Quality Control:

Microbiology
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QUALITY CONTROL FOR REGULATED BIOTECHNOLOGY

UNIVERSITY OF HOUSTON

QUALITY CONTROL
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Main Responsibilities:
Testing and release of raw materials, in process product

and final product

Environmental monitoring
Testing and release of equipment prior to use

QUALITY CONTROL
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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

Confirming Sterilization:
Biological Indicator Strips
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Cleanrooms

Cleanroom:

Material Flows

Specifications for cleanroom air quality in the

pharmaceutical industry
Maximum number of
particles/ m3 permitted
permitted
Class

0.5 micron

Maximum number of
viable microorganisms/m3

5.0 micron

A/100

3,500

Statistically less than 1

B/1,000

350,000

2,000

C/10,000

3,500,000 20,000

<100

D/100,000

Not defined Not defined

<200

<10

http://www.pmeasuring.com/moreinfo/GMP/appnotes/app41/appFile/app41pharma.pdf

LASER PARTICLE COUNTER

C
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(

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Channel sizes:
0.3, 0.5, 0.7, 1.0, 5.0, 10.0 microns

RCS (Rotor Centrifugal Sampler)

High Flow Air Sampler

Guess the Sources of EM Counts

Outside
Classroom
Warehouse
Biomanufacturing Suite
BSC with Blower

E
D
A
B
C

Outside
Classroom
Warehouse
Biomanufacturing Suite
BSC with Blower

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RODAC PLATES:
Replicate Organism Detection and Counting Plates

Rodac Plates From a Surface Test of Disinfectants

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Bioburden Testing
Water, 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 Testing

Organic 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)
Polysaccharide

O-Specific Chain

Outer Inner
Core Core

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Lipid

Lipid A

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

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Many companies work years on a therapy but

it proved difficult to combat.
Eli Lilly produces Xigris activated Protein C
Neutralizes an inhibitor of tPA
Estimated cost $5,000-$10,000/dose

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Tumor Necrosis Factor

Endotoxin

MACROPHAGE

Interleukins (1,6,8)

History of Endotoxin Testing

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1940s
Pyrogen Test:
Universally required to assure safety of injectable drugs.

History of Endotoxin Testing

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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

History of Endotoxin Testing

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1970s
Limulus Amebocyte Lysate (LAL) Test
recognized by the FDA as an alternative
method of
endotoxin testing.
Limulus Horse Shoe Crab
Amebocyte White Blood Cells
Lysate 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

Advantages of the LAL Test

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Greater Sensitivity
Rapid Assay
Less Expensive
More Reproducible
More Humane

HORSESHOE CRAB FACTs:

Horseshoe Crab Fossil

Associates of Cape Cod, Inc.

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Endotoxin
Factor C Active Factor C
Factor B Active Factor B
Factor A Active Factor A
Coagulogen

Coagulin & peptide C

Polymerization
Clot
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Preparing the Lysate

Horseshoe crabs are collected during

Spring and Summer
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Horseshoe crabs are bled through the large dorsal sinus

Blood is centrifuged to separate cells from serum
Serum is removed and cells are washed with saline

Cells are lysed with WFI water

Lysate is filtered to remove cellular debris
Filtrate is lyophilized to a powder

Filling Room

Associates of Cape Cod, Inc.

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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

Reading the Results: Gel-clot Test

Lysate clots when endotoxin is present.

Associates of Cape Cod, Inc.

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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 fluorescence
is measured at excitation/emission
wavelengths 380/440nm
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Mycoplasma Testing
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http://www.visualsunlimited.com/browse/vu197/vu197401.html

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
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Direct Culture Testing

Hoechst Stain
PCR
MycoProbe Hybridization Capture Probe
MycoAlert Luciferase ATP assay

http://www.poliklinika-harni.hr/teme/ginteme/mycoplasma.htm

Mycoplasma
Contamination

Negative
Control

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 hyorhinis

Niklas Darin, Norman Kadhom, Jean-Jacques Brire, Dominique Chretien, Ccile M Bbar, Agns Rtig, Arnold
Munnich and Pierre Rustin
BMC Biochemistry 2003, 4:15

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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

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Contamination Now What???

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

ciprofloxacin