FR2492269A2 - ELECTRODIALYSIS APPARATUS AND METHOD FOR FRACTIONING PROTEIN MIXTURES - Google Patents

Abstract

A. PROCESS FOR FRACTIONING MIXTURES OF LIQUID PROTEINS 1 INTO FRACTIONS OF INTRINSICALLY DIFFERENT COMPOSITIONS. B. PROCESS CHARACTERIZED IN THAT IT CONSISTS OF EXTRACTING ALL THE PRECIPITE TROUBLESHOOTING THE MIXTURE 1; TO PASS THIS MIXTURE THROUGH AN ELECTRODIALYSIS APPARATUS 4 WITH MEMBRANES DEFINING FLOW CHAMBERS; TO APPLY TO THIS APPLIANCE AN ELECTRIC CURRENT SUFFICIENTLY MODIFYING THE IONIC ENVIRONMENT OF THE MIXTURE TO DESTABILIZE AT LEAST ONE PROTEIN; TO LET THIS PRECIPITATE; AND EXTRACTED THIS PRECIPITE 17 BY MAINTAINING THE TEMPERATURE OF THE MIXTURE BETWEEN 0 AND 40C. C. THE INVENTION CONCERNS THE FRACTIONATION OF PROTEINS BY ELECTRODIALYSIS.

Description

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  An electrodialysis apparatus and a method for fractionating mixtures

of proteins.

  The main patent relates to a process for fractionating liquid protein mixtures containing dissolved salts, by using an electrodialysis apparatus provided with one or more pairs of concentration and dilution chambers, these chambers being defined by alternating membranes. exchange of anions and cations, located between anode terminal electrodes, and cathode, characterized in that it consists of passing the protein mixture in the dilution chambers, to apply a current cnntinu electrode terminals to reduce the salt content of this protein mixture by making

  pass the salt from the dilution chambers to the

  tration; collecting the desalted protein mixture in the dilution chambers; separating and extracting one or more protein elements from the desalted protein mixture and then passing the desalted mixture obtained into the concentration chambers, whereby the salts entering the concentration chambers from the adjacent dilution chambers reconstitute exactly the initial salt content of the mixture

desalted protein.

  The object of the present addition is to perfect the main patent and for this purpose concerns a method for fractionating liquid protein mixtures in which an aqueous protein mixture is separated into fractions.

  intrinsically different compositions.

  Biological fluids, such as the

  plasma or blood serum, milk, whey, urine etc.

  - contain a mixture of various proteins.

  For example, blood plasma contains albumin (3.5 to 4.5 g / 100 ml), fibrinogen (0.20 to 0.45 g / 100 ml), and g -globulin (0.4 to 1 g). g / 100 ml) of the globulin (0.4 to 1.8 g / 100 ml) of the immunoglobulin Ig M (0.6 to 0.25 g / 100 ml) etc ... (Prank W. Putnam, The Trace Components of Plasma, An Overview). Immunoglobulins (Ig)

  are very important because they come into play in the mechanis-

  protection and defense against infectious organisms

tious.

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  Clinical disorders characterized by imbalances in these protein systems, either in the ability to recognize invading organisms or in the ability to recognize indigenous acids from proteins or polymelic acids, have been at the origin of the understanding of clinical aspects of immunology. It is known that abnormal immunological reactions produce a wide range of

spectrum of diseases.

  Among the diseases related to reactions

  complex immunological problems, for example, the

  serum sickness, glomerulonephritis and myastemic gravis.

  Plasmaphoresis is a technique used to reduce, slow down

  or stop the immunopathological process associated with circula-

  of a humoral antibody and / or complex immunological compounds of the plasma (Glassman, Rationale for Plasmapheresis,

  "Plasma Therapy", Vol. 1, No. 1, Page 13 (1979).

  A known method is to perform the plasmaphoresis of about 4 liters of plasma during a

  period of 2 to 4 hours. Plasma thus Functioned on the

  It is usually discarded and replaced with albumin and either the physilogic serum or a Ringer's solution to obtain the equilibrium between proteins, electrolyte and water. This process however is expensive. In another method, replacement of the removed plasma is done by supplying fresh or frozen plasma. This method, although less expensive, presents the risk of transmission of hepatitis virus to the patient. The method according to the invention (called immunophoresis) makes it possible to overcome the above disadvantages of the prior art by selectively eliminating immunoglobulins, englobulins or complex mixtures of englobulins and antigens producing the disease or resulting from it. here, while at the same time reconstituting most of the albumin, the electrolyte (salt) and the water, thus restoring the patient's own plasma (practically without complex mixtures of Ig or Ig antigens). ) containing the correct dosage of protein, without risk of hepatitis because it is not necessary

  add albumin or donor plasma.

  The anti-hemophilia factor

  Antihemophilic line (Factor VIII, AHF or AHG) is one of the

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  elements involved in the coagulation of blood. A hereditary abnormality of blood coagulation, or haemophilia, causes very large bleeding at the joints, in the muscles or in the internal organs to the slightest trauma. These disorders appear to be due to an AHF specific protein deficiency of the plasma. Individuals with these disorders are most often required to receive special care after the smallest accidents. In case of surgical operations

  the defect of coagulation is corrected by plasma transfusions.

  my charges or by injections of concentrated Factor VIII, this

  last solution being preferable because it avoids hyperpro-

  malignant and renal disorders resulting from large volumes of transfusions. Processes, according to the prior art, for the production of AHF consist, for example, in using a common pool of plasma, or plasma bank, to form a cryoprecipitate, in centrifuging the precipitate consisting essentially of a mixture

  AHF and fibrinogen, and then use freeze-dried

  to produce AHF concentrate. However, these methods all have the disadvantage of being long and tedious, and also present risks of transmissions of hepatitis by the common source or plasma bank. Furthermore, the presence of the fibrinogen impurity makes it difficult to dissolve the AHF concentrate. Finally, the fact that several days elapse between the moment the plasma is given and the moment it is

  used, leads to a considerable loss of AHF activity.

  Unit AHF is defined as the activity in 1 ml. normal average human plasma less than one hour old (100% / 0 AHF level). Thus, after six hours the loss of activity of the extracorporeal liquid plasma can reach 80%. A quick treatment process for AHF

  prevents this loss of activity.

  Apparatus and processes according to

  the purpose of remedying the above-mentioned disadvantages

  continuous treatment in real time. This allows a recovery

  AHF in the range of 4 to 5 times that of current processes

much longer.

  The invention can also be adapted to a plasma bank of smaller volume, containing for example 2 to 3 elements without risk of hepatitis, of the family

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  hemophiliacs, the recovery of AHF is done after in-situ, which allows to obtain, with no risk of hepatitis AHF very high activity. Continuous methods according to the invention can also be used to recover donor Factor VIII during plasmaphoresis. The basic techniques used in the invention, that is plasmaphoresis and electrodialysis, are both well known in the art. The novel combination of the techniques described herein produces a synergistic effect, i.e., an increase in the yield of each step of the combination. This surprising increase in yield is extremely useful, especially for real-time in-situ care of patients who need to be punctured with Ug or

complex compounds of these Ig.

  The processes according to the invention will be described in the preferred examples of plasma and whey, but it is obvious that the invention is equally applicable to

  other biological fluids or other proteins without limita-

  its scope. The use of electrodialysis to saturate

  salt or on the contrary desalt the biological fluid, in order to obtain a separation of the proteins, is an extremely useful tool

  effective in the hands of protein chemists.

  For this purpose, the invention relates to a method for fractionating mixtures of liquid proteins according to the main patent according to which an aqueous mixture of proteins is separated into fractions of intrinsically different compositions, a process characterized in that it consists in removing all the disturbing precipitate. the aqueous mixture; to pass

  then this mixture at a rate of about 3 to 40 cm / sec.

  in an electrodialysis apparatus containing at least a pair of adjacent membranes defining therebetween a liquid flow chamber; to apply an electric current in this apparatus with a CD ratio of about 0.1 to 10 (CD being the current density in mA / cm and K the conductivity of the aqueous mixture in milliSiemens / cm) to thereby modify the ionic environment a mixture of a value sufficient to at least partially destabilize one or more proteins of the mixture to allow the destabilized protein to precipitate; and then removing all the precipitate obtained by maintaining the temperature of the mixture in a range of about 0 to 400 C throughout

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the separation operation.

  The invention utilizes electrodialysis to separate aqueous mixtures of proteins into fractions of intrinsically different compositions that can be distinguished by well-known physical or chemical methods. For this purpose, the invention uses the fractionation or the partial separation of protein mixtures and the subsequent reconstitution of their equilibrium with salt and water. The invention relates not only to salt depletion fractionation (desalting) but also to salt enrichment fractionation

(saturation in salt).

  Protein mixtures essentially (but not exclusively) include plasma, serum or their derivatives. The electrodialysis process used in desalting removes the dissolved salts (ions) and, therefore, the enobulins or their compounds are completely precipitated by reducing their ionic concentration, combined, if desired, with changes in temperature and / or pH. In the elimination of salts, the interaction of saline ions with the ionizable groups of proteins is apparently reduced, which allows an interaction between the molecules of englobulins

  and therefore the precipitation thereof.

  Albuibine and other proteins that are not nature-by-nature do not give precipitates

  for (low) salt concentrations suitable for

  lines, and therefore remain in solution for subsequent refunds

  to the patient or for recoveries. After removing the turbid (or precipitated) elements of englobulins, the plasma concentration can optionally be reduced to its exact initial value by using the desalinated plasma as a fluid.

  salt receiver in a column or electrodialysis module.

  The desalinated plasma is thus completely restored to the electrolytes (and water) and can therefore be returned to the donor or the patient without further modification of the salt or water content. In the form of carrying out the salt saturation process, salt is added to the protein mixture to cause precipitation one by one.

  various proteins as the ionic concentration increases.

  Saline saturation agents apparently act in this

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  group by reducing the activity of water in the mixture of

  solvents, thereby dehydrating the hydro-

  philes of protein molecules by causing precipitation

of these proteins.

  In a third embodiment of the invention is used the addition of certain agents such as for example metal ions, small anions and polyanions (polyphosphates etc ...) tending to cause precipitation by an apparently different mechanism thanks to which the electrostatic charges of a small number of critical groups of proteins seem to be affected. Since the ionization of these critical groups is necessary to maintain the normal state of hydration of protein molecules, precipitation is often produced by a simple effect of compensating for the resulting electrical charge of the protein molecules. These agents are only needed for low concentrations

  and well defined, because the mechanism is not a mass effect.

  Electrodialysis makes it possible to obtain this result

perfectly well controlled.

  The prior art uses the addition

  mass of these agents, which has strong local effects.

  ized. With electrodialysis all the embodiments of the invention described above can be easily treated and the fractionation or even

in some cases strengthen it.

  More specifically, certain embodiments of the invention employ fractionation processes of liquid protein mixtures in one or more dissolved salts, using an electrodialysis (ED) apparatus having one or more pairs of receiving or salt dilution separated from each other by

  ion selective membranes, neutral membranes (non-selective

  tives) or combinations of neutral and selective membranes

to ions.

  In a particular form of

  In accordance with the invention, the electric current is applied between the terminal electrodes so as to reduce the salt content of a protein mixture located in the salt dilution chambers by transferring the salts of these salts to adjacent receiving chambers. . This desalting continues until we

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get the precipitation.

  Precipitation can be facilitated, if desired, by prior, simultaneous or subsequent changes in pH and / or temperature. The fully desalted protein mixture from the dilution chambers is collected and processed to separate or remove one or more of the precipitating protein elements. The desalted protein mixture can then be passed optionally through the salt-receiving chambers so that salts entering these chambers from the adjacent dilution chambers essentially return the desalted protein mixture to its initial salt and water content. This return to the norm is desirable if the protein mixture

  is blood plasma that we want to restore to the donor.

  The process described above is particularly effective when mixing liquid proteins

  is blood plasma or serum, when the protective

  removed are globulins and / or their compounds, and

  when at least one of the membranes of each pair is selected

ion.

  Another method of carrying out the embodiment described above, in which at least one of the membranes of each pair is ion selective, consists in collecting the desalted protein mixture.

  from the dilution chambers of the column of electro-

  dialysis (ED), remove from the protein mixture flessalé one or more precipitated proteins, and then recycle the desalted mixture and emptied of its proteins, to the chambers

  initial dilution. A continuous current of polarity is applied

  such that the initial dilution chambers, now containing a desalted mixture, become salt concentration or reception chambers, and that the first receiving chambers containing the salts removed in the first part of the process become dilution chambers or deconcentration of salt, so as to reconstitute the

  initial salt and water content of the desalted protein mixture.

  In another embodiment

  of the invention, the precipitate can be caused by a saturated

  salt, that is to say by adding, in the mixture of

  proteins, a salt saturation agent. These agents of saturation

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  saturation include, for example, the sulfates (Na 2 SO 4, K 2 SO 4, (NH 4) 2 SO 4, Mg SO 4 etc.), the acetates (of sodium, potassium, etc.), the citrates (of sodium, of potassium, etc.). ..) chlorides (NaCl, KCl, MgC12, CaCl2, AlCl3) and other essentially non-toxic soluble salts. The electrodialysis apparatus used is the same as that of the first case described above; the salt-enriched protein mixture, in which the precipitation occurred under the action of salt saturation caused by a saturation agent such as Na 2 SO 4 for example, is collected and processed to separate and remove the salt one or more of

  protein elements causing the precipitate. The current of

  salt (desalting) must contain a salt saturation agent, or salt-enriched plasma obtained after elimination

precipitate.

  It is thus possible to carry out exactly at the same time the two operations, that is to say the removal of the precipitating or salt-saturating agent, and the addition of this

  agent to cause saturation. The protein stream of

  salt may be returned to the source, possibly after

  the equilibrium of the electrolyte at its initial

exact salt and water.

  The salt saturation process described above is particularly applicable in the case where the mixture of liquid proteins is blood plasma or serum, and the removed protein elements are -globulins and / or their complex compounds. Electrodialysis (ED) makes it possible to add the precipitation agent at a controlled rate, which is a very important factor. Slow addition of the precipitating agent leads to the formation of crystalline proteins precipitates much purer in proteins, these crystals containing many fewer pollutants absorbed than in

  the case of finer protein flakes, precipitated by ad-

  rapid joining of the precipitating agent, which may

  contain proteins that would not precipitate in case of ad-

slow junction.

  In yet another form of realization

  of the invention, the precipitation can be produced by

  pH modification by electrodialysis (ED) to bring practice--

  this pH at the isoelectric point (pI) of a given group of

  proteins such as for example g -globulins in the case of blood plasma. The saturation of salt can be done for a lower salt concentration if one works near the isoelectric point of the protein. The desalting precipitation can be done for a salt concentration more

  high when working near the isoelectric point.

  In yet another form of reali-

  In addition, the protein fractionation can be carried out by addition of zinc glycinate, for example (final concentration of about 20 mM) at a pH of about 7.2, by electrodilay (ED). The zinc ion (Sn "+) produces the

  precipitation in the plasma of the various proteins (g globul-

  Unes, fibrinogen etc ...) without causing the disappearance of albumin. This method is similar to that of the salt saturation described above in that the dilution compartment

  is emptied of zinc salt and that the compartment of concentration

  containing the protein mixture is enriched

to produce the precipitation.

  The amount of Zn ++ needed is

  very low compared to many other saturation agents.

  saline ration, because the mechanism of precipitation apparently consists in compensating simply the overall negative electric charge of the molecule; the charge balance of the remaining ion groups being at zero, the essentially neutral protein molecule is apparently not able to retain

  enough water to stay in solution.

  Some of the techniques and embodiments described above may be combined as will be apparent to the subject matter experts. In the separation of some Y - globulins from the plasma, the direct addition of the saline saturation agent can be considered as a combination with the electrodialysis to remove the added salt in order to find a solution relatively

  rich in albumin (after removal of the globulin precipitate).

  In the above examples, Na 2 SO 4 is preferably used as a saline precipitating agent,

  without in any way constituting a limitation of the invention

  other salts and mixtures thereof which may be

  be used to perform the splitting process.

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  The invention will be better understood in

  reading of the detailed description which follows and which refers

  in the accompanying drawings in which: - Figure 1 shows a first embodiment of the desalting process and apparatus according to the invention; - Figure 2 shows a second

  embodiment of the process and apparatus of

  by the desalting according to the invention, in which the same parts as those of FIG. 1 are identified by the same references; FIG.

  embodiment of the process and apparatus of

  by saline saturation according to the invention, - Figure 4 represents a second

  embodiment of the process and apparatus of

  salt saturation according to the invention,

  FIG. 5 is a diagram showing

  the results obtained by the various processes for adding Na 2 S0 4 electrolyte, and FIG.

  electrodialysis machine operating in alternating current.

  Electrodialysis (ED) is very

  used for desalting aqueous solutions. brackish water, whey (US Pat. Nos. 3,433,726, 3,447,939,

  35595-766, 3.757.005, 3.754.650 etc ...). These patents do not concern

  only the reduction of the salt content of a liquid, and not the use of the electrodialysis process in a complex fractionation process followed by a rebalancing of the salt and water content of a protein mixture. intended for example

  to a therapeutic application in case of plasmaphoresis.

  Desalting by the technology of the ion exchange column has been used in the past to cause precipitation and therefore splitting

  plasma proteins (US Pat. Nos. 3,234,199 and 3,073,744).

  This process had a limited flexibility and the

  are difficult to handle, clean and sterilize

  when used in the conditions necessary to obtain

  protein fractionation.

  It has now been discovered that electrodialysis can be used not only for protein fractionation by desalting, but also to carry out the saline saturation process and to restore the equilibrium between the electrolyte (salt) and water in the processed protein mixtures. The proteins thus obtained are ready to be returned to the donor or to a

patient, with the initial salts.

  The combination of the techniques described here includes as essential steps the electrodialysis of the

  mixture of proteins, (possibly combined with a modification

  temperature and pH), and the separation of a certain

  number of precipitated proteins, then a

  tative of the salt and water balance of the initial mixture. This new process increases the yield of each step so

  completely unexpected and makes the process extremely interesting.

  especially for therapeutic applications

  real-time, performing plasmaphoresis on patients for whom the globulins or their compounds must be eliminated while accurately restoring the initial plasma. This method not only avoids the need to replace albumin and salt, but also eliminates the risk of transmission of hepatitis related to plasma injection

  fresh or frozen from a plasma bank.

  A first type of process and

  The desalting fraction will now be described hereinafter in FIGS. 1 and 2 in which the same parts are identified by the same references. In these figures the fluid subjected to the treatment is blood plasma, but it is obvious that this fluid can be constituted by any other type of protein mixture. As shown in FIG. 1, the citrated or heparinized blood 1 is ultrafiltered and / or centrifuged 2 to separate the elements of the cell 3 or any other suspended body (called formed elements (EF)), and the remaining plasma. 15 is sent to an electrodialysis (ED) column 4 such as that sold by Ionics, Inc. Watertouq, MA. These electrodialysis equipment and their method of implementation are described more completely in US Pat. Nos. 2,848,403, 2,863,813, 3,003,940, 3,341,441 and 4,155,225.

and others.

  Such a column normally comprises one or more pairs of concentration and dilution chambers separated by alternating anion exchange membranes.

  and cations. Ion-selective membranes can also

  be replaced, under certain conditions, by membra-

  are essentially electrically neutral. Thus the anion membrane can be replaced by a neutral membrane if a reduced current efficiency for the ion transfer can be tolerated. The rooms are located between the anode (+) and a cathode (-). An electrolyte solution is preferably passed through the cathode and anode chambers to conduct the current through the concentration and dilution chambers. The electric current is passed until at least one start of precipitation, or until precipitation is achieved when the temperature is reduced and / or when

  set the pH to the pI value of the last protein.

  Generally a chamber of concentration

  isolates the electrode solutions from the product or the chambers

  dilution. Membranes are generally, but not necessarily

  chosen in such a way as to minimize the transfer of

  its low molecular weight such as blood sugars. The flow rates through the column and the applied electric current are regulated so as to avoid excessive pH changes. The plasma passes into the dilution chambers and the application of a direct current across the electrodes makes it possible to reduce the content of salt or duplasma ions as a result of the passage of salt in the concentration chambers.

  adjacent areas (as indicated by the vertical arrows of the

  ne), these rooms can be decleaved initially, if

  by a small amount of plasma or albumin.

  The completely desalted plasma obtained is collected in the dilution chambers (not shown) and passes means for separating and removing one or more proteins forming the precipitate (globulins or their compounds in this case). The separation means may for example be constituted by a heat exchanger 6 leading to lower temperatures, and by a device of

  pH and centrifugation and / or ultrafiltration adjustment 7.

  After having been separated from the globulins 17 or other proteins, the desalted mixture 8 passes into the concentration chambers (not shown) of the electrodialysis column 4, which makes it

  allows the salts from the dilution chambers to be

  adjacent areas (as indicated by the vertical arrows) and

  restore the exact initial salt content of the mixture.

  This mixture, having recovered its salt content, then optionally passes through a heat exchanger 10 for approximately adjusting the mixture to body temperature when necessary, and then the mixture receives the obtained elements 3 (such as red blood cells and whites and platelets) previously separated from the plasma. This reconstituted protein mixture 12 can then be restored to pat1int 14, with virtually no external supply of albumin or electrolyte. The process is therefore completely closed-loop, autonomous, and able to implement

  real time, in-situ, for therapeutic applications.

plasma exchange ticks.

  If, during electrodialysis, the

  The plasma temperature is maintained between approximately 0 C and 400 C, if the speed of the mixture of proteins in the dilution chambers is maintained between 3 and 40 cm / sec, and if the ratio (CD) of the

2 (K

  current density (CD) in mA / cm at the conductivity K of the protein solution in milliSiemens / cm, is maintained between 0.1

  and 10, the pH of the proteins hardly varies and the

  Thus obtained (even in the presence of a relatively complete desalting) is such that it avoids any risk of obstruction of the chambers of the electrodialysis column. (It will be noted that

  milli Siemens is equal to one millimho).

  Another embodiment of the apparatus and method according to the invention is shown in FIG. 2. The fluid is, again, plasma 1, but can be any other aqueous mixture of fluid proteins. . (Citrate or heparin may be added or not added to reduce plasma coagulation during treatment). The heparinized or citrated or untreated plasma is ultrafiltered or centrifuged to remove the precipitate and then sent to an electrodialysis column 4 similar to that described in Example I. The plasma is introduced into the dilution chambers (no represented) and the passage of a continuous current in the column makes it possible to pass the plasma salts into the concentration chambers (as indicated by the arrows

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  vertical). The desalted mixture from the dilution chambers passes through the heat exchanger 6 for cooling the plasma, and the precipitate obtained is separated by an ultrafiltration or centrifugation device 7 or by any other similar device. The desalinated liquid which floats 8 is

  then sent to the salt concentration chambers (not

  tees) of another electrodialysis column 18 shown for this second electrodialysis. In practice it may be the initial ED electrodialysis column 4 when the first concentration stream 19 of the ED 4 column is the dilution stream. The polarity of the ED 18 column can be reversed from that of the ED 4 column. This second step

  of ED 18 causes the return of the salts of the first

  centration 19 to the desalinated plasma, which restores the salt balance. This rebalanced plasma 9 then passes into a heat exchanger

  heat 10, then the blood cells 3 are added.

  The blood thus treated may be returned to the donor or to any

other patient 14.

EXAMPLE I

  This example illustrates the restitution of the electrolyte and water equilibrium of desalinated plasma, by

  use of fresh plasma in the diluted stream.

  The apparatus used is a laboratory electrodialysis column using only a single pair of cells (i.e. a dilution chamber and a chamber of concentrations defined by ion selective membranes) located between terminal electrode chambers. A solution of 0.2N.N a2SQ is used at the electrodes to conduct the direct current. A volume of 360 ml of fresh plasma or citrate is used in the dilution stream, and a volume of

  340 ml of desalinated plasma is used in the concentration stream.

  tration. The linear velocity of the dilution stream is about cm / sec, the temperature is maintained between 15 and 200 C, and the flow rates are maintained at 90 ml / min. per pair of cells. The

  Effective cell area is about 220 cm2.

  The process flow is summarized in the table below: CD / K Amp. Dilution current ConcentraUia current (min) (fresh citrated plasma) (desalinated plasma)

Conducti- Conduct-

vity (K) pH Vol. quickly (K) pH Vol.

  4,7 0 17 16,500 8.2 360 0.030 5.2 340

  4,7 6,8,8 8,600 7,4 352 8,500 7.2 347

  4.7 12 4.4 4.300 6.9 347 12.40000 7.7 350

  , 0 25 0.9 0.825 6.1 344 15.600 8.0 353

  27.5 35 0.2 0.033 5.2 342 16.400 8.3 355

  Thus, the desalinated plasma was brought back into the concentration stream at a conductivity value comparable to that of the initial unsalted citrated plasma, and

  the water balance has been restored.

* EXAMPLE II -

  Although the electrodialysis column used in Example I has ion-selective membranes (of the anion and cation types), it is also possible to use a combination of ion selective membranes with

  neutral membranes (non-selective).

  In this example, the anion-selective membrane of the column of Example I was replaced by a neutral membrane made of regenerated cellulose. Other types of neutral membranes well known in the art, such as reverse osmosis or dialysis membranes, could also be used if desired. Neutral membranes have the disadvantage of not being as effective as ion selective membranes. However, these membranes are very interesting to use in

  process where the input energy level is not a characteristic

important risk.

  In this example the column containing the regenerated cellulose membrane operates with the same solutions and in the same conditions as those of Example I. The progress of the process is summarized in the table below:

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  CD / K Amp. Dilution current Concentration current (min) (fresh plasma) (desalinated plasma)

Conducti- Conduct-

vity (K) pH Vol. quickly (K) pH Vol.

  3.3 0 12.0 16.500 8.2 360 0.050 5.3 340

  3.3 14 6.2 8.700 7.4 350 8.480 7.4 350

  3.3 27 3.1 4.300 6.9 342 12.390 7.7 355

  3.3 57 0.6 0.830 6.1 337 15.450 8.0 360

  23.3 80 0.2 0.039 5.2 334 16.420 8.2 363

  Here again the desalinated plasma was brought back to a conductivity comparable to that of the fresh initial plasma and the equilibrium in water was restored. It should be noted that the increase in operating time (80 minutes) is due to the fact that the current efficiency is considerably smaller for the combination of neutral membranes and membranes.

exchange of caions.

EXAMPLE III

  This example is similar to Example I except that the desalted plasma is used in the initial dilution stream of Example I and an aqueous solution containing the salts removed by a first desalting step is used in the process. initial concentration current. The polarity of the current is reversed (thereby transforming the initial concentration chambers into dilution chambers and the initial dilution chambers into concentration chambers) and the sls from the saltwater stream are transferred to

  desalinated plasma (which is now the current of concentra-

  to bring the plasma salts back to their original concentration.

EXAMPLE IV

  This example illustrates the extraction of

  immunoglobulin Ig as a function of the degree of desalting.

  The apparatus of Example I is used with 200 ml of heparinized human plasma in the dilution chamber. The temperature is between 10 and 26 C and the mA / Dm2 value of CD / K is about 4 S / cm. A fluid velocity of 25 cm / sec is used. The table below summarizes the results obtained and shows that approximately 50% of the total Ig are extracted while the extraction of albumin is practically not altered after

desalting 99.7%.

  Protein remaining in the supernatant liquid (mg / 100 ml) pH time Conduc% IgA IgG IgA IgM Albumin (min)

0 7.55 15.280 0 820 115 72 4.410

0o 7.55 13.720 0 750 85 50 3.900

7.35 7.020 48.80 780 80 46 3.900

8 7.10 3.710 72.96 700 75 36 3.900

6.55 1.310 90.60 630 75 24 4.100

13 5.40 0.447 96.70 570 55 12 4.000

15 5.20 0.190 98.60 450 50 10 4.000

  17 4.90 0.047 * 99.70 410 40 16 3.900

19 5.00 0.028 99.80 410 45 12 3.800

5.10 0.022 99.84 410 45 10 1.800

  * Sum of% Ig extracted after 99.7% desalting% extract% extract (corrected for water transfer) Ig G 45.3 46.7 Ig A = 52.9 55.9 Ig M: 68, 0 71.4 Total Ig = 47.3 49.7

EXAMPLE V

  This example illustrates yet another embodiment of the invention in which the variation of the pH to the isoelectric point of a protein desired is

  extract, helps to help his precipitation. The continuation of

  Salting the plasma from Example IV lowers the pH to the isoelectric point pI of albumin (about 4.9), thereby producing its precipitation. The precipitated albumin is separated by filtration and then resuspended by addition of salt. This addition is done by electrodialysis using the albumin-rich material as the salt concentration stream, giving an isotonic albumin solution of 3 to

18 2492269

  %. This albumin is practically free of immunogobulins and their compounds, and can be used to extend the plasma. This is the best method to use when extracting 50% of immunoglobulins for "immunophoresis".

  (immunoglobulin extraction) in case of autoimmune disorders

  munologie. In another embodiment of the invention, the fractionation is obtained by "saline saturation" that is to say by use of salts such as (NH4) 2 S04 Na2 SQ4 etc ... injected into the protein mixture by electrodialysis. The different proteins precipitate to

  different saline concentrations which leads them from

  same to a split. A particular advantage of this

  use of electrodialysis instead of the direct addition

  of salts, is that electrodialysis can better control

  the addition of salts and thus avoid the de-concentration gradients

  local management. "Saline saturation" by electrodialysis is also much faster than that achieved by a single dialysis in which the only drive mechanism is diffusion (without difference in electrical potential). Electrodialysis results in a fractionation comparable to that obtained by addition of salt or by dialysis, but for

  saline concentrations much lower.

  We will now describe a method and apparatus for saline saturation used according to the invention for the fractionation of blood plasma, but it is obvious

  that the invention is not limited only to plasma.

Two embodiments of

  "Saline saturation" are shown in Figures 3 and 4.

  As shown in FIG. 3, the fresh plasma 15 is pumped as a concentration stream (salt reception current) by two electrodialysis columns 4 and 11. The salt-saturated plasma obtained passes through separation means 7, centrifugal

  for example, to remove precipitated proteins 17.

  The supernatant liquid 8, which becomes the dilution stream of the electrodialysis column 11, is essentially an albumin filtrate containing the saline saturation agent (such as Na 2 SO 4 for example) from which a protein fraction 17 has been removed. precipitates (such as immunoglobulins and fibrinogen

for example).

  This current is designed to release its salt (see the direction of the vertical arrows) when a current is

  applied to the terminals of the electrodialysis column, that is to say

  say to transfer its saline saturation agent to a fresh plasma stream 15 thereby causing the precipitation of

  certain proteins (globulins) of this fresh plasma stream.

  In turn, this diluted stream 8 is impoverished in salt and tends to a simple solution of albumin. The final electrodialysis or finishing column 4 is shown separately but may be constituted by a part of a single electrodialysis column. This finishing step may not be necessary when the product 9 may contain salts not interfering with the melting. A finishing electrolyte 20 may be necessary if the electrodialysis finishing step is to be carried out. This method is therefore compatible with an implementation in situ for a therapeutic plasma exchange, and can be implemented not only in successive batches but also in

continuous diet. -

  Another embodiment of saline saturation according to the invention is shown in FIG. 4. In the latter variant, the saline saturation agent (salt solution) is added directly to the plasma to cause the precipitation of the immunoglobulins. separate 7 and extract 17 by filtering for example. The plasma 16 obtained, used as a dilution stream, is desalted by the electrodialysis column 4; the concentration stream 18 becoming essentially a solution of saturation agent

  saline. This concentrated solution 19 can be added directly

  The electrolytes 20 and the obtained elements 3 can be added to the desalinated albumin solution 9 before the solution 12 is again administered to the donor or the patient 14. Although it is preferred to use Na 2 SO 4 as a saline saturation agent, it is obvious that the invention is not limited to this agent alone. It is possible to use other salts or their mixtures such as K 2 SO 4, (NH 4) 2 SQ 4, sodium citrate, phosphates, NaCl, KCl, acetates, etc., and mixtures thereof. The amount

  added salt obviously depends on the desired fractionation.

  The following example illustrates the separation between IgG and

albumin.

EXAMPLE VI

  300 ml of plasma are heated to 28 ° C. at 370 ° C. and a saturated solution of Na 2 SO 4 (approximately 6 N) at 28 ° to 370 ° C. is added at a rate of 10 to 15 ml / min while stirring the plasma mixture rapidly and continuously. . The quantity of albumin and globulins (IgG) remaining in the supernatant liquid is determined, during the addition of salt, according to

  the salt concentration (electrolyte) of the supernatant liquid.

  FIG. 5 compares the results obtained by the various Na 2 SO 4 electrolyte addition processes, and represents the approximate fractionation of the

  proteins (albumin and IgG) for different electron

  lyte. FIG. 5 also represents in particular the curve obtained when using a mixture of salts (6 N NaCl and

6 N Na 2 SO 4).

  Finally, FIG. 5 also represents the comparison between saline saturations obtained by

  electrodialysis, by dialysis and by direct addition of salt.

  The results show that to obtain an extraction of about 80% of IgG globulins in fresh plasma, it is necessary to use a 1.8 N salt concentration (Na 2 SO 4) in the plasma (supernatant) when present in the plasma. case of the direct addition of salt. In these conditions

  however, at the same time extraction (i.e.

  say a loss) of about 15% albumin.

  When, in the case of the direct addition of salt, a mixture of salt (Na 2 SO 4 + NaCl) is used, an extraction of globulins of 80% is obtained for a salt content of about 2.05 times the normal , With albumin extraction limited to 5% only. When the addition of salt (Na 2 SO 4) is done by dialysis, an extraction of globulins of 80% is obtained for a salt content of the order of 2.3 times normal, but with a loss of albumin of about 10%. Compared with the addition of salt by ED electrodialysis, we can observe that we obtain an extraction of globulins of% / e for a salt content much closer to normal (1.2 N), with a loss of albumin less than 15 i. In summary it appears that the addition of salt by electrodialysis allows a more complete globulin extraction with less albumin loss and lower salt content. An alternative method may be, if desired, to use a combination of direct addition of salt or dialysis of

  the saline saturation agent, followed by treatment with

  trodialysis to remove added salts.

EXAMPLE VII

  This example illustrates the separation of antihemophilic factor (AHF) and fibrinogen from plasma. As the activity of the AHF is sensitive to the duration and

  temperature, the separation is done at low temperature (4 C).

  A method applicable to the separation is to lower the ionic strength of the plasma, preferably by desalting electrodialysis, to cause the precipitation (separation) of fibrinogen and AHF, and then to redissolve the precipitate in for example 0.15 N of NaCl, and again subject the redissolved liquid to electrodialysis to lower the ionic strength and thereby cause the precipitation (and separation) of the AHF from fibrinogen. In an alternative embodiment, the last separation can be produced, after redissolution, by specific absorption of AHF on the anion exchange column, or by permeabilization techniques.

by gel.

  A second method is to directly saturate the salt AHF with a suitable force. All these methods can be applied in series as well as blow by

  as in the case of immunophoresis.

Another embodiment of

  the invention consists in applying electrodialysis to the fractional

  plasma proteins using small amounts of heavy metal ions with low toxicity such as

  zinc diglycinate, used as precipitators.

  Plasma is first partially electrodialysed to remove coagulation factors that precipitate during desalting. After extracting these factors, the liquid

  remainder that passes in the compartments of concentration

  Salt in an electrodialysis column containing zinc diglycinate in the dilution compartments. By application of a difference in electric potential a controlled amount of zinc diglycinate can be transferred to the concentration compartments to obtain a strength.

  ionic about 0.1 be the normal, in the supernatant liquid.

  4, the operation is between 0 and 40 C and for a pH of about 7.0 to 7.2 This results in the formation of a precipitate consisting essentially of globulins, with a supernatant rich in albumin. This supernatant liquid can be clarified from the zinc added by desalting electrodialysis, after the pH has been lowered to about 5.1 or 5.8 by addition.

suitable buffer product.

  Aluminum chloride can also be substituted for zinc diglycinate to split

  by precipitation all the proteins except K -globulins.

  The direct addition of an equal volume of 0.1 M of AC10 at 0 ° C. to the plasma, with rapid stirring, causes a precipitate of all the other proteins which can then be redissolved.

in 0.15 N NaCl.

EXAMPLE VIII

  This example illustrates the use of AC power rather than DC power normally

  used in electrodialysis operations. The column of elec-

  trodialysis thus uses a combination of neutral membranes

  (N) and cation-selective membranes (C) in the

  FIG. 6. An electrodialysis column operating in alternating current is described in detail in FIG.

  U.S. Patent No. 2,955,999 (C.E. Tirrell).

  A metal valve niobium 4 plated with a noble metal such as platinum 5, is used as anode 1, and a metal valve 6 without a noble metal coating, is used as cathode 3. The metal valves have the property of not driving the current only when they are cathodic thus giving a kind of "rectification" of the alternating current (A / C). Such an electrodialysis column uses two pairs of cells (instead of a single pair as in Example II) removed by a bipolar central electrode 2 plated with platinum 5 on one side, to serve as anode. During the positive half-period (+) of the current, the platinum plated side 5 of the electrode 1 serves as the anode, and the unplated platinum side 4 of the electrode 2 serves as the cathode, so that the column of membranes limited by the electrodes 1 and 2 is in service while the column of membranes between the electrodes 2 and 3

23 2492269

  is inactive because the electrode 3 not platinum plated

can not work in anode.

  During the negative half-period (-)

  it is the column between the electrodes 2 and 3 which function

  This is because the platinum plated side 5 of electrode 2 serves as the anode. Such an electrodialysis column operates in the same way as in the case of Example 11 but has the advantage of being AC powered without the need for a rectifier. The neutral membrane can also be replaced by a membrane selective to

  anions, so as to increase the operating efficiency.

EXAMPLE IX

  This example illustrates the extraction of

  Factor VIII of human plasma by desalting electrodialysis.

  The membrane separation apparatus used is a Dial-A-Cell T.M electrodialysis column sold by Ionics, Inc. Watertown, Mass. and described in detail in the

U.S. Patent No. 4,220. 772.

  The column comprises two pairs of cells having an effective membrane area of 13.6 cm 2. The ion exchange membranes used are cation-selective (CR 61 CSL) and anion-selective (AR 103 QZL), both of which are sold by Ionics, Inc. 300 ml of fresh plasma is used in this process. containing ACID (anticoagulant solution consisting of a mixture of sodium citrate, citric acid and dextrose). The starting plasma exhibits an activity of

  Factor VIII about 78% of normal.

  The table below summarizes the

  results obtained by the desalting operation. When the plasma

  is desalted, Factor VIII precipitates and is thus eliminated

  born of the liquid that floats. For a level of desalting of about% the supernatant liquid retains about 5 to 10% of Factor VIII and the removed precipitate therefore contains 90 to 95% of the factor.

  VIII initially contained in the initial plasma sample.

249292269

  Time Conductivity% Desalting Activity of the Factor (min) (K) VIII in the supernatant liquid (% or normal)

O 13.6 O 78

12 11.6 14.7 7

18 8.4 38.2 63

24 6.4 52.9 42

3.6 47.6 30

33 2.8 79.4 9

36 2.2 83.8 12

39 1.8 86.8 5

  42 1, o 92.6 whey treatment for increasing protein content and reducing residues (salt) and lactose has been the subject of a wide variety of processes. L.H. Francis discloses in US Pat. No. 3,615,664 a technique in which lactose is removed from the whey by concentrating the crude whey into crystallized lactose and then electrodialysis the supernatant liquid to obtain demineralization. The same inventor describes in US Pat. No. 3,447. 930, another process in which demirkalisation is first carried out followed by elimination

lactose.

  These various methods and other methods

  of the prior art are intended to achieve as final product.

  highly refined whey with a high protein content.

  The main problems posed by the implementation of these processes are the denaturation of whey proteins (lactalbumin) during: the heating necessary for

  concentration, and crystallization. The process according to

  The invention solves these problems by saturating the whey proteins with salt, and separating and removing the precipitated proteins by centrifugation or filtering, the salt being then removed from the supernatant liquid. This supernatant liquid contains essentially lactose and can therefore be subjected to high temperatures without fear of denaturation

  proteins. It will be noted that this process not only allows

  not only to separate the proteins but also to carry out desalting by the processes described in the above examples relating to

2492269

  to the treatment of plasma proteins.

EXAMPLE X

  The electrodialysis apparatus used is similar to that described in Example I. The dilution stream consists of 500 ml of supernatant liquid obtained by a first whey treatment in which virtually all the proteins thereof are removed, that is to say saturated with salt by electrodialysis, with a sodium sulfate content of the order of 3.5 times normal, and a temperature of about 380 C.

  Concentration current is used

  300 ml concentrated whey with a solids content of 22.5% (solids: 12% protein, 80% lactose hydrate

and about 8% of residues).

  A continuous current density of about

  ASF 130 is used initially (starting CD / K-2), then, at the end of the process, when the dilution liquid is free of most of its salts, the electric current is

  set to match a CD / K of approximately 4.8.

  The operation continues until a Na2 SO4 content of about 3.5 times normal is obtained in the concentration stream where the conductivity

is about 95 milli Siemens / cm.

  The volume of the dilution stream which is reduced to about 300 ml is drained of 90% salt and can then be treated again to recover lactose.

  The concentration stream, the volume of which increases to about 500 ml, forms a fine precipitate of protein which is removed by centrifugation. The supernatant high salt liquid is then used as a dilution stream to transfer its saline saturating agent to a next batch of concentrated whey. This method of transferring the saline saturation agent is carried out in the same manner as in the above-described cases of plasma protein separation.

human.

2492269

Claims (30)

CLAIMS -   1. A process for fractionating mixtures of liquid proteins according to the main patent according to which an aqueous mixture of proteins (1) is separated into fractions of intrinsically different compositions, characterized in that it consists in removing all the precipitate disturbing the mixture. aqueous (1); to pass   then this mixture at a rate of about 3 to 40 cm / sec.   in an electrodialysis apparatus (4) containing at least a pair of adjacent membranes defining between them a liquid flow chamber; to apply an electric current in this apparatus with a CD ratio of about 0.1 to 10 (CD being the current density in mA / cK2 and K the conductivity of the aqueous mixture in milli Siemens / cm) to thereby modify the environment ionic mixture of a value sufficient to at least partially destabilize one or more proteins of the mixture; to let the destabilized protein precipitate; and then removing (6, 7) all the precipitate obtained (17) while maintaining the temperature of the mixture in a range of the order   from 0 to 400 ° C throughout the separation process.   2, - Process according to claim 1, characterized in that the ionic environment of the mixture (1) is modified by changing its ionic concentration of at least % about.   3.- Method according to any one   claims 1 and 2, characterized in that the environment   ionic mixture (1) is modified by changing its pH to bring it exactly to the isoelectric point of at least one proteins.   4. Process according to any one   Claims 1 to 3, characterized in that the membranes   of the electrodialysis apparatus (4) are non-ion selective. 5.A method according to any one   Claims 1 to 3, characterized in that at least one   contiguous pair membranes are ion selective.   6. Process according to any one   Claims 1 to 3, characterized in that one of the membra-   contiguous pairs are selective to cations.   4o 7.- Method according to any one 27 2492269   Claims 1 to 3, characterized in that one of the   contigated pair membranes is selective to cations, the other   membrane being selective to anions.   8. A process according to claim 5, characterized in that at least a significant part of the current Electric is DC.   9.- Method according to any one   Claims 6 and 7, characterized in that the current   electric and alternating current.   10. Process according to any one   Claims 1 to 3, characterized in that the environment   ionic mixture (1) is essentially modified by increasing   the concentration of non-monovalent ions.   11.- Method according to any one   Claims 1 to 3, characterized in that the environment   The ionic concentration of the mixture (1) essentially changes by increasing both the concentration of monovalent ions and non-monovalent ions. 12.A method according to any one   Claims 1 to 11, characterized in that the precipitate removed is then redissolved.   13.- Process according to any one   Claims 1 to 11, characterized in that the precipitate   removed is at least partly redissolved, and in that this redissolved portion is converted into an insoluble fraction of precipitate and a fraction of precipitate that can be redissolved   by changing the ionic environment again by electro-   lyse, the insoluble fraction and the soluble fraction being separated from each other.   14.- Process according to any one   Claims 1 to 11, characterized in that the precipitate   removed is at least partly redissolved in the electrolyte   and partly at least recovered by electrodialysis in the   protein mixture, the protein redissolved then being separated   in two elements with intrinsically distinct igoins.   15.- Method according to any one   Claims 1 to 14, characterized in that the mixture   of aqueous proteins comprises plasma proteins, and in that the precipitate removed comprises fibrinogen and a factor antihemophilic. 28 2492269   16.- Process according to any one   Claims 1 to 14, characterized in that the mixture of   The aqueous protein comprises essentially undenatured plasma, and in that the precipitate removed essentially comprises immunoglobulins. 17.- Method according to any one   Claims 1 to 14, characterized in that the mixture   protein (1) comprises plasma proteins, and in that   the precipitate removed comprises fibrinogen and an anti-   hemophiliac, the precipitate being redissolved and separated into a fraction   composition rich in fibrinogen and a fraction rich in antihemophilic factor, by contact with a material selected from a group comprising ion exchange resin granules, gel permeabilization granules, and mixtures of these granules.   18.- Process according to any one   Claims 1 to 17, characterized in that the mixture of   aqueous protein (1) comprises plasma proteins, and   what the precipitate removed includes alb-gmine.   19.- Process according to any one   Claims 1 to 18, characterized in that the precipitate   removed is redissolved to extend the plasma.   20.- Process according to any one   Claims 1 to 3, characterized in that the mixture of   aqueous protein comprises plasma proteins; in that the ionic environment of the mixture is modified by adding essentially soluble and non-toxic salts selected from a group comprising sulphates, citrates, phosphates, chlorides, acetates, perchlorates, nitrates, sulphites, thiosulfates, bromides, iodides, and mixtures of these various salts; and in that the precipitate obtained, essentially containing non-albuminoidal proteins, is removed from the mixture.   21. A process for separating an aqueous solution of proteins according to any one of   Claims 1 to 3, including englobulins and pseudo-   doglobulins to be pooled in a fraction rich in englobulins and a fraction rich in pseudoglobulins, characterized in that it consists in removing all the precipitate from the mixture (1) to pass this mixture into an electrodialysis apparatus (4) 29 2492269   comprising at least one pair of contiguous membranes defining between them a liquid passage chamber; q) plowing an electric current across the apparatus (4) to thereby increase the ionic concentration of the mixture to a level sufficient to form a precipitate phase relatively rich in englobulins and relatively low in pseudoglobulins; then removing all the second precipitate obtained; to pass the remaining aqueous mixture (8) back into an apparatus   electrodialysis (18), to re-apply an electric current   than the terminals of the apparatus (18) to further increase the con-   ionic concentration of the mixture at a sufficient point to obtain the formation of a precipitated phase relatively en lip englobulins and relatively rich in pseudoglubulins; then removing all the third precipitate obtained; to pass an electrodialysis machine again; and apply an electric current across the device to decrease   thus significantly the ionic concentration of the solu- remaining.   22. A process for separating an aqueous mixture of proteins (1) into fractions of different composition   according to any one of claims 1 to 21,   characterized in that it consists in removing all the precipitate (3) from the mixture; to then add a concentrated solution of a salt selected from a group comprising sulphates, citrates, acetates, phosphates, chlorides, perchlorates, nitrates, thiosulfates, sulphites, bromides, ionic or mixtures of these salts, to thereby modify the ionic environment of the mixture to a point sufficient to at least partially destabilize one or more proteins of the mixture; to let precipitate the unstabilized proteins; then removing all the precipitate obtained (17); to then pass the remaining liquid which floats, with a speed of the order of 3 to 40 cm / sec., in an electrodialysis apparatus (4) comprising at least one pair of contiguous membranes defining between them a passage chamber liquid; applying an electrical current across the apparatus with a CD ratio of the order of 0.1 to 10, to remove salts from the mixture; K and to maintain the temperature of this mixture in a range of   At 400 ° C. during the extraction of the salt. 2492269   23. The method of claim 22, characterized in that the ionic environment of the mixture is modified by increasing its ionic concentration by at least about 10%. 24.- Process according to any one   claims 22 and 23, characterized in that the environ-   The ionic mixture of the mixture is further modified by varying its pH to bring it exactly to the isoelectric point of one   at least proteins so as to obtain the precipitate   at least 25. Method according to any one of the preceding   Claims 22 to 24, characterized in that the addition   concentrated salt is done via a dialysis membrane. 26.- Process according to any one   Claims 22 to 24, characterized in that the addition   of salt is done via a non-selective membrane to ions.   27, - Method according to claim 22,   characterized in that the membranes of the electromechanical apparatus lysis are nonselective to ions.   28. The process as claimed in claim 22, characterized in that at least one of the contiguous membranes   of a pair is selective to ions.   29. The process as claimed in claim 22, characterized in that one of the contiguous membranes of a pair is selective to cations.   30. A process according to claim 22, characterized in that one contiguous membrane of one pair is selective and the other membrane is selective to anions.   310- Method according to claim 22, characterized in that at least a very important part of the   electric current is direct current.   32.- Process according to any one   Claims 29 and 30, characterized in that the current   Electric is AC power.   33.- Process according to any one   Claims 22 to 32, characterized in that the salts   extracts, by the electrodialysis apparatus, of the remaining liquid Z492269   supernatant, are new added to a protein blend   next to cause a precipitate.   34.- Process according to any one   Claims 22 to 33, characterized in that the mixture   aqueous protein comprises plasma proteins, and in that the precipitate extracted comprises fibrinogen and a anti-hemophilia factor.   35.- Process according to any one   Claims 22 to 34, characterized in that the mixture   aqueous protein comprises essentially undenatured plasma proteins and in that the precipitate comprises immunoglobulins.   36.- Process according to any one   Claims 22 to 35, characterized in that the mixture   aqueous protein comprises plasma proteins and in that the precipitate extracted by electrodialysis is essentially   albumin with a small percentage of salts.   37.- Process according to claim 36, characterized in that the final product obtained by electrodialysis   is virtually salt-free albumin.   38. The method of claim 37, characterized in that salt from an external source is added to the albumin free of salt, to obtain a suitable electrolyte balance suitable for applications. IV.   39.- Process according to any one   Claims 1 to 38, characterized in that the mixture   Aqueous protein consists of whey.   40.- Process according to any one   Claims 1 to 38, characterized in that the mixture   aqueous protein comprises plasma proteins and in this   that the precipitate extracted comprises an anti-hemophiliac factor. 3 1