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Research History
Pyro, meaning fire in Greek, is the root for the
term pyrogen, referring to any fever-causing sub-
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materials are different than those from living bacteria. Gasperd confirmed Panum's conclusion by
demonstrating that injections of cow's milk, beef
broth, or human urine do not elicit as strong a
reaction as do small infusions of putrid material.
Wechselmann and Mueller underscored the role
of bacterial contamination of distilled water used in
preparations. Salt Fever, the pyrexia associated with
the administration of crude saline solutions, was
not caused by salt but rather by salt solutions prepared with contaminated water. Fever often accompanied the administration of therapeutic agents.
Heat sterilization or filtration failed to eliminate the
pyrogenicity of these preparations.
In 1912, Hort and Penfold designed the standardized rabbit test, classifying bacteria into
pyrogenic and non-pyrogenic types. They determined that gram-negative (staining) bacteria are
pyrogenic and that gram-positive (staining) bacteria are non-pyrogenic. Comparing the response
from live gram-negative cultures against those
that had been killed, they correlated pyrogenicity
of water purified from sources with differing bacterial concentration.
Basic Definitions
54
IL-1
1L-2
TNF
platelet activating factor
Prostaglandins
Cyclic AMP
Monaminies
t
t
THERMOREGULATORY CENTER
Circulatory System
ENDOGENOUS PYROGEN
Transcription
Translation
Secretion
PHAGOCYTIC LEUKOCYTES
Kupffer Cells, Splenic and Alveolar Macrophages,
Neutophlls, Monocytes, Eosinophlls
t
EXOGENOUSPYROGENS
Viruses, Bacteria, Fungi, Bacterial Products, Endotoxin,
Etiocholanolone, Ag-Ab Complexes, Polynucleotides,
Antigens (via Iymphokines from sensitized
lymphocytes)
Figure 2
DIAGRAM OF A GRAM-NEGATIVE CELL MEMBRANE
. . . - "0' Antigen
Side Chains
"'-Protein
. . . - Phospholipid
Protein
Lipoprotein
_ ....._ _ _...______........t--Pepticloglycan
. . . - "Periplasmic Gap'
...-Protein
. . . - Phospholipid
"'-Protein
nuter
fembrane
Lnner
Membrane
REPRINTED FROM PYROGENS ENDOTOXlNS IAL TESTING ANQ DEPXRQGENATION P. 25 BY COURTESY OF MARCEL DEKKER, INC.
I
CH
C.
_0 - P--.----ii
Figure 3
LIPID A
HC-NH-;tM
ENDOTOXINS
FA--o-!H
Basic Definitions
Hf-O--<A
HC~
HJ _____
55
surrounding medium. Unpurified endotoxins contain lipid, carbohydrate, and protein. When protein is removed, the purified composition is
termed an LPS to emphasize its chemical composition. Endotoxins are heat-stable in solution and
may be inactivated by dry heat, alkali, acid, and
exposure to polymyxin B.
Removal
Ultrafiltration units with molecular weight cutoffs of 20,000 to 100,000 daltons often are used
to remove endotoxins from solutions .
Distillation by means of phase transformation
and liquid separation systems has been known
to effectively separate endotoxin from the
resulting vapor and subsequent distillate .
Various methods are used to detect endotoxin,
and a comparison of these tests is provided.
(See Figure 4 on page 126.)
The most efficient method for endotoxin
removal is a distillation unit that incorporates a
baffling system . Separation is effected by the
change in state from liquid to vapor to liquid. The
baffling system is used to increase the efficiency of
the phase transition. Many distillation units employ
various devices, including "the famed" Q-baffle,
spiral separators, demister pads, cyclone separators, and combinations of these.
RO systems also are used; however, they tend
to have weak links that can leak. For example,
interconnection seals, chevron seals, and membranes may be prone to disintegrate and/or foul.
These units must be monitored and sanitized regularly to be used effectively.
Ultrafiltration systems, which use plate technology or hollow fiber units employing 5,000 to
20,000 molecular weight cutoffs, are becoming the
units of choice. Though not extensively used in
the United States, these steamable ultrafiltration
units are likely to take the place of distillation in
the years to come.
Other methods include charge modified media
filtration (e.g., Pall's Posidyne filters), microporous
membrane filtration, and other membrane filtration
systems which may be either hydrophobic or
hydrophilic.
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o
~
~
Figure 4
CD
3
a
OJ
Gel-clot
Endpoint Turbidimetric:
Kinetic Turbidimetric:
Endpoint Chromogenic:
Kinetic Chromogenic
Relatively inexpensive,
widely available
instrumentation
Moderate to expensive
instrumentation
Relatively inexpensive,
widely available
instrumentation
Most expensive
instrumentation
NA
0.005 - 50 EUlml
Resolution
+/-25%
+/-25 or 50%"
+/-25%
+/-25 or 50%"
Susceptibility to
interference
Timing
Automated instrumentation
handles timing
Automated instrumentation
handles timing
Reaction vessel
Microplate"", sometimes
glass culture tubes
Other Comments
LAL-5000:
-very good temperature
control
-individually controlled
timing for each well
-samples can be added to a
test in progress -
Cost
Sensitivity
OJ
a..
oo
c::
(Q
0-
'"O::l
c::
:;
'"
May show more interference than gel-clot, but greater sensitivity gives more scope for dilution to overcome it
-~
~~~
- -- -- -
"The resolution of kinetic methods depends on the spike recovery range used. The 1987 FDA "Guideline on Validation of the Limulus Amebocyte Lysate Test...."
specifies that spikes be recovered within +1-25%. This was increased to +1-50% in the 1991 FDA "Interim Guidance for Human and Veterinary Drug Products and
Biologicals: KINETIC LAL TECHNIQUES". This change did not apply to medical devices.
""Microplates cannot be practically depyrogenated by the user. Occasional contaminated wells ("hot wells") are to be expected when used. An appropriate source of
relatively clean plates is necessary. Pyroplates are available from Associated of Cape Cod, Inc. and are provided with a certificate of analysis.
Courtesy of: Associates of Cape Cod, Inc., Woods Hole, Massachusetts
Figure 5A
O[!]
Figure5B
WFI
Storage
Still
58
WFI
Storage
Still
Figure
UV Light
l _ __
IL=.J-
~O
~O
-----<L---.JI
0
~
~
0
0
LJO---LJO ~
USP Purified
Water
t
2 Pass Reverse Osmosis
Figure 7
1.0 Micron
Backllow Preventer
Softeners
sv
____
ChIr_~-'
Filter
sv
sv
Still
sv
sv
59
Figure 8
NaOH
0.25% H20 2 in a 1%
5.25% Sodium
solution of NaOH
Hypochlorite
Ammonium Salts
Household Bleach
Mineral Acids
0.5-1.0%
(HCI;
Peracetic Acid
Household Detergents
Ozone
Figure 9
EFFECTIVENESS OF COMMON BIOCIDE AGENTS
Hydiogen" .~.-.wHydrogen
Time
(1%)
15 minutes
. 60
~.
Peroxide
(5%)
Peroxide
(10%)
2.0xio'
2.0xlo'
<10
<10
<10
o.
minutes
I
........+....
2 hours
... . ... .. . ... ...... ... .;.
; .. 12 hours
<10
' <10
LOxioJ
<it)
i
D values
!. ....
'.r..
...._........
. ........
. ,,<
... ...............
. .." .". ...
~, . ..........
. . ~, ..
. <
" ..... ..........
,
,
60
. ,
.....
<10
llDlin
. ............ ........
" .'....... ............... ..
< . . '
~.;
61
contamination during each step in the purification process. Systems must be maintained properly to produce high quality water routinely.
Sampling valves must be located strategically
throughout a system to measure its effectiveness.
It is every pharmaceutical manufacturers'
duty to produce pharmaceutical WFI that is free
of microbes, pyrogens, and chemical contamination. In so doing, companies are able to produce biomolecules and pharmaceuticals with
the purity that they desire and that the patient
deserves.
REFERENCES
1. Pyrogens, Endotoxins, LAL Testing and Depyrogenation. Dr.
Frederick C. Pearson IIi., Marcel Dekker Inc. 1985
2. LAL Update, Editor Dr. Thomas J. Novitsky, Ph.D., 1995,
Associates of Cape Cod, Inc., Woods Hole, Massachusetts
3. Drug, Device and Diagnostic Manufacturing, The Ultimate
Resource Handbook, Carol DeSain, Copyright 1991 by
Interpharm Press, Inc.
4. Bacterial endotoxin: molecular relationships of structure to
activity and function, Vol 8 February 1994 The FASEB Journal,
Ernst T. Rietschel ET AL.
5. Annu. Rev. Biochem. 1990. 59:129-70 Copyright 1990 by
Annual Reviews Inc. Biochemistry of Endotoxins, Christian R.H.
Raetz.
6. Biological Fouling of Industrial Water Systems: A problem solving approach. Marc W. Mittelman and Gill E. Geesey, Water Micro
Associates, San Diego, CA, 1987.
7. High Purity Water Preparations, Theodore H. Meltzer, Tall Oaks
Publishing, 1993.
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