Bioburden: guidance and testing

1. Prepared By:- ARPIT BANA
PQA,M.PHARM
M.S UNIVERSITY.
GUIDED BY: Dr. RAJASHREE MASHRU
Faculty of Pharmacy, M.S University.

2.  Bioburden is the population of viable
microorganism on a particular object,
formulation and/or finished product.
 It is the number of bacteria living on a surface
that has not been sterilized.
 Bioburden Testing, also known as microbial
limit testing, is performed on pharmaceutical
products and medical products for quality
control purposes.
 Bioburden is generally expressed as CFU/mL
(Colony Forming Units)

3.  The development of a microbial contamination control program
is critical to the effort to get a new facility qualified, and to
maintain the facility in a state of control once qualified.
 To determine the total number of viable microorganisms in or on
a medical device, container or component after completion of all
in-process steps before sterilization.
 To act as an early warning system for possible production
problems which could lead to inadequate sterilization or possible
product recall.
 To calculate the necessary dose for effective radiation
sterilization and to monitor product to ensure adequate dosing.
 To test the effectiveness of cleaning agent against bacteria.
 Act as an indicator of manufacturing condition. The purpose of
environmental monitoring is to correct problems before product
is placed at risk.

4.  The Legal basis for Bioburden testing lies in CFR 21
(Code of Federal Register 21) and ISO 11737 worldwide.
 21 C.F.R. 211.110 (a)(6) states that bioburden in-process
testing must be conducted pursuant to written
procedures during the manufacturing process of drug
products.
 Current good manufacturing practice (CGMP)
requirements as specified in 21 CFR Part 211.113, Control
of Microbiological Contamination, state that “Appropriate
written procedures, designed to prevent objectionable
microorganisms in drug products not required to be sterile,
shall be established and followed.”
 The United States Pharmacopeia (USP) outlines several
tests that can be done to quantitatively determine the
Bioburden of non-sterile drug products.
 It is important when conducting these tests to ensure that
the testing method does not either introduce bacteria into
the test sample or kill bacteria in the test sample.

16. Raw material: 1) warehouse
2) sampling equipment
3) sampling personal
4) sampling environment
Dispensing: 1) dispensing equipment
2) dispensing personal
3) dispensing equipment
Manufacturing:1) equipment
2) air system
3) no. of personnel
Packaging : 1) primary packaging
2) secondary packaging

17. 1. Types of Non-viable contamination:
2. Types of Viable contamination:
Fibers Particles
Clothing Dead skin
Papers Dandruff
Hair Tobacco smoke
Cleaning equipment
Microorganism Insects & pest
bacteria Rodent
Fungi Cockroach
Viruses mice

18.  Water is a frequent source of endotoxins
and bioburden, which can carry Gram
negative bacteria. It is therefore
important to examine process water
usage.
 These arise from several mfg or
processing steps- for example from
extrusion, grinding, milling or cleaning
processes, etc.

20. 1. Air-borne bioburden- organisms found
in critical environment.
2. Water-borne bioburden- due to use of
purified water as an ingredient or during
processing steps.
3. Surface-borne bioburden- organisms
present on surface of particular
equipments and devices.
4. Personnel bioburden- arising on
account of improper personnel hygiene,
clothing, cleanliness or sanitation.

21. 1. Airborne particulate bioburden:
2. Surface bioburden:
CLASS OF
AREA
ACTION
LEVEL
ALERT LEVEL
I 1 CFU 1 CFU
II 10 CFU 5 CFU
III 25 CFU 15 CFU
CLASS OF AREA ACTION LEVEL ALERT LEVEL
I 1 CFU 1 CFU
II 10 CFU 5 CFU
III 25 CFU 15 CFU

22. 3. Personnel Bioburden:
MATERIAL ACTION LEVEL ALERT LEVEL
Gloves ≥ 5 CFU/plate ≥ 2 CFU/plate
Gown ≥ total 30 CFU/ 3 plates ≥ total 20 CFU/3 plates

23. LEVEL ROOM :NOT IN
USE
ROOM:PRIO
R TO USE
ROOM: IN USE
I Once a week 3 days CL: once before start, once
during process, once after
finishing.
II Once a week 3 days SL: once during process &
once a week.
III Once every 2
weeks
3 days SL: once a day
OL: once every 2 weeks
CL: critical locations
SL: selected locations
OL: other locations

25. 1. Sampling
2. Extraction methods
3. Enumeration procedures
4. Incubation

26.  General sampling strategies and
characteristics:
The method of obtaining samples for bioburden
influences the test results. Preferred method is
to obtain random samples.
ASCEPTIC: Be clean as possible. The method of
sampling itself should not contribute to
microbial contamination, else it gives faulty
results.
RANDOM: Random samples are taken from each
area of production room and not just from one
specific location.

27. REPRESENTATIVE: Units sampled for
testing should represent each phase of
production process.
REJECTED SAMPLES: rejected samples
may be used for laboratory testing if they
have been through the same process that
the normal units have undergone.
SIMULATED PRODUCT: Special units
used for sampling made exactly similar
to the original product via the same
processing. Such units are used due to
expensive nature of original product.

28.  Goal of sampling:
• It will help to determine whether
microorganism present at various places is
affecting the individual or preparation.
• Sampling is use to identify the location of
microbes, because they are collected from
different spaces.
• It will help in identification of microorganism.
Sr.no Various system Sample site
1. Environmental
air
Near open door and/or
filled container.
2. Room air Proximal to work area.
3. Water Point of use.
4. operator Finger impression

29. sampling
Air
sampling
Culturable Non-
Culturable
Water
sampling
Nichrome
wire loop
sampling
Surface
sampling
Tape lift
surface
sampling
Rodac plate
method
Cotton
swabbing

30.  This method mainly determine the quality of
parenteral processing environment,
 It provides the information about the type of
microbes present in air.
a) Culturable air sampling: Requires culture
medium for the growth of microbes.
 slit to agar sampler
 Anderson technique
 Surface air system
 The gelatin membrane system
 Sterilization microbial sampling
 Liquid impingers

31. ANDERSON
AIR SAMPLER
GELATIN-
MEMBRANE
CULTURE SYSTEM

32. LIQUID IMPINGER
AIR SAMPLER

33. b) Non Culturable air sampling:
 In this method, measured volume of
air is passed through sampling devices.
The collector of air has a sticky surface of
glass where microbes get stick and
trapped which are further analyzed.
 Hence no use of culture medium.
 The efficiency of air sampling technique
depends on :
 The design of sampler
 Sampling rate and volume of air taken
 The nature of collection medium
 Sampling time

34.  Nichrome wire loop method-
• In this method nichrome wire loop is used which
has rounded tip and by using this tip, sample is
collected and observed under microscope or put in
a specific growth medium.
• Generally, microbiologists use inoculating loops
to transfer microorganisms to growth media.
• Loop consists of a three inch long, 25 gauge
nichrome wire with a loop at one end and
mounted on an eight inch long aluminum handle.
• It is easy to sterilize and reuse because nichrome
wire resists deterioration with repeated
heat/cooling cycles.

36. It includes the sampling from the floors, walls, machinery,
equipment, rooms etc.
a. Rodac plate method
(RODAC- Replicate Organism Detection and Counting)
In this method, a 100mm diameter agar contact plate is used in
which agar is poured. Then this plate is pressed against a flat
surface, microbes will stick to the medium.
Instead of agar plate, nylon plates are also used.
b. Cotton swab method
It can be collected by cotton Q-tip applicator that has been moist
with growth media and than send to testing laboratory.
Disadvantage is the presence of cotton fibers on its surface.
c. Tape lift surface sampling
In this cotton adhesive tape is positioned on the surface and press
gently without rubbing, and then directly observed under
microscope.

37. RODAC
PLATES
TAPE-LIFT SURFACE
SAMPLING
COTTON SWAB

38.  After sample collection, it is required that the
sample is extracted from the sampler and
transferred to a suitable medium for further
growth of microbes and analysis.
 Methods:
1. Ultrasonicating
2. Vortex mixing
3. Blending
4. Shaking
5. Agar overlaying

39. After extracting the sample, growth of microbes on
suitable media to count them.
A. Membrane Filtration
B. Plate count methods
i. Pour-plate method
ii. Surface spread method
C. Serial dilution method

41.  Membrane filters used are 50 mm in
diameter, having a nominal pore size of
0.45 µm for retaining bacteria.

44.  Suitable incubating conditions are required for
growth of microbes.

45.  The bioburden validation is a test to determine
the efficacy of a method that is used to estimate
the bioburden on the product.
 With the results of bioburden validation a
correction factor is calculated which is used in
the estimation of the product’s bioburden.
45

46.  EN-ISO 11737-1 describes two general methods
for the BV:
1. Repetitive(Exhaustive) Recovery Method
2. Inoculation Method
46

47.  In this method the extraction procedure on a single
sample product is to be repeated until there is no
significant increase in the number of recovered
microorganims.
 The goal is to recover all viable microorganisms by
washing the sample product repeatedly.
 The counts in CFU that are recovered from the first
extraction are compared to the total counts
recovered from all the washes to calculate a
percent recovery when doing just one extraction.
47

48.  The percent recovery is used to calculate a
correction factor which is then applied to the
bioburden test numbers for the product.
 In this way routine bioburden tests require
only one extraction.
 The CFU recovered from one extraction are
then simply multiplied by the correction factor
to determine the total bioburden of a product.
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49.  In this method a sterile product is inoculated
with a known amount of viable
microorganisms in order to create an artificial
bioburden.
 After inoculation, the product is allowed to dry
for a defined period of time.
 Once the inoculum has dried, the chosen
method for extracting the microorganisms from
the product is applied.
49

50.  A ratio of recovered titer to initial inoculum
count establishes the recovery efficiency and
correction factor for the product.
 Disadvantage of this method is that sterile
samples are required and viability of
microorganisms after drying process can not be
guaranteed.
50

51.  EN-ISO 11737-1 describes a number of methods that
can be combined in order to obtain the best result in
recovering the bioburden from the product.
 Elution methods for releasing of bioburden from the
product into a rinsing fluid:
1. Stomaching
2. Ultra Sonication
3. Shaking
4. Vortex Mixing
5. Flushing
6. Blending
7. Swabbing
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52.  Non elution methods for estimation of
bioburden:
1. Contact Plating
2. Agar Overlaying
 For the transfer into culture medium different
methods like membrane filtration, pour
plating, spread plating or spiral plating can be
applied.
 Best method for a specified product is
determined by the BV.
52

53.  For estimation of bioburden, mainly two
medias are used:
1. Tryptone Soya Agar(TSA)- for aerobic m.o.
with incubation of 3-5 days at 32.5 ºC ± 2.5ºC.
2. Sabouraud Dextrose Agar(SDA)- for yeast and
mould with incubation of 5-7 days at 22.5ºC ±
2.5ºC.
53

55.  When the population of microorganism is
subjected to a sterilization process, all the cells do
not die at the same time.
 The no. of surviving cells decreases exponentially
with time of exposure until viable organism can no
longer be detected. So in order to find out the
efficiency of the sterilization process some
terminologies are employed i.e.
1) D value
2) Z value
3) F value
4) Inactivation factor
1)D VALUE
 The resistance of a given organism to any specified
killing process can be characterized by the D value.
 This is the time in minutes required to reduce the
no. of organisms by 90% i.e. upto10% of the original
count.

56.  Sterilization by heating in an autoclave or by
dry heat: the D value is expressed by time in
minutes at defined temperature. The
temperature is shown as subscript Ex. D₁₂₁,
D₁₇₀.
 Sterilization by exposure to ionizing
radiation: the D value is expressed by
absorbed dose.
 Sterilization by exposure to ethylene oxide:
the D value is expressed by time in minutes.
 The D value is mathematically shown by
following equation
D = U/log N₀−log Nu
Here, U = exposure time
N₀ = initial microbial population
Nu = microbial population after receiving U
time.

57. 2) Z VALUE( THERMAL DESTRUCTION VALUE):
 Z value relates the heat resistance of a microorganism to
change in temperature.
 It is the total degrees of temperature change to produce a 10
fold reduction in D value or the temperature change required
for 1 log reduction in D-value.
 Z-value is obtained from the plot of log D value vs
temperature.
Z= T2-T1/ log D1-log D2
 Bacterial spores have Z value in range 10 to 15ºC while most
non- sporing organisms have Z value of 4 to 6ºC.
3) F VALUE
 It is the time in minutes required to kill an organism at
250ºF(121ºC)
 Thus if sterilization process said to have F value of 15min. It
implies that it has the same lethal effect on a given organism as
that of heating at 121ºC for 15min.
 F value is a measure of the lethality of total process of
sterilization.
F₀ = ∆t ∑ 10(T-To)/Z
Where, ∆t is time interval for measurement of product tempT
And To is the reference temperature
Z = 10⁰C F₀ = total lethality

58. 4) INACTIVATION FACTOR
 It is the amount by which a given
combination of temperature ,radiation dose
rate etc and time of exposure will reduce
the no. of survivors of a given organism.
 It is calculated from known D value of
organism as follows
 Inactivation factor = 10(t/D)
Where, t is the treatment time
D is D-value at that same time.
 The lower the contamination rate and
higher the inactivation factor of a
sterilization process, less is the risk of
failure.

59. Here: B=No. of org. surviving after sterilization
A= Initial no. of micro-organisms
Ft = Equivalent exposure time
Dt=Log reduction microbial contamination(D-
value)
It is desirable that ‘B’ should be as low as
possible:-
1. By reducing Bioburden on bulk product (A)
2. Increasing the exposure time (Ft)
3. Employing micro-org. with a lower D-value at
specified temperature.

60.  A pharmaceutical product has bioburden of
378 CFU. For how much time should it be
sterilized with D-value(121°C) of 1.5 min/log
so as to assure SAL of 106 ?
60

61. 61

62. Equations and Examples
62

63.  Product Contamination:
Batch bioburden= sample bioburden * (batch volume/
sample volume)
 Equipment Contamination:
1. Contact surface Bioburden= sample bioburden *
(contact area/ sample area)
2. Contact surface Bioburden=Rinse fluid * sample
bioburden (Batch volume/ rinse volume)
 Environmental Contamination:
1. Fraction contaminated= CFU * (area of opening of
container/ area of settle plate) * (time contamination
exposed/ time settle plate exposed)
2. Contamination rate= 1/ fraction contamination
63

64. 64
Total
Contamination
Risk
Product
Contamination
Equipment
Contamination
Environmental
Contamination

65.  For airborne
bioburden at max of
12 inch upstream
from point of use.
 For surface bioburden
measured per 25 sq
cm.
 For personnel
bioburden exit test.
65
Level Action
Level
Alert
Level
I 1 CFU 1 CFU
II 10 CFU 5 CFU
III 25 CFU 15 CFU
Action
Level
Alert Level
Gloves 5
CFU/plate
2
CFU/plate
Gown 30 CFU/3
plates
20 CFU/ 3
plates
Airborne and Surface
Personnel

66.  A product xyz was tested for bioburden. Batch
size is 10,000 bottles. Find the contamination
per bottle.
66
Ingredient Quantity (g) Bioburden per g
(CFU)
Active ingredient 5000 10
Preservatives 500 10
Vehicle 87400 10
Excipients 100 500

67. 67
Ingredient Quantity (g) Bioburden per g
(CFU)
Total Bioburden
(CFU)
Active
ingredient
5000 10 50,000
Preservatives 500 10 5000
Vehicle 87,400 10 8,74,000
Excipients 100 500 50,000
Grand Total 979,000
Contamination per bottle= 979,000/ 10,000≈ 98 CFU

68.  For, a container with neck area of 0.8 sq cm is
open during filling for
(a) 10 min
(b) 1 sec
and a 14 cm plate(area= 154 sq cm) is exposed
adjacent in 4 hrs, 2 microorganisms are found
to have deposited on the settle plate. Find the
contamination rate for both (a) and (b).
68

69. (a)
 Fraction contaminated
= 2* (0.8/ 154)* (10/4*60)
= 0.00043
 Contamination rate
= 1/ 0.00043
=2325.58
≈2326
(b)
 Fraction contaminated
= 2* (0.8/ 154)*
[(1/60)/(4*60)]
= 7.215 * 10-7
 Contamination rate
= 1/ 7.215 * 10-7
=1.38 * 108
≈ 1.4 * 108
69

70.  A 125 litre capacity blender has internal surface
area of 2356 sq cm. Surface bioburden was
found to be 33 CFU per 25 sq cm. Calculate the
contamination risk per 100 ml of product
mixed in the blender.
70

71.  Surface contamination
= 33 * ( 2356/ 25)
= 3109.92 CFU
 Contamination risk per 100 ml
= (100/ 125000) * 3109.92
=2.48
≈ 3 CFU
71

72.  Scott Sutton, PhD; “Bioburden Contamination Control: A Holistic Overview”;
American Pharmaceutical Review; Endotoxin Supplement; July/Aug 2015;
volume 18, issue 5.
 Microbiological Validation according to EN-ISO 11137-2:2012, Method VDmax
25
Initial Validation; Pharma Help Bag; Synergy Health.
 “Bioburden: Characterization, Method Validation and Determination”; Eurofins.
 “The Microbial Bioburden of USP 797 Compliance”; Simplifying Environmental
Quality and Control Practices for Pharmaceutical Compounding; PathCon
Laboratories; Fall 2009.
 Scott Sutton, PhD; “The Role of Bioburden in the Contamination-Control Plan”;
Equipment and Processing Report; Jan 19, 2011.
 Microbial Risk Assessment Guideline, Pathogenic Microorganisms with focus on
Food and Water; Prepared by the Interagency Microbiological Risk Assessment
Guideline Workgroup; USDA and FSIS; July 2012(001).
 S. P. Denyer, R. M. Baird; “Guide To Microbiological Control In
Pharmaceuticals”; Ellis Horwood Limited; England.
 “Glimpses of Pharma Profession: A compilation of presentations made at
different programs organized by IPA Vadodara Branch (2000-2001); The Indian
Pharmaceutical Association, Vadodara Branch.
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