Bioburden

:The Burden
on our
Biological Operations

Objective

To understand

To Implement

Issues related to Bioburden

Sources of Bioburden

Methods & Systems to Control Bioburden

Ultimate Goal

To Prevent Loss of Time, Resources and Money

Bioburden
Bioburden is normally defined as the number of bacteria living on
a surface that has not been sterilized
The term is most often used in the context of bioburden testing,
also known as microbial limit testing, which is performed on
pharmaceutical products and medical products for quality control
purposes. Products or components used in the pharmaceutical or
medical field require control of microbial levels during processing
and handling. Bioburden or microbial limit testing on these
products proves that these requirements have been met.
Bioburden testing for medical devices made or used in the USA is
governed by Title 21 of the Code of Federal Regulations and
worldwide by ISO 11737.

According to United States Pharmacopeia (USP) <1227>;

ISO 11737 PARTS 1&2
Natural bioburden is defined as:
The amount of living microorganisms on an item (e.g. tissue or
medical device) prior to and after manufacturing
Bioburden can be described both in terms of QUANTITY and TYPE
of microorganisms
Why is knowing your bioburden important?
Sterilization Dose
Aseptic processing
Process Capabilities (manufacturing of pharma and non-pharma
products like food,beverages etc.)
CONTROL!!! Prevention of infection in medical aid, hospitals,
surgery etc.)

Bioburden

Bioburden:
Population of viable microorganisms on a raw material,
component, a finished product, and/or package.
Measured in CFU (colony forming units) per unit of
product

Sterile:
Free from living organisms (Microbes)
Aseptic
Removal of Bioburden

Where all do we need Bioburden

Control ?
Raw Material: including water
Resins
Membranes
Intermediates
Drug Substance/Product Stability
Samples, Control Samples
Enzymatic reactions
Reagents pH standards, Buffers

Sources of Bioburden
Raw Material
Processing Equipment
Environment
Personnel
Packing Material

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Why Bioburden Control is

Critical

Product Degradation /Spoilage

Safety
Efficacy
Appearance

Material Degradation & Life Cycle

Reduction
Affect experimental data & Results

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Solution for Bioburden

Control
Use everything sterile
Commercially and Technically not feasible
and not required too.

Bioburden Control in
R & D Set up

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10

Material Sourcing

Low Bioburden Products- Expensive

Material inherently low in bioburden

Organic Products

Inorganic salts

Material inherently high in bioburden

Aqueous liquids- with / without salts & sugars
Natural Products Yeast Extract, Peptone

Water- Fresh Every Day

Raw Material Handling

Minimize opening and

Use clean tools for dispensing

Keep the lid onreduce moisture ingress

Refrigerate when instructed/possible

Avoid low and high bioburden processing together

Open Critical Material in Controlled air LFH,

Biosafety hood etc.

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Process Equipment

Immediately clean after use

Use bioburden reduction agents where ever

possible NaOH, Acids, Hyprochloride

Use Autoclaved glassware for critical operations

If in doubt, clean/ sanitize again

Store either in very dry state or with biostatic

liquid- no not store wet/ moist

Periodically clean equipment thoroughly for

removing soil/ grime from hard to clean areas

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Environment & Personnel

Keep Lab area clean

Use Laminar Flow Hood for all critical sample
Aliquot when possible to reduce risk
Following good handing procedures
Sanitize hands before critical material handling
Hold containers away from mouth/ brim
Keep your body and other things away from
the product

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Controlling Bioburden in
Process

Filter Buffers and solutions

Filter Intermediates- for storage
Filter intermediates before loading on to the
columns
Filter all critical DS/ DP
Avoid multiple opening and handling
Tubing are good source of bioburden Wet,
difficult to clean
Use Secondary Packing Additional Shield

Packing Material

Use Autoclaved Glassware and handling tools

Always use sterile tips and pipettes

Falcon tubes are sterile as long as you handle

them that way

Bioburden Applications

Device Bioburden
Applications

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Microbiological Testing of
Products

Quality assurance and manufacturing

controls shouldbe such that organisms
capable of proliferation and contamination
of the product are within acceptable limits.

Microbiological Limit Test

The principle of the Microbial Limit Testis that each viable
microbial cell present in a sample, will, when mixed with an
agar medium, and after incubation, form a visible separate
colony.
The enumeration of these colony-forming units (cfu), when
taking into account sample preparation will give an estimate
of the microbial population of the product under the specified
incubation conditions.
Quality control test for non sterile pharmaceuticalproduct
includes the microbiological testing of raw material ,
finalproducts .

Microbiological Limit Test

Non sterile pharmaceutical products are allowed certain
microbial bioburden based upon the product specifications
With the absence of pathogenic microorganisms that
change chemical composition by spoilage and might affect
stability and integrity of the product and package

Microbiological Limit Test

There are 2 methods used in enumerating the microbial content of
a product.
Membrane Filtration uses filtration apparatus.
Total Plate-Count Method (TPC) involves direct plating.
USP divided MLT into 2 types
Quantitative test
Determines number of bacteria, yeast and mold present in a
given pharmaceutical samples.
Qualitative test
Determines the presence of specific pathogen as salmonella spp,
Staphylococcus aureus , Ps. aeruginosa and Enterobacteriaceae.

Microbiological Limit Test

o Salmonella and E coli (toxin producers) are gram negative
rods lactose fermentors , indicates fecal contamination.
o Ps aeruginosa gram negative non fermenting rods
associated with opportunistic infection
o Staphylococcus aureus is gram positive cocci usually
associated with skin and gastrointestinal infections.
opportunistic infectionn.An infection by a microorganism
that normally does not cause disease but becomes
pathogenic when the body's immune system is impaired
and unable to fight off infection.
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Microbiological Limit Test

USP recommendations and limits
1.
2.
3.
4.

Plant animal , mineral based formula ..absence of

salmonella
Orally administered product .. absence of E coli
Topical pharmaceutical preparations absence ofS.aureus
and Ps. aeruginosa
Vaginal,rectal and urethral ..absence of molds and
yeasts

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Microbiological Limit Test

USP recommendations and limits
1.
2.
3.
4.

Plant animal , mineral based formula ..absence of

salmonella
Orally administered product .. absence of E coli
Topical pharmaceutical preparations absence ofS.aureus
and Ps. aeruginosa
Vaginal,rectal and urethral ..absence of molds and
yeasts

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Microbiological Limit Test

Preparatory testing
It is done to assure that the sample itself do not inhibit
multiplication of microorganisms under test conditions.
It includes the inoculation of diluted samples of the product with
separate viable cultures of Staphylococcus aureus ,
Ps.aeruginosa ,E.coli and Salmonella .

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Microbiological Limit Test

Result of the preparatory test (preliminary test)
Presence of growth..substance can be assayed by
this methodology under test conditions
Absence of growth ..invalidate the MLT and
Necessitates modification of the procedure by
1. An increase in the volume of the diluent, with the quantity of
test material remaining the same ex: 1gm /100ml
2. Addition of suitable inactivating agent in the diluents (0.5%
soy lecithin and 4% polysorbate 20 are used to neutralize
inhibitory substances present in the sample
3. Combination of 1 and 2

Microbiological Limit Test

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Microbiological Limit Test

Antimicrobial Effectiveness Test (AET) - USP
Antimicrobial preservatives are often added to compendial
products for the following reasons:
1. To prevent proliferation or limit microbial contamination that
may occur subsequent to the manufacturing process.
2. To inhibit growth of microorganisms during normal
conditions of storage and use when microorganisms might be
introduced inadvertently from repeated withdrawing of
individual doses from containment.
3. To prolong the life span of the product.
The Antimicrobial Effectiveness Test is performed to prove
that the added preservative provides adequate protection from
adverse effects that may arise from microbial contamination or
proliferation during storage and use.
This test is not to be used for routine control purposes but it
designed to put a challenge on a compendial preparation in its
final container with a prescribed inoculums of suitable
microorganisms.

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Microbiological Limit Test

Testing sterile pharmaceutical preparations
Sterile pharmaceutical preparations are those intended for
injections and those intended for ophthalmic ,sterile otic
and nasal preparations
Sterility tests
These tests are exclusively based upon the principle that
in case the bacteria are strategically placed in a specific
medium that contains requisite nutritive material and
water, and maintained at a favourable temperature (37
2C), the microbes have a tendency to grow, and their
legitimate presence may be clearly indicated by the
appearance of a turbidity in the originally clear medium.
Please

notice that Compliance with the tests for

sterility individually cannot certify absolute
assurance of freedom from microbial contamination.
Tests for sterility are adequately designed to reveal the presence of
microorganisms in the 'samples' used in the tests.
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Sterility testing

All products labeled sterile must pass the sterility test as they
have ben subjected to an effective process of sterilization as
per International Pharmacopoeia and USP
These tests are suitable to reveal the presence of viable forms
of bacteria, fungai and yeasts in a pharmaceutical products or
devices

Extraneous microorganisms should be excluded

throughout the test procedure and period

The sterility testing of human and veterinary products

is conducted by specific procedures

Test is based on assumptions that Microorganisms

grow on the provided culture medium

Limitations (different organism have different

nutritional requirements, temp. for growth, spores
take more time to grow)

Antimicrobial precautions
To avoid any accidental contamination
use Laminar Flow Hood
Already present Microorganisms on the
product must be killed
Working area should be monitored
periodically both

Air
Surface

Microbiological Limit Test

Tests for sterility are adequately designed to reveal the presence
of microorganisms in the 'samples' used in the tests.
However, the interpretation of results is based upon
the assumption that the contents of each and
every container in the batch, had they been
tested actually, would have complied with the
tests.
As it is not practically possible to test every
container, a sufficient number of containers must
be examined to give a suitable degree of
confidence in the ultimate results obtained of
the tests.

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Microbiological Limit Test

Batch : 'a homogeneous collection of sealed
containers prepared in such a manner, that the
risk of contamination is the same for each of
the units present in it'.
Sampling
Injectable Preparations
(a) Not more than 100 containers Either 10% or 4
containers whichever is greater.
(b) More than 100, but not more than 500 containers
10 containers.
(c) More than 500 containers. Either 2% or 20
containers whichever is less.

Microbiological Limit Test

Sterility test could be performed either by
(a) Membrane Filtration
(b) Direct Inoculation.
Membrane Filtration
This method is applied to the sample which contains antimicrobial
substances . The Membrane filtration sterility test is the regulatory
method of choice for filterable pharmaceutical products. The test is
particularly suitable for samples containing preservative,
bacteriostatic or fungistatic compounds, which inhibit microbial
growth of potential contaminants. With membrane filtration,
microorganisms are retained by a 0.45-micron pore size filter, and
all inhibiting compounds are rinsed using a suitable rinse solution.
Appropriate media, which are selected based upon their ability to
support the growth of anaerobic & aerobic microorganisms, are then
transferred to the membrane filters. A 14-day incubation period is
required to obtain final test results

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Microbiological Limit Test

Microorganisms Tested for Positive Control Tests :
(a)Bacillus

cerreus
(b) Staphylococcus aureus
(c) Klebsiella aerogenes
(d) Enterobacteria.
the membrane filtration is to be preferred exclusively in the
case of the following
(i) an oil or oil-based product,
(ii) an ointment that may be put into solution,
(iii) a non-bacteriostatic solid that does not become soluble in
the culture medium rapidly, and
(iv) a soluble powder or a liquid that essentially possesses
either inherent bacteriostatic or inherent fungistatic
characteristic features

Microbiological Limit Test

The test should be carried using
(a) a sophisticated laminar sterile airflow cabinet (having hepafilters),
(b) necessary precautionary measures taken to be such so as to
avoid contamination that they do not affect any microbes which
must be revealed duly in the test.
(c) ensuing environment (i.e., working conditions) of the laboratory
where the 'tests for sterility' is performed must always be
monitored at a definite periodical interval by :
sampling the air of the working area,
sampling the surface of the working area, and
perforing the stipulated control tests.
Once the filtration is completed, wash the membrane filter with
peptone water and then clise the device to introduce the liquid
culture medium 'Fluid Soyabean-Casein Digest Medium'*, and
incubate at 20-25C for a duration of seven days. And Fluid
Thioglycollate Medium',** and incubate at 30-35 C for seven
days.

Microbiological Limit Test

Direct Inoculation [or Direct Inoculation of Culture
Media]
The three usual methods being used for performing the 'tests for sterility' are
as enumerated under :
(a) Nutrient Broth, for the 'aerobic microorganisms'.
(b) Cooked Meat Medium for clostridium and Thioglycollate Medium, for
anaerobic
(c) Sabouraud Medium for moulds and fungi
This test is suitable for non filterable solutions like oily or viscous solution
sterile ointments Sterile containers , gloves and gauzes
1. Liquid from the 'test containers' must be removed carefully with a with a
sterile syringe.
2. Transfer aseptically the requistite prescribed volume of the substance from
each container to a vessel of the culture medium.
3. Mix the liquid with the medium carefully taking care not to aerate
excessively.
4. Incubate the 'inoculated media' for not less than 14 days

Microbiological Limit Test

Limulus Amebocyte Lysate test. (pyrogen test )
It is done to detect the presence of pyrogen in any parental pharmaceutical
preparation
A pyrogen is frequently described as a fever producing substance. The most
potent pyrogens originate from gram negative bacteria, which are common waterborne organisms.
Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells
(amoebocytes) from the horseshoe crab, Limulus polyphemus.
LAL reacts

with bacterial endotoxin or lipopolysaccharide (LPS), which is a

membrane component of Gram negative bacteria. This reaction is the basis of the
LAL test, which is used for the detection and quantification of bacterial
endotoxins.

Microbiological Limit Test

Limulus Amebocyte Lysate test. (pyrogen test )
The primary application for LAL is the testing of parenteral pharmaceuticals
and medical devices that contact blood or cerebrospinal fluid
For pharmaceutical preparations
- 1 ml of diluted solution using LAL

incubated at 60 C for 1 hr

Formation
Formerly

water is injected into LAL test tube and

of gel indicates presence of pyrogen

rabbit test was used where dilution was injected into a rabbit and
rabbit watched for pyrexia

Microbiological Limit Test

Antibiotic assay
The activity (potency) of antibiotics may be demonstrated under
suitable conditions by their inhibitory effect on microorganisms.
Two general methods are employed,
1. the cylinder-plate or "plate" assay diffusion of the
antibiotic from a vertical cylinder through a solidified agar layer in
a petri dish such that growth of the added microorganism is
prevented entirely in a circular area or "zone" around hole
containing a solution of the antibiotic.
2. the turbidimetric or "tube" assay. the inhibition of growth
of a microbial culture in a uniform solution of the antibiotic in a
fluid medium that is favorable to its rapid growth in the absence
of the antibiotic.

Microbiological Limit Test

Antibiotic assay
The potency of antibiotics is designated in either "Units" or
"M-g" of activity
The corresponding USP Reference Standard is Each antibiotic is
tested against a specific microorganisms listed used in the pharmacopeia eg
Microbe under test is inoculated in a concentration of 1 % in muller hinton
agar which do not interfere in activity of all known antibiotics the seeded agar is
left to solidify
Holes are formed using 8 mm cork borer
Then certain volume of serial dilutions of the test and standard is instilled into
the holes left for one hour to diffuse then incubated for 24 hrs the potency is
calculated through known formula by using diameter of inhibition zones

Environmental monitoring
Environmental Monitoring (E/M) is a program designed
to demonstrate the control of viable (living
microorganisms) and non-viable particles in critical
areas.
These areas include clean-rooms for drug fill/ finish,
formulation tank rooms, laminar flow hoods, molding
machines, kit assembly lines, Intravenous (IV)
compounding areas and sterile packaging.
Viable monitoring is :
Testing

for the detection and enumeration of bacteria,

yeast and mold. It includes the monitoring of personnel,
air and area surfaces for microbial contamination.
Non-viable environmental monitoring is particle
counts measured by a laser counter.

Environmental monitoring

The items that are sampled in a manufacturers clean room

include
1. Personel

2. Air

3. Surface

1.Personnel

- Personnel are the biggest source of contamination in clean areas.

Personnel harbor bacteria, carrying them with them everywhere they go.
Gowning is the most effective way to protect the cleanroom environment from
ourselves.. Personnel monitoring employs contact plates to assess microbial
contamination of clean room personnel.
How Personnel are monitored in a Clean Room
Contact Plates - Personnel in critical areas may be monitored for microbial
contamination utilizing contact plates. The contact plates monitor areas of the
body that may interact with the sterile field or product exposure areas. These
may include gloved hands, forearms, or other areas. Personnel monitoring is a
good indication of how well personnel are gowning when they enter the clean
room. .

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Microbiological Limit Test

AIR
the air in a clean room is controlled and Monitored on a regular basis (e.g., daily, weekly,
quarterly) for particle counts, viable counts, temperature and humidity.
HEPA filters are used to control the viable and non-viable particulate counts within the air.
HEPA filters have the capability to filter out particulates down to 0.2 2m in size.
These filters run continuously at a calibrated flow rate in order to maintain the required air
quality within the room. Humidity is usually kept at a low level in order to help prevent the
proliferation of microbes within the room such as bacteria and mold, which tend to prefer damp
conditions in order to replicate.
Two methods of Air sampling in a Clean Room
1. Air Samplers (active air sampling) - Air samplers draw in predetermined volumes of air. The
air is drawn over a sterile media plate, which is later incubated to reveal the number of viable
organisms per cubic feet or liter. Currently agar impaction is the method of choice throughout
the industries. Using a specially designed, and calibrated piece of equipment which holds the
media plate under a perforated lid and draws in a known amount of air one can accurately
measure the amount of viable bacteria within the air.

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Environmental monitoring
Settling plates (passive air sampling)
Petri dishes containing sterile growth media are exposed to the environment for a
specific period of time, usually between 30-60 minutes but can be exposed up to
four hours before compromising the integrity of the media itself. Viable
microorganisms which settle onto the media surface will grow after the plates are
incubated. However, passive air sampling is tending to be phased out because
it does not reflect microbial contamination with an accurately measured
volume of air.

Environmental monitoring
3. Surfaces (including floors, walls, equipment, etc.) are cleaned

and monitored on a regular basis for viable counts by using specially

designed contact plate
Two methods for surface monitoring in a Clean Room
1. Contact Plates -are special Petri dishes which contain sterile
growth medium prepared in a manner so the surface of the media
protrudes above the rim of the plate. These plates that contain a
growth medium called Trypticase Soy Agar (TSA) and Sabouraud
Dextros Agar (SDA, TSA at 30-35C which is mainly the optimal growing
temperature for most environmental bacteria, and 20-25 C which is
the optimal growing temperature for most mold and yeast species.
The contact plate is pressed against any flat surface that needs to be
sampled. Any viable microorganisms on the surface will stick to the
agar surface and will grow upon proper incubation. This technique
reveals the number of viable microorganisms on a surface.
2. Swabs - are sterile and stored in a suitable sterile liquid. The swabs
are rubbed over the test surface. The microbiologist can determine the
type of microorganisms on the swab by subculturing it to media.
Swabs are used for surfaces that are not flat, and can be used to
sample hard to reach areas of machinery that could not be sampled
with a contact plate. Swabbing is more qualitative than quantitative.