FR2777803A1 - Dialysis process with enzymatic pumping to increase speed and selectivity for removal of small solutes, particularly removal of glucose from blood - Google Patents

Abstract

Dialysis process is assisted by enzymatic pumping of at least one solute (S) across a porous membrane that separates at least two liquid compartments (CI, CII), in which CI becomes depleted, and CII enriched, in S.- DETAILED DESCRIPTION - An enzyme (Ea) is present in, or on, the face (FII) of the membrane directed towards CII, and can convert S in CII to a product (SaII), and the liquid (dialysate) in CII (preferably also that in CI) is set in motion by providing at least one non-turbulent diffusion layer (dII) on FII, with thickness (edII) controlled to be 0.1-10, preferably 0.5-3, em (em = the thickness of the membrane). Conditions are maintained such that S in CII is converted to SaII, and this conversion induces diffusion of S in CI towards CII. An INDEPENDENT CLAIM is also included for an apparatus for the process.- ACTIVITY - None given.- MECHANISM OF ACTION - None given

Description

 The field of the present invention is that of the separation and elimination / purification of small molecules (solutes) in the liquid phase, by passage through a bioactive porous membrane. This is aimed at the depollution of a given liquid compartment by eliminating unwanted solutes or, more generally, the isolation of some solutes not necessarily polluting but can be endowed with a high added value. These techniques for transporting ionic or non-ionic solutes by diffusion through a membrane, whose role is to prevent the convective transport of the solvent, are commonly referred to as dialysis.

 The invention therefore relates to an active and selective dialysis process, assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least part of two liquid compartments C, and Ca, the transfer of operating Cl that is depleted in S to C11 that is enriched in S.

 The invention also relates to a dialysis device assisted by enzymatic pumping, useful especially for the implementation of the above-mentioned method.

 Passive transmembrane diffusion dialysis has been known for a long time. It has been the subject of numerous ad hoc applications in the chemical industry and in the agri-food and pharmaceutical fields. It is essentially a slow operation which is conditioned by the existence of a concentration gradient on either side of the membrane for the solute (s) to be separated and / or eliminated. Dialysis has therefore proven to be relatively expensive at the industrial level. Thus, more radical processes, based on external driving forces (mechanical or electrical), have been much more studied and exploited, despite their higher energy cost. In fact, industrial dialysis becomes competitive only when these energy-consuming processes are ineffective, when they risk damaging the solute to be isolated or even when the slowness of the dialysis operation does not constitute a handicap. These three conditions apply to a technique that is not specifically industrial, but which at present is the most important application of dialysis: hemodialysis.

 Hemodialysis is a medical technique for subjects with partial or complete renal failure. It consists of an extracorporeal treatment of blood, providing the same functions as the kidney, thanks to a membrane process based on both dialysis and ultrafiltration. A less elegant solution than organ transplantation, it is essential because of the disproportion between the number of potential donors and the number of applicants.

The small molecules that are to be removed from blood in hemodialysis are urea, uric acid, creatinine, and other low molecular weight metabolic wastes. This elimination must be done selectively by allowing the treated blood to retain the macromolecules and metabolites essential for these functions and preserving its electrolytic and aqueous balance.

 In addition to hemodialysis, hemofiltration is also known as a method of removing certain unwanted solutes from the blood. The principle of hemofiltration is to use filtration as a driver of solute exchange.

Hemofiltration allows good purification of medium molecules.

 In general, in both of these methods, the blood is passively purified (hemodialysis) or by creating a slight difference in pressure between the blood circulating in the hollow fiber lumen and a liquid called a dialysate circulating outside the lumens. fibers (hemofiltration). In both cases, the passive or induced diffusion does not make it possible to differentiate between them the small molecules contained in the blood, according to which they are harmful and thus to eliminate or indispensable and thus to preserve.

Selective removal of certain small molecules is therefore not possible by known hemodialysis or haemofiltration techniques.

 To overcome this, it would be interesting to have a new technique to quickly and selectively eliminate a small molecule.

 Moreover, it is known that for dialysis techniques to be effective, it is imperative that there is a concentration gradient on either side of the membrane, the solute concerned circulating from the most concentrated compartment to the compartment the less concentrated in solute. This implies the implementation of large volumes of dialysate to reduce the concentrations outside the compartment to be cleaned. In addition to the difficulties of handling and congestion they cause, these large volumes of dialysate also have the disadvantage of causing leakage of molecules or blood solutes essential.

 In such a state of the art, one of the essential objectives of the present invention is to improve the known techniques for the separation or elimination of certain solutes by transmembrane diffision, by providing a forced dialysis method making it possible to lower a consistent and rapid way to concentrate the level of concentration of a solute in a given liquid compartment by transferring it through a membrane into another liquid compartment, irrespective of the level of concentration of the solute considered in the latter compartment; that is, even in the absence of a concentration gradient favorable to a diffusion flux from the most concentrated compartment to the least concentrated compartment.

 Another essential objective of the invention is to provide a forced dialysis method for extracting, for example for depollution, purification or isolation purposes, a given solute by implementing a transmembrane diffusion having a significantly kinetic superior to that of a conventional passive transmembrane diffusion process.

 Another essential objective of the present invention is to provide a differential dialysis method that is differential and therefore selective for one or more solutes.

 Another essential objective of the present invention is to provide a simple, fast and economical dialysis process.

 Another essential objective of the present invention is to provide a selective and rapid dialysis process that eliminates the need for large quantities of dialysates hitherto useful for generating concentration gradients required for passive diffusion.

 Another essential objective of the present invention is to provide a dialysis process that is simple, selective, rapid, economical and applicable in particular in hemodialysis in a high performance manner.

 Another essential objective of the present invention is to provide a dialysis device adapted to allow in particular the implementation of the aforementioned method.

 Another essential objective of the invention is to provide a dialysis device that is simple, economical, selective and fast.

Having set these objectives, the inventors had the merit of highlighting, after long and painstaking research and experimentation, that it was possible to achieve these objectives by stimulating and optimizing the phenomenon of passive diffusion, by placing of an auxiliary enzyme pumping system. This original auxiliary mechanism, discovered by the inventors, is based on the intervention of at least one specific enzyme of the solute considered and disposed on the face of a porous membrane opposite that which is opposite the liquid compartment that is being sought. to deplete in solute by transmembrane diffusion. Such an enzymatic system may comprise at least one pair of reverse enzymes, one of the enzymes of the pair being attached to one side of the membrane, while the other enzyme is immobilized on the other side of this membrane, these Moreover, the enzymes are optionally contained in the liquid medium of the compartments, excluding the porous enzymatic diffusion membrane. This system behaves like a pump that generates and maintains a solute concentration difference (s) to be transferred between the two compartments.
As a result, the present invention firstly relates to a method of dialysis assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least partly two liquid compartments Cl and Cll, (S being referred to as SI in Cl and S, in CH), this dialysis is effected from the liquid compartment C1 which is intended to deplete in S, towards the liquid compartment CII intended consequently to be enriched in S, characterized in that it consists essentially and successively or not:
to implement at least one porous membrane comprising on
and / or in its face Fn opposite CII at least one enzyme E α apt
to catalyze the reaction of transformation of the solute S11 into solute
primary product S, n;
to move at least the liquid of C11 (dialysate), and
preferably also that of Cl> by providing at least one layer
non-turbulent dll diffusion, adjacent to the face F11 of the
E & alpha carrier membrane; and next to Cll, and regulating
the edII thickness of this diffusion layer dll so that
em em
# edII # 10.em, preferably # edII # 3.em;
10 2
where em is the thickness of the membrane;
and to ensure that the operating conditions are such
qu'interviennent:
* enzymatic transformation by E α from SII to S α II,
* and correlatively the diffusion of Sl, through the membrane
from compartment C1 to compartment Cll, in particular under
the effect of enzymatic pumping, induced by the reaction

 The innovative technical principle that governs the process according to the invention can be likened to an enzymatic pumping mechanism for transferring a solute S into a liquid compartment (I), to a compartment (II) liquid arrival, by active transport through an enzymatic porous membrane. The solute S accumulates and concentrates in the arrival compartment, even if the concentration of the solute in the latter is greater than that of S in the starting compartment. Moreover, the enzymatic pumping according to the invention has the effect of maintaining a concentration difference dS = [Su] - [St]> 0.

Such an enzymatic pumping method is particularly advantageous since it allows a rapid and selective elimination of one or more solutes of a liquid compartment C1, for example blood (hemodialysis), without the use of large volumes of dialysate. Without excessive consumption of energy. This enzymatic pump essentially only requires chemical energy, which can be provided, for example, by energy-rich binding molecules, such as adenosine triphosphate (ATP).
It should be noted that the kinetic gain provided by the invention remains compatible with the threshold of duration below which blood stress is generated in the hemodialysis application.

 This is due to the fact that the non-turbulent diffusion layers proper to the method according to the invention, somewhat limit the flow velocities of blood and dialysate during hemodialysis.

 The development of the process according to the invention is rooted, in particular, in the understanding of the active transport phenomenon specific to biological membranes. This new scientific theory on biological transmembrane transport brings new blood to the state of scientific knowledge in this field. The latter can be summed up in the theory based on pores and in the theory based on the conformational transformation of proteins of biological membranes.

The novel and innovative approach according to the invention therefore consisted, in a first step, in understanding and explaining the biological phenomenon and, in a second step, in reproducing it (mimicry) by adapting it to the medical or industrial specifications of separation and concentration elimination. chemical compounds, in particular for the purpose of eliminating and isolating toxic compounds, or for recovering molecules with high added value: chemical, pharmaceutical, cosmetic or other.

In accordance with a preferred feature of the invention, one selects, on the one hand, the enzyme E which governs the production of Sa and, on the other hand, the porous membrane, so that the latter is impermeable to SalI and thus prevents, at least partially, the Sarx dispersion from C11 to C1; San and the membrane are thus preferably electric charge entities of the same sign.

The electrically charged character of SalI results from the action of the enzyme Ea on the neutral substrate S11.

The electric charge of the faces of the porous membrane can be obtained for example by immobilizing, on the membrane, the enzymes E α and Ess and polyamino acids whose pKs give the membrane, for a given pH, negative or positive charges. Thus, in the case where the polyamino acid is e.g. polylysine, then the membrane is positively charged at pH 9, whereas with a polyamino acid corresponding e.g. to a polyglutamate, the membrane is negatively charged at pH 9.

According to an alternative, one can choose membranes whose zeta potential at the optimum pH is adapted to given conditions of the process of the invention.

In the absence of means to prevent pollution of the liquid compartment C1 by the product S α II of the reaction involving SII as substrate and E α as an enzyme or to complete the action of these means, a first variant is provided for the process according to the invention in which:
at least one porous membrane comprising at least one
and / or in its face F1 opposite Cl, at least one suitable enzyme Ess
to catalyze the transformation reaction of the solute-primary product S, resulting from the diffusion through the membrane of Sa in from C11 to
Cl in solute-secondary product Sss,
being preferably the inverse enzyme of E "so that SssI = =
provision is made for the movement of Cl and C11 fluids in
providing a non-turbulent diffusion layer dl, d11 related to
each face Fl, F ", and adjusting the thicknesses edI and edII of these
layers so that:
* 0.1.em # edI / edII # 10.em,
preferably 0.5.em S edIl edII <3 .em;
operating conditions favorable to the transformation are fixed
Enzymatic by Ess of S &alpha; I in SssI, and, correlatively, in the case where
= S, the diffusion of SssI from Cl to CII, through the
membrane, in particular under the effect of a resulting enzymatic pumping induced by the reaction

 We are here in the presence of a porous membrane separating C1 and CII and carrier on each of its F1 and FII faces of enzymes, preferably reversible, Ess and E &alpha; respectively. The enzymatic pump, thus equipped with additional enzymatic means, allows the retransformation of the S & alpha metabolites, resulting from the transfer of the S &alpha; II metabolites from C11 to C1. The S metabolite, retransformed into SI in C1, can then join the S-transfer flux of Cl. towards C "through the membrane.

 According to the invention, it could be envisaged to fix Ess on a support other than the F1 face of the membrane. Ess could thus be carried by an additional membrane and / or on any other solid support dispersed or not in Cl.

According to a second variant of the method according to the invention:
. we bring in CII; at least one enzyme E capable of catalyzing
the transformation reaction of solute Sa into solute Su,
and set operating conditions conducive to the progress of this transformation

In this second variant, the S &alpha; OO metabolite is eliminated as it is formed, to further enrich CII in Su solute. In doing so, the concentration difference AS = [S "] - [Sl] is further increased, which confirms whether it is necessary for the enzyme pump according to the invention to operate by generating this AS and maintaining it.

 It is apparent from the above description that it is important according to the invention to provide a diffusion layer dl, d11 non-turbulent adjacent to each face FI, FII of the membrane or membranes, especially when the face is considered carrier of an enzyme constitutive of the enzymatic pump.

 The differences between the rates of appearance and disappearance of the solute S in the compartments Cl, CII, on either side of the membrane, depend in particular on the proper functioning of the enzyme pump and therefore in part on the thicknesses of the diffusion layers. not turbulent, but also for other parts of the compartments Cl and Cx. Thus, the volume Vu of CII is preferably greater than volume VI of Cl. Advantageously, the ratio V n (V 1 is as large as possible so as to facilitate the kinetics of the transport.

et

interviennent au voisinage de chacune des faces FI, FII de la membrane, il est non seulement important de prévoir les couches de diffusion d1 et du évoquées ci-dessus, mais également de mettre en place des conditions opératoires optimales, notamment de pH et de température, qui seront détaillés infra.For enzymatic transformations

and

are involved in the vicinity of each of the faces FI, FII of the membrane, it is not only important to provide the diffusion layers d1 and evoked above, but also to set up optimal operating conditions, including pH and temperature , which will be detailed below.

With regard to the diffusion layers, the adjustment of the stirring conditions, and thus the Edl and Ed thicknesses of the non-turbulent diffusion layers d1 and dn, can be carried out on the basis of the article by BARDELETTI, G. , B. MAISTERRENA and P.

R. COULET, 1985 "Boundary layer on product flow-splitting in an immobilized membrane membrane"; J. Membrane Sci. 24; 185-296.

According to a preferred characteristic of the invention, Ed1, EdII are between 50 and 500 micrometers, depending on the hydrodynamic conditions imposed.

 The second variant corresponds to the preferred embodiment of the invention, which combines the advantages of an enzymatic pumping system comprising two reversible enzymes fixed, respectively, one and the other on the opposite faces of a membrane. porous diffusion and a third active enzyme in the compartment C11 for enriching S solute diffusing through the membrane. This third Es enzyme is, preferably (as is Ess) the reverse enzyme of the Cn E enzyme.

The enzyme E8 is advantageously contained in C11 on a support other than the face
FII of the membrane. E8 can thus be, for example, fixed on an auxiliary membrane or any other suitable support bathed in C11 or else be dispersed in Cn.

The enzymatic pump according to the invention has an action promoting the diffusion of solute S through the membrane. This action involves the creation and maintenance of a concentration gradient AS = [S111- [Sl]> 0 on both sides of the membrane.

The retransformation in Cl and C "of the enzymatic metabolites resulting from the enzymatic reactions with E & alpha and Ess, in Si and / or S solutes, optimizes the effect of the pumping.This can be further completed by conferring on the membrane a valve effect by relative to the metabolites S &alpha; II produced in C, to prevent these from rebroadcasting in Cl.

The result of the enzymatic pumping according to the invention is the transfer of S, Cl in Cll. Once this result is achieved, it is easy to recover SII for disposal or recovery purposes.

 According to a first mode of operation of the process according to the invention [which may be described as discontinuous], the enzyme membrane is in the form of a monolayer or multilayer film, preferably substantially plane.

This discontinuous mode corresponds, for example, to the case in which there is a reactor formed by a first container containing the liquid medium in which is at least partially immersed a second container. The immersed part of this second container being constituted at least in part by the porous enzymatic membrane. These two containers define the compartments Cl and Cll.

The membrane may form all or part of the container wall. Preferably, this membrane constitutes a substantially flat portion of the partition wall between the two compartments C1 and C11.

 According to a second mode of operation of the process according to the invention (which will be described as continuous), the enzymatic membrane is in tubular form and more precisely constitutes all or part of the wall of a tube, the light of which forms one of the compartments Cl.

Advantageously, the enzymatic membrane may be made of a plurality of hollow fibers or tubular membranes, preferably bundled together (x).

 According to this second continuous mode of operation, at least a portion of this (or these) tubular membrane (s) is bathed in the liquid medium forming the other compartment C11 and a liquid to be depleted in solute is circulated. S, in this (or these) enzymatic membrane (s) tubular (s). The circulation of the liquid in the tubular membrane or membranes constituting the other compartment C1, being provided by any appropriate means such as a pump.

 On reading the foregoing, it is easy to see the importance of the liquid medium of the Cl and Cn compartments, and in particular of their physicochemical characteristics, for carrying out the process according to the invention.

Thus, according to another interesting modality of the process according to the invention, the pH and temperature parameters of the liquid media are adjusted so as to obtain an optimum in kinetics and in yield for the enzymatic transformations by Ex and Ep or even E8.

In practice, the liquid medium of Cl and / or Cli is thus prepared using a solvent - preferably substantially aqueous -, optionally comprising solutes chosen from the following group:
e pH buffers adapted to Ea's optimum operating pH
and Ess, or even E;
energy-rich binding molecules - preferably ATP or
any molecule capable of transferring a phosphate group
on a solute (for example: phosphoenol pyruvate, carbamyl
phosphate, phosphocreatine ...)
and their mixtures.

 In addition to adjusting the pH and providing a source of chemical energy necessary for enzymatic transformations Ea and Ep, or E; the temperature of the liquid media is also regulated by caloric intake or evacuation, e.g. thermostatic reactor with double wall of circulation of temperature control fluid.

 According to the invention, the diffusion layers d1, dn are obtained by setting the liquid in motion in each compartment C 1 / C 1, this setting being carried out by stirring - preferably using a rotor in the first mode of operation according to the invention and / or by circulating said liquid according to the second mode of operation of the method according to the invention.

By these means, the stirring and / or circulation conditions are adjusted so that the non-turbulent diffusion layer (s) d, and d "have, in practice, thicknesses of at least 100 clam.

 To improve the kinetics and the transfer performance of the migratory solute S, it is possible to provide, in accordance with the invention, auxiliary means for assisting the transmembrane migration, preferably constituted by an ionic and / or electrical force gradient, of on both sides of the membrane, and more preferably still by pH gradients (protomotive force).

According to a preferred arrangement of the invention:
. S = SII = SssI = SII = glucose,
. S &alpha; = glucose-6-phosphate,
E = glucose kinase,
Ep = E # alkaline phosphatase.

 Among the interesting applications of the process according to the invention are those in which the purification of physiological liquids is carried out, preferably blood, which liquid then constitutes the liquid contained in Cl. It follows that the present invention also relates to a hemodialysis process.

According to another of its aspects, the present invention aims a dialysis device assisted by enzymatic pumping of at least one solute S characterized in that it essentially comprises:
at least one container of a liquid medium;
at least one porous membrane of thickness em, bathing or
likely to bathe in the liquid medium, to delimit at
least partially a liquid Cl compartment intended to be
depleted in solute S and at least partially a compartment
CII liquid for receiving S1 solute from Cl,
C1, CII respectively containing volumes V1 and V11;
at least one enzyme Ea capable of catalyzing, in Cll, the reaction of
transformation of S into solute-product San primary, Ea being carried
by the membrane on and / or in its face opposite Cl ,,
preferably at least one enzyme Ess capable of catalyzing, in Cl, the
transformation reaction of primary solute S &alpha; 1 = S, xll in solute
secondary product Sssl, Ess being carried by the membrane on and / or
in his face F, next to Cl,
Ess being, more preferably still, the inverse enzyme E,
so that Sss1 =
preferably at least one enzyme E5 capable of catalyzing in C11, the
transformation reaction of primary solute S &alpha; II in solute S11, E #
being dispersed in CII in immobilized form;
means for moving at least liquid CII and
preferably, also C liquid;
and possibly means for adjusting the temperature of the
liquid medium.

 This device is,

 According to a second embodiment of the device according to the invention, the enzymatic membrane constitutes all or part of the wall of at least one tube whose light forms one of the compartments Cl.

 The enzyme pairs that can be used in the method and the device according to the invention are numerous. For the sake of clarity, it can be stated that these EdEB couples are mainly chosen from enzyme pairs allowing the addition / removal of a chemical group charged to a metabolite, preferably from the pairs of phosphorylation / dephosphorylation enzymes, and more preferably still among the kinases / phosphatases.

 The invention will be better understood and its advantages or other variants of the invention will emerge from the following description of two embodiments of the device that is the subject. This device description will be completed by tests of three modes of implementation of the method according to the invention using the two devices previously exemplified.

The description of the two embodiments of the device according to the invention will be made with reference to the accompanying drawings in which; - fig. 1 represents a simplified diagram of the first embodiment of the device
dialysis assisted by enzymatic pumping, according to the invention
batch); - fig. 2 is a simplified diagram illustrating the second embodiment of the
device according to the invention (continuous mode); - fig. 3 is a symbolic representation of the enzymatic membrane separating
the two compartments C1 and C of the device of FIG. 1 or 2, said
representation corresponding to an embodiment of the dialysis method
from a solute S, from compartment C, to compartment C ":

<tb><SEP> Membrane <SEP> 11 <SEP> E
<tb> IF <SEP> X <SEP> S,
<tb> - fig. 4 is a symbolic representation of the same nature as FIG. 2, at the
difference that it concerns a first variant of implementation of the method
dialysis of solute S from compartment C1 to compartment Cn:

<tb><SEP> Membrane <SEP> E <SEP> Membrane <SEP>, aIIEe <SEP> spI = <SEP> ER
<tb> s <SEP>><SEP> Sll <SEP> S, <SEP> -.k, ScrII <SEP> S11ll
<tb> - fig. S is a symbolic representation of the same nature as FIG. 2, at the
difference that it concerns a second variant of implementation of the method
dialysis of solute S from compartment C1 to compartment C11:

<tb><SEP> Membrane <SEP> Ea <SEP> Membrane <SEP> Ep
<tb> S, <SEP> SCCI, <SEP> p <SEP> Sal <SEP> S <SEP>><SEP> 5a1
<tb> m
<tb> - fig. 6 is a simplified cross-sectional view along line VI-VI of FIG.
fig. 2; - fig. 6a is a magnified view of one of the elements of the tubular membrane seen
in section in FIG. 6; - fig. 7 represents a graph of the concentrations of SI (#), $ SII (#), $ S &alpha; II (#) in CI,
C11, as a function of the time t in min in the test carried out according to the topography of FIG. 3, - fig. 8 shows a graph of the concentrations SI (#), Sii <>, S i (o), S &alpha; II (#), in mM, in
function of the time t in min, in the test carried out according to the topography of FIG. 4; - Fig. 9 represents a graph of the experimental concentrations SI (#), SII (#), S x (O),
S &alpha; II (#)) and theoretical Si (curve a) in mM versus time t in min, in the test
realized according to the topography of fig. 5.

 The device for separating and concentrating at least one solute S to be dialysed, according to the first embodiment of the invention, is designated in FIG. 1 by the general reference 1. This device or reactor 1 is constituted by a thermostatically controlled tank 2 having a double wall, provided for the circulation of a thermoregulatory fluid, between the inlet 2 and the outlet 22.

 This vessel 2 has, for example, a generally hollow cylindrical general shape and can be made from any suitable material, metal, plastic (polymer e.g. polymethacrylate type).

This tank 2 contains a liquid medium not referenced in the drawing and is also equipped with symbolically represented stirring means 3 which are advantageously constituted by a rotor, which may be, for example, a magnetic bar operating with a magnetic stirrer, or else a conventional stirring helix.

 A tubular body 4 also containing liquid medium, partially bathes in the liquid medium in the tank 2. This tubular body 4 advantageously has a circular cross section. The submerged lower end of this tubular body 4 is closed by an enzymatic membrane 5 inserted between two annular peripheral seals 6, consisting for example of paraffinic plastic material. The membrane 5 is secured to the tubular body 4 by means of a lip 7 or an annular flange formed at the free end immersed of said tubular body. The fastening means used are for example screws and nuts 8, e.g. Teflon.

The tubular body 4 is equipped with stirring means 9, of the blade stirring type. The liquid levels in the tank 2 (1st container) and in the tubular body 4 (2nd container) are adjusted so that they are identical.

 The end enzyme membrane delimits with the wall of the tube 4 the compartment referenced by Cl in FIG. 1. This membrane 5 also separates said compartment C1 from the compartment Cn constituted by the tank 2 filled with liquid medium.

In this example, the internal volume Vj of the compartment C1 = 1.5 cm3, while the volume V11 of the compartment Cl1 = 300 cm3.

According to the invention, the membrane 5 is made of modified or unmodified natural polymer or of synthetic polymer. In this case, it is a polyamide membrane marketed by Pall Europe Limited, Portsmouth England under the reference Pall NAZ being in the form of nonwoven material, having a thickness of 100 μm. The porosity of this membrane is defined by the cutoff threshold, the latter being naturally chosen according to the size of the molecules that one wishes to migrate through the membrane to overconcentrate in a compartment. Expressed in maximum molecular weight for the molecules capable of migrating through the pores, this cut-off threshold is advantageously between 500 and 10,000 D, depending on the size of the molecules that one wants to overconcentrate.

According to one variant, this membrane may also consist of regenerated cellulose and is in the form of a non-woven film with a thickness of between 10 and 100 μm.

 Each of the faces Fn, F1 of the membrane 5 opposite, respectively, of the compartment C11 and the compartment C1, was subjected to a grafting enzyme Ea / Ess, forming a pair of reversible enzymes in which the enzyme E is capable of catalyzing the transformation of Su into SD and the enzyme Ex, the transformation of Sa into SI.

The grafting of the enzyme may be of a covalent nature or even a simple inclusion of the enzyme by adsorption within the fibrous polymer matrix.

Covalent grafting can be carried out according to conventional techniques. The following examples give an illustration of enzymatic grafting.

The second embodiment of the device according to the invention is shown in FIG. 2, in which it is designated by the general reference 10. This dialysis device 10 C1 o C IX of a solute S is able to operate in a continuous mode
This device 10 comprises a thermostated chamber 11 of the same type as that (2) described in the first embodiment of FIG. 1. The references 11, and 11z denote respectively the inlet and the outlet of the circulation circuit of a thermoregulatory fluid in the double jacket of the tank 11.

 This tank 11 contains the liquid medium initially containing the solute S and constituting the liquid compartment C11. Means 12 for mechanical agitation are provided within this liquid medium forming Cll. These stirring means 12 are of the same type as those described for the first embodiment shown in FIG. 1 and designated by the reference 9 in this figure.

 In this second embodiment, the enzymatic membrane consists of a plurality of tubes 13 with a porous wall and grouped together to form a beam 14, part of which is immersed in the liquid medium of the tank 11 (Cl.

In fact, the lights of the tubes 13 constitute as many liquid compartments C, involved in the process according to the invention.

The arrows shown in fig. 1 in the beam 13 gives the direction of flow of the compartments C ,.

The section of fig. 6 shows the bundle 14 of tubular enzymatic membranes 13.

This beam 14 has a curved portion U-shaped, the base of which plunges into the liquid compartment Cn defined by the tank 11. This tubular bundle 14 is intended to allow the circulation of liquid medium to deplete solute S by active transmembrane migration. For this purpose, the beam 14 is equipped with circulation means 15 constituted, for example, by a pump.

Fig. 6a is a cross-sectional view of a tubular enzymatic membrane 13 constituting the beam 14 and delimiting via its wall, on the one hand, an internal compartment C1 (tube lumen), and secondly as regards the immersed portion of the beam 14, an outer compartment C11, defined by the tank 11. Like the membrane 5 of FIG. 1, the wall of the tube 13 comprises on each of these inner and outer faces an enzyme Ea and Eg respectively, constituting a pair of reversible enzymes. The methods for fixing and immobilizing the enzymes are the same as those described above. In addition, the constituent materials of these tubular membranes are also of the same nature as those mentioned for the membrane 5 of FIG. 1. In practice, this will for example be regenerated cellulose of a porous nature, having a cutoff threshold which may vary from 500 to 10,000 D.

 The stirring means 12 of the tank 11 and the movement of the liquid C1 in the hollow fibers 13 by means of the pump 15 make it possible to define the diffusion layers d1 and d11, shown in FIG. 6 bis. The latter also shows the internal diameter D of the hollow fiber 13, as well as the thickness em of the membrane wall loaded with enzymes E fEss.

 The sum of the volumes of the compartments Cl immersed in CII is less than the volume Vu of the compartment Cll. Report VI / VII is as low as possible.

 The tubular membranes 13 may be electrically charged in positive or negative, so as to avoid parasitic diffusion of S &alpha; II in Cl.

This second embodiment of the device according to the invention is adapted to the second continuous mode of implementation of the dialysis process. We find the same methodology used for the first discontinuous mode of implementation, with the difference that the liquid medium of the compartments C1 circulates in the hollow fibers 13 enzymatic, the active transmembrane transport of S occurring naturally at the level of the submerged part of the beam 14 in the compartment
Liquid CII of the tank 11.

 The circulation velocity of the liquid medium of the compartments C1 is adapted to the total surface area of the exchange zone in the immersed part, the enzymatic transformation kinetics EC, / Ep, as well as the transmembrane diffusion rates of the solute S.

 The symbolic representation of fig. 3 shows the membrane 5 carrying in its face Fn opposite Cn the enzyme E ,. This membrane has a thickness em. It is adjacent by its face Fn with a diffusion layer dII and its face FII opposite Cl with another diffusion layer dI, thickness edl. Depending on whether the solute S is in C1 or in Cn, the reference S comprises the subscript I or II separated by a comma from the parameter t corresponding to the time. Depending on whether the solute S is in a diffusion layer or in the rest of the compartment C1 or Cn, the reference S comprises by exposing the letters d and b (b for "bulk" in English) respectively. The second exponent of the reference S corresponds to the negative (-) or neutral (o) charge of the solute.

At time t = 0 we have: Sbt, f O; SII, 1b = S &alpha; II, t = S 5b =
At time t, solute SI migrated through the membrane to pass from Cl to Cn.

And it is in the diffusion layer dn of Cn that Sn is transformed into Sali by Ea The part of Sn which is not metabolized by E &alpha; diffuses into the compartment Cn. Part of S n crosses the membrane to pass into the compartment C1, so that the latter comprises solute Sd in its diffusion layer and in the rest of the compartment. Part of S n also diffuses into the entire compartment Cn.

Fig. 4 is a synoptic representation of the topography proper to the first variant embodiment of the method according to the invention.

The faces Fn and F1 of the membrane are respectively carriers of the reversible enzymes E &alpha; / Ess The solute S1 crosses the membrane to pass from C1 to Cn and to be partly metabolized in the diffusion layer dn adjacent to the face Fn of
Cn, in S solute, by the enzyme Ed. Part of S n diffuses across the Cn membrane to Cl and the compound S &alpha; I having thus diffused and transformed into SI in the Si-doping diffusion layer, by the enzyme E ,. Sn and S n diffuse into Cn, just as Sai diffuses into Cl. The membrane is preferably electrically charged with the same sign as S n, in this case negatively, so as to limit the diffusion of S n in Cl.

Fig. 5 corresponds to the second alternative embodiment of the method according to the invention. We find the same pattern as that of fig. 4, with the difference that the compartment Cn comprises an enzyme E8 capable of metabolizing S n to Su, this transformation taking place preferably in the part of Cn different from the non-turbulent diffusion layer dn.

In the implementations corresponding to the diagram of FIGS. 3 to 5, the enzymatic pumping, ensured by E, E, and E #, causes the formation of a concentration gradient A [S] = [S2] - [Sl]> 0.

 The examples which follow illustrate the process according to the invention in its three preferred modes of implementation. These examples will make it possible to better understand the meaning of the invention and to grasp all its advantages and variants of implementation.

EXAMPLES
EXAMPLE 1: DIALYSIS ASSISTE BY ENZYMATIC PUMPING OF A
SOLUTE S = GLUCOSE, OF A LIQUID COMPARTMENT C1,
TOWARDS A CII COMPARTMENT ACCORDING TO THE FIRST MODE OF IMPLEMENTATION
PROCESS OF THE INVENTION AND USING THE
DEVICE OF THE INVENTION IN ITS FIRST MODE OF
PRODUCTION.

1. 1.Material
The device used is that described above and shown in FIG. 1.

The dialysis membranes used are membranes of the Pall NAZ type marketed by the company PALL EUROP LIMITED. They are polyamide membranes with a thickness equal to 100 μm of active transfer surface (Aw) of 12% and referenced under number 09025.

Regenerated cellulose dialysis membranes of thickness 40 clam, sold under the name spectra / Por. 3500 MWCO - ref 132723 by the company spectrum. These dialysis membranes are those that do not carry enzymes.

The enzyme Ea = glycerinkinase - GK - (EC2.7.1.30) from the cellulomonas species (52 units.mg ', ref 6142).

 Ess = = E # alkaline phosphatase -PA- (EC3.1.3.1.) Extracted from bovine intestinal mucosa (1200 Units.mgl, P 6672).

The enzymes Ew Ep and Es defined above are marketed by SIGMA.

The immobilization of the enzymes on the membrane takes place as follows:
The membranes are in the form of discs 5 cm in diameter which is activated with a sulfuric acid solution in methanol under reflux for 6 hours. They are then subjected to the action of a 2% V / V glutaraldehyde solution, as described in the article by Michalon, P., Couturier, R., Hacques, MF.
Favre-Bonvin, G., Ville, A. & Marion, C. (1990) Biochem. Biophys. Res. Common, 167, 9-15.

The grafting of the enzymes Ea = GK and Ep = E8 = PA is carried out by immersing the discs at room temperature for 3 hours in 5 cm 3 of an enzyme solution titrating 2 mg. cm-3 and 0.2 mg. cm-3 for GK and phosphatase, respectively, in 0.1 M borate buffer, pH 8.5.

The volume of Cl = 1.5 cm3 and the volume of C11 = 300 cm3.

1.2. Operating conditions
The liquid media in Cl and CII have the same composition with the exception of the concentrations of compound A = glycerol-3-phosphate2- and compound B = glycerol.

 The temperature of the liquid medium is thermostatically controlled by the tank at 25 C.

The other operating conditions vary according to the tests carried out.

1.3. Tests according to the topography of fig. 3
In this test, ClQ liquid media contain 0.1M borate buffer in an amount such that the pH is of the order of 9 as well as ATP Mg Cl2 - 7 mM.

S = SI = 5u = glucose.

 = S = glucose -Sphosphate (G-6-P).

E, = GK.

The enzymatic reactions used in the examples are as follows:

<tb> MgCl2 / ATP <SEP> + <SEP> glucose <SEP> GK <SEP> glucose-6-phosphate <SEP> + ADP / MgCl2
<tb><SEP> PA
<tb><SEP> G-6-P <SEP> t <SEP> glucose <SEP> + <SEP> phosphate
<Tb>
The glucose concentrations are determined from samples taken from the compartments Cl and CII, using a sensor formed by an enzymatic electrode composed of a platinum amperometric probe associated with a nylon membrane loaded with glucose oxidase, this probe being coupled to a polarimeter of the PRGE type of the company RADIOMETER (VILLEURBANNE,
FRANCE), (THEVENOT et al), 1979. This biosensor allows glucose concentration determinations according to the following reaction:

glucose <SEP> + <SEP> O2 <SEP> glucose <SEP> oxidase
<tb> D-glucose <SEP> + <SEP> 2 <SEP>><SEP> acid <SEP> gluconic acid <SEP> + <SEP> H202
<tb><SEP> Puanode (650mV) VsAg / Agcl
<tb><SEP> H202 <SEP> p <SEP> 2 <SEP> + 2H ++ 2e ~
<Tb>
The anode current thus obtained is proportional to the concentrations of H2O2, which are themselves proportional to the glucose concentrations.

The thickness em of the dialysis membrane loaded with GK with respect to CII is 240 clam.

The stirring conditions of this test are respectively 180 revolutions I min and 100 revolutions / min in CI and CII.

These conditions make it possible to obtain the diffusion layer thicknesses d1 and dII which are: - ed1 = 100 μm.

- edII = 180 m.

During the course of the tests, samples of G-6-P- are taken from the two compartments Cl and CII and analyzed with G-6-P-dehydrogenase by measuring the increase of the absorption at 340 nm associated with the reduction of NADP + (Lang and Michal, 1974).

The enzymatic activities of NAZ membranes loaded with GK and PA are determined in the following manner.

The NAZ disks loaded with GK are immersed in one ml of 0.1 M borate buffer pH 8.5 containing 7 mM ATP / MgCl 2 in the presence of a saturating glucose concentration, that is to say 0.2 M at 250 C. Samples are taken each
S min for 20 min. In these samples, the concentration of G-6-P- is measured so as to determine the GK activity.

In the same way, NaZ disks loaded with PA are immersed in 5 ml of 0.1M borate buffer, pH 8.5 in the presence of 33 mM G-6-P- (saturating concentration). Samples are taken every 5 min for 20 min and the glucose concentration is measured therein to derive PA activity.

The liquid medium of C1, C11 is constituted by 0.1 M phosphate buffer containing 7 mM ATP / MgCl2 at pH 8.5 and 25 C.

IF, tb, o = SI, td, o = S2, td, o = S2, tb, o = 2mM.

Fig. 7 clearly shows the decrease of the IF concentration in glucose in the compartment which is much more marked than the decay of Sn, although the enzymatic reaction promoted by E = GK occurs only in CII. On the other hand, the concentration S in G-6-P- in CII grows less rapidly than the concentration Sai in G6-P- in the nonenzymatic compartment C1.

EXAMPLE 2 TESTS ACCORDING TO THE TOPOGRAPHY OF FIG. 4.

In these tests, the dialysis membrane comprises an NAZ membrane loaded with E &alpha; = GK with respect to CII, a NAZ membrane loaded with Ess = PA opposite Cl and a spectra / Por membrane sandwiched between these two enzymatic membranes.

The dialysis membrane has a thickness em = 240 clam.

The stirring conditions are 180 revolutions / min and 100 revolutions / min in CI and CII respectively.

Such conditions determine thicknesses for d1 and du which are: - edI = 100 m - dII = 180 m.

The other experimental conditions are identical to those described above in Example 1.

Fig. 8 clearly shows that G-6-P- produced in Cn (S0, n) does not pollute Cl; S is constant and is equal to 0. Moreover, the concentration Si in glucose in Cl decreases rapidly in 500 min. Su concentration in glucose in C11 decreases less rapidly than S1 while S &alpha; II increases steadily.

EXAMPLE 3: DIALYSIS TESTS OF GLUCOSE ACCORDING TO THE TOPOGRAPHY OF
LAIIG. 5.

In this test, we find the same topography as in fig. 4, with the difference that the compartment Cn contains an enzyme Es = PA, which is supplied via 4 NAZ membrane disks loaded with PA. The latter is intended to transform glucose-6-phosphate present in Cn. The experimental conditions are the same as in Examples 1 and 2 above.

The stirring conditions are the same as in Examples 1 and 2 above.

These conditions determine a thickness edi = 100 m for the diffusion layer dn and edII = 180 μm for the diffusion layer dn.

The thickness of the dialysis membrane is em = 240 m,
Fig. 9 shows the results obtained and it appears that no pollution of compartment I by the G-6-P- produced in CII is involved. Similarly, the concentration S &alpha; II remains constantly zero in Cn. Moreover from t = 500 min, a constant AS = [SII] - [SI] difference of between 0.5 and 1 mM is observed.

The comparison of the SIth and SI = f (t) curves shows a good correlation between the theoretical model and the experimental model. As a result of the above enzyme-assisted glucose dialysis resulting from an enzymatic and cyclic mechanism of phosphorylation / dephosphorylation, on both sides of the membrane in the inlet C11 and starting Cl compartments. is highly efficient, selective and offers good yields without requiring the use of large volumes of dialysates. This mechanism is further optimized when G-6-P- dephosphorylation is predicted out of the dn diffusion layer of the Cn compartment. Phosphorylation / dephosphorylation occur respectively in D1 and D1.
In all these tests, the membrane is negatively charged like G-6-P- so as to limit the diffusion thereof from C11 to C1.

Claims (18)

CLAIMS:  1 - Method of dialysis assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least partly two liquid compartments Cl and Cxl, (S being referenced as SI in Cl and SII in CII), this dialysis takes place from the liquid compartment C, which is intended to deplete in S, to the liquid compartment Cx1 intended consequently to enrich itself in S,  characterized in that it consists essentially and successively or not:  to implement at least one porous membrane comprising on  and / or in its face Fn with respect to CII at least one enzyme Ea apt  to catalyze the conversion reaction of solute SII into solute  primary product S &alpha;II;  to move at least the liquid of CII (dialysate), and  preferably also that of Cl, providing at least one layer  non-turbulent dD diffusion, adjacent to the FII face of the  E & alpha carrier membrane; and next to CII, and regulating  the edii thickness of this diffusion layer dII so that  em em # edII # 10.em, preferably # edII # 3.em;  10 2  where em is the thickness of the membrane;  and to ensure that the operating conditions are such  qu'interviennent:  * enzymatic transformation by E of SII into  * and correlatively the diffusion of S ,, through the membrane  from compartment C1 to compartment CII, in particular under  the effect of enzymatic pumping, induced by the reaction

 2 - Process according to claim 1, characterized in that one selects, on the one hand, the enzyme E &alpha; which governs the production of S &alpha; II and, on the other hand, the porous membrane, so that the latter is impermeable to S, and thus prevents, at least partially, the diffusion of S &alpha; II from CII to CI; S l and the membrane being preferably, electric charge entities of the same sign.  3 - Process according to claim 1 or claim 2 characterized in that:  at least one porous membrane comprising at least one  and / or in its face F1 opposite Cl, at least one suitable enzyme Ess  to catalyze the reaction reaction of the solute-primary product  S &alpha; I derived from diffusion across the membrane of S &alpha; II from CII to  Cl in solute-secondary product  being preferably the inverse enzyme of E so that Spl = =  provision is made for the movement of Cl and CII fluids into  leaving a diffusion layer dl, dII non-turbulent connected to  each face FI, Full, and adjusting the edl and edII thicknesses of these  layers so that: * 0.1. em # edI / edII # 10.em,  * preferably 0.5. em # edI / edII # 3.em;  operating conditions favorable to the transformation are fixed  Enzymatic by Ess of S, in SssI, and, correlatively, in the case where = = Sj, the diffusion of SssI from Cl to Ctl, through the  membrane, in particular under the effect of a resulting enzymatic pumping induced by the reaction

 4 - Process according to any one of claims 1 to 3, characterized in that:  at least one enzyme E capable of catalyzing is used in CII  the reaction of transformation of the solute S &alpha; II in solute SII,  and set operating conditions conducive to the progress of this transformation

 5 - Process according to any one of claims 1 to 4, characterized in that the enzymatic membrane is in the form of a monolayer or multilayer film, preferably substantially planar.  6 - Process according to any one of claims 1 to 4, characterized in that the enzymatic membrane constitutes all or part of the wall of at least one tube whose light forms one of the compartments CI, CII,  in that at least a portion of this membrane is bathed  tubular in the liquid medium forming the other compartment CII C;  and in that the liquid to be depleted is circulated in solute SI, in  this or these tubular enzymatic membranes, preferably  in at least one bundle.  7 - Process according to any one of claims 1 to 6, characterized in that one therefore develops the liquid medium of Cl and / or CII by using a solvent - preferably substantially aqueous - optionally comprising solutes selected from the following group:  pH buffers adapted to Ea's optimum operating pH  and E, even Es;  energy-rich binding molecules - preferably ATP or  any molecule capable of transferring a phosphate group  on a solute;  and their mixtures.  8 - Process according to any one of claims 1 to 7, characterized in that the pH and temperature parameters of the liquid media are adjusted so as to obtain an optimum in kinetics and yield for enzymatic transformations by Ea and / or Ep and / or Es.  9 - Process according to any one of claims 1 to 8, characterized in that the setting in motion of the liquid in at least one of the compartment Cl, C, occurs by stirring and / or by circulation of the liquid.  10 - Process according to any one of claims 1 to 9, characterized in that one uses means for assisting the transmembrane migration of S, said means being preferably constituted by an ionic strength gradient and / or electrical, on both sides of the membrane, and more preferably still pH gradients (protomotive force).  11 - Process according to any one of claims 1 to 10, characterized in that:  S = Sl = Sx = Sn = glucose,  Sa = = glucose-6-phosphate,  Ea = glucose kinase,  Ep = E; alkaline phosphatase.  12 - Process according to any one of claims 1 to 11 characterized in that the liquid of Cl is a physiological liquid, preferably blood.  13 - Dialysis device assisted by enzymatic pumping of at least one solute S characterized in that it essentially comprises:  at least one container of a liquid medium;  at least one porous membrane of thickness em, bathing or  likely to bathe in the liquid medium, to delimit at  less partially a liquid Cl compartment intended to be  depleted in solute S and at least partially a compartment  a liquid C "intended to be a receiver of the solute S1 from C1,  CI, CII respectively containing volumes VI and VII;  at least one enzyme E &alpha; able to catalyze, in Cll, the reaction of  transformation of S into solute-product S, primary, Ea being carried  by the membrane on and / or in its face opposite CII,  preferably at least one enzyme Ess capable of catalyzing, in C1, the  reaction of transformation of the primary solute Sas, = S ,, "in solute  secondary product SssI, Ess being carried by the membrane on and / or  in its F1 face next to CX,  Ep being, more preferably still, the inverse enzyme Et, p of  so that SssI = SI;  preferably at least one enzyme E8 capable of catalyzing in CII, the  Transformation reaction of S &alpha; II primary solute into SII solute, E #  being dispersed in CII in immobilized form;  means for moving at least liquid CII and  preferably, also Cl liquid;  and possibly means for adjusting the temperature of the  liquid medium.  14 - Device according to claim 13, characterized in that S and the membrane are entities of opposite electrical charges, the (or) membrane (s), being charged (s) on at least one of its faces, preferably on F11 carrying E &alpha; and even more preferably on both.  15 - Device according to claim 13 or 14, characterized in that the enzymatic membrane is a porous matrix in and / or on both sides FII / FI which are included and immobilized enzymes E, Ess respectively, this matrix being formed by at least one macromolecular compound, preferably chosen from proteins, polysaccharides, synthetic (co) polymers and mixtures thereof and / or alloys, cellulose and its derivatives, as well as (co) polyamides being particularly preferred.  16 - Device according to any one of claims 13 to 15, characterized in that the enzyme membrane (5) is in the form of a monolayer or multilayer film, preferably substantially planar.  17 - Device according to any one of claims 13 to 15, characterized in that the membrane constitutes all or part of the wall of at least one tube whose light forms one of the compartments CI / CII.  18 - Device according to any one of claims 13 to 17, characterized in that the pair of reversible enzymes E &alpha; / Ess is chosen mainly from the couples of enzymes allowing the additionlèvement of a chemical group loaded on a metabolite preferably from the pairs of phosphorylation / dephosphorylation enzymes, and even more preferably from the kinases / phosphatases.