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QUALITY CONTROL
MICROBIOLOGY
Maggie Bryans
Sheila Byrne
John Hasyn
BIOMAN 2011
MiraCosta College
Oceanview, California
Quality
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 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-viable
3) Air monitoring – viable
4) Microbial surface testing using RODAC plates,
5) Gown and fingertip RODAC testing.
Air Monitoring
Particulate (Non Viable)
Air Monitoring
How large is a human hair?
How large is a particulate?
Cross section
of a hair
Guidelines for Clean Rooms
Federal Standard 209
FS 209 E
ISO 14644-1
ISO 14644-2
1963
First comprehensive guideline to
clean room classification.
English units.
1992
Fifth revision added metric or SI
units
FS 209 Class 1 to 6
2001
International Society for
Standardization
ISO Class 1 to 9
Classification of Clean Rooms - Federal Standard 209
≥ 0.1µm
≥ 0.2µm
≥ 0.3µm
3
3
Particles/ft3 Particles/ft Particles/ft
≥ 0.5µm
Particles/ft3
Class
1
35
7.5
3
1
Class
10
350
75
30
10
750
300
100
Class
100
≥ 5.0µm
Particles/ft3
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 Class Class
10
100
1,000
Class Class
10,000 100,000
ISO
14644-1
Classes
Class
3
Class Class Class
4
5
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
Facilities Maintenance System
Portable Particle Counter
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 209
CLASS
ISO
>0.5
CLASS
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
Acceptable / Action / Alert - Levels
Environmental Monitoring Testing Results
Action Level:
a test result that is
___?__ .
140
120
Action Level
Alert Level: indicates ___?__.
100
80
Alert Level
60
Passing Level:
are __ ? __ results.
40
20
0
Test
#1
Test
#2
Test
#3
Test
#4
Test
#5
Test
#6
Test
#7
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
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•
•
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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
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•
•
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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®
API Strips®
Miniaturized biochemical tests
API Strips® - Method
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•
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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®
Genotypic Methods
•
Molecular Microbiology
(genotypic methods) is
the wave of the future.
•
No single method or
system is ideal for all
identifications
Endotoxin Testing
Endotoxin
What is it?
A lipopolysaccharide
Where does it come from? The outer
bacteria.
membrane of Gram
negat
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
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, paranitro 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
Chromogenic
Endpoint
Chromogenic
Kinetic
Turbidimetric
Semiquantitative
Quantitative
Quantitative
Quantitative
Simple, Least
expensive,
Requires 37°C bath
Requires
spectrophotometer
or plate reader
Requires
incubating plate or
tube reader
Requires
incubating plate or
tube reader
Manually read and
recorded
Manual or can be
automated,
allows electronic
data storage
Is automated,
allows electronic
data storage
Is automated,
allows electronic
data storage
Sensitive down
to 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)