Transcript Quality Control Toolbox - Northeast Biomanufacturing

QUALITY CONTROL IN
BIOMANUFACTURING
Kevin Lampe, Sheila Byrne, Laura
Roselli, Melanie Lenahan and Linda
Rehfuss
BIOMAN 2007, Portsmouth, NH
Quality Control Toolbox
• Endotoxin Testing
• Air Monitoring
• Microbial
Identification
• Pipette
Calibration Check
Quality
Ensure purity- # 1 goal
Also identity and strength
Quality Assurance
• A unit that is part of the whole
• US CFR – United States Code of
Federal Regulations- defines the quality
unit’s job
• Approve or reject all materials
associated with production of any
product – includes containers,
components, labels, other packaging
materials
Quality Control (QC) within
QA
• QC is a testing function
• Defined as fitness for use
• Need to be integrated throughout
process
Endotoxin Testing
Endotoxin
What is it?
a lipopolysaccharide
Where does it come from?
the cell membrane of Gram negative bacteria
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
LAL Test
• Developed in 1960’s by Drs. Bang
and Levin
• Based on clotting reaction of
horseshoe crab blood to endotoxin
• Faster, more economical, more
sensitive than rabbit pyrogen test
Types of LAL Test
• 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.
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
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Can be 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)
Air Monitoring
Particle Counting
Clean Rooms
are separate environments designed to keep particle
contamination at known controlled levels.
• Pharmaceutical
Manufacture
• Microchip
Manufacture
Clean rooms
Types of possible particles
Inert
Viable
Controlled by
Filters and laminar flow
Gowning - “People are prolific particle
generators.”
Guidelines on Clean Rooms
Federal Standard 209
FS 209 E
ISO 14644-1
ISO 14644-2
First comprehensive
guideline to clean room
classification. English
units.
1963
Fifth revision added
metric or SI units
FS 209 Class 1 to 6
1992
International Society for
Standardization
ISO Class 1 to 9
2001
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
Classification of Clean Rooms
ISO 14644-1
CLASS
Number of Particles per Cubic Meter by Micrometer Size
0.1 µm
0.2 µm
0.3 µm
0.5 µm
ISO 1
10
2
ISO 2
100
ISO 3
1 µm
5 µm
24
10
4
1,000
237
102
35
8
ISO 4
10,000
2,370
1,020
352
83
ISO 5
100,000
23,700
10,200
3,520
832
29
ISO 6
1,000,000 237,000
102,000
35,200
8,320
293
ISO 7
352,000
83,200
2,930
ISO 8
3,520,000
832,000
29,300
ISO 9
35,200,000 8,320,000 293,000
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.
Portable Particle
Counter
Air Monitoring
Microbiological Air Testing
Pharmaceutical Applications
Trend analysis of aseptic filling areas
Determine microbiological quality of
laminar flow hoods
Assess decontamination procedures
Sample Collection Methods
Passive - Settle plates are exposed for > 1
hour.
Active - Electric pump draws preset
sample volume of air onto nutrient media
plate.
 After samples are
collected on nutrient
media, the plates are
incubated at 30-35
degrees C. for 48 hrs. to
promote growth of
bacteria, yeast and mold.
 The plate colonies are
counted and reported as
colony forming units 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
Microbial Identification
What Do We Identify?
Bacteria
Yeast
Mold
What Is An Identification?
Determination of the species - Escherichia coli
Where Do They Come From?
• Products
• Raw Materials /
Water
• Manufacturing
Environment
• Manufacturing
Personnel
When Do We Identify?
• When the # of microorganisms exceeds
an acceptable level
• When a microorganism is recovered
from a presence/absence test
Identification Methods / Systems
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
Gram Stain
• Gram stain
reaction
• Size and shape of
the cells
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®
API Strips®
Miniaturized biochemical tests
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
Limitations
• Convenient
• Easy to use
• Low cost per ID ($6)
• Small database
• Subjective
• Dependent on the
bacteria’s ability to
use the
biochemicals
Automated Identification Systems
• Vitek®
• Biolog®
Pipette Calibration

Why check
pipettes?_________________
________________________E
very scientist and laboratorian
understands the impact of
unnecessary inaccuracy
and imprecision on scientific
data yet pipettes and pipetting
technique tend to be the least
controlled process in the
laboratory.
Pipettes and GMP Compliance

Regulatory Guidelines 21 Code of
Federal Regulations Parts 210 and 211
The calibration of instruments... at suitable
intervals in accordance with an established
written program containing specific directions,
schedules, limits for accuracy/precision, and
provisions for remedial action in the event
accuracy and/or precision limits are not met.
Instruments... not meeting established
specifications shall not be used.2
The ARTEL PCS

In the case of the PCS
Pipette Calibration
System, a photometer
with extraordinarily low
noise is coupled with
NIST-traceable reagents
to measure liquid
delivery colorimetrically.
• Molecular
Microbiology is the
wave of the future.
• No single method or
system is ideal for
all identifications.
www.biomanufacturing.org
QUESTIONS ??
Minuteman Regional
High School