Environmental Monitoring In

The Pharmaceutical Industry

  Environmental cleanliness and hygiene

Manufacturing
Environment
Microbiological
Quality of • Building (Ceiling, Wall, Floor)
1
Pharmaceutical
Product • Equipment used in production
2

3
• Atmosphere of production floor
Materials used

4
• Person operating the process

• Final container & packaging

5
Objectives of cleanliness and hygiene
 Environmental cleanliness and hygiene
Limit of microorganisms for different pharmaceutical preparations:
 FINAL
PRODUCTS

STERILE NON-STERILE

Non- Pathogens
pathogens

Pyrogenic
substance
 Sources of contamination in production zone
Atmosphere

Microbial content
Air is not a natural environment for the growth and reproduction of
microorganisms. Because-
o Lack of necessary amount of moisture
o Lack of nutrients in utilizable form.
o Continuous desiccation and
o Exposure to UV and sunlight.
o However, almost any sample of untreated air contains suspended bacteria,
moulds and yeasts due to their ability to tolerate desiccation and the
continuing dry state.
 Sources of contamination in production zone
Atmosphere
Microorganisms commonly isolated from air are as following-

• The spore-forming bacteria:

Bacillus spp. and
Clostridium spp.,

• The non-sporing bacteria:

Staphylococcus spp.,
Streptococcus spp. and
Corynebacterium spp.

• The moulds:
Penicillium spp.,
Cladosporium spp.,
Aspergillus spp. and
Mucor spp., as well as

• The yeast
Rhodotorula spp.
 Sources of contamination in production zone
Atmosphere

The number of organisms in the atmosphere depends on

• The activity in the environment.
• The amount of disturbed dust.
• Humidity and the size of the particle.
• Nature of raw materials and packaging components.

Reduction of microbial count

The microbial count of air may be reduced by
- Filtration,
- Chemical disinfection and
- Ultraviolet (UV) light.
 Sources of contamination in production zone
Water

Water has multiple uses in Pharmaceutical Industry.

1. Functions as a constituent of many products as well as
2. Used for various washing and cooling processes.

Water could be a source of contamination due to two main aspects-

1. The quality of the raw water and any processing it receives and
2. the distribution system.

The microorganisms present in the water could be of three types.

1. Indigenous
2. Introduced
3. Sewage contamination
 Sources of contamination in production zone
Water

1. Indigenous 3. Sewage contamination

Microorganisms indigenous Contamination by sewage

to fresh water include results in the presence of
 Pseudomonas spp., 2. Introduced  Proteus spp.,
 Alcaligenes spp., Bacteria which are introduced  Escherichia coli
 Flavobacterium spp., as a result of soil erosion, heavy and other enterobacteria,
 Chromobacter spp. and rainfall and decaying plant  Streptococcus faecalis
 Serratia spp. matter include- and

 Bacillus subtilis,  Clostridium spp.

 B. megaterium,
 Enterobacter aerogenes and
 Enterobacter cloacae.
 Sources of contamination in production zone
Personnel
Skin and respiratory tract flora
• Natural Skin Flora:
o In hand, face and deep layer of skin:
Staphylococcus aureus - most undesirable
o In the moist regions:
Sarcina spp., Diphtheroids, Acinetobacter spp., and Alcaligenes spp.
o In the fatty and waxy sections, scalp, glabrous skin, ear secretions etc.:
Pityrosporum ovale, P. orbiculare, Epidermophyton spp., Microsporon spp., and Trichophyton spp. and other
saprophytic bacteria.
Nasal passages and the nasopharynx:
o S. aureus (large amount), S. albus (limited)
o Streptococcus salivarius (commonly secreted during normal respiratory function and speech), N. pharynges
o Haemophilus influenzae and Klebsiella pneumoniae (Occasionally present)

Poor personal hygiene associated organism:

S. aureus, micrococci, enterococci, α- haemolytic and non-haemolytic streptococci, Clostridium spp., Bacillus
spp. and Gram-negative intestinal bacteria, Clostridium perfringens, coliforms, Proteus spp. and Pseudomonas
aeruginosa.
 Sources of contamination in production zone
Raw Materials
 Raw materials account for a high proportion of microorganism in pharmaceutical
products.
 Selection of material can control contamination level in both product and
environment.
 There are two sources of raw materials:
1) Natural source
2) Synthetic raw material
• Natural sources support extensive and varied microflora. These may be-

Name of Material Organism

source
Animal Gelatine, Thyroid, E. coli, Salmonella and animal
Pancreas borne pathogen
Plant Gum acacia, Agar, Erwinia spp., Pseudomonas
Starches, Powdered spp., Bacillus spp.
rhubarb

 Synthetic raw materials are usually free from all but incidental microbial
contamination.
 Sources of contamination in production zone
Packaging components
 Packaging material has dual role. These are:
Contain the product
Prevent the entry of microorganism
 Microflora of packaging materials depend on:
Its composition
Storage condition
 Packaging materials are glass containers, plastic, cardboard, paperboard,
pulpboard etc.
 Glass containers Contamination during storage and transport Mould
spores of Penicillium spp., Aspergillus spp. and bacteria such as Bacillus spp.
 Plastic bottles If transported in a non-sanitary packaging materials
Mould spores.
 Untreated Cardboard and paperboards mould spores of Cladosporium spp.,
Aspergillus spp. and Penicillium spp. and bacteria such as Bacillus spp. and
Micrococcus spp..
Sources of contamination in production zone
Building

Wall and Ceilings

Building Floors and Drains

Doors, Windows and Fittings

 Sources of contamination in production zone
Building

1. Wall and ceilings

Most common flora: moulds especially Cladosporium spp., Aspergillus spp.,

Penicillium spp. and Aureobasidium (Pullularia) spp. etc.

2. Floors and drains:

Microorganisms of floors and drains are basically determined and

influenced by air, cleanliness and personnel activities.

2. Doors, windows and fittings:

Microorganisms of doors, windows and fittings are also determined and

influenced by air, cleanliness and personnel activities.
Sources of contamination in production zone
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, especially the temperature
and pH.
 Microbiological environmental monitoring involves the collection of data
relating to the numbers of microorganisms present in a clean room or clean
zone.

 These microorganisms are recovered from surfaces, air, and people.

 Nonviable particle counting, a physical test, is often included within the

program because this function has often resided with the microbiology
department to perform and due to the theoretical relationship between high
numbers of nonviable particles and viable counts.

 The main aim of microbiological environmental monitoring is to assess the

monitoring of trends over time and the detection of an upward or downward
movement, within clean areas.
The levels of microbial contamination in the manufacturing areas are
monitored on a regular basis to confirm that the numbers do not exceed the
specified limits. The viable count aspect of environmental monitoring
consists enumerating the numbers of microorganisms present in a clean
room by collection results by using the following sample types:
1. Settle plate techniques- passive air-sampling
2. Active air sampling- volumetric air-sampling
3. Surface monitoring-
a. Swab sampling
b. Contact plate
c. Finger plates
d. Plates of sleeves/ gowns.
Settle plates
A settle plate is an agar plate, placed in a defined location. The exposure time of the settle
plate can be varied, although there is probably little value in exposing plates for less than 1
h. For consistency of sampling, for aseptic filling, the EU GMP Guide recommends a 4h
exposure time. This time should not be exceeded without strong justification, and even then
there will probably be a challenge from the regulatory authority.
For exposure times under 4h, such as when a shorter activity is being monitored, the result
obtained should be extrapolated using the simple equation:
𝐶𝑜𝑢𝑛𝑡
cfu / 4h = x240
𝑇𝑖𝑚𝑒 𝑒𝑥𝑝𝑜𝑠𝑒𝑑 (𝑚𝑖𝑛)

The risk from any exposure is desiccation. The depth and condition of the agar are the key
variables, as is the cleanroom environment. The agar in the plate will dry out faster if the
airflow is excessively high or if the air humidity is low. Therefore, the exposure time of
settle plates under the conditions of use.
Active air samples
Active (or volumetric or bioaerosol) air samplers are a slightly different
measure of microorganisms in air than settle plates. As indicated
earlier, the settle plate indicates the number of microorganisms that
may deposit onto a surface; whereas, the active air-sampler indicates
the number of microorganisms present in a given volume of air within
the range of the air-sampler. Both of these approaches have merits and
any comprehensive program will use both active air samples and settle
plates. Active air-samplers sample a defined quantity of air. The volume
of air sampled is normally one cubic meter of air. This allows the data
to be quantified as cfu/m3.
Surface sampling methods
Contact plates
Surface contact plates are a common test for surface contamination. Contact plates are
Petri-dishes filled with microbiological agar. The plate is filled to a level above the rim of
the plate so that the agar surface extends upwards when dry. The plate has a typical
diameter of 50–55 mm and a surface area of 25 cm2.
The raised surface allows the agar to be pressed onto a surface. The design of the contact
plate is, therefore, different to the standard Petri-dish, where the agar is contained within
the Petri-dish. The contact plate is a quantifiable method, because the contact between the
plate and the surface provides a ―mirror image‖ of the surface. Following incubation, this
image transfer provides information relating to the number of microbial colonies and their
relative position. The quantification is derived from the recording the number of colony
forming units (cfu) per square centimeter. This act of replication gives the contact plate its
alternative name: RODAC. This is an acronym for ―Replicate Organism Detection and
Counting,‖ and the term is more commonly applied in North America.
In Grades A and B cleanrooms, in relation to aseptic processing, contact plates are taken
from personnel hands (during processing activities) and from clothing. Personnel gown
monitoring should be carried out at the end of a shift. This is because the sampling damages
the fabric integrity of the suit through the moisture of the agar plate.
Swab sampling

Swabs are typically made up of sterile cotton tips, although swabs vary in the materials
used for the applicator stick (either wood or plastic) and the tip (where materials like
cotton, viscose, alginate, and so on are used). Some types of swabs require prewetting
using a diluent (such as Ringer’s solution, phosphate-buffered saline, sodium
hexametaphosphate, sterile water, etc.) before use. Other types of swabs are contained
with a transport medium. They are either contained within a transport medium or require
prewetting with a suitable recovery medium. Swabbing is performed by rubbing a surface
while rotating the swab—so that all parts of the tip are exposed—through a number of
strokes (typically between 10 and 25). Following the sampling, the swab can either be
streaked out onto an agar plate (which is the least efficient method) or, where the
appropriate tip has been used, dissolved in a diluent and tested either by pour plate or by
membrane filtration (of which the latter is the most efficient method). The membrane
filter will be either 0.45 or 0.22μm, and this technique carries a further advantage In that
disinfectant residues can be overcome through rinsing the filter.