CA2037727A1

CA2037727A1 - Removing lps pyrogen from aqueous solution

Info

Publication number: CA2037727A1
Application number: CA 2037727
Authority: CA
Inventors: Burton W. Blais; Hiroshi Yamazaki
Original assignee: Individual
Current assignee: Individual
Priority date: 1991-03-07
Filing date: 1991-03-07
Publication date: 1992-09-08

Abstract

ABSTRACT OF THE DISCLOSURE

A method for removing LPS pyrogens from aqueous solutions is provided herein. The method comprises passing such aqueous solution through a filter comprising a macroporous hydrophobic cloth which has been coated with an oligopeptide, e.g., a hydrophobic polypeptide, or a polymyxin, preferably polymyxin B, thereby to have such oligopeptide, e.g., a hydrophobic polypeptide, or a polymyxin, preferably polymyxin B, stably bound thereto, whereby LPS pyrogens are adhered to the filter.

Description

- 2 3 ~ ~ 7 2 7 : 1 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 : 'f~ ~
.' :

., :.. , ' ~. . , : . , ' ' '.`~' . ' . . .. - ' ' ' ' ' ' . ' ' . ' . .' . , ~ ' ',: ' : .

,.. ' .. .'., ~' ... : ~ ' .. -,': .. ' . , ' ' ", - ' :

7 ~ ~

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
, ., - , . . .
~ : . . -, . . ~ . .

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;
~, .
. 1 .

.

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.

/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 , . .
.~, .

. ...

:,~
., , :

~ . i . i ~ . , . .. . , , :

~ ~ 7 ri 2 1 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 ' :

~ ., - . . . .
- ' . .:
;: ` - - . .

, 7 i~, ~

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 - 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.
-, ~. .

~.
., .

~772 ~ :

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 . ~i .. ; ' '. ', ' . ' ` ' . ' - - . -' . ' ' . ' ':: : ' ' . `., ' ' . ' ' :. .: ' . " ' "

: . , . ' , . . ` . .: ' ' ' ` ` . . :
i' ` '. ` : ' ', ' , ` ` ` . ::, ~ ~3 .~ h 1' 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
' ':
. ~ .
':, ,,.: - . ' ~ ... . . ~ ,, - . , : . .
.. . , , ., . ,,, ~ , . . .. . .

. . . :, ., , .. , .. - ... : ~
-... : - . ~ :, .. . .. , .. , , . .. ~ ... ..

. . . ;: . :
., - , , .. . . . . . . . ..

J ri ~

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 .

.~ .

' ' . , ' ~ ' :

2 1~ ~ r~J 7 ~ ~

media and other solutions where the presence of pyrogens is undesirable.

"

... .
-~., .~' , ' ,i' .

. .
:,~

~ :

~ .
.~
~' ` .

. ~ . .
;:
~i i ~
~i . .

~ !
'`' . ~ . ~. ' , ' , ' ' . ' ' '"' ' '

Claims

1. A method for removing LPS pyrogen from aqueous solution which method comprises: passing said aqueous solution through a filter comprising a macroporous hydrophobic cloth which has been coated with an oligopeptide, a hydrophobic polypeptide, or a polymyxin, thereby to have said oligopeptide, or said hydrophobic polypeptide said polymyxin stably bound thereto, whereby LPS pyrogens are adhered to said filter.

2. A method for removing LPS pyrogen from aqueous solution which method comprises: passing said aqueous solution through a filter comprising a macroporous hydrophobic cloth which has been coated with polymyxin B, thereby to have said polymyxin B stably bound thereto, whereby LPS pyrogens are adhered to said filter.

3. The method of claim 2 wherein said cloth is formed from a hydrophobic fibre selected from the group consisting of polyester, polyethylene, polypropylene and nylon.

4. The method of claim 2 wherein said cloth is packed into an upright cylindrical column provided with a control valve.

5. The method of claim 4 wherein the flow rate is 25 to 200 ml/hr.

6. The method of claim 5 wherein the flow rate is 50 ml/hr.

7. A filter comprising a macroporous hydrophobic cloth which has been coated with an oligopeptide, a hydrophobic polypeptide, or a polymyxin, thereby to have said oligopeptide, or said hydrophobic polypeptide said polymyxin stably bound thereto.

8. A filter comprising a macroporous hydrophobic cloth which has been coated with polymyxin B, thereby to have said polymyxin B stably bound thereto.

9. The filter of claim 8 wherein said cloth is formed from a hydrophobic fibre selected from the group consisting of polyester, polyethylene, polypropylene and nylon.