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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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Confirming Sterilization:Biological Indicator Strips

Cleanroom: Cleanrooms

Material Flows

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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

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RCS (Rotor Centrifugal Sampler) High Flow Air Sampler

OutsideClassroomWarehouseBiomanufacturing SuiteBSC with Blower

Environmental Monitoring with MetOne Laser Particle Counter

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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

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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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Identifying the “Bug”

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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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LAL Assay

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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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Endotoxin MACROPHAGE

Tumor Necrosis Factor

Interleukins (1,6,8)

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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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HORSESHOE CRAB FACTs:

Associates of Cape Cod, Inc.

Horseshoe Crab Fossil

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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

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Preparing the LysateHorseshoe crabs are collected during Spring and Summer

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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ENDOTOXIN LIMIT FOR WFI IS 0.25EU/ml

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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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Mycoplasma Testing

http://www.visualsunlimited.com/browse/vu197/vu197401.html

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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

Contamination – Now What???

$ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $

Throw it away (most conservative path)Treat with mycoplasma specific antibiotics

such as ciprofloxacin