Note: Descriptions are shown in the official language in which they were submitted.
- 2 3 ~ ~ 7 2 7
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This invention relates to the removal of pyrogens
from solutions.
Pyrogens are toxic substances which cause fever in
humans as well as in other animals, and which display
toxicity to many living cell types, e.g. mammalian
cells maintained ln vitro in cell or tissue cultures.
Pyrogens must be removed from solutions or any product
destined for injection (e.g. pharmaceuticals, e.g.
injectable saline, pharmacological drugs and
lo antibiotics~ into humans or animals, or for use in the
preparation of culture media for the propagation or
mammalian cell or tissue cultures. Gram-negative
; bacterial lipopolysaccharides (LPS) (also termed
"endotoxins"), a major constituent of the outer cell
wall, are the main types of pyrogens encountered.
` Almost all Gram-negative bacteria found in nature
- produce pyrogenic LPS, including (among numerous
~ others), Escherichia coli and Salmonella species.
-`~ Several methods have been developed for the removal
of LPS pyrogens from solutions, including one method
which is based on the affinity of the antibiotic
~ polymyxin B for the LPS molecule. Issekutz, in the
.~ Journal of Immunological Methods (1983) 61: 275 - 281,
described the use of polymyxin B covalently bound to
agarose ~eads to remove LPS pyrogens from solutions in
a packed column operation. However, the method for
preparing the polymyxin-agarose support is laborious
cl and may be subject to batch variations, si~ce it
.~ involves chemical treatment in order to immobilize the
polymyxin B on the agarose beads, and the extent and
uniformity of the treatment may vary from batch-to-
~'r batch. Such method is also costly, since the
-~ ~aterials, quality control measures and chemical
~-1 treatments required are relatively expensive. Indeed,
3S products currently available on the market to remove
pyrogens ~rom solutions are costly, and this cost in
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turn affects the cost of the final products (e.g.
pharmaceuticals) from which pyrogens must be removed.
Boehringer Mannheim has now provided polymyxin B-
SEPHAROSE, namely polymyxin B bound covalently to
5SEPHAROSE 4B. This product is said to be available in
gel form and is said to be able to remove endotoxin
impurities. It is also said that the gel can be
regenerated by washing with deoxycholate solutions.
This pyrogen removal agent su~fers from the same
lodisadvantages as disclosed above with respect to the
polymyxin B covalently bound to agarose beads. It is
manifest that a commercially-attractive process cannot
be conceived using this gel filter.
Alerchek Inc. has also now provided an affinity
15filter in the form of a derivatized PVC-silica
composite with polymyxin B sulfate and an LPS binding
co-peptide covalently immobilized to the silica.
Since this filter is microporous, filtering is
effected using a tangential filter apparatus/ which is
20a more expensive technique.
An object of the broad aspect of this invention is
the provision of a method for the removal of LPS
pyrogens fxom water in which the LPS pyrogen-removal
agent can be prepared without the need for any
25chemical modification of the substrate used in such
pyrogen removal.
An object of another aspect of this invention is the
provision of a method for the removal of LPS pyrogens
. from water in which the method for preparing such LPS
30pyrogen-removal agent is such that the quality of the
LPS pyrogen-removal agent has minimal batch-to-batch
variations.
An object of still another aspect of this invention
is the provision of a method for the removal of LPS
~35pyrogens from ~ater in which the method for preparing
-~such LPS pyrogen-removal agent is such that the LPS
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stability of the LPS pyrogen-removal agent is
enhanced.
An object of yet another aspect of this invention
is the provision of a method for the removal of LPS
pyrogens from water in which the method is such that
it enables the low cost preparation of such LPS
pyrogen-removal agent.
By a- broad aspect of this invention a method i5
provided for removing LPS pyrogens from an at~ueous
solution which comprises passing such at~ueous solution
through a filter consisting essentially of a
macroporous hydrophobic cloth which has been coated
with an oligopeptide, e.g. a polymyxin, preferably
with polymyxin B, or with any other hydrophobic
polypeptide, which is stably bound thereto.
- Polymyxin is an oligopeptide which has a high
affinity for lipopolysaccharides (LPS) in aqueous
buffers. Polymyxin is an antibody complex produced by
Bacillus polymyxa:
'Y 1 2
L-DAB - D-X ~ L-Y
5 _
L-DAB -~L-Thr -~Z-~L-DAB
~-NH2 L-Thrt L-DA8- L-D
. ' = = I = ~
,," . y-NH2 'Y-NH2
DAB = ~ diaminobutyric acid
~ . .
~A Polymyxin B. Mixture of polymyxin~ Bl and B2.
~ 35 Polymyxin Bl. C56H~N16013 R = (+)-6-methyloctano-yl;
'~ X = phenylalanine; Y = leucine; Z = L-DAB.
~, PolYmyxin B2. C55~6N16013, R = 6-methylheptanoyl;
~, .
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2 ~ 3 J 7 2 7
X = phenylalanine; Y = leucine; Z = L-DAB.
Polymyxin D1- CsoHs3N1s1s~ R = (+)-6-methyloctanoyl;
X ~ leucine; Y = threonine; Z = D-serine.
Polymyxin Dz~ C4~91N1sO,5. R = 6-mathylheptanoyl;
X = leucine; Y = threonone; Z = D-serine.
Colistin.
H2
/L-DAB--D-L~u L-~ ,e~
L-D~a L-Thr ~ L-DA8 - L-DA8
1 0 = I = = I ~ ~ ~ :
; ~H2 ~-~2 L-Tllr-- L-D.~8~ L-D,~
~ H2 `~-~H2
DAB = o,~-diaminobutyric acJd
.. .
Colistin A. Cs3H1ooN16013, polymyxin E1. R = (+)~6-
methyloctanoyl.
; Polymyxin E C52H98N16O13. R = 6-methylheptanoyl.
The cloth is preferably formed from a hydrophobic
fibre selected from the group consisting of polyester,
polyethylene, polypropylene and nylon.
; It is preferred that the cloth be packed into an
. .
upright cylindrical column fitted with a valve. The
flow ratè used is from 25 to 200 ml/hr, but the rate
preferably used is 50 ml/hr.
This invention also provides a filter comprising a
macroporous hydrophobic cloth which has been coated
with an oligopeptide, a hydrophobic polypeptide, or a
^ 30 polymyxin, thereby to have the oligopeptide, or the
hydrophobic polypeptide the polymyxin stably bound
thereto. Preferably the filter comprises a
macroporous hydrophobic cloth which has been coated
, with polymyxin B, thereby to have the polymyxin B
stably bound thereto. Still more preferably, the
cloth is formed from a hydrophobic fibre selected from
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the group consisting of polyester, polyethylene,
polypropylene and nylon.
The term "macroporous" as applied to cloths when
used herein is intended to mean textiles composed of
hydrophobic synthetic polymeric fibres, which are
either woven or non-woven into a physically
structurally stable cloth of more than about 200 ~m
thickness, such that the pores (i.e. spaces between
the fibres) exceed about 20 ~m in diameter.
Macroporous cloths may be made substantially
completely from synthetic fibres selected from the
group consisting of polypropylene, polyethylene,
nylon, and polyester.
Such macroporous cloths have the following
characteristics: they can accommodate a larger volume
of the oligopeptide, i.e. polymyxin, e.g. polymyxin B
per area; they have a larger surface area for binding
to the oligopeptide, i.e. polymyxin, e.g. polymyxin B;
they have minimum flow resistance; and they have both
strength and durability. Such macroporous cloths, by
virtue of their hydrophobic characteristics, have been
found to adsorb and absorb the oligopeptide, i.e.
polymyxin, e.g. polymyxin B since they provide a large
surface area for the oligopeptide, i.e. polymyxin,
e.g. polymyxin B ~apture. Macroporous cloths all have
such minimum flow resistance.
Such macroporous hydrophobic cloths made of, e.g.
polypropylene and polyester, are readily commercially
available and are moderately priced because of their
large commercial demand as textiles and filters.
~acroporous 100% nylon cloth is commercially available
` as a generic product and may be acquired locally in
the Ottawa, Canada area. Macroporous woven polyester
cloth is commercially available as a generic product
and may be acquired locally in the Ottawa, Canada
area. Macroporous non-woven polypropylene filter
cloth is available as a generic product and may be
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purchased from Aldrich Chemical Co. A variety of non-
woven, macroporous polyester cloths may be obtained
from DuPont, and are known by the trade-mark SONTARA.
One preferred embodiment of such SONTARA is SONTARA
5 81O0T~, which has the following chemical and physical
characteristics.
Typical Physical Properties of SONTARA 8100 are:
(in English Units)
U!JIT Tn'lC~NESSS~IE I G;lAEI ,.q~ OlD , .UU~ 'N ~ .q AIR ROLL SIZ`=
WEIGHT T_NSIL_ T--AP, D~URST ~ .IEA21UTr ~7-' ID COAQ
o:~ya i~) Imils) (Ibsl (Ib~ (P-i~ (CFMI~l~ in. 11n.
MO XD UO XD @ 05'~ ~,O) 0Ø yd
S~yle
.'~10tt~' Polyest~r
8100 40 40 70 45 3540 120 215 44 1700
- Frazier Air Permeability is described in ASTM D737-
. 75, and is attached hereto as Appendix I.
20The term "hydrophobic" as applied to cloths when
used herein is intended to mean that the cloths repel
water, the degree of repelling being dependent on the
- pore size and the inherent polymeric properties.
The term "non-woven" when referring to the cloth is
25intended to mean a cloth formed ~rom a random
arrangement of natural or synthetic fibres by
adhesives, heat and pressure, or needling techniques.
Polyester cloth can passively (hydrophobically)
adsorb the oIigopeptide, i.e. plymyxin, e.g. polymyxin
303 using a simple coating procedure. Such -
oligopeptide, i.e. polymyxin, e.g. polymyxin B-coated ~-
cloth can effectively capture LPS antigens.
- Accordingly, by this invention it is now taught that -
the oligopeptide, i.e. polymyxin, e.g. polymyxin B-
35coated polyester cloth may be a suitable support in a
method for the removal of LPS pyrogens from solutions.
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By the present invention, it has been found thatthe oligopeptide, i.e. polymyxin, e.g. polymyxin B-
coated polyester cloth has several advantages over the
use of other LPS pyrogen removal devices. Some
advantages are (1) the ease with which polymyxin B-
cloth can be prepared without the need for any
chemical modifications of the cloth or the antibiotic;
(2) th`e minimum batch-to-batch variation in the
guality of the oligopeptide, i.e. polymyxin, e.g.
polymyxin B-cloth preparation, due to the simple
method for its preparation; (3) the stability of the
oligopeptide, i.e. polymyxin e.g. polymyxin B-
~ polyester cloth hydrophobic interaction; and (4) the
-~ low cost of its preparation, which should help to
reduce the commercial cost of pharmaceuticals or ~ther
solutions to which it is applied.
It is also believed that other oligopeptides, e.g.
hydrophobic polypeptides which bind to LPS pyrogens
would also bind to hydrophobic macroporous cloths to
provide a filter for use in the method of this
invention.
The following Examples are given with respect to the
preparation of polymyxin B-coated cloth and the use of
` such cloth.
Polymyxin B sulfate (No. P-1004), Salmonella
typhimurium lipopolysaccharide (LPS) (No. L-6511~ and
... .
-~ ~ the E-TOXATETM system for measuring LPS pyrogens (No.
210-A) were from Sigma Chemical Co.
Polyester cloths used were SONTARA obtained from
~; 30 DuPont and having the following characteristics:
: EXAMPLE A
Pre~aration of PolYmyxin-Coated Polyester Cloth
` All glassware used herein was de-pyrogenized by
heating for 20 h at 250C~ Pyrogen-free distilled
' 35 water was used as a universal solvent.
Polyester cloth (DuPont, Sontara 8100~M) was cut
into 6 x 6 mm segments and wetted by vigorously
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sha~ing in doubly distilled water, and then blotted.
One gram of the cloth segments ("wet" weight) was
placed in 5 ml of polymyxin B solution [10 mg
polymyxin/ml in 0.01 M phosphate-buffered (pH 7 . 2~ -
- 5 0.85% NaCl (PBS)] and incubated for 16 h at room
temperature. The polymyxin-coated segments were then
washed with 100 ml of PBS on a filter under suction
and stored in P8S at 4C until use.
EXAMPLE I
Removal of_ LPS Pyroaens from Solution Usina
Polymyxin-Coated Polyester Cloth
one gram of polymyxin B-coated polyester cloth
segments (prepared as Example A) was packed into a
' small disposable polypropylene QUIK-SEPTM column
(Isolab. Inc., No. QS-U) fitted with a valve. A
solution of pyrogens consisting o~ Salmonella
typhimurium LPS dissolved in PBS was then allowed to
flow through the polymyxin B-cloth column by gravity
flow, with the flow rate adjusted using the valve as
requiredO The effluent from the column was c.ollected
', and its pyrogen content measured using the E-TOXATETM
~ kit for pyrogen detection according to the
','' manufacturer's directions.
EXAMPLE II
Effect of LPS Pyrogen Concentration on its Removal
from Solution
The ability of polymyxin B coated polyester cloth
~, to remove LPS pyrogens from solution was studied using
' polymyxin B-cloth packed into a column through which
' 30 the pyrogen solution could be "trickled" or filtered
', to effect the pyrogen removal. As an example, the
removal of S. typhimurium LPS was studied.
Fifty millilitres of solutions containing various
concentrations of S.,typhimurium LPS were passed at a
' 35 flow rate of 50 ml/h through a column packed with
`' polymyxin B-cloth. The total effluent from the column
was collected and then assayed for remaining LPS
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pyrogen using the E-TOXATET~ kit. Table 1 shows that
for input LPS concentrations of less than lO ~g/ml,
the polymyxin B-cloth column efficiently removed the
majority of the LPS from the solution. The LPS in the
solution at an input concentration of 10 ~g/ml (or
greater) might be more effectively removed by either
decreasing the flow rate through the column or
increasing the quantity of polymyxin B-cloth segments
packed in the columnO
~ TABLE 1. Effect of LPS Pyroqen Concentration on its
: Removal from Solution by Polymyxin-Coated
Polyester Cloth
: Input Effluent
: 15 LPS Concentration LPS Concentration
(~q/ml) (~q/ml)
0.1-1.0
2 < 0.05
,~
1 < 0.05
0.1 < 0.05
EXAMPLE III
Effect of Flow Rate on the Removal of LPS Pyrogens
` from Solution .-
It is expected that the rate at which the LPS
solution i5 passed through the polymyxin B-cloth
`~ column will affect the efficiency of the column in
. removing the LPS from solution. Therefore, the effect
- of flow rate on LPS removal was studied.
Fifty millilitres of a 1 ~g/ml solution of LPS was
35 passed through the polymyxin B-cloth column at various
flow rates, obtained by adjusting the valve on the
.~ column as reguired. The effluent from the column was
then collected and measured for LPS pyrogens as above.
Table 2 shows that a maximum flow rate of 50 ml/h
achieved efficient removal of the LPS from solution,
.~ -,.
.
. .
whereas flow rates greater than this resulted in some
LPS remaining in the effluent solution. Since high
flow rates would be desirable from the point of view
of the time saved in LPS removal, the use of longer
columns with more polymyxin B-cloth packing may be
necessary to achieve more effective removal of the LPS
at the desired higher flow rates.
TABL~ 2. Effect of Flow Rate on the Removal of LPS
from Solution by Polymyxin-Cloth
Flow Rate Effluent
(ml/h LPS Concentration
- (~q/ml)
200 0.1-10
- 15 100 0.1
< 0-05
< 0.05
.
These results demonstrate that polymyxin B-coated
20 polyester cloth is applicable to the removal of 1PS -
pyrogens from solutions. This system was efficient,
rapid and inexpensive, which should make it a
commercially attractive alternative to other systems
in current use. -~
Thus, by the present invention, macroporous
polyester cloth coated with polymyxin B sulfate was
packed into small plastic disposable columns, and
solutions of phosphate-buffered saline containing
various concentrations of S. tY~himurium
30 lipopolysaccharide pyrogens were filtered through the
packed columns by gravity flow. Passage of the
pyrogen solutions through the packed columns at a flow
rate of 50 ml/h effectively removed the majority of
the pyrogens from the solutions, as determined by the
', 35 limulus amoebocyte lysate detection system. Polymyxin
B-coated macroporous hydrophobic cloth should be
useful for the removal of pyrogens from pharmaceutical
solutions (e.g. injectable solutions), cell culture
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media and other solutions where the presence of
pyrogens is undesirable.
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