Depyrogenation refers to the removal of pyrogens from solution, most commonly
from injectable pharmaceuticals. A pyrogen is defined as any substance
that can cause a fever. Bacterial pyrogens include endotoxins and
exotoxins, although many pyrogens are endogenous to the host. Endotoxins
include lipopolysaccharide molecules found as part of the cell wall of
Gram-negative bacteria, and are released upon bacterial cell lysis. Endotoxins
may become pyrogenic when released into the bloodstream or other tissue where
they are not usually found. Although the colon contains Gram-negative bacteria in
abundance, they do not cause a pyrogenic effect as the bacteria are not
undergoing gross lysis, and the immune system is not exposed to free endotoxin
while the colonic wall is intact. When LPS is released upon bacterial cell
lysis, the lipid A component is first bound by serum LPS-Binding Protein and
then transferred to CD14. This monomerises the aggregated LPS, as the
LPS receptor Toll-like Receptor 4 cannot recognise LPS while aggregated.
Monomeric LPS is then transferred to MD-2 pre-complexed with TLR4 on
macrophages and monocytes. This leads to release of pro-inflammatory cytokines
and nitric oxide, which may lead ultimately to septic shock depending on
the strength of response. Vascular endothelial cells also express TLR4 and
MD-2 and so respond to LPS directly, as well as via cytokines and nitric oxide.
Bronchial epithelial cells and colonic epithelial cells also express TLR4, but
as they do not express MD-2 they rely on LPS precomplexed with serum MD-2 in
order to signal to LPS. Maximum acceptable endotoxin level
Because endotoxin molecular weight may vary a great deal, endotoxin levels are
measured in "endotoxin units". One EU is approximately equivalent to 100 pg of E.
coli lipopolysaccharide—the amount present in around 105 bacteria. Humans
can develop symptoms when exposed to as little as 5 EU/kg body weight. These
symptoms include, but are not limited to, fever, low blood pressure, increased
heart rate, and low urine output; and even small doses of endotoxin in the
blood stream are often fatal. The FDA has set the following maximum
permissible endotoxin levels for drugs distributed in the United States:
Drug - 0.2 EU/kg body weight Drug - 5 EU/kg body weight
Sterile water - 0.25-0.5 EU/ml Pyrogen detection
Rabbit Test Early endotoxin detection was
accomplished by injecting rabbits with the sample and observing the response in
their body temperature. Rabbits have similar endotoxin tolerance to humans,
and were thus an ideal choice. However, this method was costly, time consuming,
and prompted protests from animals rights advocates. But perhaps the
biggest drawback of this test was its inability to quantify the endotoxin
level. LAL test
Currently, the method of choice for endotoxin detection is the Limulus
Amebocyte Lysate test. This test is based on Dr. Frederik Bang's [1]
observation that horseshoe crab blood forms clots when exposed to endotoxins.
Amoebocyte extract from horseshoe crab blood is mixed with a sample suspected
of endotoxin contamination, and a reaction is observed if endotoxins are
present. The FDA has approved four variations of the LAL test: gel-clot,
turbidimetric, colorimetric, and chromogenic assay. The differences in
these variations refer to the characteristics of the
amoebocyte/endtoxin reaction. This test is fast and highly sensitive. However,
because it only detects LPS endotoxins, some pyrogenic materials can be missed.
Also, certain conditions can lead to false negatives. Glucans from
carbohydrate chromatography matrices can also lead to false positives.
Pyrogen removal Pyrogens can often be difficult to
remove from solution due to the high variability of their molecular weight.
Pyrogens are also relatively thermally stable and insensitive to pH changes.
However, several removal techniques exist.
Ion exchange chromatography Endotoxins are negatively charged, and
will bind to an anion exchanger. If the target substance is not also negatively
charged, it will pass through the column before the endotoxin, and an effective
separation can be achieved. This method is sometimes used in the purification of
albumins. Ligands of known affinity to endotoxins can be coupled to an anion
exchange system to increase its endotoxin binding strength and further
improve the purity of the final product. Typical examples of endotoxin binding
ligands include histamine, nitrogen-containing heterocyclic
compounds, and polymyxin B. However, polymyxin B is known to induce
production of interleukin-1, an exogenous pyrogen, and thus must be
shown to be absent in the final product if used.
Example of using anion exchange chromatography to purify albumin:
2% of the endotoxin does not bind to the column. However, this 2% washes out
before the albumin peak, and can thus be removed simply by starting collection
after this 2% has washed out. 10% of the endotoxin that does bind to
the column will eventually wash out after the albumin peak. This can be
prevented from entering the final product by stopping collection before
this happens. The remaining 90% of the bound endotoxin
must be cleaned off the column using NaOH
An alternative to anion exchange is cation exchange chromatography, in which
positively charged solutes bind to the solid chromatographic media. In this
method, the target binds to the column instead of the endotoxin. The endotoxin
then washes through the column, and a pure target is later eluted off the
column. Cation exchange chromatography has been shown to effectively purify
β-interferon. Ultrafiltration
Because the molecular weight of endotoxins is usually over 10 kD,
ultrafiltration can sometimes be used to perform as a size based separation. Due
to the high variability of endotoxin size, it can be difficult to select the
correct membrane, hence this method is best used only when all endotoxins
present are larger than 300,000 Da. Commercially available ultra filters
have been shown to remove pyrogens to a level below 0.001 EU/ml.
Distillation This method is also based on the large
molecular weight and heat stability of endotoxins. Low molecular-weight
solvents can be easily purified by boiling and collecting the condensed
vapor in an endotoxin free vessel. The large LPS molecules do not easily
vaporize, and are thus left behind in the heating vessel. This is the method
of choice for the purification of water. Inactivation/destruction
Because pyrogens are often difficult to remove, inactivation or destruction of
the LPS molecule can sometimes be preferable.
Acid-base hydrolysis This method has been shown to cleave Lipid A from the
polysaccharide in the LPS molecule. The lipid moiety alone is not soluble in
water. Thus unable to bind to endothelial cells, it is rendered
inactive. However, acid-base hydrolysis can denature a target protein, and is
thus unsuitable when purifying a protein.
Oxidation Oxidation using hydrogen peroxide is
often used as a low cost pyrogen destroying solution. The mechanism for
this destruction is unknown, but hydrogen peroxide can easily be removed
further downstream in the purification process, and is therefore a useful
method of pyrogen removal. However, like acid-base hydrolysis, it is not suitable
when purifying proteins. Heating
Heating methods are often used to ensure that glass and other lab equipment are
free of pyrogenic material. Heat is applied by baking in a dry heat oven
that is designed specifically for the depyrogenation process. Although
endotoxins are relatively thermally stable, sufficient heating results in a
3log reduction of endotoxin levels. Due to the high temperature levels, this
method is also not suitable when purifying proteins.
Sodium Hydroxide When purifying proteins, sodium
hydroxide can be used safely and effectively. It is also widely used for
depyrogenation of non-autoclavable equipment and chromatography columns. In
fact, when using an anion exchanger to remove pyrogens, it is necessary to
clean the column with NaOH after each batch.
Preventive methods Because virtually all raw materials
involved in a production process, including factory employees, can be
potential sources of pyrogen contamination, raw material screening
and depyrogenation can often go a long way to ensuring the final product is
free of pyrogens and does not require costly removal or inactivation methods.
Ultrafiltration of chemicals and buffer solutions, applying appropriate hygienic
practices, and performing regular tests can all be helpful.
See also Endotoxin
Downstream processing References
LAL Update Depyrogenation LAL Update
Sofer, G.; Hagel, L.. Handbook of Process Chromatography: A guide to
Optimization, Scale-up, and Validation. Academic Press, 158-161. ISBN
0-12-654266-X FDA Office of Regulatory Affairs:
Inspection Technical Guide, Bacterial Endotoxins/Pyrogens
Textbook of Bacteriology Horseshoe Crab Medical Uses
Chromatographic Removal of Endotoxins and/or Ethanol from Albumin. Application
Note 206. Pharmacia Biotech., Uppsala, 1990.
Dembinski, W.; O'Malley, J.A.; Sulkowski, E. Large Scale Purification
Procedure for Human Fibroblast Interferon. Interferon Scientific
Memoranda, Jan./Feb., 6. Tours, N. and Sandle, T. Comparison of
dry-heat depyrogenation using three different types of Gram-negative
bacterial endotoxin, European Journal of Parenteral and Pharmaceutical Sciences,
Volume 13, No.1, 2008, pp17–20