Introduction to Pharmaceutical Microbiology

A brief intro to microbiology lab in pharmaceutical industry, scope, activities and career path of microbiologist.

1. Introduction to
Pharmaceutical Microbiology
Mohamed Abo-Elgheit
Pharmaceutical Microbiology Expert
Quality System Supervisor in Egypt Otsuka Pharmaceuticals
Microbiology Supervisor in Egypt Otsuka Pharmaceuticals
BS. Microbiology, 2010 -TQM Diploma, 2018
Email: [email protected]

2. Aim of this lecture
•To understand the role of microbiology lab in the pharmaceutical
industry.
•To get an overview on what is conducted in the pharmaceutical
microbiology lab
•To be familiar with microbiological tests.
•To get to know career path and qualifications of the
pharmaceutical microbiologist.
Mohamed Aboelgheit [email protected] 2

3. Content
•PART 1: Microbiology Lab
•PART 2: Activities in Microbiology Lab
•PART 3: To be a Microbiologist.
Mohamed Aboelgheit [email protected] 3

4. PART 1: Microbiology Lab
•What is Microbiology?
•What is Pharmaceutical Microbiology?
•Pharmaceutical Microbiology Lab
•Aseptic Technique
Mohamed Aboelgheit [email protected] 4

5. (1) What is Microbiology?
•Microbiology is the study of
microscopic microorganisms:
•Bacteriology(the study of bacteria)
•Mycology (the study of fungi)
•Phycology (the study of algae)
•Virology (the study of viruses)
Mohamed Abo-Elgheit
[email protected]
5

6. Microbiology in Tree of
Life
Mohamed Abo-Elgheit
[email protected]
6

7. Bacteria
•Bacteriaare
prokaryotic single cell
organisms
•Prokaryote == lack
nucleus, no nuclear
membrane around the
chromosomal DNA.
Mohamed Abo-Elgheit
[email protected]
7

8. Bacteria: Gram Stain and
Cell Wall
•Bacteriaare 2 groups:
•Gram +veBacteria:
•High peptidoglycan in cell
wall
•Retain the stain
•Gram –veBacteria:
•Low peptidoglycan
•High lipopolysaccharide
in cell wall
•Don’t retain the stain
Mohamed Abo-Elgheit
[email protected]
8

9. Bacteria: Gram Stain and
Cell Wall
Mohamed Abo-Elgheit
[email protected]
9
Staphylococcus
auresus
E.coli

10. Bacteria: Aerobic vs
Anaerobic
•Aerobic bacteria utilize oxygen to make energy.
•Anaerobic bacteria don’t require oxygen to make their
energy so they can survive without oxgen.
•Some anaerobic bacteria cannot survive in presence of
oxygen, also called “obligate anaerobic bacteria”
Mohamed Abo-Elgheit
[email protected]
10

11. Fungi
•Fungiare a group of eukaryotic living organisms that
include, but not limited to, yeasts and molds.
•Eukaryote == has a true nucleus
•Yeasts:single cell eukaryotic organisms
•Molds:multicellular filamentous eukaryotic organism.
Mohamed Abo-Elgheit
[email protected]
11

12. Fungi: yeast and molds
Mohamed Abo-Elgheit
[email protected]
12
Candidiaalbicans
Rhizopussp.

13. Normal Flora
•Normal Flora is present in:
•Skin e.g. Staph. aureus
•Upper respiratory tract e.g. Staph.aureus
•Oral Cavity e.g. Viridansstreptococcus
•Intestine e.gE.coli
•Vaginal Tract e.g. Lactobacillusspp
Mohamed Abo-Elgheit
[email protected]
13

14. (2) What is Pharmaceutical
Microbiology?
•Microbiologyis the biology branch
involved with the study of microscopic
organisms e.g. bacteria, fungi, algae,
viruses.
•Pharmaceutical Microbiology is an
applied sub-branch of microbiology
concerned with the study of
microorganisms associated with the
manufacture of pharmaceuticals.
Biology
Microbiology
Pharmaceutical
microbiology
Mohamed [email protected] 14

15. Pharmaceutical
Microbiology Scope
•Study of microorganisms associated with the
industry:
•control and monitor microorganism through the process to
ensure the safe manufacturing of pharmaceutical products:
•Microbiological Analysis Tests
•Environmental monitoring
Mohamed Aboelgheit [email protected] 15

16. Flowchart of Pharmaceutical
Manufacturing
Mohamed Aboelgheit [email protected] 16
Aseptic
Environment
control
Environment
control

17. (3) Microbiology Lab
Microbiology lab
Clean Areas
Washing Media
Preparation
Sterilization
Testing
Areas
MLT area
Sterility Area
BET Area
Live Culture Areas
Growth
Promotion
Strain maintenance
Microbial
Identification
Decontamination
Area
Mohamed Aboelgheit [email protected] 17

18. Microbiology lab Equipment
•Glassware
•Weighing
balance
•pH meter
•Filtration System
•Oven
Mohamed Aboelgheit [email protected] 18
•Autoclave
•Laminar Air Flow
•Safety Cabinet
•Incubator

19. Autoclave (steam
sterilization)
Mohamed Aboelgheit [email protected] 19
Steam Sterilization
•Media
•Glassware
•Gowns
Decontamination
•Incubated Cultures
•Contaminated items

20. Autoclave: mechanism
Mohamed Abo-Elgheit
[email protected]
20

21. Laminar Air Flow (LAF)
Mohamed Aboelgheit [email protected] 21

22. Laminar Air Flow: air
direction
Mohamed Abo-Elgheit
[email protected]
22

23. Microbiological Incubators
Mohamed Aboelgheit [email protected] 23
Bench Incubator
Walk-in Incubator

24. Good Manufacturing
Practices (GMP)
•Good Manufacturing Practices (GMP) are a set of
practices which ensures that pharmaceutical products
are consistently produced and controlled according to
the quality standards (WHO).
•Areas under GMP are: facilities, equipment,
production, process control, etc.
Mohamed Abo-Elgheit
[email protected]
24

25. Good Laboratory Practices
(GLP)
•Good Laboratory Practices (GMP) are a set of
practices which ensures reliability and reproducibility in
the lab.
•Areas under GLP: testing facilities, personnel training,
equipment, records, reports, etc.
Mohamed Abo-Elgheit
[email protected]
25

26. GLP in Microbiology Lab
•Personnel:trained,
qualified:
•Aseptic technique
•colony counting
•plate pouring
•media preparation, etc.
Mohamed Abo-Elgheit
[email protected]
26

27. GLP in Microbiology Lab
•Lab Environment:
•Separation of clean and dirty activities
•Dedicated spaces for samples, media, reference
microorganisms.
•Disinfection and sterilization can be effectively done
Mohamed Abo-Elgheit
[email protected]
27

28. (4) Aseptic Technique
•Bacteriaareeverywhere.Itisimportanttotake
measurestoavoidcontaminatingyourworkwith
undesiredbacteria.
Disinfection of working areas, and hand sanitation
Use of flames to kill bacteria that may enter vessels
openings.
Mohamed Aboelgheit [email protected] 28

29. Aseptic Technique
Mohamed Aboelgheit [email protected] 29

30. PART 2: Activities in
Microbiology lab
•Media Preparation
•Media Growth Promotion
•Microbiological Analysis Tests:
•Microbial Limit Tests
•Sterility Test
•Bacterial Endotoxin Test
•Viable Environmental Monitoring
•Rapid Microbiological Methods
Mohamed Aboelgheit [email protected] 30

31. (1) Media Preparation
Mohamed Abo-Elgheit
[email protected]
31

32. Media Preparation
Mohamed Aboelgheit [email protected] 32

33. Media Preparation
Weighing
Adding
water
Dissolving
SterilizationDispensingStorage
Mohamed Aboelgheit [email protected] 33

34. Media Preparation
Mohamed Abo-Elgheit
[email protected]
34

35. Agar Media
Mohamed Abo-Elgheit
[email protected]
35
•Solid media usually poured in plates
•Used for: enumeration, isolation and purification
•e.g. TSA, Mac. Agar, SDA, R2A, etc

36. Broth Media
•Liquid media usually dispensed in tubes, beakers, etc.
•Used for: enrichment
•e.g. TSB, FTM, Mac. broth,..etc.
Mohamed Abo-Elgheit
[email protected]
36

37. (2) Growth Promotion Test
•Growth promotion test is a QC test of prepared media to
ensure the media are able to enhance the microbial
growth as intended.
Mohamed Aboelgheit [email protected] 37
Inoculation
(> 100 CFU)
Incubation Inspection
Growth 50-
200%
Growth <50%
or >200%
PASS
FAIL

38. Reference microorganism
in GPT for TSA
ReferenceMicroorganism Incubationperiod and conditions
Staphylococcus aureusATCC 6538 30°–35°≤3 days
Pseudomonas aeruginosasuch ATCC 9027 30°–35°≤3 days
Bacillus subtilisATCC 6633 30°–35°≤3 days
Candida albicansATCC 10231 30°–35°≤5 days
AspergillusbrasiliensisATCC 16404 30°–35°≤5 days
Mohamed Abo-Elgheit
[email protected]
38

39. (3) Microbiological Tests
•Microbial Limit Test
•Enumeration test
•Test for specified microorganisms
•Sterility Test
•Bacterial Endotoxin Test
Mohamed Abo-Elgheit
[email protected]
39

40. (3.1) Microbial Limit Test
MLT
Microbial
Enumeration Test
Membrane
Filtration
Plate Count
Method
Test for specified
microorganism
Determination for
presence of absence
of objectionable
microorganism
Mohamed Abo-Elgheit
[email protected]
40

41. (3.1.1) Microbial Enumeration
Test (bioburden)
•In this test, a general solid (agar) media is used to
enhance the growth of all microorganisms in a sample and
hence these microorganisms can be enumerated.
•Thistestisconductedfor:
•Non-sterilefinishedproduct
•WaterSamples
•Intermediatestagesofsterileproducts
Mohamed Aboelgheit [email protected] 41

42. Microbial Enumeration Test:
Membrane Filtration
Mohamed Aboelgheit [email protected] 42

43. Microbial Enumeration Test:
Plate Count Methods
Mohamed Aboelgheit [email protected] 43
Sample

44. Membrane Filtration VS Pour
plate:think microbiologically!
Membrane Filtration Pour Plate
Sample sizeWholesample 0.1 –1.0 mL
Accuracy Higher Lower
Sample
form
Soluble samples
Sampleswith low
bioburden
Solubleor insoluble
Samples with high
bioburden
Tools Membrane filtration
system
MFS is not required
Mohamed Aboelgheit [email protected] 44

45. Microbial Enumeration
Test: Media
Mohamed Abo-Elgheit
[email protected]
45
Purpose Media
TotalAerobic
Microbial
Count (TAMC)
TSA (trypticsoy
Agar)
R2A
Total Yeast
and Mold
Count (TYMC)
SDA (Sabouraud
Dextrose Agar)

46. (3.1.2)Tests for Specified
Microorganisms
•In such test, a solid media is used to enhance the
growth of a specified microorganism e.g. test for E. coli
•Thistestisconductedfor:
•Non-sterilefinishedproduct
•WaterSamples
Mohamed Abo-Elgheit
[email protected]
46

47. Example: Tests for E.coli
Mohamed Abo-Elgheit
[email protected]
47
90 ml diluent
+ 10 gram
sample
1
TSB
10 ml
(= 1 g
sample)
Incubation
2
30 –35ᴼC
18-24
hrs.
4
1 ml
(Mac.Broth)
Incubation
42 –44ᴼC
24-48 hrs.
5
Streak
Incubation
30 –35ᴼC
18-24 hrs.
6
7
3

48. Test for specified
microorganism
Test Media used
Escherichiacoli MacConkeyAgar (MCA) 42-44 ⁰C
MacConkeyBroth (MCB) 30-35⁰C
Salmonellaspp. Rappaport(RVB) 30-35⁰C
PseudomonasaeruginosaCetrimideAgar 30-35⁰C
StaphylococcusaureusMannitolSalt Agar 30-35⁰C
Clostridiaspp. ReinforcedMedium for Clostridia 30-35⁰C
Columbia Agar 30-35⁰C
Candidaablicans SabouraudDextrose Broth (SDB) 30-35⁰C
SabouraudDextrose Agar (SDA) 30-35⁰C
Mohamed Aboelgheit [email protected] 48

49. MLT Acceptance Criteria
(USP, ch.1111)
Dosageform TAMCTYMC Specified microorganism
Nonaqueousoral use10
3
10
2
Absenceof E. coli
Aqueousoral use10
2
10
1
Absenceof E. coli
Rectal Use 10
3
10
2
NA
Cutaneoususe 10
2
10
1
Absenceof S. aureus
Absenceof P. aeruginosa
Vaginal use 10
2
10
1
Absenceof S. aureus
Absenceof P. aeruginosa
Absenceof C. albicans
Mohamed Abo-Elgheit
[email protected]
49

50. (3.2) Sterility Test
•Sterilitytest:isaqualitativetestforpresenceorabsenceof
microorganisms.(sterile==nomicrobialpresencedetected)
•Inthistest:abroth(liquid)mediaisusedtoenhancethe
growthofanymicroorganisminthesampleandhencethese
microorganismscanbedetectedasaturbidityintheculture
media.
Mohamed Aboelgheit [email protected] 50

51. Sterility Test
•Aseptictechniqueisverycriticalto
thistest,asasinglebacterialcell
growthfailsthetest.
•TheTestisdoneinClassAwith
backgroundClassB.
•Thistestisconductedfor:Sterile
finishedproduct.
Mohamed Aboelgheit [email protected] 51

52. Sterility Test: media
Media Used
Incubation
Conditions
Targeted microorganisms
TrypticSoy Broth
(TSB)
20 –25⁰C Aerobicbacteria and fungi
Fluid Thiglycolate
Medium
30 -35 ⁰C Anaerobicbacteria (mainly)
Mohamed Abo-Elgheit
[email protected]
52

53. Sterility Test
Mohamed Abo-Elgheit
[email protected]
53
Filter the
sample
solution
Incubate
for 14 days
Inspect
Clear Turbidity
PASS FAIL

54. Sterility Test: closed
system
Mohamed Aboelgheit [email protected] 54

55. Rapid Microbiological
Methods
Rapid Microbiological method are microbiological
testing method alternative to the traditional ones where
microorganism detection requires days or weeks.
Mohamed Aboelgheit [email protected] 55
Traditionalmethods Rapid methods
Upfrontcost Low Very high
Speed
Very low (daysor
weeks)
Real time / minutes
orhours
Sensitivity Low High

56. (3.3)Bacterial Endotoxin
Test (BET)
•Whengramnegativebacteriaarekilled,the
disintegrationofcellwallresultsin
lipopolysaccharideswhichcausefeverifintroduced
intotheblood.Thisgroupoflipopolysaccharides
producedasbyproductofcellwalldisintegrationare
knownasbacterialendotoxins.
Mohamed Aboelgheit [email protected] 56

57. Bacterial Endotoxin Test
(BET)
•Inthistest,aspecificreagentcalledLimulus
AmebocyteLysate(LAL)ismixedwiththe
sampletodetectorquantifytheendotoxin
content.
•Methods:GelclotBET&QuantitativeBET
•Thistestisconductedforsterileinjectable
solutionsandforwaterfeeds.
Mohamed Aboelgheit [email protected] 57

58. BET : Gel Clot Method
Mohamed Aboelgheit [email protected] 58
incubation at 37◦C
for 60 min
sample + LAL reagent (Gel) Firm Gel (+) i.e. Endotoxin detected
then inverting 180◦C
No firm gel (-) i.e. No endotoxin detected

59. BET: Quantitative Methods
Mohamed Aboelgheit [email protected] 59
sample + LAL reagent (Turb)
incubation at 37⁰C
Measuring the turbidity
Calculation of endotoxin amount using
the standard curve

60. (4) Viable Environmental
Monitoring
Mohamed Aboelgheit [email protected] 60

61. Viable Environmental
Monitoring
•Pharmaceuticalmanufacturingmustbeoperatedinair-
controlledroom,socalled“cleanrooms”.
•EMisconcernedwith:
•Nonviableparticlese.g.dustandfineparticles(0.5–5µm)
•Viableparticlese.g.bacteriaandfungi.
•ViableEMroleismonitoringnumberandtypesof
microorganismsintheair,onthesurfacesandwithinthe
personnelinthecleanrooms.
Mohamed Aboelgheit [email protected] 61

62. Clean Room Classes (WHO)
Grade 0.5 µm 5 µm Operation
A 3520 20 Filling of product
B 3520 29 ----
C 352000 2900 Preparation of solution
D 3520000 29000
Handling of components
after washing
Mohamed Abo-Elgheit
[email protected]
62

63. Viable Environmental
Monitoring
Mohamed Aboelgheit [email protected] 63
Viable EM
Air samples
Personnel samplesSurface
samples

64. Viable Environmental
Monitoring Techniques
CategorySampletitle Description Photo
Air
samples
Passive
Air
Sample
Settle Agarplates left
open for a specific
period in specific
locations
Active
Air
Sample
Using a pumpto
collect a specific
amount of air.
Mohamed Aboelgheit [email protected] 64

65. Viable Environmental
Monitoring Techniques
CategorySampletitle Description Photo
Surface
samples
Surface
swab
Using a cotton swab
to collect the
microbes from a
surface
Contact
plate
sample
platesfilled with
agar to the highest
level contacting the
surface of
examination.
Mohamed Aboelgheit [email protected] 65

66. Viable Environmental
Monitoring Techniques
Category Sampletitle Description Photo
Personnel
samples
Finger
plates
Agarplates contacting the
fingers of the personnel
hand
Gown
swab
Using a cotton swabto
collect the microbes from
the personnel gown
Mohamed Aboelgheit [email protected] 66

67. PART 3: To be a
Microbiologist or Not to be
•to be a pharmaceutical microbiologist
•Pharmaceutical Microbiologist Career Path
Mohamed Aboelgheit [email protected] 67

68. To be a pharmaceutical
microbiologist
•You must have BSc. in Microbiologyor in Pharmaceutical
Science or any relevant field.
•Love to work with microbes
•Detail-oriented
•Logical thinking
•Problem solving
Mohamed Aboelgheit [email protected] 68

69. Pharmaceutical
Microbiologist Career Path
•Lab Microbiologist
•Lab Supervisor/ Section Head/ Manager
•Microbiology Consultant
•Quality Control Manager
Mohamed Aboelgheit [email protected] 69

70. References
•WHOTechnicalreportno.961,2011:
•Annex2:WHOgoodpracticesfor
pharmaceuticalmicrobiologylaboratories
•Annex6:WHOgoodmanufacturing
practicesforsterilepharmaceutical
products
Mohamed Abo-Elgheit
[email protected]
70

71. References
•UnitedStatesPharmacopoeiaUSP:
•<1117>MicrobiologicalBestLaboratoryPractices
•<61>MicrobiologicalExaminationofNonsterile
Products:MicrobialEnumerationTests
•<62>MicrobiologicalExaminationofNonsterile
Products:TestsForSpecifiedMicroorganisms
•<71>SterilityTests
•<1111>MicrobiologicalExaminationofNonsterile
Products:AcceptanceCriteriaForPharmaceutical
PreparationsAndSubstancesForPharmaceuticalUse
•<85>BacterialEndotoxinsTest
Mohamed Abo-Elgheit
[email protected]
71

72. Other references
•British Pharmacopoeia (BP)
•Japanese Pharmacopoeia (JP)
•European Pharmacopoeia (Ph.Eur.)
•ISO Standards
Mohamed Abo-Elgheit
[email protected]
72

73. Mohamed Aboelgheit [email protected] 73