Bioburden

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. Bioburdenisthepopulationofviable
microorganismonaparticularobject,
formulationand/orfinishedproduct.
Itisthenumberofbacterialivingonasurface
thathasnotbeensterilized.
BioburdenTesting,alsoknownasmicrobial
limittesting,isperformedonpharmaceutical
productsandmedicalproductsforquality
controlpurposes.
BioburdenisgenerallyexpressedasCFU/mL
(ColonyFormingUnits)

3. Thedevelopmentofamicrobialcontaminationcontrolprogram
iscriticaltotheefforttogetanewfacilityqualified,andto
maintainthefacilityinastateofcontroloncequalified.
Todeterminethetotalnumberofviablemicroorganismsinoron
amedicaldevice,containerorcomponentaftercompletionofall
in-processstepsbeforesterilization.
Toactasanearlywarningsystemforpossibleproduction
problemswhichcouldleadtoinadequatesterilizationorpossible
productrecall.
Tocalculatethenecessarydoseforeffectiveradiation
sterilizationandtomonitorproducttoensureadequatedosing.
Totesttheeffectivenessofcleaningagentagainstbacteria.
Actasanindicatorofmanufacturingcondition.Thepurposeof
environmentalmonitoringistocorrectproblemsbeforeproduct
isplacedatrisk.

4. TheLegalbasisforBioburdentestingliesinCFR21
(CodeofFederalRegister21)andISO11737worldwide.
21C.F.R.211.110(a)(6)statesthatbioburdenin-process
testingmustbeconductedpursuanttowritten
proceduresduringthemanufacturingprocessofdrug
products.
Currentgoodmanufacturingpractice(CGMP)
requirementsasspecifiedin21CFRPart211.113,Control
ofMicrobiologicalContamination,statethat“Appropriate
writtenprocedures,designedtopreventobjectionable
microorganismsindrugproductsnotrequiredtobesterile,
shallbeestablishedandfollowed.”
TheUnitedStatesPharmacopeia(USP)outlinesseveral
teststhatcanbedonetoquantitativelydeterminethe
Bioburdenofnon-steriledrugproducts.
Itisimportantwhenconductingtheseteststoensurethat
thetestingmethoddoesnoteitherintroducebacteriainto
thetestsampleorkillbacteriainthetestsample.

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 Deadskin
Papers Dandruff
Hair Tobacco smoke
Cleaning equipment
MicroorganismInsects & pest
bacteria Rodent
Fungi Cockroach
Viruses mice

18. Wateris 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 ≥ total30CFU/ 3 plates ≥ total 20 CFU/3plates

23. LEVELROOM :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 every2
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 strategiesand
characteristics:
Themethodofobtainingsamplesforbioburden
influencesthetestresults.Preferredmethodis
toobtainrandomsamples.
ASCEPTIC:Becleanaspossible.Themethodof
samplingitselfshouldnotcontributeto
microbialcontamination,elseitgivesfaulty
results.
RANDOM:Randomsamplesaretakenfromeach
areaofproductionroomandnotjustfromone
specificlocation.

27. REPRESENTATIVE:Unitssampledfor
testingshouldrepresenteachphaseof
productionprocess.
REJECTEDSAMPLES:rejectedsamples
maybeusedforlaboratorytestingifthey
havebeenthroughthesameprocessthat
thenormalunitshaveundergone.
SIMULATED PRODUCT:Specialunits
usedforsamplingmadeexactlysimilar
totheoriginalproductviathesame
processing.Suchunitsareuseddueto
expensivenatureoforiginalproduct.

28. Goalofsampling:
•Itwillhelptodeterminewhether
microorganismpresentatvariousplacesis
affectingtheindividualorpreparation.
•Samplingisusetoidentifythelocationof
microbes,becausetheyarecollectedfrom
differentspaces.
•Itwillhelpinidentificationofmicroorganism.
Sr.noVarious 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 Fingerimpression

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. Thismethodmainlydeterminethequalityof
parenteralprocessingenvironment,
Itprovidestheinformationaboutthetypeof
microbespresentinair.
a)Culturableairsampling:Requiresculture
mediumforthegrowthofmicrobes.
slittoagarsampler
Andersontechnique
Surfaceairsystem
Thegelatinmembranesystem
Sterilizationmicrobialsampling
Liquidimpingers

31. ANDERSON
AIRSAMPLER
GELATIN-
MEMBRANE
CULTURE SYSTEM

32. LIQUID IMPINGER
AIR SAMPLER

33. b)NonCulturableairsampling:
 Inthismethod,measuredvolumeof
airispassedthroughsamplingdevices.
Thecollectorofairhasastickysurfaceof
glasswheremicrobesgetstickand
trappedwhicharefurtheranalyzed.
Hencenouseofculturemedium.
Theefficiencyofairsamplingtechnique
dependson:
Thedesignofsampler
Samplingrateandvolumeofairtaken
Thenatureofcollectionmedium
Samplingtime

34. Nichromewireloopmethod-
•Inthismethodnichromewireloopisusedwhich
hasroundedtipandbyusingthistip,sampleis
collectedandobservedundermicroscopeorputin
aspecificgrowthmedium.
•Generally,microbiologistsuseinoculatingloops
totransfermicroorganismstogrowthmedia.
•Loopconsistsofathreeinchlong,25gauge
nichromewirewithaloopatoneendand
mountedonaneightinchlongaluminumhandle.
•Itiseasytosterilizeandreusebecausenichrome
wireresistsdeteriorationwithrepeated
heat/coolingcycles.

36. Itincludesthesamplingfromthefloors,walls,machinery,
equipment,roomsetc.
a.Rodacplatemethod
(RODAC-ReplicateOrganismDetectionandCounting)
Inthismethod,a100mmdiameteragarcontactplateisusedin
whichagarispoured.Thenthisplateispressedagainstaflat
surface,microbeswillsticktothemedium.
Insteadofagarplate,nylonplatesarealsoused.
b.Cottonswabmethod
ItcanbecollectedbycottonQ-tipapplicatorthathasbeenmoist
withgrowthmediaandthansendtotestinglaboratory.
Disadvantageisthepresenceofcottonfibersonitssurface.
c.Tapeliftsurfacesampling
Inthiscottonadhesivetapeispositionedonthesurfaceandpress
gentlywithoutrubbing,andthendirectlyobservedunder
microscope.

37. RODAC
PLATES
TAPE-LIFT SURFACE
SAMPLING
COTTON SWAB
TECHNIQUE

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. Membranefiltersusedare50mmin
diameter,havinganominalporesizeof
0.45µmforretainingbacteria.

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

45. Thebioburdenvalidationisatesttodetermine
theefficacyofamethodthatisusedtoestimate
thebioburdenontheproduct.
Withtheresultsofbioburdenvalidationa
correctionfactoriscalculatedwhichisusedin
theestimationoftheproduct’sbioburden.
45

46. EN-ISO11737-1describestwogeneralmethods
fortheBV:
1.Repetitive(Exhaustive)RecoveryMethod
2.InoculationMethod
46

47. Inthismethodtheextractionprocedureonasingle
sampleproductistoberepeateduntilthereisno
significantincreaseinthenumberofrecovered
microorganims.
Thegoalistorecoverallviablemicroorganismsby
washingthesampleproductrepeatedly.
ThecountsinCFUthatarerecoveredfromthefirst
extractionarecomparedtothetotalcounts
recoveredfromallthewashestocalculatea
percentrecoverywhendoingjustoneextraction.
47

48. Thepercentrecoveryisusedtocalculatea
correctionfactorwhichisthenappliedtothe
bioburdentestnumbersfortheproduct.
Inthiswayroutinebioburdentestsrequire
onlyoneextraction.
TheCFUrecoveredfromoneextractionare
thensimplymultipliedbythecorrectionfactor
todeterminethetotalbioburdenofaproduct.
48

49. Inthismethodasterileproductisinoculated
withaknown amount ofviable
microorganismsinordertocreateanartificial
bioburden.
Afterinoculation,theproductisallowedtodry
foradefinedperiodoftime.
Oncetheinoculumhasdried,thechosen
methodforextractingthemicroorganismsfrom
theproductisapplied.
49

50. Aratioofrecoveredtitertoinitialinoculum
countestablishestherecoveryefficiencyand
correctionfactorfortheproduct.
Disadvantageofthismethodisthatsterile
samplesarerequiredandviabilityof
microorganismsafterdryingprocesscannotbe
guaranteed.
50

51. EN-ISO11737-1describesanumberofmethodsthat
canbecombinedinordertoobtainthebestresultin
recoveringthebioburdenfromtheproduct.
Elutionmethodsforreleasingofbioburdenfromthe
productintoarinsingfluid:
1.Stomaching
2.UltraSonication
3.Shaking
4.VortexMixing
5.Flushing
6.Blending
7.Swabbing
51

52. Nonelutionmethodsforestimationof
bioburden:
1.ContactPlating
2.AgarOverlaying
Forthetransferintoculturemediumdifferent
methodslikemembranefiltration,pour
plating,spreadplatingorspiralplatingcanbe
applied.
Bestmethodforaspecifiedproductis
determinedbytheBV.
52

53. Forestimationofbioburden,mainlytwo
mediasareused:
1.TryptoneSoyaAgar(TSA)-foraerobicm.o.
withincubationof3-5daysat32.5ºC±2.5ºC.
2.SabouraudDextroseAgar(SDA)-foryeastand
mouldwithincubationof5-7daysat22.5ºC±
2.5ºC.
53

55. Whenthepopulationofmicroorganismis
subjectedtoasterilizationprocess,allthecellsdo
notdieatthesametime.
Theno.ofsurvivingcellsdecreasesexponentially
withtimeofexposureuntilviableorganismcanno
longerbedetected.Soinordertofindoutthe
efficiencyofthesterilizationprocesssome
terminologiesareemployedi.e.
1)Dvalue
2)Zvalue
3)Fvalue
4)Inactivationfactor
1)DVALUE
Theresistanceofagivenorganismtoanyspecified
killingprocesscanbecharacterizedbytheDvalue.
Thisisthetimeinminutesrequiredtoreducethe
no.oforganismsby90%i.e.upto10%oftheoriginal
count.

56. Sterilizationbyheatinginanautoclaveorby
dryheat:theDvalueisexpressedbytimein
minutesatdefinedtemperature.The
temperatureisshownassubscriptEx.D₁₂₁,
D₁₇₀.
Sterilizationbyexposuretoionizing
radiation:theDvalueisexpressedby
absorbeddose.
Sterilizationbyexposuretoethyleneoxide:
theDvalueisexpressedbytimeinminutes.
TheDvalueismathematicallyshownby
followingequation
D=U/logN₀−logN
u
Here,U=exposuretime
N₀=initialmicrobialpopulation
Nu=microbialpopulationafterreceivingU
time.

57. 2)ZVALUE(THERMALDESTRUCTION VALUE):
Zvaluerelatestheheatresistanceofamicroorganismto
changeintemperature.
Itisthetotaldegreesoftemperaturechangetoproducea10
foldreductioninDvalueorthetemperaturechangerequired
for1logreductioninD-value.
Z-valueisobtainedfromtheplotoflogDvaluevs
temperature.
Z=T2-T1/logD1-logD2
BacterialsporeshaveZvalueinrange10to15ºCwhilemost
non-sporingorganismshaveZvalueof4to6ºC.
3)FVALUE
Itisthetimeinminutesrequiredtokillanorganismat
250ºF(121ºC)
ThusifsterilizationprocesssaidtohaveFvalueof15min.It
impliesthatithasthesamelethaleffectonagivenorganismas
thatofheatingat121ºCfor15min.
Fvalueisameasureofthelethalityoftotalprocessof
sterilization.
F₀=∆t∑10
(T-To)/Z
Where,∆tistimeintervalformeasurementofproducttempT
AndT
oisthereferencetemperature
Z=10⁰CF₀=totallethality

58. 4)INACTIVATION FACTOR
Itistheamountbywhichagiven
combinationoftemperature,radiationdose
rateetcandtimeofexposurewillreduce
theno.ofsurvivorsofagivenorganism.
ItiscalculatedfromknownDvalueof
organismasfollows
Inactivationfactor=10(t/D)
Where,tisthetreatmenttime
DisD-valueatthatsametime.
Thelowerthecontaminationrateand
highertheinactivationfactorofa
sterilizationprocess,lessistheriskof
failure.

59. Here: B=No. of org. surviving after sterilization
A= Initial no. of micro-organisms
F
t= Equivalent exposure time
D
t=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 (F
t)
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 10
6
?
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) Bioburdenper g
(CFU)
Active ingredient5000 10
Preservatives 500 10
Vehicle 87400 10
Excipients 100 500

67. 67
Ingredient Quantity (g) Bioburdenper g
(CFU)
Total Bioburden
(CFU)
Active
ingredient
5000 10 50,000
Preservatives500 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 * 10
8
≈ 1.4 * 10
8
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

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MicrobiologicalValidationaccordingtoEN-ISO11137-2:2012,MethodVD
max
25
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QualityandControlPracticesforPharmaceuticalCompounding;PathCon
Laboratories;Fall2009.
ScottSutton,PhD;“TheRoleofBioburdenintheContamination-ControlPlan”;
EquipmentandProcessingReport;Jan19,2011.
MicrobialRiskAssessmentGuideline,PathogenicMicroorganismswithfocuson
FoodandWater;PreparedbytheInteragencyMicrobiologicalRiskAssessment
GuidelineWorkgroup;USDAandFSIS;July2012(001).
S.P.Denyer,R.M.Baird;“GuideToMicrobiologicalControlIn
Pharmaceuticals”;EllisHorwoodLimited;England.
“GlimpsesofPharmaProfession:Acompilationofpresentationsmadeat
differentprogramsorganizedbyIPAVadodaraBranch(2000-2001);TheIndian
PharmaceuticalAssociation,VadodaraBranch.
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