CA2249548A1

CA2249548A1 - Endotoxin-specific membranes

Info

Publication number: CA2249548A1
Application number: CA002249548A
Authority: CA
Inventors: Birger Anspach; Dagmar Petsch; Thomas Beeskow; Wolf-Dieter Deckwer
Original assignee: Individual
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 1996-03-11
Filing date: 1997-03-11
Publication date: 1997-09-18
Also published as: JP2002514970A; WO1997033683A1; EP0888172A1; DE19609479A1

Abstract

The invention concerns a microfiltration membrane for separating endotoxins from liquid media, in particular water, protein solutions or parenteralia. The microfiltration membrane is characterized by covalently bonded ligands for endotoxins, the ligands being carried by a polymer which is applied to the membrane.

Description

CA 02249~48 1998-09-11 Title: Endotoxin-Specific Membranes Description The invention relates to a microfiltration membrane for the separation of endotoxins from liquid media as well as the use of such microfiltration membranes.

Endotoxins are lipopolysaccharides from the outer cell membrane of Gram-negative bacteria which act as pyrogens. Due to the omnipresence of bacteria, such endotoxins are ubiquitous.
However, in contrast to bacteria they cannot be removed or rendered harmless by standard methods such as sterile filtration or autoclave processing (1). For this reason, sterile cannot be equated with endotoxin-free. The presence of endotoxins in injection or infusion solutions (paranteralia) is particularly critical, since intervenous application can induce fever already in an amount 1 ng per kg body weight. With increasingly higher dosings (e.g. in high volume parenteralia) the symptoms range up to severe shock and death (2, 3). For this reason, apart from sterilization, almost all pharmacopeia prescribe strict upper limits of endotoxins, e.g. 0.2 EU per mg chloramphenicol for injection or only 0.003 EU per heparin unit (4). The attainment of these requirements is difficult in practice. In particular, the production of biological medicaments cannot take place free of endotoxins in all steps.
The main sources of endotoxins include:

CA 02249~48 1998-09-11 - raw materials such as plasma or tissue, which could already be contaminated with bacteria.
- for recombinant products the introduction of host-specific endotoxins can be expected.
- bacterial contamination of devices, filters or additives during the production.

The usual heat-decontamination for thermally stable agents (30 minutes at 250 ~C) is just as unsuitable for the preparations as is the treatment with acids, alkaline solutions or strong oxidizing agents (H2O2) (1).

When using active coal or depth filters such as ZETA PLUS, considerable product loss is often the result, so that their use remains limited to treatments in aqueous solution (5, 6).

The ultrafiltration as a very gentle method has achieved great popularity in the field of endotoxin removal. Here one works with cut-offs of 5000 or 10000 in order to also effectively separate monomer components (MW ca. 14000), which are also present apart from high molecular aggregates (up to a molecular weight of several million). Despite the above, reoccuring problems arise with low molecular break-up components which also have pyrogenic effects (e.g. Lipid A). This especially concerns hemodialysis. Although the dialysis buffer is ultrafiltrated, 400000 dialysis patients annually develop septic symptoms (7) for example in the USA. In addition, the neccessity of lower cut-offs restricts the use of ultrafiltration to the decontamination of low molecular substances (8).

In the case of high molecular products such as pharmaceutical proteins, albumin preparations or heparin, great difficulties still exist. If endotoxin contamination occurs in these preparations, the only remaining possibility at the moment is CA 02249~48 1998-09-11 to perform reprocessing to meet the requirements of the FDA, USP or EP.

To avoid this extensive procedure, but still meet product requirements, the possibility has been investigated to selectively decontaminate impure products with chromatographic sorbents having endotoxin specific ligands. This procedure has also not provided the desired result. Despite high to very high association constants at high endotoxin starting concentrations, the reported affinity sorbents having His, Him or PMB as the ligands have not proven to be suitable (9). In addition, in the presence of proteins competing protein adsorption was observed, which led to reduced endotoxin removal rates and in part to high protein loss (in particular for acidic proteins such as BSA).

Literature (1) S.K. Sharma Endotoxin detection and elimination in biotechnology Biotechnol. Appl. Biochem. 8 (1986), 5 - 22 (2) N. Haeffner-Cavaillon, J.M. Cavaillon, L. Szabo Cellular receptors for endotoxin Handbook of Endotoxins, Vol. 3: Biology of Endotoxins, Elsevier Science Publishers B.V. (1985) (3) D.C. Morrison, J.L. Ryan Endotoxins and disease mechanisms Ann. Rev: Med. ~8 (1987), 417 - 32 (4) USP XXII Suppl. 5 (Nov. 1991) CA 02249~48 1998-09-11 (5) K.C. Hou, R. Zaniewski Depyrogenation by endotoxin removal with positively charged depth filter cartridge J. Paranteral Sci. Tech., Vol. 44, No. 4 (1990), 204 -(6) CUNO Newsletter for Pharmaceuticals (Oct. 1995), p. 3 (7) B.P. Smollich, D. Falkenhagen, J. Schneidewind, S. Mitzner, H. Klinkmann Importance of endotoxins in high-flux dialysis Nephrol. Dial. Transplant 3 (Suppl.) (1991) 83 - 85 (8) E. Flindt Pyrogenentfernung mittels Ultrafiltration Memoscript CONCEPT-Symposium "Pyrogene II"
(June 1983), p. 54 - 60 (9) F.B. Anspach, O. Hillbeck Removal of endotoxins by affinity sorbents J. Chromatogr. A 711 (1995), 81 - 92 This prior art is improved according to the present invention by a microfiltration membrane for seperation of endotoxins from liquid media, in particular water, protein solutions or parenteralia, where the microfiltration membrane is characterized by covalently bonded ligands for endotoxins and where the ligands are carried on a polymer which is applied to the membrane.

With respect to membrane technology and also membrane production reference is made to Ho ~ Sirkar (Editors), Membrane Handbook, van Norstrand Reinhold, New York, 1992.

CA 02249~48 1998-09-11 The covalently bonded ligands can include (a) an endotoxin specific ligand, preferably histamine, histidine, polyethylene imine, poly-L-lysine or polymyxin B and/or (b) a ligand which is not endotoxin speciflc per se, preferably diaminohexane, diethylaminoethyl or desoxycholate.

The membrane material for the microfiltration membrane according to the invention can be regenerated cellulose, cellulose acetate, polysulfone, polyethylene vinyl alcohol or polyamide, preferably Nylon.

The polymer, which according to the invention is applied to the microfiltration membrane, can include a hydrophilic polymer, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethylcellulose.

These hydrophilic polymers can be water soluble, swellable in water or insoluble in water.

The polymers can be carried on the microfiltration membrane according to the invention with the aid of a spacer. The covalently bonded ligand can also be carried by a spacer. These spacers can include those derived from bisoxirane, glutardialdehyde, epihalogenhydrin or diisocyanate, optionally after oxidative activation.

Concerning activation and immobilization chemistry, also with respect to endotoxin specific ligands and spacers, reference is made for example to Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992.

CA 02249~48 1998-09-11 According to the invention, microfiltration membranes are thus provided, which have suitably modified surfaces and which separate endotoxins from water and aqueous solutions tbuffers, protein solutions). The surface modification can comprise the application of a bi-functional covalently bonded spacer, which is reacted with a hydrophilic polymer, whereby non-specific interactions of the membrane are reduced, especially with proteins. The covalently bonded hydrophilic polymer can be reacted with the endotoxin specific ligand, optionally via a further spacer. The principle of the surface modification of the membrane is shown in Fig. 1.

The endotoxin removal in the presence of proteins can be dependent upon the net load of proteins. By optimizing the conditions (pH value), acid proteins (such as BSA and mouse IgG
1) can be completely decontaminated, namely without appreciable loss of proteins. For alkaline proteins (for example lysozyme and bFGF) high removal rates can also be achieved.

Polymer-coated microfiltration membranes according to the invention with covalently bonded endotoxin specific ligands can remove endotoxins in one pass, also from highly loaded solutions (6000 EU ml~1).

In principle, the structure of the membranes is shown in Fig.
1. Initially, a hydrophilic polymer is applied over a spacer, which is then combined with a endotoxin specific ligand, optionally via a spacer. Particularly suited for the membrane material is:

- cellulose - polysulfone - PEVA (polyethylene vinyl alcohol) - polyamide (especially Nylon, such as for example N66).

CA 02249~48 1998-09-11 .

Reactive bi-functional compounds are suitable as the spacer.
Particularly suited are:

- bisoxirane - glutardialdehyde - epihalogenhydrin - diisocyanate.

For activation of the vicinal diol bond resulting from the use of bisoxirane and epihalogenhydrin, oxidation through periodate can be employed, where an aldehyde group results. The spacer bonded to the membrane is further reacted with a hydrophilic polymer. Such polymers preferably include:

- dextran - polyvinyl alcohol (PVA) - modified celluloses, especially hydroxyethylcellulose (HEC).

The further reaction takes place either directly with the endotoxin speclfic ligand or again via an intermediate spacer as mentioned above, optionally after its oxidative activation.
The endotoxin specific ligands include (see list of abbreviations): DAH, Him, His, PEI, PLL, PMB. In addition, the ligands normally not specific to endotoxin, such as DEAE and DOC, are found to be highly specific in the membrane configuration, at the same time with a high passage of the proteins.

The performance of the developed membranes can be taken from the given examples. The endotoxin removal can almost always be considered complete. It is normally below 1 EU ml~1, often below the detection limit with the LAL test.

CA 02249~48 1998-09-11 No endotoxin depletion was found with the control membranes (Nylon without modification and with attached hydrophilic polymer with or without a spacer) without endotoxin specific ligands.

The new membranes can be employed for endotoxin removal from water and parenteralia. Good results are also achieved in the presence of proteins. However, in the case of alkaline proteins, it should be considered that interactions of the proteins with the endotoxins can arise, which can lead to a~
endotoxin masking. Endotoxin bound to the protein can not be clearly detected by the LAL test. In this conjunction it should be mentioned that it is not finally clarified whether protein-bonded endotoxin is still toxic.

The membranes according to the invention have numerous applications.

Medical and pharmaceutical fields:
- hemodialysis.
- safe infusion and injection solutions (parenteralia).
- safe diagnostic materials (e.g. antibodies).
In biotechnology:
- production of pharmaceutical products - endotoxin separation in process water and raw materials.
- decontamination of products (measures for processing are eliminated).

Methods 1. Production of the membranes CA 02249~48 1998-09-11 Hydrophilic polymers, in particular dextran, polyvinyl alcohol and hydroxyethylcellulose are covalently bonded on microfiltration membranes based on Nylon (preferably 0.45 um or larger). In the next step, endotoxin specific ligands are applied to the polymers. Fig. 1 illustrates the structure of the mebranes.

1.1. Membrane coating as an example with dextran The Nylon membranes were first activated with bisoxirane. For this, they were shaken for 16 hours at 80 ~C in a mixture of 9 ml bisoxirane, 1 ml ethanol and 1 ml 25 mM sodium carbonate buffer (pH 11) (Fig. 2a). After thorough washing, each membrane was incubated with 5 ml of a 20 ~ dextran 40000 solution (pH
11) for 15 minutes at room temperature (Fig. 2b). The membranes were then dried for 14 hours at 120 ~C. To remove non-specific bonded dextran, the membranes were washed three times with a 0.1 M caustic soda solution and a further three times with water.

As Fig. 3 shows, the coated membranes display a significantly reduced non-specific interaction, which is expressed through the adsorbed amount hemoglobin.

As also shown in Fig. 3, a single dextran coating cannot achieve the same effect as with PVA and HEC. With a second layer, an improved result can be achieved, while a third layer only has a small effect. Dextran was therefore always used in a double coating.

1.2. Immobilization of endotoxin specific ligands The ligands PLL, PMB and PEI were immobilized either directly on the periodate-activated coating polymers or after incorporation of a periodate-oxidizable spacer (bisoxirane).
The procedure is illustrated in Fig. 4 by way of example. DEAE

CA 02249~48 1998-09-11 .

was coupled to the matrix directly without a spacer, the other low molecular ligands were bonded via epibromidehydrin.

1.2.1. PEI immobilization via bisoxirane For activation, the membranes coated with hydrophilic polymer were incubated for 3 hours at room temperature in a mixture of 100 mg sodium borohydride, 5 ml bisoxirane and 45 ml 1 M
caustic soda solution. After hydrolysis of the free oxirane ring (30 minutes incubation at pH 2.5) and periodate oxidation of the resulting vicinal diol (90 minutes incubation in 0.2 M
sodium periodate), the membranes were reacted for 2 hours at room temperature in a solution of 0.5 g PEI (MW 50000) in 0.1 M
phosphate buffer, which was held at a pH of 8, so that the structure shown in Fig. 1 is produced. Finally, washing was performed with 1 M sodium chloride solution and water.

1.2.2. Histidine immobilization Histadine was immobilized over DAH on a coated membrane activated with epibromidehydrin. Epibromidehydrin activation was carried out as described for bisoxirane. Immobilized DAH
was activated through reaction for 8 minutes with a mixture of 5 ml epibromidehydrin and 5 ml 4 M caustic soda solution at 90 ~C and immediately reacted with L-histidine at 80 ~C (0.5 g L-histidine in 20 ml water, pH 12). The finished membrane was washed with 1 M sodium chloride solution and water.

The corresponding procedures were used for the coating with other polymers and the covalent bonding with the other endotoxin specific ligands.

2. Separation Experiments CA 02249~48 1998-09-11 All investigations on the adsorption behaviour of endotoxins on the membranes were carried out at room temperature in the dead-end mode.

Each individual membrane piece was fixed on the floor of an ultrafiltration cell (membrane surface of 13.4 cm2) and washed with a 30 % ethanol 0.1 M caustic soda solution, 1.5 M sodium chloride solution and pyrogen-free water to remove traces of endotoxin. After equilibration of the membrane, 20 ml of contaminated solution was filtered through each membrane at a flow rate of 2 ml/min. The filtrate was collected and examined in the LAL test.

3. Endotoxin Test To quantify the endoxin in the starting solution and in the filtrate, a chromogenic Limulus Amebozyte Lysate test (LAL
test) was used. The test is based on the fact that the endotoxin induces the release of the chromogen p-nitroaniline, whereby a linear relationship exists between the released amount of p-nitroaniline and the given endotoxin concentration in the range of 0 to 1.2 EU/ml. With the photometric determination of p-nitroaniline, the endotoxin concentration in the sample can be derived with the aid of a calibration line (standard endotoxin E. coli Olll:B4).

The LAL test was introduced in Europe in 1995 from the European Pharmaceutical Handbook Commission for detection of endotoxins and since 1989 has also replaced the rabbit test in the monograph entitled "Wasser fur Injektionszwecke".

Sample Applications CA 02249~48 1998-09-11 1. (Fig. 5) Separation from highly-loaded buffer solutions.
Feed: 20 ml 20 mM phosphate buffer (pH 7) with 6000 EU/ml added thereto.

The membranes marked with -d represent membranes not incorporating a spacer.

2. (Fig. 6 to 7) Separation from endotoxin-enriched BSA
solutions Feed: 20 ml 20 mM phosphate buffer (pH 4.66) with 1 mg/ml BSA and 6610 EU/ml added thereto.

Protein recovery: BSA

3. (Fig. 8) Separation from commercial BSA
Feed: 20 ml 20 mM phosphate buffer (pH 4.66) with 1 mg/ml BSA
Endotoxin concentration 65 EU/ml 9. (Fig. 9 to 10) Separation from commercial lysozyme Feed: 20 ml 20 mM phosphate buffer (pH 7) with 1 mg/ml lysozyme Endotoxin concentration 134 EU/ml Protein recovery: lysozyme 5. (Fig. 11 to 12) Separation from MAX 16 H 5 Feed: 20 ml 20 mM phosphate buffer (pH 5.5) with 3 mg/ml protein Endotoxin concentration 62.5 EU/ml Protein recovery: IgG

CA 02249~48 1998-09-11 -6. Separation from previously purified bFGF
Feed: 5 ml bFGF containing 9 EU/ml The separation was studied with a PEI membrane. In the filtrate, 0.202 EU/ml was still detectable.

7. Separation from milli-Q water containing endotoxin Feed: 1 l water containing 270 EU/ml A PEI and a DAHHis membrane were used for separation.
PEI filtrate: < 0.015 EU/ml DAHHis filtrate: 0.07 EU/ml CA 02249~48 1998-09-11 Abbreviations BSA bovine serum albumin bFGF alkaline fibroplast growth factor DAH diaminohexane DEAE diethylaminoethyl DEX dextran DEX/2 a membrane coated twice successively with dextran DEX/3 a membrane coated three times successively with dextran DOC desoxycholate EP European Pharmacopeia EU endotoxin unit FDA Food and Drug Administration HEC hydroxyethylcellulose Him histamine His histidine MW molecular weight N66 untreated Nylon membrane PEI polyethylene imine PLL poly-L-lysine PMB polymyxin B
PVA polyvinyl alcohol USP US Pharmacopeia

Claims

1. Microfiltration membrane for separation of endotoxins from liquid media, in particular water, protein solutions or parenteralia, characterized by covalently bonded ligands for endotoxins, the ligands being carried by a hydrophilic polymer, which is water soluble itself and which is covalently applied to the membrane.

2. Microfiltration membrane of claim 1, characterized by (a) histamine, histidine, polyethylene imine, poly-L-lysine or polymyxin B as the endotoxin specific ligand, and/or (b) diaminohexane, diethylaminoethyl ligand or desoxycholate as a ligand not specific to endotoxin per se.

3. Microfiltration membrane of claim 1 or 2, characterized by regenerated cellulose, cellulose acetate, polysulfone, polyethylene vinyl alcohol or polyamide, in particular Nylon, as membrane material.

4. Microfiltration membrane of any one of the preceding claims, characterized by dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethylcellulose, as the hydrophilic polymer.

5. Microfiltration membrane of any one of the preceding claims, characterized in that the polymer is carried by the membrane with the aid of a spacer.

6. Microfiltration membrane of claim 5, characterized by a spacer derived from bisoxirane, glutardialdehyde, epihalogenhydrin or diisocyanate.

7. Microfiltration membrane of any one of the preceding claims, characterized in that the ligands are covalently carried by the polymer with the aid of a spacer.

8. Microfiltration membrane of claim 7, characterized by a spacer derived from bisoxirane, glutardialdehyde, epihalogenhydrin or diisocyanate or optionally oxidatively activated bisoxirane.

9. Use of a microfiltration membrane according to any one of the preceding claims for separation of endotoxins from liquid media, in particular from water, protein solutions or parenteralia.