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Q U A L I T Y C O N T R O L F O R R E G U L AT E D B I O T E C H N O L O G Y
UNIVERSITY OF HOUSTON
Quality Control:Microbiology
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QUALITY CONTROL
Main Responsibilities:
Testing and release of raw materials, in process product and final product
Environmental monitoring
Testing and release of equipment prior to use
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QUALITY CONTROL
QC Microbiology -
Responsible for environmental sample collection and analysis to identify sources of contamination. Includes sterilization equipment, air, surfaces (including personnel), water, and raw material monitoring.
QC Biochemistry
Responsible for determining concentration and purity of protein as well as purity of raw materials and cleaning processes
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Specifications for cleanroom air quality in the pharmaceutical industry
Maximum number of Maximum number of particles/ m3 permitted viable microorganisms/m3
permitted
Class 0.5 micron 5.0 micron
A/100 3,500 0 Statistically less than 1
B/1,000 350,000 2,000 <10
C/10,000 3,500,000 20,000 <100
D/100,000 Not defined Not defined <200
http://www.pmeasuring.com/moreinfo/GMP/appnotes/app41/appFile/app41pharma.pdf
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LASER PARTICLE COUNTER
Channel sizes (µm)
Channel sizes: 0.3, 0.5, 0.7, 1.0, 5.0, 10.0 microns
OutsideClassroomWarehouseBiomanufacturing SuiteBSC with Blower
Environmental Monitoring with MetOne Laser Particle Counter
9,446,640
0
16,713,420
66,864,310
16,886,920
0
20,000,000
40,000,000
60,000,000
80,000,000
A B C D E
Area
0.5
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Environmental Monitoring with RCS Plus
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45
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135
0
50
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150
A B C D E
Area
Via
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Guess the Sources of EM Counts
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OutsideClassroomWarehouseBiomanufacturing SuiteBSC with Blower
Environmental Monitoring with MetOne Laser Particle Counter
9,446,640
0
16,713,420
66,864,310
16,886,920
0
20,000,000
40,000,000
60,000,000
80,000,000
A B C D E
Area
0.5
um
pa
rtic
les
pe
r c
ub
ic
me
ter
Environmental Monitoring with RCS Plus
5
45
0 7
135
0
50
100
150
A B C D E
AreaV
iab
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EDABC
12Rodac Plates From a Surface Test of Disinfectants
RODAC PLATES:Replicate Organism Detection and Counting Plates
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Bioburden TestingWater, buffers, media and culture, and raw materials
Milliflex PLUS Vacuum Pump
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TOC- Total Organic Carbon TestingOrganic carbon reflects biological input and indicates the presence of bacteria
Can be placed inline with water purification system to provide rapid real-time assessment of water purity.
Principle: Analyzer oxidizes carbon under acidic conditions at elevated temperatures to produce CO2. The CO2 is then purged from the water by a N2 stream and quantified by a calibrated IR detector.
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On the outer surface of Gram negative bacteria are lipid polysacharides (LPS)
O-Specific Chain Outer Core
Inner Core
Lipid A
Polysaccharide Lipid
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ENDOTOXINS are pyrogenic molecules that come from Gram negative bacteria.
Pyro means heat
genic means production of
Pyrogens cause fevers when injected into the bloodstream. Higher levels can lead to septic shock and death
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SEPSIS is the “HOLY GRAIL” of Biotech
Many companies work years on a therapy but it proved difficult to combat.
Eli Lilly produces Xigris – activated Protein CNeutralizes an inhibitor of tPAEstimated cost $5,000-$10,000/dose
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History of Endotoxin Testing
1940sPyrogen Test:
Universally required to assure safety of injectable drugs.
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History of Endotoxin Testing
1950s
Johns Hopkins Scientist Frederick Bang and Jack Levin observed that bacteria endotoxin acts on the circulating blood cells of Limulus and forms a clot.
Prepared extract from the amebocytes elicits the same response.
© The National Aquarium in Baltimore
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History of Endotoxin Testing
1970s Limulus Amebocyte Lysate (LAL) Test recognized by the FDA as an alternative method of endotoxin testing.Limulus – Horse Shoe CrabAmebocyte – White Blood CellsLysate – Contents of cells when lysed
1971 First Large scale facility for producing LAL for industrial use built in Chincoteague, VA
1983 LAL test registered in the US Pharmacopia
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Advantages of the LAL Test
Greater Sensitivity
Rapid Assay
Less Expensive
More Reproducible
More Humane
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Endotoxin
Factor B Active Factor B
Polymerization
Clot
Coagulogen Coagulin & peptide C
Factor A Active Factor A
Factor C Active Factor C
Horseshoe crabs are bled through the large dorsal sinusBlood is centrifuged to separate cells from serumSerum is removed and cells are washed with saline
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Associates of Cape Cod, Inc.
Filling Room
Cells are lysed with WFI waterLysate is filtered to remove cellular debrisFiltrate is lyophilized to a powder
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STEPS in the GEL CLOT LAL ASSAY
1. Make various dilutions of sample in LRW
2. Transfer dilutions to test tubes
3. Add LAL and mix
4. Incubate at 37oC UNDISTURBED
5. Carefully remove tubes
6. Carefully invert tubes.
7. Record the presence or absence of a gel clot
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A gel clot must remain completely intact upon inversion to be scored as positive
Associates of Cape Cod, Inc.
Reading the Results: Gel-clot Test
Lysate clots when endotoxin is present.
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OTHER TYPES of LAL ASSAYS
Kinetic Turbidimetric - increase in turbidity. Measure rate of increased turbidity
Chromogenic - addition of a peptide that when cleaved during the assay turns a distinct color.
Endpoint - Measure absorbance after a definitive time period.
Kinetic - Measure rate of increaded absorbance.
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sells PyroGene, a Recombinant Factor C
rFC is activated by endotoxin
Activated rFC cleaves a synthetic peptide releasing a fluorogenic molecule
After a 1 hour incubation the amount of fluorescenceis measured at excitation/emission wavelengths 380/440nm
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The incidence of mycoplasma contamination ranges from 5% - 87% of cell cultures examined.
Mycoplasma contamination may adversely effect:
Metabolism
Growth
Viability
DNA, RNA and protein synthesis
Morphology
Virus Propagation
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Mycoplasma Testing
Direct Culture TestingHoechst StainPCRMycoProbe – Hybridization Capture ProbeMycoAlert – Luciferase ATP assay
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In situ fluorescent stain of Mycoplasma hyorhinis. The Hoechst 33258 fluorescent stain was used on Positive and negative control slides from Sigma Mycoplasma Stain Kit (MYC-1; 3T6-Swiss Albino; ATCC*CCL96).
REF: Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinisNiklas Darin, Norman Kadhom, Jean-Jacques Brière, Dominique Chretien , Cécile M Bébéar, Agnès Rötig , Arnold Munnich and Pierre Rustin
BMC Biochemistry 2003, 4:15
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Mycoplasma Contamination
NegativeControl
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Amplifies the DNA sequence of the conserved spacer region between 16S and 23S rRNA genes of Mycoplasma genome.
The sequence and length of this spacer region differs among different species of mycoplasma.
Size variations of the PCR products may be used for species identification.
Lane M: pHY Marker
1: M. hyopneumoniae
2: M. neurolyticum
3: M. fermentans
4: M. pulmonis
5: M. hyorhinis
6: M. orale
7: M. capricolum
8: M. arthritidis
9: M. salivarium
10: M. hominis
11: M. arginini
12: U. urealyticum
13: human DNA
14: mouse DNA