Process and device for separating electrically charged macromolecular compounds by forced-flow membrane electrophoresis

Claim 2

A process for the continuous separation of electrically charged macromolecular compounds by forced-flow membrane electrophoresis, which comprises conducting said process in an electrophoretic cell comprising (1) a membrane compartment consisting of a feed and a permeate cell which are divided by a micro- or ultrafiltration membrane permeable to said macromolecular compound, (2) at both sides thereto electrode compartments being partitioned from the cells of the membrane compartment with ion-exchange membranes, being impermeable to said macromolecular compound and having no or very low permeability to salts, and (3) means to control the flow rate to the permeate, by the process steps of (a) recycling aqueous solutions containing mixtures of electrically charged macromolecular compounds, and having a pH-value between the isoelectric points of said macromolecular compounds, through said feed cell and subjecting them to a pressure-driven membrane separation treatment, (b) simultaneously applying an electric field across the solutions and the membranes to induce electrophoretic migration of at least one of the macromolecular compounds through the micro- or ultrafiltration membrane or away from the membranes, (c) adjusting the flow rate of the permeate to a predetermined value, (d) withdrawing at least one of the macromolecular compounds either from the feed or permeate cell, and (e) recycling a dilute buffer solution through the permeate cell to sweep away macromolecular materials which have permeated through the membrane; the dilute solution may be at a pH equal, lower or higher than the pH of the feed solution, the recycle rate in the permeate and feed cells being many times the rate of feed solution-permeation and the residence time of the solution in the cells being very short.

Claim 3

A process according to claim 1, wherein the electrode compartments comprise anode and cathode compartments, the anode compartment is bordering the membrane compartment by an anion-exchange membrane and the cathode compartment is bordering the membrane compartment by a cation-­exchange membrane.

Claim 4

A process according to any one of claims 1 or 2, wherein the feed and the permeate cell are divided by inorganic or organic polymeric micro- or ultrafiltration membranes.

Claim 5

A process according to any one of claims 1 to 3, which comprises carrying out steps (a) to (e) by adjusting the pH-value of the aqueous solutions containing the electrically charged macromolecular compounds first below (above) and then above (below) the isoelectric point of the macromolecular compound to be separated.

Claim 6

A process according to any one of claims 1 to 3, wherein the pH-­value is maintained at the pI of the macromolecule to be separated and subsequently applying the electric field first in one direction and then in the other one, causing all other charged material to permeate through the membrane while the desired macromolecule is retained.

Claim 7

A process according to any one of claims 1 to 5, wherein the aqueous solutions are circulated under pressure in the feed and in the permeate cells and the electric field causes the electrophoretic migration of at least one of the electrically charged macromolecular compound causing it to pass through the membrane into the permeate cell from which it is withdrawn, or to retain it in the feed cell, preventing its passage into the permeate cell.

Claim 8

A process according to any one of claims 1 to 6, wherein the aqueous solutions are buffered aqueous solutions of optionally colloidal biopoly­mers, preferably proteins.

Claim 9

A process according to any one of claims 1 to 7, wherein the membrane compartment contains dilute buffer solutions and the electrode compart­ments highly concentrated electrolyte solutions.

Claim 10

A process according to claim 8, wherein the buffers used in the diluted buffer solutions are monovalent species of the type HA/A⁻ and KB⁺/B with a high buffer capacity per unit conductivity.

Claim 11

A process according to claim 1, wherein the recycle rate through each cell is more than 100 times greater than the permeation rate.

Claim 12

A forced-flow membrane electrophoresis apparatus for carrying out the continuous separation process according to claim 1 which comprises - electrode compartments (6, 7) spaced from each other and having the anode (4) and cathode (5) located therein, - a membrane compartment of a feed (2) and a permeate cell (3) being divided by a micro- or ultrafiltration membrane permeable to elec­trically charged macromolecular compounds, being located between said anode and cathode compartment and partitioned therefrom with ion-exchange membranes (8, 9), impermeable to said macromolecular compounds, and having no or very low permeability to salts, - an inlet (11) for supplying the aqueous solutions (10) containing the electrically charged macromolecular compounds into the feed cell (2) of the membrane compartment, - an inlet (12) for supplying buffer solution to sweep through the permeate cell (3) of the membrane compartment, - inlets and outlets (13, 14) for electrolyte and washing liquid circu­lation which are pumped through the anode (6) and cathode compartment (7), - outlets (15, 16) for withdrawing (a) separated solution(s) of the macro­molecular compounds and by-products, - means for controlling and maintaining the permeate flow at a pre­determined rate (23), and - other means, including pumps for supplying and circulating the aqueous solutions, storage tanks and monitoring devices (17-22, 24-34).

Claim 13

A forced-flow membrane electrophoresis apparatus according to claim 11 which comprises - an anode compartment (6) with an anode (4) and a cathode compartment (7) with a cathode (5) located therein, - a membrane compartment divided by a micro- or ultrafiltration mem­brane permeable to electrically charged macromolecular compounds (1) into a high pressure (feed) cell (2) and a low pressure (permeate) cell (3), being located between said anode and cathode compartments and partitioned therefrom by an anion-exchange membrane (8) which is bordering the anode compartment (6) and a cation-exchange membrane (9) which is bordering the cathode compartment (7), both the ion-exchange membranes are impermeable to said macromolecular compounds and having no or very low permeability to salts, - an inlet (11) for supplying the aqueous solutions (10) containing the electrically charged macromolecular compounds into the feed cell (2) of the membrane compartment, - an inlet (12) for supplying buffer solution to sweep through the permeate cell of the membrane compartment, - inlets (13) and outlets (14) for electrolyte and washing liquid circu­lation which are pumped through the anode (6) and cathode compart­ment (7) by pump (33) from tank (34), - outlets (15, 16) for the components being retained on the feed side and on the permeate side of the membrane (1), respectively, - means for regulation of the feed solution (10) by use of a buffer system with or without external pH-control (17-22), - a pump (23) for controlling the permeate flow at a predetermined rate, - a back pressure valve (24) to control the flux through the membrane, optionally by an automatic controller (25), - a permeate reservoir (26), equipped with a pH-electrode (27) connected to a pH-controller (28) which effects the controlled addition of buffer solution from tank (29) through pump (30), the reservoir optionally being combined with a weigh scale (31), and - a monitoring device (32), comprising a microcomputer and a monitor.

Claim 14

A process according to claim 1 for the continuous separation of an electrically charged macromolecular compound by forced-flow membrane electrophoresis, which comprises the simultaneous purification of said macromolecular compound from an aqueous mixture of other charged macromolecular compounds with pI-values both greater and less than that of the macromolecular compound to be separated, in a forced-flow membrane electrophoresis apparatus of two stacks, piggy-backed one on the top of the other, comprising - a membrane compartment of a feed cell (2) flanked by an anodic buffer cell (6a) on one side and a cathodic buffer cell (7a) on the other, both being partitioned from the feed cell (2) by micro- or ultra­filtration membranes (1) whic are permeable to electrically charged macromolecular compounds, and - electrode compartments (6, 7) spaced from each other and having the anode (4) and cathode (5) located therein, being partitioned from the anodic and cathodic buffer cells (6a, 7a) by an anion- and cation-­exchange membrane (8, 9), respectively, which are impermeable to said electrically charged macromolecular compounds, and having no or very low permeability to salts.

Claim 15

A process according to claim 13, which comprises maintaining the pH of the aqueous mixture of charged macromolecules at a value equal to the pI of the macromolecule to be separated and recycled in the feed cell (2), keeping the pH of the buffer solution in the anodic buffer cell (6a) slightly lower than the pI of the macromolecule to be separated, and the pH of the buffer solution in the cathodic cell (7a) slightly higher than said pI, applying an electric field to case migration of positively charged macromolecules from the feed cell (2) into the cathodic buffer cell (7a) and of negatively charged macromolecules from the feed cell (2) into the anodic buffer cell (6a), and retaining the macromolecules to be separated in the feed cell (2).

Claim 16

A forced-flow membrane electrophoresis apparatus of two stacks, piggy-backed one on the top of the other for carrying out the conti­nuous separation process according to claim 13, which comprises - a membrane compartment of a feed cell (2) flanked by an anodic buffer cell (6a) on one side and a cathodic buffer cell (7a) on the other, both being partitioned from the feed cell (2) by micro- or ultra­filtration membranes (1) which are permeable to electrically charged macromolecular compounds, and - electrode compartments (6, 7) spaced from each other and having the anode (4) and cathode (5) located therein, being partitioned from the anodic and cathodic buffer cells (6a, 7a) by an anion- and cation-­exchange membrane (8, 9) respectively, which are impermeable to said electrically charged macromolecular compounds, and having no or very low permeability to salts.

Claim 17

An electrophoresis apparatus according to any one of claims 11, 12 and 15, wherein the membrane compartment contains turbulence pro­moting grids.