SE452554B - MEMBRANE AND DEVICE FOR REMOVING A SUBSTANCE FROM A DISPOSAL - Google Patents

Description

.. \ _ 452 554 10 15 20 25 30 processes, where a solution containing the current substance brought into contact with the biologically active substance for transformation of the substance into the desired association. Accordingly, although the invention has a broad and diverse range of uses, however, it has proven particularly valuable in the treatment of whole blood at so-called enzyme therapy, and will in the following be described with the emphasis placed on this latter application cation. rsxnrxsns sråunrnsxr Enzyme therapy is a form of treatment according to which one strives to replace a lost or deteriorated one enzymatic function in a patient, for example in connection with genetically determined diseases or as in some forms of cancer. eg lymphoblastic leukemia, add one desired enzymatic function. The treatment is generally available so that one lets the enzyme. for example asparaginase in the treatment of patients with lymphoblastic leukemia, come in contact with the patient's blood in the bloodstream for enzymatic degradation of an aubstrst, in this example asparaginas. The enzyme can then break down asparagus somiâr essential for tumor cells, which means that tumor cells the cells die due to malnutrition. This can be done by a solution containing aaparaginase injicers intravenously in the patient.

The form of treatment described above is unsatisfactory in several respects, mainly due to the fact that one usually does not have access to human enzymes but is referred to use enzymes from, for example, mammals or bacteria. When such non-human enzymes come into contact with the blood cells in the patient's blood, antibodies against the enzyme in question, whereby the enzymes become ineffective. because the antibodies will quickly stop the enzyme 10 15 20 25 30 452 554 feature. In some cases, anaphylactoid reactions may also occur occur in the patient as a result of the reaction between antibody and enzyme (the antigen). Even in cases where you have access to human enzymes for the treatment, the problems are great, because the stability of enzymes in the patient's bloodstream is often low and it the enzymatic activity thereby becomes difficult to maintain a satisfactory therapeutic level.

I Chemical Engineering Communications 2 (l975) = 1. pp. 37-47, a technique is proposed according to which one uses of a microporous, semipermeable, asymmetric membrane with a biologically active substance (enzyme) containing closed in the pores of the membrane for diffusion contact with a solution containing a substance to be degraded or similarly removed from the solution. A disadvantage with this technology is that it can be used exclusively in associated with very large molecular enzymes, because less enzymes can penetrate the membrane pores and thereby get free access to the solution, which may for example be one body fluid. Another disadvantage may be redistribution of the enzyme (axial enzyme transport) involving a pre-fermented »Manner and earned lower efficiency. ' The object of the present invention is therefore that provide an improved membrane and device for removal of a substance from a solution according to the technique described in the above-mentioned Chemical Engineering Communication cations. In particular, the invention aims to achieve a new form of enzyme digestion according to which those described above the inequalities are avoided, whether human enzymes or enzymes from mammals or bacteria were used.

The above objects are achieved according to the invention with by means of the diaphragm and the device defined in subsequent claims and which will be described in more detail below. .. __..._.._. 1 .... .-. aaw-.híøa-.a -... a-a-í. 10 15 20 25 30 35 40 l4s2 sslwi DESCRIPTION OF THE INVENTION Thus, according to the invention, a micro- porous, semipermeable, asymmetric membrane for use in connection with the removal of a substance from a solution, which membrane comprises an immobilized biologically active substance enclosed in the pores of the membrane for diffusion contact with said solution. The membrane is characterized therefrom, that the biologically active substance is immobilized on and / or in microparticulate gel granules and that of the hollow fibers minimum pore size is smaller than the size of the enclosed microparticulate gel beads (16) with immobilized bio- logically active substance (14).

Asymmetric membranes are known per se for example, U.S. Patent 3,615,024 and therefore shall not described in more detail here. For more information regarding such membranes are instead referred to U.S. Patent 3,615 024. To facilitate the understanding of the invention, however, it is mentioned that asymmetric membranes are built up on the one hand side a very.thin layer with small pores (barrier layer) and on the other hand, a thicker layer with large pores (supporting layer), the pores having gradually increasing size from the barrier layer against the support layer, where the pores are thus as largest.

According to the invention, the asymmetric membrane should have sufficiently large pores at the backing layer for it immobilized biologically active substance, preferably an enzyme, should be able to easily penetrate deep into the membrane pores from this side, and at the same time have sufficiently small pores on the barrier layer side to prevent the substance from completely penetrating the membrane. In addition the membrane according to the invention must be sufficiently small pores on the barrier layer side to exclude unwanted components, when the membrane is intended for use in connection with the treatment of whole blood in enzyme therapy.

While avoiding contact between the immo- bilized, entrapped biologically active substances and the solution containing said substance by means of the membrane according to the invention, the advantage is also achieved that much short diffusion distances between the solution containing 10 15 20 25 30 35 452 554 said substance and the immobilized active substance can obtained by the immobilized substance can penetrate deep into the pores of the membrane against the barrier of the membrane layer side, which is intended to face the solution containing containing said substance.

Examples of suitable pore sizes of the membrane according to the invention may be> 1 μm, preferably> 10 μm on the support layer side, while the barrier layer side of the membrane may have one cut-off of less than 150,000 Daltons, preferably 20,000 - 50,000 Daltons.

The home membrane according to the invention may be in the form of a flat foil or hose, but is preferably in form of semi-fibers. wherein the solution containing said substance is intended to be guided through the axial cavity thereof fibers.

Examples of materials used for the membrane according to the invention may be polyamide, cellulose acetate, poly- sulfone, polycarbonate, polyacrylonitrile, preferably polyamide. For other useful materials, see to the aforementioned U.S. Patent 3,615,024.

Suitable microparticulate gel granules may be those described in, for example, U.S. Patent 4,061,466 moreover, these gel granules are spherical particles of polyacrylic derivatives, agarose or polystyrene, preferably polycrylic amid.

According to the invention, said gel granules have a diameter of <10 μm, preferably 0.1 - 0.5 μm.

Examples of useful biologically active substances may be antibodies, antigens, enzymes, protein A, killed bacteria or fragments thereof, preferably enzymes.

According to the invention, a device is also provided removing a substance from a solution, which device comprises a microporous, semipermeable, asymmetric membrane with a biologically active substance hilicated inside the membrane pores for diffusion contact with ...___ .......-....... dn. «- a..1.-.... 10 15 20 25 30 35 452 554 6 said solution. The device is characterized in that it the biologically active substance is immobilized on and / or in microparticulate gel granules and that the hollow fiber minimum pore size is smaller than the size of the enclosed micro- particulate gel beads (16) with immobilized biological active substance (14).

According to the preferred embodiment of the device according to the invention, the membrane is used in the form of hollow fibers enclosed within an outer casing with a first inlet and a first outlet in connection with the axial cavities in the fibers. Furthermore, the housing may comprise a second inlet or a second outlet in connection with an outer closed fluid circuit for a fluid which is intended to be passed through said casing on the outside of the half-fibers, as will be described below.

Preferably, the outer fluid crete comprises a pump to pump said liquid through with pulsating flow the casing on the outside of the hollow fibers. Through the pulsating the flow, albeit with small pulse variations, comes alternately overpressure and negative pressure to be created inside the housing, whereby the transport of permeable fractions of it through it axial cavity of the hollow fibers flowing solution containing said substance through the semi-fibrous walls for contact with the enclosed immobilized biologically active sub- punching is facilitated.

When the device according to the invention is intended for treatment of whole blood includes the external fluid circuit preferably also a detector for sensing any leakage of whole blood inside the casing.

BRIEF DESCRIPTION OF FIGURES The invention will be described in the following more detailed with reference to the attached schematic drawings, there 10 15 20 25 30 35 452 554 Fig. 1 shows a block diagram of a preferred one embodiment of the device according to the invention, Fig. 2 shows a preferred embodiment of a such detail in the membrane included in the device shown in Fig. 1 according to the invention, Fig. 3 is an enlarged cross-sectional view of the nembrane of Figs. 2, taken along the line III-III and Fig. 4 shows a practical embodiment of the outer casing enclosing a fire according to the invention and intended to be used as a detail in the device shown in Fig. 1 according to the invention.

DETAILED DESCRIPTION OF THE INVENTION As shown schematically in Fig. 1, the device comprises according to the invention an asynthetic nenbran (shown schematically with dashed lines 1, l ', 1 ") down indefinitely outer casing 2 provided with a first inlet 3 and a first outlet 4 and a second inlet 5 and a second outlet 6.

The first inlet 3 of the housing 2 is connected with a source 7 for the solution containing said inside via a conduit 8 comprising a puncture 9, below the first the outlet 4 of the housing 2 is connected down said kills 7 via a return line 10 for returning the solution to this source. The second inlet 5 of the housing 2 is in connection to the second outlet 6 via a line 11 comprising a puncture 12 and a detector 13 during formation of a closed external fluid circuit. as shown in Figs. 2 and 3, there is asyn- the metric nanbrane preferably in the form of hollow fibers 1, preferably of polyamide, down the biologically active substance 14, for example an enzyme, immobilized on a carrier 16 of nicroparticulate polyacrylanide gel granules down a such a size that they can easily penetrate the pores 15 through the pore openings on the support layer side 17 of the hollow fiber 1 to very short distances from the axial cavity 18 10 15 20 25 30 35 _ un ... ..._ _. 452 554 inner wall 19 (barrier layer). Pore opening on the hollow fiber barrier layer side 19 is then small enough to prevent these gel granules 16 with stabilized biologically active substance 14 from penetrating completely through the pores 15.

In the treatment of, for example, whole blood in connection with enzyme therapy, these gel grains 16 are less than 10 in size μm preferably 0.1 - 0.5 μm, while the pores of the hollow fiber support layer side 17 may be larger than 1 μm, preferably greater than 10 μm. The barrier fiber barrier layer 19 may have one cut-off of less than 150,000 Daltons, preferably 20,000 - 50,000 Daltons, causing unwanted substances in whole blood, as 18, with are intended to be guided through the axial cavity of the hollow fibers prevented from penetrating the pores 15 for contact the entrapped biologically active substance, e.g. vis asparaginas.

From the above it thus appears that the first the inlet 3 is connected to the first outlet 4 via the axial cavity 18 of the half-fibers 1, 1 ', 1 "inside the housing 2, and that the second inlet 5 is connected with the second outlet 6 via that space. which is formed between the housing Z and the outside of the half fibers 1, 1 ', 1 ".

Fig. 4 shows a practical embodiment of it in Fig. 1 shows the outer casing 2 enclosing a symmetrical membrane. The same reference numerals as in Figs. 1-3 for corresponding components have therefore been used in Fig. 4, but with the addition of the letter "a". As shown, the housing 2a may have in the form of a conventional fiber dialyzer provided with a first inlet 3a and a first outlet 4a and a second inlet Sa and a second outlet Ga. The inlet 3s is in connection to the outlet 4s via the axial cavity of these fibers, while the inlet 5s is connected to the outlet 6a via the space formed between the housing 2s and the outside of the enclosed hollow fibers 1a, 1'a, 1 "s. I The device works as follows. Solution containing the said substance, which shall undergo the EU enaymatic conversion, is pumped by means of the pump 9 from its source 7 10 15 20 25 30 452 554 to the inlet 3 of the housing 2 via the conduit 8 and through it the axial cavity of the fibers 1, 1 ', 1 "back to source 7 via outlet 4 and line 10. Simultaneously pumped a liquid, for example a physiological saline solution, in it closed the outer fluid circuit by means of the pump 12 through the housing 2 via the inlet 5 and the outlet 6 on the outside of the hollow fibers 1, 1 ', 1 ". During the passage through the axial the cavity of the hollow fibers of the hollow fibers 1, 1 ', 1 " will permeable fractions en solution containing said substance to penetrate into the pores 15 for contact with it biologically active substance 14, preferably an enzyme. for an enzymatic conversion of said substance. Unwanted substances in the solution, such as antibodies associated with whole blood, is prevented from penetrating the pores 15, varying by direct contact between these substances and the entrapped biologically active substance was prevented.

The penetration of the permeable fractions of the solution is facilitated by the pulsating flow in the outer the hydraulic crete, which is effected by means of the pump 12. Hed with the help of the detector 13, any leakage is quickly detected solution, for example whole blood, inside the casing 2. wherein the system is stopped.

The invention will be further elucidated with the aid of the following concrete embodiments. In all examples used a hollow fiber type haemofiltration unit (polyamide) from Gambro.

Examples 1-3 In these examples 1-3 it was biologically active the substance of the enzyme asparaginase from E. coli (Craanitin, Bayer), stabilized on microparticulate polyamide grains (0.2 0.5 μm) enclosed in the hollow fiber pores in a manner written above. The plasma asparagine was determined according to a fluorometric method (D.A. Cooney, R.L. Capizzi and 1.3.- Glovemaker, Cancer Research 30, 929-935. 1970). 10 15 20 25 30 35 452 554 10 In Example 1, a dog (weight 12 kg) was treated with a device containing 500 U L-asparaginase. L-aaparaginas- plasma levels were reduced from 45 nmol / ml after 225 U minutes of treatment. Blood flow was 20 ml / min.

In Example 2, the same hemofiltration unit as in Example 1 with an L-asparagine solution (2 L) containing 50 nmol L-asparagine per ml. The L-asparagine content was reduced practically complete within 30-40 minutes at one flow of 150-200 ml / min.

In Example 3, sheep (weight 40 kg) were treated with a hemofiltration unit of the same type as in Examples 1 and 2, but containing 2,000 U L-asparaginase in microparticles polyacrylamide grains. L-asparagine content reduced from normal 40-50 nmol / ml to <10 nmol / ml after 120 minutes treatment at a blood flow of 150-200 ml / min.

As further examples of the use of present invention in also purely industrial processes the following can be mentioned.

Example 4 - manufacture of stereospecific amino acids with using bacterial enzymes.

Stereospecific transformation of ammonium sulphate to L-aspartic acid by an amination reaction. Reaction catalyzed by the enzyme sspartase from E. coli. Bnrymet immobilized in microparticles (0.25 - 0.5 μm) from above described type and installed in the outer space of a hemofiltration device, for example of the type used in Examples 1-3 with asymmetric membranes. A solution included containing said substance, which in this example is ammonium fumarate, is pumped through the unit, the substance being diffused through sion comes in contact with the aspartase and is converted to L ~ aspartic acid which is then isolated.

Example Q - manufacture of 6-aminopenicillanic acid (6-APA). r The biologically active substance, in this example. IN penicillanacylase from E. coli, immobilized in asylum metric membranes in a hemofiltration unit, such as written, after which a solution containing crude penicillins 10 15 20 25 30 35 452 554 ll pumped through the unit. Râpenicillins, obtained from Penicillum cultures, are converted in the reactor to 6-amino- penicillanic acid, which is then isolated and can be used as starting materials for specific synthetic penicillins.

Example 6 - manufacture of glucose and galactose from lactose.

This example uses 3-galactosidase from B.coli or yeast as a biologically active substance according to the invention and is carried out in the same manner as above. One solution containing lactose will thereby form glucose and galactose on contact with the immobilized biologically active substance inside the haemofiltration unit.

INDUSTRIAL USE The device and the membrane according to the invention are useful in connection with, for example, one-rheumatic transformation of a substance present in a solution, such as therapeutic treatments or analyzes of body fluids as well as for purely industrial processes in which an enzyme risk conversion included.

It is especially valuable in connection with different types of treatments or processes, where one wishes to remove a substance from a solution either by adsorption or chemical binding of the substance in question in a biological active substance, or by chemical or biological transformation of the substance by contact with the biologically active the substance. Examples of the previous application can be in in connection with a therapeutic treatment of blood, where undesirable the substances, eg antibody / antigen complexes in the blood are removed by binding to the biologically active substance, eg protein A. Examples of the latter application may be in connection with a.k. enzyme therapy according to which an undesirable substance in, for example, a body fluid, especially whole blood; 7 removed by enzymatic transformation in contact with it biologically active substance (enzyme). Another one examples may be in connection with pure industrial pre- 452 554 12 parative processes, in which the subject in question is in contact with the biologically active substance is converted by a biological or chemical conversion reaction to a striving association.

Claims (14)

10 15 20 25 30 35 13 452 554 PATENT REQUIREMENTS A microporous, semipermeable, asymmetric membrane for use in removing a substance from a solution, which membrane comprises an immobilized biologically active substance (14) enclosed in the pores of the membrane for diffusion contact with said solution, characterized in that the biological the active substance (14) is immobilized on and / or in microparticulate gel beads (16) and that the minimum pore size of the hollow fibers is smaller than the size of the enclosed microparticulate gel beads (16) with immobilized biologically active substance (14). Membrane according to claim 1, characterized in that it is in the form of half-fibers, the solution containing said substance being intended to be passed through the axial cavity (18) of these fibers. A hembran according to claim 2, characterized in that thereof; that the pore size on the side (17) of the hollow fibers, which is intended to wett from the solution containing said substance, is> ln m, preferably 101¿m¿ Home membrane according to one of Claims 2 to 3, characterized in that the hollow fibers have a cut-off of <150,000 Daltons, preferably 20,000 - 50,000 Daltons. Membrane according to any one of claims 1-4, characterized in that said gel granules (16) have a diameter of <10 μm, preferably 0.1 - 0.511 m. The membrane according to any one of claims 1-5, characterized in that said gel granules (16) are selected from polyacrylamide, agarose and polystyrene, preferably polyacrylamide. Hembrane according to any one of claims 1-6, characterized in that it is selected from polyamide, cellulose acetate, polysulfone, polycarbonate, polyacrylonitrile, preferably polyamide. 10 15 20 25 30 35 452 554 14 '8. Membrane according to one of the preceding claims, characterized in that the biologically active substance is selected from antibodies, antigens, protein A, killed bacteria or fragments thereof, and enzymes such as asparaginase, aspartase from E. coli, penicillanic acid from E. coli , Β-galactosidase from E. coli and yeast. Device for removing a substance from a solution, said device comprising a microporous, 1 "; 1a, 1'a, 1" a) immobilized semipermeable, asymmetric membrane (1, 1 ') with a biologically active substance (14). inside the pores of the membrane for diffusion contact with said solution, characterized in that the biologically active substance is immobilized on and / or in microparticulate gel beads (16) and that the minimum pore size of the hollow fibers is smaller than the size of the enclosed microparticulate biologically active beads ( substance (14). Device according to claim 9, characterized in that said membrane is in the form of hollow fibers, the solution containing said substance being intended to be passed through the axial cavity (18) of these fibers. Device according to claim 10, characterized in that said hollow fibers are enclosed within an outer casing (2; 2a) with a first inlet (3; 3s) and a first outlet <4; aa) 1 fafb1nae1se with the axial nsxignene of in the fibers. Device according to claim 11, characterized in that said housing (2; Za) comprises a second inlet (5; Sa) and a second nt10PP (6: 6a), respectively, in connection with an outer liquid circuit for a liquid, which is intended to passed through said casing on the outside of the hollow fibers. Device according to claim 12, characterized in that said outer liquid circuit comprises a pump (12), preferably a so-called roller pump, for circulating said liquid in this circuit. _ Device according to claim 12 or 13, characterized in that said outer circuit comprises a detector (13) for detecting any leakage of solution containing said substance within the housing (2; 2a). '