained in laminar flow units and in complete suites to ensure that a positive pressure
from clean to less clean areas is always maintained.
The integrity of the air-filtration system must be checked regularly, and the most
common method is by counting the particulate matter both in the working area and
across the surface of the filter. For systems which have complex ducting or where the
surfaces of the terminal filters are recessed, smoke tests using a chemical of known
particle size may be introduced just after the main fan and monitored at each outlet.
The test has a twofold application as both the terminal filter and any leaks in the ducting
can be checked. These methods are useful in conjunction with those for determining
the microbial air count as given earlier.
Microorganism ecology and the pharmaceutical industry 341
Chemical disinfectants are limited in their use as air sterilants because of their
irritant properties when sprayed. However, some success has been achieved with
atomized propylene glycol at a concentration of 0.05-0.5 mgH and quaternary
ammonium compounds (QACs) at 0.075% may be used. For areas which can be
effectively sealed off for fumigation purposes, formaldehyde gas at a concentration of
1-2 mg H of air at a relative humidity of 80-90% is effective.
Ultraviolet (UV) irradiation at wavelengths between 280 and 240 nm (2800 and
2400 A) is used to reduce bacterial contamination of air, but is only active at a relatively
short distance from source. Bacteria and mould spores, in particular those with heavily
pigmented spore coats, are often resistant to such treatment.
2.3 Compressed air
Compressed air has many applications in the manufacture of pharmaceutical products.
A few examples of its uses are the conveyance of powders and suspension, providing
aeration for some fermentations and as a power supply for the reduction of particle size
by impaction. Unless it is sterilized by filtration or a combination of heat and filtration,
microorganisms present will be introduced into the product. The microbial content of
compressed air may be assessed by bubbling a known volume through a nutrient liquid
and either filtering through a membrane, which is then incubated with a nutrient agar
and a total viable count made, or the microbial content may be estimated more rapidly
using techniques developed to detect changes in physical or chemical characteristics in
the nutrient liquid.
3 Water
The microbial ecology of water is of great importance in the pharmaceutical industry
due to its multiple uses as a constituent of many products as well as for various washing
and cooling processes. Two main aspects are involved: the quality of the raw water and
any processing it receives and the distribution system. Both should be taken into
consideration when reviewing the hazards to the finished product and any critical control
points.
Microorganisms indigenous to fresh water include Pseudomonas spp., Alcaligenes
spp., Flavobacterium spp., Chromobacter spp. and Serratia spp. Such bacteria
are nutritionally undemanding and often have a relatively low optimum growth
temperature. Bacteria which are introduced as a result of soil erosion, heavy rainfall
and decaying plant matter include Bacillus subtilis, B. megaterium, Klebsiella aerogenes
and Entewbacter cloacae. Contamination by sewage results in the presence of Proteus
spp., Escherichia coli and other enterobacteria, Streptococcus faecalis and Clostridium
spp. Bacteria which are introduced as a result of animal or plant debris usually die as a
result of the unfavourable conditions.
An examination of stored industrial water supplies showed that 98% of the
contaminants were Gram-negative bacteria; other organisms isolated were Micrococcus
spp., Cytophaga spp., yeast, yeast-like fungi and actinomycetes.
342 Chapter 17
Raw or mams water
The quality of the water from the mains supply varies with both the source and the
local authority, and whilst it is free from known pathogens and from faecal contaminants
such as E. coli, it may contain other microorganisms. When the supply is derived from
surface water the flora is usually more abundant and faster-growing than that of supplies
from a deep water source such as a well or spring. This is due to surface waters receiving
both microorganisms and nutrients from soil and sewage whilst water from deep sources
has its microflora filtered out. On prolonged storage in a reservoir, water-borne organisms
tend to settle out, but in industrial storage tanks the intermittent through-put ensures
that, unless treated, the contents of the tank serve as a source of infection. The bacterial
count may rise rapidly in such tanks during summer months and reach 10
5
-10
6
ml
_I
.
One of the uses of mains water is for washing chemicals used in pharmaceutical
preparations to remove impurities or unwanted by-products of a reaction, and although
the bacterial count of the water may be low, the volume used is large and the material
being washed may be exposed to a considerable number of bacteria.
The microbial count of the mains water will be reflected in both softened and
deionized water which may be prepared from it.
Softened water
This is usually prepared by either a base-exchange method using sodium zeolite, by a
lime-soda ash process, or by the addition of sodium hexametaphosphate. In addition to
the bacteria derived from the mains water, additional flora of Bacillus spp. and
Staphylococcus aureus may be introduced into systems which use brine for regeneration
and from the chemical filter beds which, unless treated, can act as a reservoir for bacteria.
Softened water is often used for washing containers before filling with liquid or
semi-solid preparations and for cooling systems. Unless precautions are taken, the
microbial count in a cooling system or jacketed vessel will rise rapidly and if
faults develop in the cooling plates or vessel wall, contamination of the product may
occur.
Deionized or demineralized water
Deionized water is prepared by passing mains water through anion and cation exchange
resin beds to remove the ions. Thus, any bacteria present in the mains water will also
be present in the deionized water, and beds which are not regenerated frequently with
strong acid or alkali are often heavily contaminated and add to the bacterial content of
the water. This problem has prompted the development of resins able to resist
microbiological contamination. One such resin, a large-pore, strong-base, macroreticular,
quaternary ammonium anion exchange resin which permits microorganisms to enter
the pore cavity and then electrostatically binds them to the cavity surface, is currently
being marketed. The main function is as a final cleaning bed downstream of conventional
demineralizing columns.
Deionized water is used in pharmaceutical formulations, for washing containers
and plant, and for the preparation of disinfectant solutions.
Microorganism ecology and the pharmaceutical industry 343
Distilled water
As it leaves the still, distilled water is free from microorganisms, and contamination
occurs as a result of a fault in the cooling system, the storage vessel or the distribution
system. The flora of contaminated distilled water is usually Gram-negative bacteria
and since it is introduced after a sterilization process, it is often a pure culture. A level
of organism up to 10
6
mH has been recorded.
Distilled water is often used in the formulation of oral and topical pharmaceutical
preparations and a low bacterial count is desirable. It is also used after distillation
with a specially designed still, often made of glass, for the manufacture of parenteral
preparations and a post-distillation heat sterilization stage is commonly included in
the process. Water for such preparations is often stored at 80°C in order to prevent
bacterial growth and the production of pyrogenic substances which accompany such
growth.
Water produced by reverse osmosis
Water produced by reverse osmosis (RO) is forced by an osmotic pressure through a
semi-permeable membrane which acts as a molecular filter. The diffusion of solubles
dissolved in the water is impeded, and those with a molecular weight in excess of 250
do not diffuse at all. The process, which is the reverse of the natural process of osmosis,
thus removes microorganisms and their pyrogens. Post-RO contamination may occur
if the plant after the membrane, the storage vessel or the distribution system is not kept
free from microorganisms.
Distribution system
If microorganisms colonize a storage vessel, it then acts as a microbial reservoir and
contaminates all water passing through it. It is therefore important that the contents of
all storage vessels are tested regularly. Reservoirs of microorganisms may also build
up in booster pumps, water meters and unused sections of pipeline. Where a high positive
pressure is absent or cannot be continuously maintained, outlets such as cocks and taps
may permit bacteria to enter the system.
An optimum system for reducing the growth of microbial flora is one that ensures
a constant recirculation of water at a positive pressure through a ring-main without
'dead-legs' (areas which due to their location are not regularly used) and only very
short branches to the take-off points. In addition there should be a system to re-sterilize
the water, usually by membrane filtration or UV light treatment, just prior to return to
the main storage tank.
Some plumbing materials used for storage vessels, pipework and jointing may
support microbial growth. Some plastics, in particular plasticized polyvinylchlorides
and resins used in the manufacture of glass-reinforced plastics, have caused serious
microbiological problems when used for water storage and distribution systems. Both
natural and synthetic rubbers used for washers, O-rings and diaphragms are susceptible
to contamination if not sanitized regularly. For jointing, packing and lubricating
materials, PTFE and silicone-based compounds are superior to those based on
natural products such as vegetable oils or fibres and animal fats, and petroleum-based
compounds.
3.7 Disinfection of water
Three methods are used for treating water, namely chemicals, filtration or UV light.
1 Chemical treatment is applicable usually to raw, mains and softened water, but is
also used to treat the storage and distribution systems of distilled and deionized water
and of water produced by reverse osmosis (section 3.5).
Sodium hypochlorite and chlorine gas are the most common agents for treating the
water supply itself, and the concentration employed depends both upon the dwell time
and the chlorine demand of the water. For most purposes a free residual chlorine level
of 0.5-5 p.p.m. is adequate. For storage vessels, pipelines, pumps and outlets a higher
level of 50-100 p.p.m. may be necessary, but it is usually necessary to use a descaling
agent before disinfection in areas where the water is hard. Distilled, deionized and RO
systems and pipelines may be treated with sodium hypochlorite or 1 % formaldehyde
solution. With deionized systems it is usual to exhaust the resin beds with brine before
sterilization with formaldehyde to prevent its inactivation to paraformaldehyde. If
only local contamination occurs, live steam is often effective in eradicating it. During
chemical sterilization it is important that no 'dead-legs' remain untreated and that all
instruments such as water meters are treated.
2 Membrane filtration 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, the water is continually being returned to the storage tank and refiltered. As many
water-borne bacteria are small, it is usual to install a 0.22-^um pore-size membrane as
the terminal filter and to use coarser prefilters to prolong its life. Membrane filters
require regular sterilization to prevent microbial colonization and 'grow through'. They
may be treated chemically with the remainder of the storage/distribution system or
removed and treated by moist heat. The latter method is usually the most successful for
heavily contaminated filters.
3 UV light at a wavelength of 254 nm is useful for the disinfection of water of
good optical clarity. Such treatment has an advantage over chemical disinfection as
there is no odour or flavour problem and, unlike membrane filters, is not subject to
microbial colonization. The siting in the distribution system is important since any
insanitary fittings downstream of the unit will recontaminate the water. Industrial
in-line units with sanitary type fittings which replace part of the water pipeline are
manufactured.
One of the most useful techniques for checking the microbial quality of water is by
membrane filtration, since this permits the concentration of a small number of organisms
from a large volume of water. When chlorinated water supplies are tested it is necessary
to add an inactivating agent such as sodium thiosulphate. Although an incubation
temperature of 37°C may be necessary to recover some pathogens or faecal contaminants
from water, many indigenous species fail to grow at this temperature, and it is usual to
incubate at 20-26°C for their detection.
Microorganism ecology and the pharmaceutical industry 345
Skin and respiratory-tract flora
Microbial transfer from operators
Microorganisms may be transferred to pharmaceutical preparations from the process
operator. This is undesirable in the case of tablets and.powders, and may result in
spoilage of solutions or suspensions, but in the case of parenterals it may have serious
consequences for the patient. Of the natural skin flora organisms, Staph, aureus is
perhaps the most undesirable. It is common on the hands and face and, since it resides
in the deep layers of the skin, is not eliminated by washing. Other bacteria present are
Sarcina spp. and diphtheroids, but occasionally Gram-negative rods such as Mima spp.
(Acinetobacter) and Alcaligenes spp. achieve resident status in moist regions. In the
fatty and waxy sections of the skin, lipophilic yeast are often present, Pityrosporum
ovale on the scalp and P. orbiculare on glabrous skin. Various dermatophyte fungi
such as Epidermophyton spp., Microsporon spp. and Trichophyton spp. may be present.
Ear secretions may also contain saprophytic bacteria.
Bacteria other than the natural skin flora may be transferred from the operator
as a result of poor personal hygiene, such as faecal organisms from the anal region
or bacteria from a wound. Open wounds without clinical manifestation of bacterial
growth often support pathogenic bacteria and Staph, aureus has been found in
20%; other contaminants include micrococci, enterococci, a-haemolytic and non-
haemolytic streptococci, Clostridium spp., Bacillus spp. and Gram-negative intestinal
bacteria. Clostridium perfringens in such circumstances is usually present as a
saprophyte and dies fairly rapidly. Wounds showing signs of infection may support
Staph, aureus, Strep, pyogenes, enterococci, coliforms, Proteus spp. and Pseudomonas
aeruginosa.
The nasal passages may contain large numbers of Staph, aureus and a limited number
of Staph, albus, whilst the nasopharynx is often colonized by streptococci of the viridans
group, Strep, salivarius or Neisseria pharyngis. Occasionally, pathogens such as
Haemophilus influenzae and K. pneumoniae may be present. The most common
organisms secreted during normal respiratory function and speech are saprophytic
streptococci of the viridans group.
The hazard of the transfer of microorganisms from humans to pharmaceutical
preparations may be reduced by comprehensive training in personal hygiene coupled
with regular medical checks to prevent carriers of pathogenic organisms from coming
in contact with any product.
Hygiene and protective clothing
Areas designed for the manufacture of products intended for injection and eye or ear
preparations usually have washing facilities with foot-operated taps, antiseptic soap
and hot-air hand driers at the entrance to the suite, which must be used by all process
operators. For the manufacture of such products it is also necessary for the operators to
wear.sterilized clothing including gowns, trousers, boots, hoods, face masks and gloves.
For the production of products for oral and topical use, staff should be made to wash
their hands before entering the production area. The requirements for protective clothing