1. QUALITY CONTROL MICROBIOLOGY
Maggie Bryans
Sheila Byrne
John Hasyn
BIOMAN 2011
MiraCosta College
Oceanview, California 1

2. Quality
The degree to which a set of inherent properties of a product, system or process fulfills requirements FDA Guidance for Industry Q Quality Risk Management

3. Quality Control (QC):
Testing performed during biopharmaceutical manufacturing to verify that appropriate standards of product quality are attained
The actual organizational group (QC unit) who execute this work

4. Biopharm QC Testing Scheme
Figure 7-2 P-257

5. “ Quality should be built into the product and testing alone cannot be relied on to ensure product quality.”

6. Testing Performed by QC Microbiology
Environmental monitoring
Utility testing
Sterility testing
Microbial content testing
Bioburden
Microbial Limit
Bacterial Endotoxin (LAL)
Microbial identification
Antimicrobial Effectiveness Testing
Cleaning validation
Media fills

7. Environmental monitoring includes…..
1) Water and clean steam monitoring,
2) Air monitoring- non-viable
3) Air monitoring – viable
4) Microbial surface testing using RODAC plates,
5) Gown and fingertip RODAC testing.
Presenter: Review the slide.
Ask the question and continue to the next slide….

8. Air Monitoring Particulate (Non Viable) Air Monitoring
Quality Control in Biomanufacturing
Monday July 23rd, 2007
Air Monitoring
Particulate (Non Viable)
Air Monitoring
How large is a human hair?
How large is a particulate?
Cross section
of a hair
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9. Guidelines for Clean Rooms
Quality Control in Biomanufacturing
Monday July 23rd, 2007
Guidelines for Clean Rooms
Federal Standard 209
1963
First comprehensive guideline to clean room classification. English units.
FS 209 E
1992
Fifth revision added metric or SI units
FS 209 Class 1 to 6
ISO
ISO
2001
International Society for Standardization
ISO Class 1 to 9
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10. Classification of Clean Rooms - Federal Standard 209
Quality Control in Biomanufacturing
Monday July 23rd, 2007
Classification of Clean Rooms - Federal Standard 209
≥ 0.1µm
Particles/ft3
≥ 0.2µm Particles/ft3
≥ 0.3µm Particles/ft3
≥ 0.5µm Particles/ft3
≥ 5.0µm
Class 1 35
7.5 3 10
350 75 30
100
750
300
1000
1,000 7
10,000 70
100,000
700
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11. Selected Equivalent Classes
FS Classes
Class 1 10
100
1,000
10,000
100,000
ISO Classes 3 4 5 6 7 8
ISO Class is equivalent to FS 209 Class above it.

12. Particle Detection The validation of a clean room is ongoing
The air quality of a clean room must be monitored
An optical particle counter is used to monitor air quality
“Real-time” test results 12

13. 13

14. 14

15. Types of Particle Counters
Facilities Maintenance System
Portable Particle Counter

16. Microbial Air Monitoring
Quality Control in Biomanufacturing
Microbial Air Monitoring
Monday July 23rd, 2007
Passive - Settle plates are exposed for specified time period.
Active - Electric pump draws preset sample volume of air onto nutrient media plate.
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17. Pharmaceutical Applications
Quality Control in Biomanufacturing
Monday July 23rd, 2007
Pharmaceutical Applications
Trend analysis of aseptic filling areas
Determine microbiological quality of laminar flow hood air
Assess decontamination procedures
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18. Inspection of Agar Plate and Count
Total microbial count
Bacteria
Mold
The colonies are counted and reported as colony forming units (CFU) per cubic meter of air

19. FDA Guidance For Aseptic Processing
Quality Control in Biomanufacturing
Monday July 23rd, 2007
FDA Guidance For Aseptic Processing
FS 209
CLASS
ISO
>0.5
PARTICLES/m3
ACTION LEVELS
cfu/m3
100 5
3520 1
1000 6
35200 7
10,000
352000 10
100,000 8
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20. RODAC Plate

21. Acceptable / Action / Alert - Levels
Environmental Monitoring Testing Results
Action Level: a test result that is ___?__ .
Alert Level: indicates ___?__.
Passing Level: are __ ? __ results.
Action Level
Environmental testing results fall into one of three categories: Passing, Alert Level, or Action Level. Alert and Action Levels are assigned for each room classification and each type of test performed.
1) A Passing Level indicates that the result was within acceptable established levels.
2) An Alert Level indicates an excursion above the environmental norm for the site and is usually set at 10-50% of the Action Level. It is based upon historical statistical data, and is reevaluated on a yearly basis. (Note: An alert level is a passing result but we still need to investigate.) An Alert Level alerts you to a potential problem.
3) An Action Level is a test result that reaches or exceeds the area classification as established from industry/regulatory guidelines (ISO , Aseptic Guidelines, USP, etc.) and/or a statistical analysis of historical data. An Action Level indicates a possible problem and requires you to take action. An investigation is conducted to determine product impact and the root cause.
Reference: SOP X “Response to Alert and Action Levels” provides an outline of the actions taken for each result at the action or alert level.
Alert Level

22. Knowledge Management
ICH Q10

23. Microbial Identification
What Do We Identify?
- Bacteria
- Yeast
- Mold 23

24. What Is An Identification?
Determination of the genus and species, e.g. Escherichia coli 24

25. When Do We Identify?
When the # of microorganisms exceeds an acceptable level
Class 100
Class 10,000
When a microorganism is recovered from a presence/absence test 25

26. Identification Methods / Systems (Phenotypic Methods)
Bacteria
Conventional Method
Standardized Identification Systems
Automated Identification Systems 26

27. Conventional Method Colony morphology and Gram stain
Series of biochemical tests
Read reactions
Refer to Bergey’s Manual 27

28. Size, shape, texture, and color
Colony Morphology
Size, shape, texture, and color 28

29. Biochemical Tests Fermentation of carbohydrates Production of catalase
Production of indole
Production of hydrogen sulfide gas 29

30. Limitations of Conventional Method
Time consuming / labor intensive
Dependent on the bacteria’s ability to use the biochemicals
Requires a high level of technical knowledge 30

31. Standardized Identification Systems
API Strips®
Enterotube® 31

32. Miniaturized biochemical tests
API Strips®
Miniaturized biochemical tests 32

33. API Strips® - Method Gram stain Prepare a suspension of the bacteria
Inoculate with the suspension
Incubate strip
Read the pattern of reactions (color changes)
Refer to index 33

34. API® Strips Limitations Benefits Small database Convenient Subjective
Dependent on the bacteria’s ability to use the biochemicals
Benefits
Convenient
Easy to use
Low cost per ID ($6) 34

35. Automated Identification Systems
Vitek®
Biolog® 35

36. Genotypic Methods
Molecular Microbiology (genotypic methods) is the wave of the future.
No single method or system is ideal for all identifications 36

37. Endotoxin Testing 37

38. Endotoxin
What is it? A lipopolysaccharide Where does it come from? The outer membrane of Gram negative bacteria.

39. Endotoxin Testing Which products are tested?
injectable drugs and medical devices which will contact blood or spinal fluid
includes raw materials, water and in process monitoring 39

40. The USP now recognizes two tests –
The Pyrogen Test conducted with rabbits
Bacterial Endotoxins Test, also termed the Limulus Amebocyte Lysate (LAL) Test.

41. Pyrogen Assay
USP XIX considers a solution to be pyrogenic when 10 ml/kg is injected into a rabbit and there is a rise of temperature of 0.6 C or more for any rabbit, or a total rise of more than 1.4 C for three rabbits in a three rabbit test group.

42. LAL Test
Limulus amebocyte lysate test - based on clotting reaction of horseshoe crab (Limulus polyphemus) blood cell (amebocyte) lysate to endotoxin
Developed in 1960’s by Drs. Bang and Levin
Faster, more economical, more sensitive than rabbit pyrogen test 42

43. Types of LAL Tests Gel Clot Turbidimetric Colorimetric
All accepted by the FDA, USP, EP,& JP 43

44. Gel Clot Method Original method The official “referee test”
The specimen is incubated with LAL of a known sensitivity. Formation of a gel clot is positive for endotoxin. 44

45. Chromogenic Method
Endotoxin concentration is measured as a function of color intensity
LAL contains enzymes that are activated in a series of reactions in the presence of endotoxin. The last enzyme activated in the cascade splits the chromophore, para-nitro aniline (pNA), from the chromogenic substrate, producing a yellow color.

46. Turbidimetric Method
In the presence of endotoxin LAL becomes turbid and, under appropriate conditions,
forms a solid gel-clot.
In the kinetic turbidimetric LAL method, endotoxin concentration is measured as a function of either the rate of increase in turbidity or the time taken to reach a particular level of turbidity.

47. Comparison of Methods Gel Clot Chromogenic Endpoint Kinetic
Turbidimetric
Semi-
quantitative
Quantitative
Simple, Least expensive,
Requires 37°C bath
Requires
spectrophotometer
or plate reader
incubating plate or tube reader
Manually read and recorded
Manual or can be automated,
allows electronic
data storage
Is automated,
Sensitive down
to 0.03 EU/ml
to 0.1 EU/ml
to .005 EU/ml
to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions) 47