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

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are usually less stringent but include clean overalls, hair covering and gloves, and

where possible, face masks are an advantage.

5 Raw materials

Raw materials account for a high proportion of the microorganisms introduced during

the manufacture of pharmaceuticals, and the selection of materials of a good

microbiological quality aids in the control of contamination levels in both products

and the environment. It is, however, common to have to accept raw materials which

have some non-pathogenic microorganisms present and an assessment must be made

as to the risk of their survival to spoil the finished product by growing in the presence

of a preservative system, or the efficacy of an in-process treatment stage to destroy or

remove them. Whatever the means of prevention of growth or survival by chemical or

in-process treatment, it should be regarded as critical and controlled accordingly.

Untreated raw materials which are derived from a natural source usually support

an extensive and varied microflora. Products from animal sources such as gelatine,

desiccated thyroid, pancreas and cochineal may be contaminated with animal-borne

pathogens. For this reason some statutory bodies such as the British Pharmacopoeia

(1993) require freedom of such materials from Escherichia coli and Salmonella spp. at

a stated level before they can be used in the preparation of pharmaceutical products.

The microflora of materials of plant origin such as gum acacia and tragacanth, agar,

powdered rhubarb and starches may arise from that indigenous to 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., and non-mycelated yeasts, or those introduced during cultivation. For example,

the use of untreated sewage as a fertilizer may result in animal-borne pathogens such

as Salmonella spp. being present. Some refining processes modify the microflora of

raw materials, for example drying may concentrate the level of spore-forming bacteria

and some solubilizing processes may introduce water-borne bacteria such as E. coli.

Synthetic raw materials are usually free from all but incidental microbial

contamination.

The storage condition of raw materials, particularly hygroscopic substances, is

important, and since a minimum water activity (A

) of 0.70 is required for osmophilic

yeasts, 0.80 for most spoilage moulds and 0.91 for most spoilage bacteria, precautions

should be taken to ensure that dry materials are held below these levels. Some packaging

used for raw materials, such as unlined paper sacks, may absorb moisture and may

itself be subject to microbial deterioration and so contaminate the contents. For this

reason polythene-lined sacks are preferable. Some liquid or semi-solid raw materials

contain preservatives, but others such as syrups depend upon osmotic pressure to prevent

the growth of osmophiles which are often present. With this type of material it is

important that they are held at a constant temperature since any variation may result in

evaporation of some of the water content followed by condensation and dilution of the

surface layers to give an A

value which may permit the growth of osmophiles and

spoil the syrup.

The use of natural products with a high non-pathogenic microbial count is possible

if a sterilization stage is included either before or during the manufacturing process.

Microorganism ecology and the pharmaceutical industry 347

Such sterilization procedures (see also Chapter 20) may include heat treatment, filtration,

irradiation, recrystallization from a bactericidal solvent such as an alcohol, or for dry

products where compatible, ethylene oxide gas. If the raw material is only a minor

constituent and the final product is adequately preserved either by lack of A

chemically

or by virtue of its pH, sugar or alcohol content, an in-process sterilization stage may

not be necessary. If, however, the product is intended for parenteral or ophthalmic use

a sterilization stage is essential.

The handling of contaminated raw materials as described previously may increase

the airborne contamination level, and if there is a central dispensing area precautions

may be necessary to prevent airborne cross-contamination, as well as that from infected

measuring and weighing equipment. This presents a risk for all materials but in particular

those stored in the liquid state where contamination may result in the bulk being spoiled.

6 Packaging

Packaging material has a dual role and acts both to contain the product and to prevent

the entry of microorganisms or moisture which may result in spoilage, and it is therefore

important that the source of contamination is not the packaging itself. The microflora

of packaging materials is dependent upon both its composition and storage conditions.

This, and a consideration of the type of pharmaceutical product to be packed, determine

whether a sterilization treatment is required.

Glass containers are sterile on leaving the furnace, but are often stored in dusty

conditions and packed for transport in cardboard boxes. As a result they may contain

mould spores of Penicillium spp., Aspergillus spp. and bacteria such as Bacillus spp. It

is commonplace to either airblow or wash glass containers to remove any glass spicules

or dust which may be present, and it is often advantageous to include a disinfection

stage if the product being filled is a liquid or semi-solid preparation. Plastic bottles

which are either blow- or injection-moulded have a very low microbial count and may

not require disinfection. They may, however, become contaminated with mould spores

if they are transported in a non-sanitary packaging material such as unlined cardboard.

Packaging materials which have a smooth, impervious surface, free from crevices

or interstices, such as cellulose acetate, polyethylene, polypropylene, poly vinylchloride,

and metal foils and laminates, all have a low surface microbial count. Cardboard and

paperboard, unless treated, carry mould spores of Cladosporium spp., Aspergillus spp.

and Penicillium spp. and bacteria such as Bacillus spp. and Micrococcus spp.

Closure liners of pulpboard or cork, unless specially treated with a preservative,

foil or wax coating, are often a source of mould contamination for liquid or semi-solid

products. A closure with a plastic flowed-in linear is less prone to introduce or support

microbial growth than one stuck in with an adhesive, particularly if the latter is based

on a natural product such as casein. If required, closures can be sterilized by either

formaldehyde or ethylene oxide gas.

In the case of injectables and ophthalmic preparations which are manufactured

aseptically but do not receive a sterilization treatment in their final container the

packaging has to be sterilized. Dry heat at 170°C is often used for vials and ampoules.

Containers and closures may also be sterilized by moist heat, chemicals and irradiation,

but consideration for the destruction or removal of bacterial pyrogens may be necessary.

348 Chapter 17

Regardless of the type of sterilization, the process must be validated and critical control

points established.

Buildings

Walls and ceilings

Moulds are the most common flora of walls and ceilings and the species usually found

are Cladosporium spp., Aspergillus spp., in particular A. niger and A. flavus, Penicillium

spp. and Aurebasidium (Pullularia) spp. They are particularly common in poorly

ventilated buildings with painted walls. The organisms derive most of their nutrients

from the plaster on to which the paint has been applied and a hard gloss finish is more

resistant than a softer, matt one. The addition of up to 1% of a fungistat such as

pentachlorophenol, 8-hydroxyquinoline or salicylanilide is an advantage. To reduce

microbial growth, all walls and ceilings should be smooth, impervious and washable

and this requirement may be met by cladding with a laminated plastic. In areas where

humidity is high, glazed bricks or tiles are the optimal finish, and where a considerable

volume of steam is used, ventilation at ceiling level is essential.

To aid cleaning, all electrical cables and ducting for other services should be installed

deep in cavity walls where they are accessible for maintenance but do not collect dust.

All pipes which pass through walls should be sealed flush to the surface.

Floors and drains

To minimize microbial contamination, all floors should be easy to clean, impervious to

water and laid on a flat surface. In some areas it may be necessary for the floor to slope

towards a drain, in which case the gradient should be such that no pools of water form.

Any joints in the floor, necessary for expansion, should be adequately sealed. The

floor-to-wall junction should be coved.

The finish of the floor usually relates to the process being carried out and in an area

where little moisture or product is liable to be split, poly vinylchloride welded sheeting

may be satisfactory, but in wet areas or where frequent washing is necessary, brick

tiles, sealed concrete or a hard ground and polished surface like terazzo is superior. In

areas where acid or alkaline chemicals or cleaning fluids are applied, a resistant sealing

and jointing material must be used. If this is neglected the surface becomes pitted and

porous and readily harbours microorganisms.

Where floor drainage channels are necessary they should be open if possible, shallow

and easy to clean. Connections to drains should be outside areas where sensitive products

are being manufactured and, where possible, drains should be avoided in areas where

aseptic operations are being carried out. If this cannot be avoided, they must be fitted

with effective traps, preferably with electrically operated heat-sterilizing devices.

Doors, windows and fittings

To prevent dust from collecting, all ledges, doors and windows should fit flush with

walls. Doors should be well fitting to reduce the entry of microorganisms, except where

Microorganism ecology and the pharmaceutical industry 349

a positive air pressure is maintained. Ideally, all windows in manufacturing areas should

serve only to permit light entry and not be used for ventilation. In areas where aseptic

operations are carried out, an adequate air-control system, other than windows, is

essential.

Overhead pipes in all manufacturing areas should be sited away from equipment to

prevent condensation and possible contaminants from falling into the product. Unless

neglected, stainless steel pipes support little microbial growth, but lagged pipes present

a problem and unless they are regularly treated with a disinfectant they will support

mould growth.

8 Equipment

Each piece of equipment used to manufacture or pack pharmaceuticals has its own

peculiar area where microbial growth may be supported, and knowledge of its weak

points may be built up by regular tests for contamination. The type and extent of growth

will depend on the source of the contamination, the nutrients available and the

environmental conditions, in particular the temperature and pH.

The following points are common to many pieces of plant and serve as a general

guide to reduce the risk of microbial colonization.

1 All equipment should be easy to dismantle and clean.

2 All surfaces which are in contact with the product should be smooth, continuous

and free from pits, with all sharp corners eliminated and junctions rounded or coved.

All internal welding should be polished out and there should be no dead ends. All

contact surfaces require routine inspection for damage, particularly those of lagged

equipment, and double-walled and lined vessels, since any crack or pinholes in the

surface may allow the product to seep into an area where it is protected from cleaning

and sterilizing agents, and where microorganisms may grow and contaminate subsequent

batches of product.

3 There should be no inside screw threads and all outside threads should be readily

accessible for cleaning.

4 Coupling nuts on all pipework and valves should be capable of being taken apart

and cleaned.

5 Agitator blades and the shaft should preferably be of one piece and be accessible

for cleaning. If the blades are bolted onto the shaft, the product may become entrained

between the shaft and blades and support microorganisms. If the shaft is packed into a

housing and this fitting is within a manufacturing vessel it also may act as a reservoir

of microorganisms.

6 Mechanical seals are preferable to packing boxes since packing material is

usually difficult to sterilize and often requires a lubricant which may gain access to

the product. The product must also be protected from lubricant used on other moving

parts.

7 Valves should be of a sanitary design, and all contact parts must be treated during

cleaning and sanitation, and a wide variety of plug type valves are available for general

purpose use. For aseptically manufactured and filled products valves fitted with steam

barriers are available. If diaphragm valves are used, it is essential to inspect the

diaphragm routinely. Worn diaphragms can permit seepage of the product into the seat

350 Chapter 17

of the valve, where it is protected from cleaning and sterilizing agents and may act as a

growth medium for microorganisms, in addition if diaphragm valves are used in very

wet area, a purpose-made cover may be useful to prevent access of water and potential

microbial growth occurring under the diaphragm.

8 All pipelines should slope away from the product source and all process and storage

vessels should be self-draining. Run-off valves should be as near to the tank as possible

and sampling through them should be avoided, since any nutrient left in the valve may

encourage microbial growth which could contaminate the complete batch. A separate

sampling cock or hatch is preferable.

9 If a vacuum exhaust system is used to remove the air or steam from a vessel, it is

necessary to clean and disinfect all fittings regularly. This prevents residues which

may be drawn into them from supporting microbial growth, which may later be returned

to the vessel in the form of condensate and contaminate subsequent batches of product.

If air is bled back into the vessel it should be passed through a sterilizing filter.

10 If any filters or straining bags made from natural materials such as canvas, muslin

or paper are used, care must be taken to ensure that they are cleaned and sterilized

regularly to prevent the growth of moulds such as Cladosporium spp., Stachybotrys

spp., and Aureobasidium (Pullularia)pullulans, which utilize cellulose and would impair

them.

8.1 Pipelines

The most common materials used for pipelines are stainless steel, glass and plastic,

and the latter may be rigid or flexible. Continuous sections of pipework are often

designed to be cleaned and sterilized in place by the flow of cleansing and

sterilizing agents at a velocity of not less than 1.5ms

through the pipe of the largest

diameter in the system. The speed of flow coupled with a suitable detergent removes

microorganisms by a scouring action. To be successful, stainless steel pipes must be

welded to form a continuous length and must be polished internally to eliminate any

pits or crevices which would provide a harbour for microorganisms. However, as

soon as joints and cross-connections are introduced they provide a harbour for

microorganisms, particularly behind rubber or teflon O-rings. In the case of plastic

pipes, bonded joints can form an area where microorganisms are protected from cleaning

and sterilizing agents.

The 'in-place' cleaning system described for pipelines may also be used for both

plate and tubular types of heat exchange units, pumps and some homogenizers. However,

valves and all T-piece fittings for valves and temperature and pressure gauges may

need to be cleaned manually. Tanks and reaction vessels may be cleaned and sterilized

automatically by rotary pressure sprays which are sited at a point in the vessel where

the maximum area of wall may be treated. If spray balls are incorporated into a system

which re-uses the cleansing-in-place (CIP) fluids, then it may be necessary to incorporate

a filter to remove particles which may block the pores of the spray ball. Fixtures such

as agitators, pipe inlets, outlets and vents may have to be cleaned manually. The nature

of many products or the plant design often renders cleaning in place impracticable and

the plant has to be dismantled for cleaning and sterilizing.

Microorganism ecology and the pharmaceutical industry 351

Cleansing

There are several cleansing agents available to suit the product to be removed, and the

agents include acids, alkalis and anionic, cationic and non-ionic detergents. The agent

selected must fulfil the following criteria.

1 It must suit the surface to be cleaned and not cause corrosion.

2 It must remove the product without leaving a residue.

3 It must be compatible with the water supply.

Sometimes a combined cleansing and sterilizing solution is desirable, in which case

the two agents must be compatible.

Disinfection and sterilization

Equipment may be sterilized or disinfected by heat, chemical disinfection or a

combination of both. Many tanks and reaction vessels are sterilized by steam under

pressure, and small pieces of equipment and fittings may be autoclaved, but it is

important that the steam has access to all surfaces. Equipment used to manufacture and

pack dry powder is often sterilized by dry heat. Chemical disinfectants commonly

include sodium hypochlorite and organochlorines at 50-100p.p.m. free residual chlorine,

QACs (0.1-0.2%), 70% (v/v) ethanol in water and 1% (v/v) formaldehyde solution.

The method of disinfection may be by total immersion for small objects or by spraying

the internal surfaces of larger equipment. When plant is dismantled for cleaning and

sterilizing, all fittings such as couplings, valves, gaskets and O-rings also require

treatment. The removal of chemical disinfectants is very important in fermentation

processes where residues may affect sensitive cultures.

All disinfection and sterilization processes for equipment should be validated, for

preference using a microbiological challenge with an organism of appropriate resistance

to the disinfectant, sterilant or sterilizing conditions. Once the required log reduction

of the challenge organism has been achieved, physical and/or chemical parameters can

be set which form the critical control points for the process.

Microbial checks

Either as part of an initial validation or as an ongoing exercise, the efficacy of CIP

systems can be checked by plating out a sample of the final rinse water with a nutrient

agar, or by swab tests. Swabs may be made of either sterile cotton wool or calcium

alginate. The latter is used in conjunction with a diluent containing 1% sodium

hexametaphosphate which dissolves the swab and releases the organisms removed from

the equipment; these organisms may then be plated out with a nutrient agar or alternative

methods of evaluation used. Swabs are useful for checking the cleanliness of curved

pieces of equipment, pipes, orifices, valves and connections, but unless a measuring

guide is used the results cannot be expressed quantitatively. Such measurement can be

made by pressing a nutrient agar against a flat surface. The agar is usually poured into

specially designed Petri dishes or contact plates, or is in the form of a disc sliced from

a cylinder of a solid nutrient medium. The nutrient agar or plate or section, when

incubated, replicates the contamination on the surface tested. Since this technique leaves

a nutrient residue on the surface tested, the equipment must be washed and resterilized

before use. The development of methods for the rapid detection of microorganisms

has advantages over more traditional methods if quantitative results are used as part

of a critical control programme, but not all methods lend themselves to identifying the

contaminant, and it may be necessary to use a combination of methods if qualitative

determinations are required.

Cleaning equipment and utensils

The misuse of brooms and mops can substantially increase the microbial count of

the atmosphere by raising dust or by splashing with water-borne contaminants. To

prevent this, either a correctly designed vacuum cleaner or a broom made of synthetic

material, which is washed regularly, may be used. Hospital trials have shown that,

when used, a neglected dry mop redistributes microorganisms which it has picked up,

but a neglected wet mop redistributes many times the number of organisms it picked

up originally, because it provides a suitable environment for their growth. In order to

maintain mops and similar non-disposable cleaning equipment in a good hygienic

state, it was found to be necessary first to wash and then to boil or autoclave the items,

and finally to store them in a dry state. Disinfectant solutions were found to be

inadequate.

Many chemical disinfectants (see also Chapter 10), in particular the halogens, some

phenolics and QACs, are inactivated in the presence of organic matter and it is essential

that all cleaning materials such as buckets and fogging sprays are kept clean. Halogens

rapidly deteriorate at their use-dilution levels and QACs are liable to become

contaminated with Ps. aeruginosa if stored diluted. For such reasons it is preferable to

store the bulk of the disinfectant in a concentrated form and to dilute it to the use

concentration only as required.