METHOD FOR PURIFYING HIGH MOLECULAR WEIGHT ADENOSINE-BASED COENZYMES BY TANGENTIAL DIAFILTRATION

Abstract

The present disclosure relates to the field of making high molecular weight adenosine-based coenzymes available on a large scale. In particular, it relates to a method for purifying high molecular weight adenosine-based coenzymes by implementing a tangential diafiltration, or even dia-ultrafiltration step. This method is, for example, applicable to the purification of coenzyme A disulfide ((CoAS).sub.2), coenzyme A (CoA), nicotinarnide adenine dinucleotide (NAD+), nicotinarnide adenine dinucleotide phosphate (NADP.sup.+) or flavin adenine dinucleotide (FAD).

Claims

1. A method for purifying a high molecular weight adenosine-based coenzyme comprising implementing a tangential diafiltration of a solution comprising the high molecular weight adenosine-based coenzyme.

2. The method of claim 1, wherein the solution comprises an aqueous washing solution or any other aqueous buffer solution.

3. The method of claim 2, wherein the solution comprises deionized water.

4. The method of claim 3, wherein the diafiltration is a tangential dia-ultrafiltration.

5. The method of claim 4, wherein the high molecular weight adenosine-based coenzyme is coenzyme A disulfide ((CoAS).sub.2), coenzyme A (CoA) or a derivative thereof, or nicotinamide adenine dinucleotide phosphate (NADP.sup.+).

6. The method of claim 5, wherein the high molecular weight adenosine-based coenzyme comprises a derivative of coenzyme A having a formula (I): ##STR00002## in which: R.sub.1 represents a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, acyl group, with or without a carboxylic acid, alcohol and/or amine group in a terminal position, or branched; a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, alkyl group, with or without a carboxylic acid, alcohol and/or amine group in the terminal position, or branched; a benzoyl or benzyl group; and R.sub.2 represents an H or a phosphate group.

7. The method of claim 6, wherein the tangential diafiltration involves separating a permeate from a retentate using a membrane comprising a polyethersulfone (PES) polymer or a membrane comprising a modified polyamide polymer.

8. The method of claim 7, further comprising applying a pressure to the solution adjacent the membrane during the diafiltration, the pressure being between 4 and 20 bar.

9. The method of claim 1, wherein the diafiltration is a tangential dia-ultrafiltration.

10. The method of claim 1, wherein the high molecular weight adenosine-based coenzyme is coenzyme A disulfide ((CoAS).sub.2), coenzyme A (CoA) or a derivative thereof, or nicotinamide adenine dinucleotide phosphate (NADP.sup.+).

11. The method of claim 10, wherein the high molecular weight adenosine-based coenzyme comprises a derivative of coenzyme A having a formula (I): ##STR00003## in which: R.sub.1 represents a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, acyl group, with or without a carboxylic acid, alcohol and/or amine group in a terminal position, or branched; a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, alkyl group, with or without a carboxylic acid, alcohol and/or amine group in the terminal position, or branched; a benzoyl or benzyl group; and R.sub.2 represents an H or a phosphate group.

12. The method of claim 1, wherein the tangential diafiltration involves separating a permeate from a retentate using a membrane comprising a polyethersulfone (PES) polymer or a membrane comprising a modified polyamide polymer.

13. The method of claim 1, wherein the tangential diafiltration involves separating a permeate from a retentate using a membrane, and wherein the method further comprises applying a pressure to the solution adjacent the membrane during the tangential diafiltration, the pressure being between 4 and 20 bar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1: Structures of (CoAS).sub.2, CoA and NADP.sup.+

[0027] FIG. 2: HPLC range of (CoAS).sub.2

[0028] FIG. 3: Filtration pilot according to a closed-circuit tangential configuration

[0029] FIG. 4: HPLC range of NADP.sup.+

[0030] FIG. 5: Tangential diafiltration pilot

DETAILED DESCRIPTION

[0031] In a first aspect, the present disclosure relates to a method for purifying a high molecular weight adenosine-based coenzyme by implementing a tangential diafiltration.

[0032] Thus, by virtue of the diafiltration, the coenzyme of interest is retained, while the smaller molecules, which are coproducts of the coenzyme synthesis process, are eliminated in the permeate.

[0033] In a preferred embodiment of the disclosure, the filtration is a dia-ultrafiltration.

[0034] “Coenzyme” means a cofactor that is required for the activity of an enzyme (protein). A coenzyme is a non-protein organic molecule. The high molecular weight adenosine-based coenzymes include coenzyme A dimer (coenzyme A disulfide or (CoAS).sub.2), coenzyme A (CoA) or the derivatives thereof, nicotinamide adenine dinucleotide (NAD.sup.+), nicotinamide adenine dinucleotide phosphate (NADP.sup.+), or flavin adenine dinucleotide (FAD). Within the meaning of the disclosure, a high molecular weight coenzyme is a compound of which the molecular weight is greater than or equal to 600 Da.

[0035] In a preferred embodiment of the disclosure, the high molecular weight adenosine-based coenzyme is a coenzyme A dimer.

[0036] In another preferred embodiment of the disclosure, the high molecular weight adenosine-based coenzyme is coenzyme A or one of the derivatives thereof.

[0037] Within the meaning of the disclosure, “derivatives of coenzyme A” means a compound of formula (I):

##STR00001##

in which: [0038] R.sub.1 represents a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, acyl group, with or without a carboxylic acid, alcohol and/or amine group in the terminal position, or branched; a C.sub.1 to C.sub.22 linear, cyclic or branched, saturated or unsaturated, alkyl group, with or without a carboxylic acid, alcohol and/or amine group in the terminal position, or branched; a benzoyl or benzyl group [0039] R.sub.2 represents an H or a phosphate group.

[0040] In another preferred embodiment, the high molecular weight adenosine-based coenzyme is NADP.sup.+.

[0041] In the case of the coenzyme A, on account of a stability problem during the preparation thereof, it is preferable to prepare a CoA dimer, i.e., CoA disulfide ((CoAS).sub.2). Indeed, the CoA must be stored at −20° C., while the CoA dimer can be kept at ambient temperature. A simple disulfide bridge reduction reaction makes it possible to obtain CoA. Reducing agents suitable for this reaction are, for example, 2-mercaptoethanol, DTT or TCEP. It is also possible to carry out this reduction by means of biocatalysis, using a CoA disulfide reductase.

[0042] The purification method according to the disclosure has the dual advantage of being inexpensive and ecological. Indeed, it is based on the implementation of diafiltration carried out on an aqueous buffer solution, which can be purified using deionized water, or any other aqueous buffer solution. The solution that allows for the purification may be selected depending on the coenzyme to be purified and on the material of the membrane. A person skilled in the art would be able to make this choice based on his general knowledge.

[0043] The material of the membrane is selected depending on the coenzyme to be purified. This is preferably a filtration membrane.

[0044] The diafiltration is preferably carried out in the absence of an organic solvent, and, on the contrary, in the presence of an aqueous solvent, preferably an aqueous buffer solution, in particular, with water, such as deionized water.

[0045] In a preferred embodiment, the membrane used is made of a polymer of the polyethersulfone (PES) type, or a modified polyamide (such as provided by the suppliers), and the solution allowing for the purification is deionized water. These parameters are applicable, in particular, for purifying a CoA dimer, coenzyme A, and NADP.sup.+.

[0046] As mentioned above, a high molecular weight coenzyme according to the disclosure is a compound of which the molecular weight is greater than or equal to 600 Da.

[0047] Thus, with the aim of purifying high molecular weight adenosine-based coenzymes, the cut-off of the membrane makes it possible to retain the coenzymes of which the molecular weight is greater than or equal to 600 Da. It should be noted that the cut-off of the membranes stated by the manufacturers does not necessarily reflect the reality, and that other factors such as the conformation of the compound, the chemical functions present (in particular, when they are charged), and the membrane-compound interactions, may influence whether or not the compound passes through the membrane. For information, the molecular weight of the CoA dimer is in the region of 1500 Da, and that of the CoA and NADP.sup.+ is in the region of 750 Da.

[0048] A functional definition of a membrane according to the present disclosure is that the membrane must allow the passage of the molecules having a molecular weight of less than 600 Da, while retaining the coenzyme of interest.

[0049] The principle of membrane filtration involves applying a motive transfer force, in this case a pressure difference, to the membrane, in order to carry out the separation. Within the context of the present disclosure, the pressure applied to the coenzyme solution to be purified is typically of between 4 and 35 bar. In a preferred embodiment, the pressure applied is of between 4 and 20 bar.

[0050] The diafiltration makes it possible to recirculate the solution containing the retentate, in order to eliminate the undesirable small molecules present in the retentate, by means of washing, and thus to improve the purity of the coenzymes.

[0051] The method is advantageously carried out continuously, which makes it possible to purify and then concentrate the coenzyme. This also makes it possible to use little solvent and to reuse it (recycling).

[0052] Thus, in a particular embodiment in which the method makes it possible to purify the CoA dimer, the CoA and the NADP.sup.+, the conditions are as follows: the membrane used is made of PES polymer or a material similar to polyamide (modified polyamide) to which a pressure of between 4 and 20 bar is applied, and the diafiltration is carried out continuously, using deionized water.

[0053] In a particular embodiment of the disclosure, the membrane is made of PES polymer, and a pressure of 20 bar is applied.

[0054] In a preferred embodiment of the disclosure, the membrane is made of modified polyamide, and a pressure of 4 bar is applied.

[0055] The coenzyme A dimer treated by the method according to the disclosure can be purified to at least 83%. In a particular embodiment, it is purified to 88%, 90%, 95%, or indeed 98% or 99%.

[0056] The coenzyme A treated by the method according to the disclosure can be purified to at least 45%. In a particular embodiment, it is purified to 88%, 90%, 95%, or indeed 98% or 99%.

[0057] The NADP.sup.+ treated by the method according to the disclosure can be purified to at least 59%. In a particular embodiment, it is purified to 88%, 90%, 95%, or indeed 98% or 99%.

[0058] The method according to the disclosure makes it possible to have access to high molecular weight adenosine-based coenzymes in large quantities and at a reasonable price, by means of an ecological and industrially viable method. This method thus opens the path to high-potential new markets.

EXAMPLES

Example 1: Study of the Effect of Different Membranes on the Separation of (CoAS).SUB.2 .Molecules by Means of Tangential Filtration

[0059] A reference scale was first implemented in HPLC with 85% commercial CoAS.sub.2 at different concentrations, in order to be able to precisely measure the amount of the target molecule in the different fractions of the purification method (feed, retentate, permeate). The results obtained are set out in FIG. 2.

[0060] A medium containing (CoAS).sub.2, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), in a final volume of 700 ml milli-Q water, was used to carry out the screening of the membranes.

[0061] A tangential membrane filtration technique was investigated, in order to separate the molecules present in the mixture. Different types of commercial membranes: NP010, GE, GH, GK, GR15PP, HydraCore50-PS 7450 and HydraCore70pHT Series 7470PHT (Table 1), were studied according to a closed-circuit tangential configuration (FIG. 3).

[0062] The features of the membranes studied, as described by the suppliers, are summarized in Table 1.

TABLE-US-00001 TABLE 1 Main properties of the membranes studied Cut-off pH Maximum Manufacturer Type Membrane (Da) range pressure Suez UF GE 900 for 1-11 40 bar PEG UF GH 1400 for 1-11 27 bar PEG UF GK 1500 for 1-11 27 bar PEG Microdyn- NF NP010 1000 0-14 40 bar Nadir Alfa Laval UF GR95PP 2000 1-13 10 bar Hydronautics- UF HydraCore50-PS7450 1000 2-11 41 bar Nitto NF HydraCore70pHT 720 .sup. 1-13.5 41 bar Series 7470PHT

[0063] Four different pressures were studied, depending on the maximum pressure that can be applied to each membrane (according to the manufacturer's recommendations). The percentage retention of the different compounds, for each pressure and membrane, was calculated according to the following equation:

Retention.sub.compound=(1−C.sub.p compound/C.sub.r compound)×100

where C.sub.p compound and C.sub.r compound are, respectively, the concentrations of the compound studied, in the permeate and the retentate.

[0064] The results obtained at a given time are shown in Table 2, below.

[0065] The best results were obtained using the membrane GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11. The maximum pressure recommended for this membrane is 27 bar. The differential retention of (CoAS).sub.2 is at a maximum at 4 bar, but also very satisfactory at the other pressures tested, i.e., up to 10 bar.

[0066] The other membrane that allows for purification of (CoAS).sub.2 is the membrane NOP10, i.e., a membrane made of a polymer of the polyethersulfone (PES) type, which has a cut-off of 1000 Da and which can be used at a maximum pressure of 40 bar. The best result was obtained at a pressure of 20 bar.

Example 2: Study of the Effect of Different Membranes on the Separation of CoA Molecules by Means of Tangential Filtration

[0067] A solution containing CoA, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and 2-mercaptoethanol, in a final volume of 700 ml milli-Q water, was used for carrying out screening of the membranes.

[0068] A tangential membrane filtration technique was investigated, in order to separate the molecules present in the mixture. Different types of membrane: GE, GH, GK were studied according to a closed-circuit tangential configuration (FIG. 3).

[0069] The features of the membranes studied are summarized in Table 1.

[0070] Four different pressures were studied, depending on the maximum pressure that can be applied to each membrane (according to the manufacturer's recommendations). The percentage retention of the different compounds, for each pressure and membrane, was calculated according to the following equation:

Retention.sub.compound=(1−C.sub.p compound/C.sub.r compound)×100

where C.sub.p compound and C.sub.r compound are, respectively, the concentrations of the compound studied, in the permeate and the retentate.

[0071] The results obtained at a given time are shown in Table 3, below.

[0072] The best results were obtained using the membrane GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11. The maximum pressure recommended for this membrane is 27 bar. The differential retention of CoA is at a maximum at 4 bar, but also very satisfactory at the other pressures tested, i.e., up to 10 bar.

Example 3: Study of the Effect of Different Membranes on the Separation of NADP.SUP.+ Molecules by Means of Tangential Filtration

[0073] A reference scale was first implemented in HPLC with 98% commercial NADP.sup.+ at different concentrations, in order to be able to precisely measure the amount of the target molecule in the different fractions of the purification method (feed, retentate, permeate). The results obtained are set out in FIG. 4.

[0074] A solution containing NADP.sup.+, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), in a final volume of 700 ml milli-Q water, was used to carry out the screening of the membranes.

[0075] A tangential membrane filtration technique was investigated, in order to separate the molecules present in the mixture. Different types of membrane: GE, GH, GK were studied according to a closed-circuit tangential configuration (FIG. 3).

[0076] The features of the membranes studied are summarized in Table 1.

[0077] Four different pressures were studied, depending on the maximum pressure that can be applied to each membrane (according to the manufacturer's recommendations). The percentage retention of the different compounds, for each pressure and membrane, was calculated according to the following equation:

Retention.sub.compound=(1−C.sub.p compound/C.sub.r compound)×100

where C.sub.p compound and C.sub.r compound are, respectively, the concentrations of the compound studied, in the permeate and the retentate.

[0078] The results obtained at a given time are shown in Table 4, below.

[0079] The best results were obtained using the membrane GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11. The maximum pressure recommended for this membrane is 27 bar. The differential retention of NADP.sup.+ is at a maximum at 4 bar, but also very satisfactory at the other pressures tested, i.e., up to 10 bar.

Example 4: Separation of (CoAS).SUB.2 .Molecules by Means of Tangential Dia-Ultrafiltration

[0080] Tangential dia-ultrafiltration (FIG. 4) was carried out using a feed solution containing (CoAS).sub.2, ATP, ADP and AMP, in a final volume of 200 ml milli-Q water. The purity of the starting solution in terms of (CoAS).sub.2 is 45%.

[0081] The membrane used is GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11.

[0082] The washing solution is deionized water.

[0083] The expected losses and purities are predicted by the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where R.sub.i is the retention rate of each type i in solution (between 0 and 1), where V.sub.0 is the feed volume, and where V.sub.t is the volume of washing solution over time:

C.sub.R,i,t/C.sub.A,i,0=e.sup.(−(1-R.sup.i.sup.)×V.sup.t.sup./V.sup.0.sup.)

[0084] The losses are calculated using the following equation, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t:

Losses.sub.i(%)=((C.sub.A,i,0−C.sub.R,i,t)/C.sub.A,i,0)×100

[0085] The purity is calculated using the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, and i, j, k, etc., are the types in solution:

Purity.sub.i(%)=(C.sub.R,i,t/(C.sub.R,i,t+C.sub.R,j,t+C.sub.R,k,t))×100

[0086] A diafiltration volume (VD) corresponds to the feed volume.

[0087] The feed solution was washed using a VD of 5, with a membrane surface area of 39 cm.sup.2. 88% of the ATP, 89% of the ADP, and 100% of the AMP were eliminated, in order to obtain a (CoAS).sub.2 purity of equal to 83%. The final purity is a relative value that depends on the initial purity and the number of washes.

[0088] Since the permeability is the capacity of a membrane to allow a solution to pass through, this was evaluated for deionized water before and after the dia-ultrafiltration.

[0089] The permeability L.sub.p(L/h/m.sup.2/bar) to water was calculated according to the following formula, where J.sub.v is the flow of water (L/h/m.sup.2), P is the pressure applied to the system at the intake (bar), V.sub.p is the permeate volume, t is a time interval (h), and S is the surface area of the membrane (m.sup.2):

L.sub.p=J.sub.v/P where J.sub.v=V.sub.p/(t×S)

[0090] The difference in permeability to water before and after the dia-ultrafiltration was less than 1%, the feed solution of the dia-ultrafiltration has not, therefore, blocked the membrane, which can be reused.

Example 5: Separation of CoA Molecules by Means of Tangential Dia-Ultrafiltration

[0091] Tangential dia-ultrafiltration (FIG. 3) was carried out using a feed solution containing CoA, ATP, ADP, AMP and 2-mercaptoethanol, in a final volume of 200 ml milli-Q water. The purity of the starting solution in terms of CoA is 13%.

[0092] The membrane used is GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11.

[0093] The washing solution is deionized water.

[0094] The expected losses and purities are predicted by the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where R.sub.i is the retention rate of each type i in solution (between 0 and 1), where V.sub.0 is the feed volume, and where V.sub.t is the volume of washing solution over time:

C.sub.R,i,t/C.sub.A,i,0=e.sup.(−(1-R.sup.i.sup.)×V.sup.t.sup./V.sup.0.sup.)

[0095] The losses are calculated using the following equation, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t:

Losses.sub.i(%)=((C.sub.A,i,0−C.sub.R,i,t)/C.sub.A,i,0)×100

[0096] The purity is calculated using the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, and i, j, k, etc., are the types in solution:

Purity.sub.i(%)=(C.sub.R,i,t/(C.sub.R,i,t+C.sub.R,j,t+C.sub.R,k,t))×100

[0097] A diafiltration volume (VD) corresponds to the feed volume.

[0098] The feed solution was washed using a VD of 4, with a membrane surface area of 39 cm.sup.2. 54% of the ATP, 56% of the ADP, 67% of the AMP, and 97% of the 2-mercaptoethanol were eliminated, in order to obtain a CoA purity of equal to 45%. The final purity is a relative value that depends on the initial purity and the number of washes.

[0099] Since the permeability is the capacity of a membrane to allow a solution to pass through, this was evaluated for deionized water before and after the dia-ultrafiltration.

[0100] The permeability L.sub.p (L/h/m.sup.2/bar) to water was calculated according to the following formula, where J.sub.v is the flow of water (L/h/m.sup.2), P is the pressure applied to the system at the intake (bar), V.sub.p is the permeate volume, t is a time interval (h), and S is the surface area of the membrane (m.sup.2):

L.sub.p=J.sub.v/P where J.sub.v=V.sub.p/(t×S)

[0101] The difference in permeability to water before and after the dia-ultrafiltration was less than 10%, the feed solution of the dia-ultrafiltration has not, therefore, blocked the membrane, which can be reused.

Example 6: Separation of NADP.SUP.+ Molecules by Means of Tangential Dia-Ultrafiltration

[0102] Tangential dia-ultrafiltration (FIG. 3) was carried out using a feed solution containing NADP.sup.+, ATP, ADP and AMP, in a final volume of 200 ml milli-Q water. The purity of the starting solution in terms of NADP.sup.+ is 52%.

[0103] The membrane used is GK, i.e., using a membrane made of modified polyamide, which has a cut-off of 1500 Da for PEG, and which can be used at a pH of between 1 and 11.

[0104] The washing solution is deionized water.

[0105] The expected losses and purities are predicted by the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where R.sub.i is the retention rate of each type i in solution (between 0 and 1), where V.sub.0 is the feed volume, and where V.sub.t is the volume of washing solution over time:

C.sub.R,i,t/C.sub.A,i,0=.sup.(−(1-R.sup.i.sup.)×V.sup.t.sup./V.sup.0.sup.)

[0106] The losses are calculated using the following equation, where C.sub.A,i,0 is the concentration in the feed of each type i in solution at time t=0, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t:

Losses.sub.i(%)=((C.sub.A,i,0−C.sub.R,i,t)/C.sub.A,i,0)×100

[OM] The purity is calculated using the following equation, where C.sub.R,i,t is the concentration in the retentate of each type i in solution at time t, and i, j, k, etc., are the types in solution:

Purity.sub.i(%)=(C.sub.R,i,t/(C.sub.R,i,t+C.sub.R,j,t+C.sub.R,k,t))×100

[0107] A diafiltration volume (VD) corresponds to the feed volume.

[0108] The feed solution was washed using a VD of 7, with a membrane surface area of 39 cm.sup.2. 50% of the ATP, 47% of the ADP, and 93% of the AMP were eliminated, in order to obtain a NADP.sup.+ purity of equal to 59%. The final purity is a relative value that depends on the initial purity and the number of washes. Although in this case the increase in purity is small, this result nonetheless demonstrates the possibility of purifying the NADP.sup.+. The purity can be improved by optimization, for example, by increasing the number of washes.

[0109] Since the permeability is the capacity of a membrane to allow a solution to pass through, this was evaluated for deionized water before and after the dia-ultrafiltration.

[0110] The permeability L.sub.p (L/h/m.sup.2/bar) to water was calculated according to the following formula, where J.sub.v is the flow of water (L/h/m.sup.2), P is the pressure applied to the system at the intake (bar), V.sub.p is the permeate volume, t is a time interval (h), and S is the surface area of the membrane (m.sup.2):

L.sub.p=J.sub.v/P where J.sub.v=V.sub.p/(t×S)

[0111] The difference in permeability to water before and after the dia-ultrafiltration was less than 10%, the feed solution of the dia-ultrafiltration has not, therefore, blocked the membrane, which can be reused.