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QUALITY CONTROL MICROBIOLOGY
Maggie Bryans Sheila Byrne John Hasyn
BIOMAN 2011 MiraCosta CollegeOceanview, California
The degree to which a set of inherent properties of a product, system or process fulfills requirements. FDA Guidance for Industry Q9 Quality Risk Management
Quality
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
Biopharm QC Testing Scheme
• Figure 7-2 P-257
“ Quality should be built into the product and testing alone cannot be relied on to ensure product quality.”
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
Environmental monitoring includes…..
1) Water and clean steam monitoring,2) Air monitoring- non-viable3) Air monitoring – viable4) Microbial surface testing using RODAC plates,5) Gown and fingertip RODAC testing.
Air Monitoring
Particulate (Non Viable) Air Monitoring
Cross sectionof a hair
How large is a human hair?How large is a particulate?
Guidelines for Clean Rooms
Federal Standard 2091963
First comprehensive guideline to clean room classification. English units.
FS 209 E1992
Fifth revision added metric or SI units
FS 209 Class 1 to 6
ISO 14644-1
ISO 14644-2
2001
International Society for Standardization
ISO Class 1 to 9
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
Particles/ft3
Class
1 35 7.5
3 1
Class
10 350 75
30 10
Class
100
750
300 100
Class
1000
1,000 7
Class
10,000
10,000 70
Class
100,000
100,000 700
Selected Equivalent Classes
FS 209 Classes
Class
1
Class
10
Class
100
Class
1,000
Class
10,000
Class
100,000
ISO 14644-1 Classes
Class
3
Class
4
Class
5
Class
6
Class
7
Class
8
ISO 14644-1 Class is equivalent to FS 209 Class above it.
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
Types of Particle Counters
Portable Particle Counter
Facilities Maintenance System
Microbial Air Monitoring
• Passive - Settle plates are exposed for specified time period.
• Active - Electric pump draws preset sample volume of air onto nutrient media plate.
Pharmaceutical Applications
• Trend analysis of aseptic filling areas
• Determine microbiological quality of laminar flow hood air
• Assess decontamination procedures
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
FDA Guidance For Aseptic Processing
FS 209CLASS
ISOCLASS
>0.5 PARTICLES/m3
ACTION LEVELS
cfu/m3
100 5 3520 1
1000 6 35200 7
10,000 7 352000 10
100,000 8 3520000 100
RODAC Plate
0
20
40
60
80
100
120
140
Test#1
Test#2
Test#3
Test#4
Test#5
Test#6
Test#7
Action Level: a test result that is ___?__ .
Alert Level: indicates ___?__.
Passing Level: are __ ? __ results.
Acceptable / Action / Alert - LevelsEnvironmental Monitoring Testing Results
Alert Level
Action Level
Knowledge Management
ICH Q10
Microbial Identification
What Do We Identify?
- Bacteria- Yeast- Mold
What Is An Identification?
Determination of the genus and species, e.g. Escherichia coli
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
Identification Methods / Systems (Phenotypic Methods)
• Bacteria• Conventional Method• Standardized Identification Systems• Automated Identification Systems
Conventional Method
• Colony morphology and Gram stain• Series of biochemical tests • Read reactions • Refer to Bergey’s Manual
Colony Morphology
Size, shape, texture, and color
Biochemical Tests
• Fermentation of carbohydrates
• Production of catalase• Production of indole • Production of hydrogen
sulfide gas
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
Standardized Identification Systems
• API Strips®• Enterotube®
Miniaturized biochemical tests
API Strips®
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
API® Strips
Benefits• Convenient• Easy to use• Low cost per ID
($6)
Limitations• Small database• Subjective• Dependent on the
bacteria’s ability to use the biochemicals
Automated Identification Systems
• Vitek®• Biolog®
• Molecular Microbiology (genotypic methods) is the wave of the future.
• No single method or system is ideal for all identifications
Genotypic Methods
Endotoxin Testing
What is it? A lipopolysaccharide
Where does it come from? The outer membrane of Gram negative bacteria.
Endotoxin
Which products are tested?
• injectable drugs and medical devices which will contact blood or spinal fluid
• includes raw materials, water and in process monitoring
Endotoxin Testing
The USP now recognizes two tests –
• The Pyrogen Test conducted with rabbits
• Bacterial Endotoxins Test, also termed the Limulus Amebocyte Lysate (LAL) Test.
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.
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
Types of LAL Tests
• Gel Clot
• Turbidimetric
• Colorimetric
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.
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.
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.
Comparison of Methods
Gel Clot ChromogenicEndpoint
ChromogenicKinetic
Turbidimetric
Semi-quantitative
Quantitative Quantitative Quantitative
Simple, Least expensive,Requires 37°C bath
Requiresspectrophotometeror plate reader
Requiresincubating plate or tube reader
Requiresincubating plate or tube reader
Manually read and recorded
Manual or can be automated,allows electronicdata storage
Is automated,allows electronicdata storage
Is automated,allows electronicdata storage
Sensitive downto 0.03 EU/ml
Sensitive down to 0.1 EU/ml
Sensitive down to .005 EU/ml
Sensitive down to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)