Separation of Biomolecules and/or Demineralization of Solutions Containing Biomolecules and Ions by Electrochemical Ion Exchange

Abstract

Methods and systems are provided for separation and/or purification of biomolecule(s) from a solution and for demineralization of a solution containing biomolecule(s) and ion(s). The methods and systems described herein include one or more ion exchange membrane(s), (e.g., ion exchange membrane(s) capable of water splitting), in an electrochemical cell, from which bound biomolecule(s) or ion(s) may be recovered from the membrane and separated from other components present in the solution by reversing the polarity of the electrodes. Using methods and systems described herein, biomolecules (e.g., whey proteins and lactose), and ions (e.g., milk salts), are able to be separated and recovered in their native forms with high biological value as premium food grade products. Methods and systems described herein offer a significant advantage over traditional processes used (e.g., in the food and beverage industry), for cost effective and chemical-free processing/extraction of valuable products from complex solutions such as whey.

Claims

1. A method for separation of a biomolecule from a solution, comprising: applying a voltage to an electrochemical cell that comprises: (a) a housing comprising an inlet and an outlet; (b) first and second electrodes: (c) at least one water-splitting ion exchange membrane between the first and second electrodes, wherein the water-splitting membrane comprises: (i) a cation exchange layer facing the first electrode and comprising a bound cation; and (ii) an anion exchange layer facing the second electrode and comprising a bound anion; (d) a solution that comprises the biomolecule, wherein the solution flows through a continuous channel from the inlet to the outlet of the cell and contacts the first and second electrodes and the cation and anion exchange layers of the water-splitting membrane, wherein the biomolecule binds to the cation exchange layer or the anion exchange layer on the water splitting membrane or flows through the electrochemical cell as an unbound molecule.

2.-3. (canceled)

4. A method according to claim 1, wherein the biomolecule is a charged cationic and/or anionic polypeptide, an uncharged polypeptide, a tagged polypeptide, a polynucleotide, a mono-, di-, or oligosaccharide, a sugar alcohol, or a glycol.

5.-15. (canceled)

16. A method according to claim 15, wherein the solution that comprises the biomolecule comprises whey or a derivative of whey selected from a retentate and a permeate of whey ultrafiltration, microfiltration, and/or nanofiltration.

17.-18. (canceled)

19. A method according to claim 2, wherein the biomolecule is charged in the solution that comprises the biomolecule, and wherein the charged biomolecule replaces either a bound cation on the cation exchange layer or a bound anion on the anion exchange layer of the water-splitting membrane, said method further comprising reversing the polarity of the first and second electrodes, wherein the bound charged biomolecule is expelled from the membrane, and wherein the charged biomolecule exits through an outlet of the electrochemical cell in a regeneration solution.

20.-33. (canceled)

34. A method according to claim 18, wherein the biomolecule does not foul the membrane as the solution flows through the electrochemical cell and wherein at least a portion of the recovered biomolecule remains in solubilized or precipitated form.

35.-38. (canceled)

39. A method for demineralization of a solution that comprises at least one biomolecule and at least one ion, comprising: applying a voltage to a first electrochemical cell that comprises: (a) a first housing comprising an inlet and an outlet; (b) first and second electrodes: (c) at least one first water-splitting ion exchange membrane between the first and second electrodes, wherein the water-splitting membrane comprises: (i) a cation exchange layer facing the first electrode and comprising a bound cation; and (ii) an anion exchange layer facing the second electrode and comprising a bound anion; (d) a solution that comprises at least one biomolecule and at least one ion, wherein the solution flows through a continuous channel from the inlet to the outlet of the cell and contacts the first and second electrodes and the cation and anion exchange layers of the water-splitting membrane, wherein at least one ion binds to the cation exchange layer or the anion exchange layer on the first water splitting membrane, thereby causing demineralization of the solution by removing or reducing the concentration of at least one ion.

40.-41. (canceled)

42. A method according to claim 39, wherein the concentration, form, and/or biological activity of the biomolecule is substantially unchanged in the solution that exits the electrochemical cell with respect to the solution that entered the cell.

43.-56. (canceled)

57. A method according to claim 39, further comprising reversing the polarity of the first and second electrodes, wherein the bound ion is expelled from the membrane, and wherein the ion exits through an outlet of the electrochemical cell in a regeneration solution.

58. (canceled)

59. A method according to claim 57, wherein the solution that comprises at least one biomolecule and at least one ion is whey or a derivative thereof that comprises cationic and anionic ions, and wherein the cationic and anionic ions are recovered as milk minerals in substantially equivalent ratios and at higher concentrations than in the solution that entered the electrochemical cell.

60.-67. (canceled)

68. A lactose powder, prepared according to the method of claim 39, wherein the solution that comprises at least one biomolecule and at least one ion is a permeate of whey filtration, and wherein the lactose powder is prepared from the solution that flows through the electrochemical cell as a demineralized solution.

69. A milk mineral powder comprising cationic and anionic milk minerals separated from other components in whey according to the method of claim 59.

70. A demineralized powder of whey or a derivative thereof, comprising charged and/or uncharged polypeptides, lactose, and/or fat that have been separated from milk minerals of whey according to the method of claim 39, wherein the solution that comprises at least one biomolecule and at least one ion comprises whey or a derivative thereof, wherein the whey or derivative thereof further comprises fat, lactose, and/or charged and/or uncharged polypeptides, wherein substantially all of the fat and lactose and at least a portion of the charged and/or uncharged polypeptides flow through the electrochemical cell as unbound molecules, and wherein the flow from the electrochemical cell is a demineralized solution comprising substantially all of the fat, lactose, and/or at least a portion of charged and/or uncharged polypeptides of whey.

71.-81. (canceled)

82. A method according to claim 39, wherein the solution that comprises at least one biomolecule and at least one ion comprises a beverage.

83.-87. (canceled)

88. A method according to claim 82, wherein the solution exits the electrochemical cell as a demineralized beverage, wherein the demineralized beverage comprises a reduction in at least one ion comprising chloride, nitrate, phosphate, malate, tartrate, sulfite, sulfate, sodium, potassium, magnesium, calcium, manganese, cadmium, lead, copper, iron and/or mercury.

89. (canceled)

90. A method according to claim 39, wherein the voltage applied to an electrochemical cell controls the degree of demineralization.

91.-99. (canceled)

100. A method according to claim 1, wherein the biomolecule has a binding affinity towards a specific ion, and wherein the biomolecule binds to and exchanges with that specific ion on the cation exchange layer or the anion exchange layer of the water-splitting ion exchange membrane.

101.-115. (canceled)

116. A system for demineralization of a solution that comprises at least one biomolecule and at least one ion, comprising: (a) a housing comprising an inlet and an outlet; (b) first and second electrodes; (c) at least one water-splitting ion-exchange membrane between the first and second electrodes, wherein the water-splitting membrane comprises: (i) a cation exchange layer facing the first and second electrode and comprising a bound cation; and (ii) an anion exchange layer facing the second electrode and comprising a bound anion; (d) a solution that comprises the at least one biomolecule and at least one ion, wherein the system is configured such that the solution flows through a continuous channel from the inlet to the outlet of the cell and contacts the first and second electrodes and the cation and anion exchange layers of the water-splitting membrane, and wherein the system is configured such that when a voltage is applied to the electrochemical cell, the at least one ion binds to the cation exchange layer or the anion exchange layer on the water-splitting membrane, thereby causing demineralization of the solution by removing or reducing the concentration of at least one ion, and wherein the system is configured such that when the polarity of the first and second electrodes is reversed, the at least one bound ion is expelled from the membrane and exits through the outlet of the electrochemical cell in a regeneration solution.

117-137. (canceled)

138. A method according to claim 1, wherein at least one water splitting ion exchange membrane comprises any combination of a strong or weak acid cation membrane layer abutting a strong or weak base anion membrane layer.

139.-144. (canceled)

145. A system according to claim 116, wherein the electrochemical cell comprises two or more water-splitting ion exchange membranes, wherein the water-splitting membranes comprise the same or different ion exchange functional groups and/or the same or different ion exchange capacities.

146. A method according to claim 39, wherein at least one water splitting ion exchange membrane comprises any combination of a strong or weak acid cation membrane layer abutting a strong or weak base anion membrane layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG. 1 shows schematically a method for demineralization of a solution that contains one or more biomolecule(s) and one or more ion(s) such as whey (or derivative of whey such as but not limited to, ultrafiltration permeate or ultrafiltration retentate) as described in Example 1.

[0073] FIG. 2 shows schematically a method for separating charged biomolecules (e.g., proteins) from a demineralized solution containing charged and uncharged biomolecules (e.g., fat and lactose in whey (e.g., demineralized whey), or a derivative of whey (e.g., demineralized derivative of whey) such as but not limited to, ultrafiltration retentate) as described in Example 2.

[0074] FIG. 3 shows schematically a method for controlled removal of ions from a solution containing charged and uncharged biomolecules (e.g., wine) as described in Example 3.

[0075] FIG. 4 shows schematically a method for separation of a specific protein from a demineralized solution that contains one or more biomolecule(s) such as demineralized whey (or derivative of whey such as but not limited to ultrafiltration retentate) using targeted binding as described in Example 4.

DETAILED DESCRIPTION

[0076] Methods and systems for separation and/or purification of biomolecules from a solution are provided. Methods and systems for separation and/or purification of biomolecules and ions from a solution are also provided. Methods and systems for demineralization of a solution that contains one or more biomolecule(s) and one or more ion(s) are also provided.

[0077] Exemplary, but non-limiting embodiments are depicted below:

[0078] In some embodiments, a solution which contains one or more biomolecule(s) and one or more ion(s) can be demineralized by controlling operational parameters such as, but not limited to, flow rate, membrane composition (cation and/or anion resins used for extrusion of the water-splitting membrane) and pH. Although not wishing to be bound by theory, during demineralization, ions may have a competitive advantage to preferentially bind to the membrane due to their smaller size, abundance and/or strong ionic binding or electrostatic attraction compared to the binding strength of the remaining charged biomolecules in solution (e.g., proteins). This competitive ionic binding may lead to displacement of any bound proteins in preference for smaller and highly charged ions present in the solution. In some embodiments, the demineralized solution containing one or more biomolecule(s) can further be subsequently processed by controlling operational parameters such as, but not limited to, flow rate, membrane composition (cation and/or anion resins used for extrusion of the water-splitting membrane) and pH, to separate charged biomolecule(s) from uncharged biomolecule(s). Methods and systems are described for demineralization and/or separation of biomolecules from biomolecule-containing solutions.

[0079] The methods and systems disclosed herein include an electrochemical cell that contains at least one bipolar ion exchange membrane that is capable of water splitting and is situated between two electrodes, and to which both cationic and anionic biomolecules and/or ions bind when an electrical voltage is applied to the cell. Bound biomolecules and/or bound ions may be removed from the membrane by reversing the polarity of the electrodes. In some embodiments, both cationic and anionic biomolecules and/or ions are recovered simultaneously from a solution using the methods and systems described herein. In some embodiments, polypeptides may be recovered in native (e.g., non-denatured) configurations. In some embodiments, a specific cationic or anionic biomolecule (e.g., a polypeptide) is bound and purified from a solution using the methods and systems described herein. In some embodiments, both cationic and anionic ions are recovered simultaneously from a solution that contains one or more biomolecule(s) and one or more ion(s) using the methods and systems described herein. In some embodiments, the ions may be recovered in substantially their naturally occurring states and ratios (e.g., milk mineral salt ratios found in whey).

Definitions

[0080] Numeric ranges provided herein are inclusive of the numbers defining the range.

[0081] Unless otherwise indicated, nucleic acids are written left to right in 5 to 3 orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0082] A, an and the include plural references unless the context clearly dictates otherwise.

[0083] Biomolecule refers to a molecule that is produced by a living system (e.g., animal plant, fungus, bacterium, virus), including but not limited to polypeptides, polynucleotides, mono- and polysaccharides, lipids, amino acids, nucleotides, vitamins, primary metabolites, secondary metabolites, and natural products. In one embodiment, biomolecule refers to a recombinantly expressed polypeptide, e.g., a polypeptide produced from a recombinant construct with or without a tag, such as a his tag or other tag. Ions refer to an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving the atom a net positive or negative electrical charge. In chemical terms, if a neutral atom loses one or more electrons, it has a net positive charge and is known as a cation. If an atom gains electrons, it has a net negative charge and is known as an anion. An ion consisting of a single atom is an atomic or monatomic ion; if it consists of two or more atoms, it is a molecular or polyatomic ion.

[0084] Ion refers to an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving the atom or molecule a net positive or negative electrical charge. In chemical terms, if a neutral atom or molecule loses one or more electrons, it has a net positive charge and is known as a cation. If a neutral atom or molecule gains electrons, it has a net negative charge and is known as an anion. An ion consisting of a single atom is an atomic or monatomic ion; if it consists of two or more atoms, it is a molecular or polyatomic ion.

[0085] Mineral refers to naturally-occurring inorganic elements, typically having a crystalline structure.

[0086] Milk minerals refers to minerals or milk salts, which include, but are not limited to, cations and anions that are in milk, for example, calcium, phosphate, magnesium, sodium, potassium, citrate, and chlorine, typically at concentration of about 5 to about 40 mM, and Vitamins A, B6, B12, C, D, K, E, thiamine, niacin, biotin, riboflavin, folates, and pantothenic acid, classified as milk trace elements or minerals.

[0087] Salts refer to ionic compounds that can result from the neutralization reaction of an acid and a base. They are composed of related numbers of cations (positively charged ions) and anions (negative ions) so that the product is electrically neutral (without a net charge)

[0088] An ion exchange membrane is a structure that includes a cation exchange surface and/or an anion exchange surface. In one embodiment, the ion exchange membrane contains a cation exchange surface and an anion exchange surface in combination. In some embodiments, the ion exchange membrane, also termed bipolar, double, or laminar, contains anion and cation exchange surfaces on opposite sides of the membrane, e.g., abutting anion and cation exchange surfaces on opposite sides of the membrane. In one embodiment, the ion exchange membrane is capable of water splitting, such that in a sufficiently high electric field produced by application of a voltage to two electrodes, water is dissociated or split into component ions H.sup.+ and OH.sup.?. In some embodiments, the water-splitting membrane contains anion and cation exchange surfaces on opposite sides of the membrane, e.g., abutting anion and cation exchange surfaces on opposite sides of the membrane, and the dissociation of water occurs most efficiently at the boundary between the cation and anion exchange surfaces, with the resultant H.sup.+ and OH.sup.? ions migrating through the ion exchange surfaces in the direction of the electrode having an opposite polarity (e.g., H.sup.+ migrates toward the negative electrode and OH.sup.? migrates toward the positive electrode). In some embodiments, an anion or cation exchange surface may be an anion or cation exchange layer on the ion exchange (e.g., water-splitting) membrane. Cation exchange refers to a material that exchanges positively-charged ions or molecules (cations) on the surface of an immobile material for cations in a solution, e.g., with no permanent change to the immobile exchange material. For example, a cation exchange material may contain an acidic group (e.g., COOM, SO.sub.3M, PO.sub.3M, or C.sub.6H.sub.4OM, where M is a cation (e.g., hydrogen, sodium calcium, or copper ion)). In some embodiments, an ion exchange membrane (e.g., a membrane capable of water splitting) as described herein includes a strong acid cation exchange surface (e.g., a strong acid cation exchange layer on the membrane). Strong acids are highly ionized in both the acid (e.g., RSO.sub.3H) and salt (e.g., RSO.sub.3Na) forms. The hydrogen and sodium forms of strong acids are highly dissociated and the exchangeable Na.sup.+ and H.sup.+ ions are readily available for exchange over the entire pH range. A nonlimiting example of a strong acid cation exchange group is a sulfonic acid group on the surface of the ion-exchange membrane (SO.sub.3M). In some embodiments, a cation exchange group (e.g., a sulfonic acid group) may interact with basic groups in a biomolecule, such as histidine, lysine and arginine side chains of a net-positively charged polypeptide. In order to keep the basic groups (e.g., basic side chains of a polypeptide) protonated, the mobile phase may be buffered to maintain the pH below 6 or 7. At higher pH, the basic groups may deprotonate, decreasing retention. In some embodiments, an ion exchange membrane (e.g., a membrane capable of water splitting) as described herein includes a weak acid cation exchange surface (e.g., a weak acid cation exchange layer on the membrane). A nonlimiting example of a weak acid cation exchange group is a carboxylic acid (COOH). The degree of dissociation of a weak acid is strongly influenced by the solution pH.

[0089] Anion exchange refers to a material that exchanges negatively-charged ions or molecules (anions) on the surface of an immobile material for anions in a solution, e.g., with no permanent change to the immobile exchange material. For example, an anion exchange material may contain a basic group (e.g., NR.sub.3A, NR.sub.2HA, PR.sub.3A, SR.sub.2A, or C.sub.5H.sub.5NHA, where A is an anion (e.g., hydroxide, bicarbonate, or sulfonate)). In some embodiments, an ion exchange membrane (e.g., a membrane capable of water-splitting) as described herein includes a strong base anion exchange surface (e.g., a strong base anion exchange layer on the membrane). A nonlimiting example of a strong base anion exchange group is a quaternary ammonium group, (NR.sub.3A). In some embodiments, an anion exchange group (e.g., a quaternary ammonium group) may interact with acidic groups in a biomolecule, such as aspartic acid or glutamic acid side chains of a net-negatively charged polypeptide. In order to keep the acidic groups (e.g., acidic side chains of a polypeptide) deprotonated, the mobile phase may be buffered to maintain the pH above 4.4. At lower pH, the acidic groups may protonate, decreasing retention. Strong bases are readily available for exchange over the entire pH range. In some embodiments, an ion exchange membrane (e.g., a membrane capable of water-splitting) as described herein, includes a weak base anion exchange surface (e.g., a weak base anion exchange layer on the membrane). The degree of ionization of a weak base is strongly influenced by solution pH.

[0090] Ionic Binding refers to formation of a type of chemical bond that involves the electrostatic attraction between oppositely charged ions. These ions are atoms that have lost one or more electrons (cations) or atoms that have gained one or more electrons (anions). Electrostatic attraction between ions of opposite charge (cations and anions) may result in an ionic bond.

[0091] As used herein, polypeptide refers to a composition comprised of amino acids and recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms polypeptide and protein are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0092] As used herein, the term polynucleotide refers to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides which encode a particular amino acid sequence. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2-O-Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin or fluorescent labels. The term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms polynucleotide, nucleic acid, and oligonucleotide are used herein interchangeably. Polynucleotides may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR.sub.2 (amidate), P(O)R, P(O)OR, CO or CH.sub.2 (formacetal), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (O) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions.

[0093] Whey refers to the liquid solution remaining after milk has been curdled (coagulated protein or casein), typically by, but not limited to, addition of enzymes (e.g., rennet), acids or salts. In its raw form, whey contains proteins, fat, cholesterol, vitamins, minerals, and lactose (milk sugar). Whey herein may also refer to a derivative of whey, such as pre-processed whey, including, but not limited to, ultrafiltration retentate or permeate.

[0094] Deproteinized whey refers to the liquid solution remaining after some portion of the whey proteins have been removed/separated from whey (using a process such as, but not limited to ultrafiltration or ion exchange). This solution includes carbohydrates (e.g., lactose (milk sugar)), lipids, and milk minerals (ions), and may also contain a small portion of whey proteins.

[0095] Lactose refers to the disaccharide carbohydrate formed by the condensation of glucose and galactose to give a ?-(1.fwdarw.4) linked product, which is present in milk and whey.

[0096] Demineralization refers to the process of removing some portion of the cationic and/or anionic monovalent and/or multivalent minerals (ions) and/or mineral salts from a solution. Demineralization grades are determined by the percentage of total ions removed from a solution (e.g., D50 refers to a 50% reduction of ions compared to the pretreated source solution).

[0097] Ultra/Micro/Nano-filtration refers to membrane filtration processes in which forces such as pressure or concentration gradients leads to the separation of molecules through a semipermeable membrane primarily based on size. Suspended solids and solutes of higher molecular weight than the filter cut-off are retained in the retentate, while water and lower molecular weight solutes pass through the membrane in the permeate.

[0098] Retentate refers to suspended solids in a filtration process that do not cross the membrane due to their size being larger than the filter cut-off. The pore size of the membrane determines which molecules are retained and which molecules pass through.

[0099] Permeate refers to suspended solids in a filtration process that cross the membrane due to their size being smaller than the filter cut-off. The pore size of the membrane determines which molecules are retained and which molecules pass through as permeate.

[0100] Conductivity refers to specific conductance of an electrolyte solution as a measure of its ability to conduct electricity. The SI unit of conductivity is Siemens per meter. A conductivity measurement provides a fast, inexpensive and reliable way of measuring the ionic content in a solution that contains one or more biomolecule(s) and one or more ion(s). Conductivity is linked directly to the total dissolved solids (T.D.S.) present in a solution. For reference, typical drinking water lies within the range of 5-50 ?S/cm, while sea water is about 5 mS/cm (i.e., sea water's conductivity is one million times higher than that of deionized water). The typical conversion of conductivity to a total dissolved solids (TDS) value is done assuming that the solid in solution is sodium chloride, and therefore 1 ?S/cm is then assumed to be an equivalent of about 0.6 mg of NaCl per kg of water.

[0101] The term derived from encompasses the terms originated from, obtained from, obtainable from, isolated from, and created from, and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to the another specified material.

[0102] A solution refers to a homogenous or substantially homogeneous mixture in which the particles of one or more substance(s) (the solute(s)), are distributed uniformly or substantially uniformly throughout another substance (the solvent). A solution as described herein can be at any pH (e.g., 1.0-14.0).

[0103] A biological solution refers to a solution that is derived from a biological source or origin.

[0104] A stabilized solution (including, but not limited to, a stabilized beverage, such as stabilized wine), refers to a solution with decreased volatility, for example, as a result of removing particles and/or ions that may cause unwanted chemical changes in the solution).

[0105] A his tag refers to a polyhistidine sequence, for example, six histidine (His) residues. In some embodiments, a his tag may be located at the N- or C-terminus of a protein, encoded by repetitive histidine codons (e.g., CAT or CAC) right after the START or before the STOP codon.

[0106] Flow rate refers to the volume of fluid that flows through a measured distance over a specific unit of time (e.g., liters per minute).

[0107] Polarity refers to electrical polarity (positive and negative), which is present in electrical circuits. Electrons flow from the negative pole to the positive pole. In a direct current (DC) circuit, one pole is negative, the other pole is positive, and electrons flow in one direction only. In an alternating current (AC) circuit, flow of electric charge periodically reverses direction as the two poles alternate between negative and positive.

[0108] Reverse polarity refers to switching the negative electrode (in a given electrochemical cell as described herein), to positive and switching the positive electrode to negative. By doing so, the electrostatic bond between a cation exchange layer and an attached cation or an anion exchange layer and an attached anion can be broken or displaced.

[0109] Denaturation refers to a process in which a biologically active molecule such as a protein or nucleic acid loses its native state or configuration by disruptions in its quaternary, tertiary and/or secondary structures. Denaturation may occur due to the application of an external stress or compound such as exposure to a strong acid or base, a salt (e.g., a concentrated inorganic salt), an organic solvent (e.g., alcohol or chloroform), radiation or heat, causing a range of issues from loss of solubility to protein aggregation and/or precipitation. In some cases, denaturation is reversible (e.g., a protein can fold back into its native state when the denaturing influence is removed). This process is referred to as renaturation.

[0110] Precipitation refers to the formation of a solid substance within a solution. For example, a substance may precipitate from solution due to a change in the composition of the solvent, which diminishes the solubility of the substance. Precipitation of proteins may be effected by a change in the pH of a biological solution. Precipitation of proteins due to change in a solution's pH may be governed by the protein's isoelectric point (pI). At a solution pH above the pI of a protein, it is negatively charged; at a solution pH below the pI of a protein, it is positively charged, and at a pH equal to the pI of the protein, the net primary charge of a protein becomes zero, at which point the protein may denature and precipitate out of solution.

[0111] Biologically active refers to a biomolecule, such as a protein, that is capable of performing the function that it typically performs in a biological system (e.g., including but not limited to structural, enzymatic, or regulatory functions). The native state of a biomolecule, such as a protein, is its properly folded and/or assembled form, which is operative and functional. For example, if a protein unfolds from its native configuration or becomes compromised in some way (e.g., denatured), it may no longer be able to perform its primary function and may be considered to have lost its biological or native activity.

[0112] Membrane refers to a permeation-selective barrier (e.g., porous, non-porous, symmetric, neutral or charged), which may effect separation under the influence of a driving force (e.g., pressure difference, concentration difference, electrical potential difference). Components can either pass through the barrier (permeate) or be retained (rententate), allowing selective separation of components based on some criteria (e.g., size or charge).

Electrochemical Cell

[0113] An electrochemical cell is provided for separation and/or purification of biomolecules as disclosed herein. In one embodiment, the electrochemical cell contains:

(a) a housing that includes at least one inlet and at least one outlet;
(b) a first electrode and a second electrode;
(c) at least one water-splitting ion exchange membrane capable of water splitting, between the first and second electrodes, with a cation exchange surface facing the first electrode and an anion exchange surface facing the second electrode;
(d) at least one channel from the inlet to the outlet, configured for a solution to flow from the inlet to the outlet of the cell, and configured such that the solution flows in contact with at least one of the cation and anion exchange surfaces of the water-splitting membrane.

[0114] In some embodiments, the water-splitting ion exchange membrane(s) is (are) bipolar, with the cation exchange surface on the opposite side of the membrane from the anion exchange surface.

[0115] In some embodiments, the solution channel is continuous or substantially continuous from the inlet to the outlet of the electrochemical cell. In some embodiments, the channel is configured such that the solution flowing through the channel is in contact with both the cation exchange surface and the anion exchange surface of the membrane.

[0116] When an electrical voltage is applied to the electrodes, positively-charged molecules in a solution that is flowing through the channel, bind to the cation exchange surface of the membrane and negatively-charged molecules in the solution bind to the anion exchange surface of the membrane. Uncharged molecules may flow through the outlet of the cell without binding to either the cation exchange surface or the anion exchange surface of the membrane.

[0117] In some embodiments, the cell includes a plurality of water-splitting ion exchange membranes, configured such that the cation exchange surface of each membrane faces the first electrode and the anion exchange surface of each membrane faces the second electrode.

[0118] In some embodiments, the cell includes a plurality of channels that flow from a single inlet to a single outlet in the cell, or that flow from a plurality of inlets to a plurality of outlets, with each channel flowing from an inlet to an outlet of the cell and configured such that the solution flows in contact with at least one of the cation and anion exchange surfaces of one of a plurality of water-splitting membranes. In some embodiments, each channel is configured such that the solution flows in contact with both the cation exchange and anion exchange surfaces of one of a plurality of water-splitting membranes.

[0119] A flow controller, such as a pump, and/or an external flow sensor, may be used to control the flow rate of the solution from a solution source through the channel and into a treated solution container. In embodiments in which the electrochemical cell includes a plurality of solution channels, the treated solution from each channel may flow into the same or separate treated solution containers.

[0120] The electrodes may be powered by a voltage supply that supplies a voltage across the electrodes (e.g., anode and cathode electrodes). Exemplary, nonlimiting electrical parameters for powering the cell include 100-120 vac (volts alternating current), 50/60 Hz (cycles per second frequency), and 6 Amps (current).

[0121] Electrodes may be constructed of any suitable material for use in the methods described herein for separation and/or purification of biomolecules. For example, electrodes may be fabricated from an electrically-conductive material, such as a metal or metal-containing material, which is resistant to corrosion in a biomolecule-containing solution from which separation and/or purification of at least one biomolecule is desired. Non-limiting examples of suitable materials for construction of electrodes include copper, aluminum, or steel cores, which may be coated with a corrosion-resistant material, such as platinum, titanium, or niobium. The shape of the electrodes may be adapted to the design of the electrochemical cell and the conductivity of the biomolecule-containing solutions which will flow through the cell. Desirably, the electrodes should provide a uniform voltage across the surfaces of the water-splitting ion exchange membrane(s).

[0122] In some embodiments, a single solution stream may be introduced into one inlet and exit via one outlet. This is in contrast to electrodialysis systems that contain monopolar ion exchange membranes or water-splitting membranes and have separate waste and product solution streams. Electrodialysis systems in continuous operation require two separate solution streams, a product stream from which ions are removed and a waste stream into which the ions are deposited. The separation of the two solution streams is often provided by monopolar ion exchange membranes. In some embodiments, the electrochemical cell does not contain a monopolar ion exchange membrane, e.g., does not contain a monopolar ion exchange membrane between adjacent water-splitting membranes. The batch mode operation of the electrochemical cell described herein eliminates the need for monopolar ion exchange membranes to separate solution streams.

[0123] In some embodiments, a plurality of bipolar water-splitting membranes, with cation and anion exchange surfaces on opposite sides of the membranes, is configured with their cation exchange surfaces facing the first electrode and their anion exchange surfaces facing the second electrode. Thus, all water-splitting membranes within a cell may be operating in either production mode (e.g., deionization and binding of charged molecules) or regeneration mode. With all cation and anion exchange surfaces of the membranes facing the first and second electrodes, respectively, the electric field will have a direction which is transverse or normal, or substantially transverse or normal, to the surfaces of the water-splitting membranes, resulting in a water-splitting reaction that is perpendicular to the surfaces of the membranes and providing the shortest pathway through the membrane to increase the efficiency of ion exchange. Alternatively, ions may be drawn in from solution or into the ion exchange surfaces of the water-splitting membranes normal or substantially normal to their surfaces. This may provide full utilization of the ion exchange materials by preventing divergent electric fields or current fluxes that would bypass portions of the ion exchange surfaces. A uniform electric field or current flux that is directly transverse to the ion exchange surfaces of the membranes provides the most uniform current distribution and ion flow pattern through the water-splitting membranes.

[0124] In some embodiments, a plurality of water-splitting ion exchange membranes in an electrochemical cell include the same or different functional groups, and/or may include the same or different ion exchange capacities.

[0125] In one embodiment, the electrochemical cell contains one or more water-splitting membranes in a spiral wrapped arrangement. For example, at least one water-splitting membrane may be wrapped or rolled with an adjacent separator or spacer within a housing that is in a circular or substantially circular shape. In one embodiment, one of the electrodes (e.g., first or second electrode) may be configured in the center of the spirally-wrapped membrane(s) and the other electrode may be configured at the outside of the membrane spiral. A biomolecule-containing solution may enter the cell via an inlet through the outer wall of the housing and may flow through solution channels that simultaneously contact the anion exchange and cation exchange surfaces of the water-splitting ion exchange membrane(s), exiting through one or more outlet(s), for example, an outlet located at the top of the cell. In one embodiments, the solution channel(s) are continuous from the inlet to the outlet of the cell.

[0126] Nonlimiting examples of electrochemical cells and components thereof that may be suitable for separation and/or purification of biomolecules as disclosed herein are provided in U.S. Pat. Nos. 5,788,826, 7,344,629, 7,780,833, 7,959,780, 8,293,085, and U.S. Patent Application Nos. 2006/0138997, 2007/0108056, and 2007/0175766, which are incorporated by reference herein in their entireties. Other configurations and materials that are suitable for the electrochemical cell are contemplated within the scope of the methods and systems disclosed herein and will be apparent to those of skill in the art.

Water-Splitting Ion Exchange Membrane

[0127] An electrochemical cell as described herein includes one or more (e.g., one or a plurality of) bipolar ion exchange membrane(s), such as ion exchange membrane(s) capable of water splitting. A bipolar ion exchange membrane (e.g., water-splitting membrane) herein includes a cation-exchange surface (e.g., a cation-exchange layer) that includes a cation-exchange material (e.g., acidic cation-exchange group(s)) and an anion-exchange surface (e.g., an anion-exchange layer) that includes an anion-exchange material (e.g., anion-exchange group(s)). In some embodiments, a bipolar ion exchange membrane (e.g., water-splitting membrane) herein includes a cation-exchange surface and an anion-exchange surface on opposing sides of the membrane, for example, abutting cation and anion exchange surfaces on opposing sides of the membrane. In some embodiments, the cation exchange layer is constructed with a strong acid resin in combination with an anion layer constructed with a weak or strong base resin. In some embodiments, the cation exchange layer is constructed with a weak acid resin in combination with an anion layer constructed with a weak or strong base resin.

[0128] A water-splitting membrane herein may be any structure that includes a cation-exchange surface and an anion-exchange surface in combination such that under a sufficiently high electric field, e.g., produced by application of a voltage to two electrodes, water is dissociated into its component ions H.sup.+ and OH.sup.? in the membrane. Hydronium ions (H.sub.3O.sup.+ may also be created, e.g., during a process of self-ionization. Although not wishing to be bound by theory, the dissociation of water may occur most efficiently at the boundary between the cation and anion exchange surfaces in the membrane, or in the volume between them. The resulting H.sup.+ and OH.sup.? ions may migrate through an ion exchange layer in the direction of the electrode having an opposite polarity. For example, H.sup.+ will migrate toward the negative electrode (cathode) and OH.sup.? will migrate toward the positive electrode (anode).

[0129] In some embodiments, a water-splitting membrane herein contains abutting cation and anion exchange surfaces. For example, cation and anion exchange layers may be secured or bonded to each other to provide a water-splitting membrane with a unitary laminate structure. The cation and anion exchange layers may be in physical contact without a bond securing them together, or the water-splitting membrane may include a non-ionic middle layer, such as, but not limited to, a water-swollen polymer layer, a porous layer, or a solution-containing layer.

[0130] A cation-exchange surface in a water-splitting membrane herein may include one or more acidic functional groups capable of exchanging cations. Nonlimiting examples of such acidic functional groups include COOM, SO.sub.3M, PO.sub.3M, and C.sub.6H.sub.4OM, where M is a cation (e.g., hydrogen, sodium calcium or copper ion). Cation exchange materials may also include neutral groups or ligands that bind cations through coordinate rather than electrostatic or ionic bonds, including but not limited to, pyridine, phosphine, or sulfide groups. Other groups suitable for use in a cation exchange material include those that include complexing or chelating moieties, including but not limited to, those derived from aminophosphoric acid, aminocarboyxlic acid, or hydroxamic acid. As described in Example 4, a metal ion (e.g., Fe.sup.3+) may be exchanged for the cation exchange material on the membrane prior to exposure to a solution containing a metal binding molecule (such as but not limited to lactoferrin which is an iron binding molecule).

[0131] An anion-exchange surface in a water-splitting membrane herein may include one or more basic functional groups capable of exchanging anions. Nonlimiting examples of such basic functional groups include NR.sub.3A, NR.sub.2HA, PR.sub.3A, SR.sub.2A, and C.sub.5H.sub.5NHA (pyridinium), where R is an alkyl, aryl, or other organic group and A is an anion (e.g., hydroxide, bicarbonate, chloride, or sulfate ion).

[0132] In some embodiments, a water-splitting membrane herein may contain a textured surface (e.g., at least a portion of the cation and/or anion exchange surface(s)) with spaced apart peaks and valleys.

[0133] Nonlimiting examples of water-splitting membranes and configurations thereof that may be suitable for separation and/or purification of biomolecules as disclosed herein are provided in U.S. Pat. Nos. 5,788,826, 7,344,629, 7,780,833, 7,959,780, 8,293,085, and U.S. Patent Application Nos. 2006/0138997, 2007/0108056, and 2007/0175766, which are incorporated by reference herein in their entireties. Other configurations and materials that are suitable for the water-splitting membranes are contemplated within the scope of the methods and systems disclosed herein and will be apparent to those of skill in the art.

[0134] In some embodiments, a water-splitting membrane may be prepared using any method, to construct heterogeneous and homogenous membranes. For example, homogeneous membranes include polymerized monomers to which ion exchange groups are added chemically, e.g., to ensure the charged groups are uniformly distributed through the membrane. An example of a homogeneous membrane is cross-linked polystyrene that has been either sulfonated, using sulphuric acid (cation), or formation of quaternary amines using trimethylamine (anion). Heterogeneous membranes may be constructed, for example, using finely powdered cation or anion monomer exchange particles, e.g., uniformly dispersed in a polymer such as, for example, polyethylene or polypropylene. In some embodiments, heterogeneous membranes may be more stable, due to structural support provided by the polymers.

[0135] In some embodiments, a water-splitting membrane can include more than one cation exchange and/or more than one anion exchange layer, and the ion exchange functional groups and/or ion exchange capacities of each respective layer can be the same or different. For example one cation exchange layer may contain a strong acid (e.g., derived from a sulfonic acid group (HSO.sub.3.sup.?, which is ionizable over a broad pH range, and hence has a large ion exchange capacity), and the secondary cation exchange layer may contain a weak acid (e.g., derived from a carboxylic group (COOH), which is ionizable over a much narrower pH range and hence has a lower ion exchange capacity). In another example, one anion exchange layer may contain a strong base (e.g., derived from a quaternary ammonium group which is ionizable over a broad pH range, and hence has a large ion exchange capacity), and the secondary anion exchange layer may contain a weak base (e.g., derived from a primary (RNH.sub.2), secondary (RNHR), or tertiary (RNR2) amine group, which is ionizable over a much narrower pH range, and hence has a lower ion exchange capacity).

Biomolecules

[0136] Using the methods and systems described herein, one or more biomolecule(s) may be separated or purified from a solution. For example, in an electrochemical cell as described herein, both positively-charged biomolecules and negatively-charged biomolecules in a solution or buffer (e.g., an aqueous biomolecule-containing solution, for example, at any pH (e.g., 1.0-14.0)) that flows through the electrochemical cell and in contact with a water-splitting ion exchange membrane that contains both anion exchange and cation exchange surfaces may be separated from other components of the solution. The charged biomolecules bind to the water-splitting membrane when a voltage is applied and may be recovered from the membrane by reversing the polarity of the electrodes as described herein. Neutrally-charged biomolecules may also be separated or purified from a solution in the flow through.

[0137] In some embodiments, positively-charged biomolecules bind to a cation exchange surface of a water-splitting ion exchange membrane and are separated or purified from a solution. In some embodiments, negatively-charged biomolecules bind to an anion exchange surface of a water-splitting ion exchange membrane and are separated or purified from a solution. In some embodiments, both positively-charged and negatively-charged biomolecules bind to cation exchange and anion exchange surfaces, respectively, and are simultaneously separated or purified from a solution. In some embodiments, neutrally-charged biomolecules are recovered or purified from a solution in the flow through. Any charged or neutral biomolecule that binds to a water-splitting ion exchange membrane under a voltage or that does not bind and flows through an electrochemical cell as described herein may be separated or purified from a solution.

[0138] In some embodiments, biomolecules that bind to and are released from the water-splitting membrane are concentrated in comparison to the biomolecule-containing solution from which they originated.

[0139] Examples of biomolecules that may be separated or purified from a solution according to a method as disclosed herein include, but are not limited to, proteins, peptides, amino acids, nucleic acids, oligonucleotides, nucleotides, lipids, fatty acids, carbohydrate molecules (e.g., mono-, di-, and polysaccharides), glycolipids, vitamins, metabolites, co-factors, and hormones.

[0140] Examples of biomolecule-containing solutions that may be introduced into an electrochemical cell in a method as described herein include, but are not limited to, cell extracts, plant extracts, body fluids, biomolecule synthesis mixtures, fermentation broth (e.g., microbial growth medium that contains one or more microbially-produced bioproduct(s), including but not limited to a biomolecule, such as, but not limited to, a recombinant polypeptide), and food, beverage, or beverage-derived liquids (including but not limited to milk and whey). In some embodiments, the solution may be diluted or concentrated prior to introduction into the electrochemical cell, in view of the capacity of the membrane(s), flow rate of the solution, or other operating parameters.

[0141] For example, a biomolecule-containing solution may contain a plurality of positively-charged and negatively-charged proteins. In some embodiments, both positively-charged and negatively-charged proteins are simultaneously separated from the solution in a method as described herein. In some embodiments, both positively-charged and negatively-charged proteins are simultaneously separated from the solution in native (e.g., non-denatured) configurations. In some embodiments, recovered proteins retain their inherent biological activity. For example, recovered enzymes retain enzymatic activity.

[0142] In another example, a biomolecule-containing solution may contain a plurality of positively-charged and negatively-charged proteins, and uncharged lipid and/or carbohydrate molecules. A nonlimiting example of such a solution is whey, which contains positively-charged proteins (e.g., lactoferrin, lactoperoxidases, and immunoglobulins), negatively-charged proteins (e.g., bovine serum albumin (BSA), beta-lactoglobulin, and alpha-lactalbumin), lactose minerals and fats. The positively-charged and negatively-charged proteins may bind to the water-splitting membrane and the uncharged lipid and/or carbohydrate molecules (e.g., lactose in whey) may be recovered in the flow through. The positively-charged and negatively-charged proteins may be recovered when the system is regenerated by reversing the polarity of the electrodes. In some embodiments, positively- and negatively-charged proteins are recovered from whey via regeneration of the water-splitting ion exchange membrane as disclosed herein in their native, biologically active configurations. In some embodiments, positively- and negatively-charged proteins are recovered from whey via regeneration of the water-splitting ion exchange membrane as disclosed herein at higher concentrations than in the whey solution that was introduced into the electrochemical cell.

[0143] In some embodiments, one or more biomolecule(s) to be separated and/or purified according to a method described herein contain a tag, for example, to further assist in purification, visualization, and/or quantitation of the biomolecule(s). Nonlimiting examples of tags include a his tag, a glutathione-S-transferase (GST) tag, a flurorescent tag, a green fluorescent protein (GFP) tag, or an maltose-binding protein (MBP) tag. In one embodiment, one or more biomolecule(s) contains a cationic or anionic tag that is positively or negatively charged, respectively, in the solution that contains the biomolecule(s). A cationic or anionic tag may bind to the cation or anion exchange surface, respectively, of the water-splitting ion exchange membrane as the solution containing the biomolecule(s) flows through the electrochemical cell. In one embodiment, the tag includes a cationic polyhistidine sequence (i.e., a his tag). In one embodiment, the biomolecule is a protein that includes one or more tag(s). In one embodiment, the biomolecule is a protein that includes one or more his tag(s).

[0144] In one embodiment, one or more biomolecule(s) to be separated and/or purified according to a method described herein has a strong binding affinity for a specific ion, to further assist in purification, visualization, and/or quantitation of the biomolecule(s). A nonlimiting example includes purification of lactoferrin which has a strong affinity for binding Fe.sup.3+ ions as described in Example 4. As an example, Fe.sup.3+ ions may be introduced/exchanged on the cation exchange surface of the water-splitting ion exchange membrane prior to the solution containing the biomolecule(s) of interest entering the electrochemical cell, to assist in binding of the specific iron binding protein (e.g., lactoferrin).

Demineralization

[0145] Using the methods and systems described herein, one or more ion(s) may be separated from a solution that contains one or more biomolecule(s) and one or more ion(s). The solution may be an aqueous solution or buffer that contains one or more biomolecule(s) and one or more ion(s), for example, a solution or buffer at pH 1.0-14.0. For example, in an electrochemical cell as described herein, both positively-charged ions and negatively-charged ions in a solution that contains one or more biomolecule(s) and one or more ion(s) (e.g., an aqueous biomolecule-containing solution such as whey at any given pH, for example, pH 1.0-14.0) that flows through the electrochemical cell and is in contact with a water-splitting ion exchange membrane that contains both anion exchange and cation exchange surfaces may be separated from other components of the solution. The charged ions bind to the water-splitting membrane when a voltage is applied and may be recovered from the membrane by reversing the polarity of the electrodes as described herein. In some embodiments, a portion of the charged biomolecules also bind to the membrane and are recovered along with the bound ions by reversing the polarity of the electrodes. In other embodiments, all or substantially all of the charged biomolecules flow through the electrochemical cell, along with neutrally charged molecules. In some embodiments, neutrally-charged biomolecules, such as but not limited to, lactose, may be separated or purified from the flow through (demineralized).

[0146] Methods are provided for demineralizing a solution that contains one or more biomolecule(s) and ion(s). The methods disclosed herein include contacting an ion exchange membrane (e.g., an ion exchange membrane capable of water splitting, such as a bipolar membrane) with a solution to be demineralized (a solution that contains at least one biomolecule and at least one ion), under a voltage sufficient to effect exchange of cationic ions with a cation on a cation-exchange surface of the membrane and/or exchange of anionic ions with an anion on an anion-exchange surface of the membrane. In some embodiments, cationic and/or anionic ions bind to the cationic or anionic surface of the membrane, respectively, as the solution containing at least one biomolecule and at least one ion (e.g., in nonlimiting examples, whey, UFR, UFP or deproteinized whey), flows through an electrochemical cell that contains at least one water-splitting ion exchange membrane and electrodes, under a voltage sufficient for the binding to occur, thereby producing a demineralized solution which flows through the electrochemical cell. The demineralized solution may contain charged and uncharged biomolecules. The ions may be extracted or expelled from the membrane by reversing the polarity of the electrodes (regeneration).

[0147] In some embodiments, positively-charged ions bind to a cation exchange surface of a water-splitting ion exchange membrane and are separated or purified from a solution. In some embodiments, negatively-charged ions bind to an anion exchange surface of a water-splitting ion exchange membrane and are separated or purified from a solution. In some embodiments, both positively-charged and negatively-charged ions bind to cation exchange and anion exchange surfaces, respectively, and are simultaneously separated or purified from a solution. In some embodiments, the solution also contains both charged biomolecules and neutrally-charged biomolecules which are recovered in the flow through, considered to be demineralized. Any charged or neutral biomolecule or ion that binds to a water-splitting ion exchange membrane under a voltage or that does not bind and flows through an electrochemical cell as described herein may be separated or purified from the flow through solution.

[0148] In methods described herein, cationic and anionic ions may be recovered together from a starting solution that contains both cationic and anionic ions.

[0149] In some embodiments, ions that bind to and are released from the water-splitting membrane are concentrated in comparison to the biomolecule-containing solution (e.g., biological solution) from which they originated.

[0150] In some embodiments, over 80% of the ions present in the original biomolecule and ion-containing solution are bound and separated from the flow through or demineralized solution, regardless of charge (positive or negative) or valence (monovalent or multivalent).

[0151] In some examples of the methods herein, the solution that contains one or more biomolecule(s) and one or more ion(s) for demineralization, can be at any pH (e.g., 1.0-14.0).

[0152] In some examples of the methods herein, the solution used to expel the one or more ions from the water-splitting membrane during demineralization can be at any pH (e.g., 1.0-14.0).

[0153] Examples of ions that may be separated or purified from a solution that contains one or more biomolecule(s) and one or more ion(s) according to a method as disclosed herein include, but are not limited to, Calcium (e.g., Ca.sup.2+), Magnesium (e.g., Mg.sup.2+), Phosphate (e.g., PO.sub.4.sup.2?) Potassium (e.g., K.sup.+), Chloride (e.g., Cl.sup.?), Sodium (e.g., Na.sup.+), Zinc (e.g., Zn.sup.2+), Iron (e.g., Fe.sup.2+, Fe.sup.3+), Iodine (e.g., I.sup.?), Copper (e.g., Cu.sup.2+, Cu.sup.3+), and Sulfate (SO.sub.4.sup.2?).

[0154] Examples of solutions that contain one or more biomolecule(s) and one or more ion(s) that may be introduced into an electrochemical cell in a method as described herein include, but are not limited to, cell extracts, plant extracts, body fluids, biomolecule synthesis mixtures, fermentation broth (e.g., microbial growth medium that contains one or more microbially-produced bioproduct(s), including but not limited to a biomolecule, such as, but not limited to, a recombinant polypeptide), and food, beverage, or beverage-derived liquids (including but not limited to milk and whey). In some embodiments, the solution that contains one or more biomolecule(s) and one or more ion(s) may be diluted or concentrated prior to introduction into the electrochemical cell, in view of the capacity of the membrane(s), flow rate of the solution, or other operating parameters.

[0155] For example, a solution that contains one or more biomolecule(s) and one or more ion(s) may contain a plurality of positively-charged and negatively-charged ions. In some embodiments, both positively-charged and negatively-charged ions are separated from the solution in a method as described herein. In some embodiments, both positively-charged and negatively-charged ions are bound and simultaneously separated from the solution while remaining biomolecules such as proteins and lactose remain in their native (e.g., non-denatured and biologically active) configurations in the flow through as a demineralized solution.

[0156] In another example, a solution that contains one or more biomolecules and one or more ion(s) may contain a plurality of positively-charged and negatively-charged proteins, and uncharged lipid and/or carbohydrate molecules as well as charged ions. A nonlimiting example of such a solution is whey, which contains positively-charged proteins (e.g., lactoferrin, lactoperoxidases, and immunoglobulins), negatively-charged proteins (e.g., bovine serum albumin (BSA), beta-lactoglobulin, and alpha-lactalbumin), uncharged carbohydrates (e.g., lactose) and milk fats, as well as charged ions (e.g., milk minerals).

[0157] By controlling variables such as pH, the flow rate, membrane composition (cation and/or anion resins used for extrusion of the water-splitting membrane) and other operating parameters of the system, the positively-charged and negatively-charged ions preferentially bind to the water-splitting membrane and the uncharged lipid and/or carbohydrate molecules (e.g., lactose in whey) as well as a significant portion of the proteins (regardless of charge), may be recovered in the flow through as demineralized whey. If ions are present in a biomolecule-containing solution, they may have a competitive advantage to preferentially bind to the membrane due to their strong ionic binding or electrostatic attraction to the membrane, compared to the binding strength of the remaining charged biomolecules in solution (e.g., proteins). This competitive ionic binding may lead to displacement of any bound proteins in preference for smaller and highly charged ions present in the solution. Once the ions are bound and removed, other charged molecules remaining in the demineralized solution can subsequently bind to the membrane without ionic competition and may be extracted or purified from uncharged biomolecules in the demineralized solution. The positively-charged and negatively-charged ions may be recovered when the system is regenerated. In some embodiments, positively- and negatively-charged ions are recovered from whey as milk minerals via regeneration of the water-splitting ion exchange membrane as disclosed herein as milk minerals. In some embodiments, positively- and negatively-charged ions are recovered from whey via regeneration of the water-splitting ion exchange membrane as disclosed herein at higher concentrations than in the whey solution that was introduced into the electrochemical cell.

[0158] In one embodiment, the solution that contains one or more biomolecule(s) and one or more ion(s) is whey (or a derivative of whey such as but not limited to deproteinized whey/UFP or UFR), which may contain cationic and anionic polypeptides, and/or uncharged carbohydrates (e.g., lactose), and/or lipids and/or other micronutrients including milk minerals (e.g., ions). At least a portion of the charged cationic and anionic ions bind to the cationic and anionic surfaces, respectively, of the water-splitting ion exchange membrane(s), and at least a portion of proteins, and/or uncharged carbohydrates (e.g., lactose) and/or lipids exit the electrochemical cell in the flow through as a demineralized solution. Both cationic and anionic, monovalent and multivalent ions may be recovered together from whey (or a derivative of whey such as but not limited to deproteinized whey/UFP or UFR), and separated from protein, and/or lactose and/or lipids, which exit in the flow through as a demineralized solution. In some embodiments, one or more ion(s) in whey or deproteinized whey is recovered at a higher concentration than the whey solution from which it was derived.

[0159] In one embodiment, the composition of a solution containing one or more biomolecule(s) and one or more ion(s) for the process of demineralization includes a food and/or beverage liquid such as a milk-derived solution (e.g., whey and whey derivatives including but not limited to, UFR and deproteinized whey/UFP). Ion(s) separated and removed from whey and its derivatives may include, but are not limited to Calcium (e.g., Ca.sup.2+), Magnesium (e.g., Mg.sup.2+), Phosphate (e.g., PO.sub.4.sup.2?) Potassium (e.g., K.sup.+), Chloride (e.g., Cl.sup.?), Sodium (e.g., Na.sup.+), Zinc (e.g., Zn.sup.2+), Iron (e.g., Fe.sup.2+, Fe.sup.3+), Iodine (e.g., I.sup.?), Copper (e.g., Cu.sup.2+, Cu.sup.3+), and Sulfate (SO.sub.4.sup.2?).

[0160] In one embodiment, the composition of a solution containing one or more biomolecule(s) and one or more ion(s) for the process of demineralization includes a biomolecule-containing solution which has been previously treated ((including but not limited to ultra/nano filtration e.g., deproteinized whey/UFP or UFR). The composition may include, for example, one or more polypeptide(s), and/or one or more carbohydrate(s), and/or one or more lipid(s), and/or one or more ion.

Methods for Separation of Biomolecules

[0161] Methods are provided for separating and/or purifying biomolecules from a solution. The solution may contain ions, or may be demineralized prior to separation of biomolecules.

[0162] In some embodiments, a solution that contains one or more biomolecule(s) and one or more ion(s) is demineralized in a first process (e.g., a first pass through an electrochemical cell as described herein), and then charged biomolecules, such as cationic and anionic polypeptides (e.g., charged biomolecules in the flow through of the first pass through the electrochemical cell), are separated from the demineralized solution (e.g., flow through from the first pass through the electrochemical cell) in a second process (e.g., a second pass through an electrochemical cell as described herein).

[0163] In some embodiments, a specific cationic or anionic biomolecule (e.g., a polypeptide) is bound and purified from a solution using the methods and systems described herein. In some embodiments, the specific biomolecule (e.g., a polypeptide) is recovered at a higher concentration than the starting solution from which it was derived. In some embodiments, the specific biomolecule (e.g., a polypeptide) is recovered in its native (e.g., non-denatured and/or biologically active) configuration.

[0164] The methods disclosed herein include contacting an ion exchange membrane (e.g., an ion exchange membrane capable of water splitting, such as a bipolar membrane) with a solution that contains at least one biomolecule to be separated and/or purified, under a voltage sufficient to effect exchange of cationic biomolecules with a cation on a cation-exchange surface of the membrane and/or exchange of anionic biomolecules with an anion on an anion-exchange surface of the membrane. In some embodiments, cationic and/or anionic biomolecules bind to the cationic or anionic surface of the membrane, respectively, as the biomolecule-containing solution flows through an electrochemical cell that contains at least one water-splitting ion exchange membrane and electrodes, under a voltage sufficient for the binding to occur. The biomolecules may be extracted from the membrane by reversing the polarity of the electrodes (regeneration).

[0165] In one embodiment, the method includes applying a voltage to an electrochemical cell that contains: (a) a housing with at least one inlet and at least one outlet; (b) first and second electrodes; (c) at least one water-splitting membrane between the first and second electrodes and including: (i) a cation exchange surface facing the first electrode and containing a bound cation, and (ii) an anion exchange surface facing the second electrode and containing a bound anion; and (d) a biomolecule-containing solution. In some embodiments, the biomolecule-containing solution contains one or more biomolecule(s) and one or more ion(s). In some embodiments, the biomolecule-containing solution is a demineralized solution. The solution flows through a continuous channel from an inlet to an outlet of the cell and contacts the cation and/or anion exchange surfaces of the membrane. In some embodiments, biomolecule(s) in the solution bind to the cation exchange surface or the anion exchange surface or exit the electrochemical cell through an outlet as an unbound molecule.

[0166] Bound biomolecules may be recovered in a regeneration process that includes reversing the polarity of the first and second electrodes and flowing a solution (e.g., water or an aqueous or other suitable solution in which the biomolecules are soluble (e.g., at any pH (e.g. 1.0-14.0)) through the electrochemical cell. The bound cationic and/or anionic biomolecules are displaced by H.sup.+ or OH.sup.? that is produced in the water-splitting reaction, and the biomolecules then exit with the solution through an outlet in the electrochemical cell.

[0167] In methods described herein, cationic and anionic biomolecules may be recovered simultaneously from a starting solution that contains both cationic and anionic biomolecules. In some embodiments, biomolecules are recovered at a higher concentration than the starting solution from which they were derived. In some embodiments, biomolecules such as polypeptides are recovered in their native (e.g., non-denatured and/or biologically active) configuration.

[0168] In various embodiments, charged biomolecules that bind to the cation exchange and/or anion exchange surface(s) of the water-splitting ion exchange membrane(s) are selected from polypeptides and polynucleotides, carbohydrates (e.g., monosaccharides, disaccharides, and/or polysaccharides) and lipids. In various embodiments, uncharged biomolecules that exit the electrochemical cell in the flow through are selected from polypeptides, polynucleotides, monosaccharides, polysaccharides, and lipids. In one embodiment, charged polypeptides bind to the cation exchange and/or anion exchange surface(s) of the water-splitting ion exchange membrane(s), and uncharged carbohydrate molecules, lipids and other micronutrients exit the electrochemical cell in the flow through.

[0169] In one embodiment, the biomolecule-containing solution is whey (or a derivative of whey such as, but not limited to, demineralized whey or UFR), which contains cationic and anionic polypeptides, uncharged carbohydrates (e.g., lactose), lipids and other micronutrients. In some embodiments, whey (or a derivative of whey such as, but not limited to ultrafiltration retentate or permeate) is demineralized in a first process (e.g., a first pass through an electrochemical cell as described herein), after which, the remaining charged biomolecules, such as cationic and anionic polypeptides (e.g., charged biomolecules in the flow through of the first pass through the electrochemical cell), are separated from the demineralized whey (e.g., flow through from the first pass through the electrochemical cell) in a second process (e.g., a second pass through an electrochemical cell as described herein). At least a portion of the charged cationic and anionic polypeptides bind to the cationic and anionic surfaces, respectively, of the water-splitting ion exchange membrane(s), and at least a portion of uncharged carbohydrates (e.g., lactose) and lipids exit the electrochemical cell in the flow through. The cationic polypeptides may include, but are not limited to, lactoferrin, lactoperoxidases, and/or immunoglobulins. The anionic polypeptides may include, but are not limited to, bovine serum albumin, beta lactoglobulin, alpha lactalbumin, and/or glycomacropeptide. Both cationic and anionic polypeptides may be recovered together from whey (or a derivative of whey such as, but not limited to, demineralized whey or UFR), and separated from lactose and lipids, which exit in the flow through. In some embodiments, one or more polypeptide(s) in whey is recovered at a higher concentration than the whey solution from which it was derived. In some embodiments, one or more polypeptide(s) is recovered from whey in its native conformation and possessing at least a portion of its original biological activity.

[0170] In one embodiment, the biomolecule-containing solution is whey (or a derivative of whey such as, but not limited to, demineralized whey or UFR), from which a specific cationic or anionic polypeptide is separated by exploiting an inherent binding capability of the biomolecule. In one embodiment, the specific polypeptide is lactoferrin which binds to the cation exchange layer of the water-splitting membrane due to its strong binding affinity for Fe.sup.3+ ions as described in Example 4.

Systems

[0171] Systems are provided for practice of the methods described herein. For example, a system for separating or purifying one or more biomolecule(s) from a biomolecule-containing solution may include an electrochemical cell and other equipment required for the operation of the cell, including, but not limited to, equipment for providing a voltage to the electrodes, such as a power supply; equipment for introducing and/or regulating flow of a solution through the cell, such as a pump; container(s) to house incoming solution; sensor(s) to measure output, adjust incoming flow rate, regulate voltage, etc.; and/or equipment for collecting solution that exits the cell through the outlet(s). A system herein may contain a biomolecule-containing fluid within the electrochemical cell and in contact with at least one surface (e.g., cation exchange and/or anion exchange surface(s)) of at least one water-splitting ion exchange membrane.

[0172] Systems that contain two or more electrochemical cells, of any scale in fluid communication with one another and capable of concurrent operation are also provided. Two or more electrochemical cells of any scale may be connected in fluid communication in parallel and/or in series for concurrent operation within a system. In some embodiments, one or more cells, of any scale, may operate in production mode and one or more cells may operate in regeneration mode, simultaneously within the same system, with the cells in fluid communication and connected in parallel and/or in series within the system.

Compositions

[0173] Compositions are provided that include one or more biomolecule(s) separated or purified from a solution, according to methods as described herein. Also provided are solutions from which one or more biomolecule(s) have been removed, according to a method as described herein.

[0174] Compositions are also provided that include one or more ion(s) separated or purified from a solution containing one or more biomolecule(s) and one or more ion(s) according to methods as described herein. Also provided are solutions from which one or more ion(s) have been removed, according to methods as described herein.

[0175] In some embodiments, the composition includes one or more biomolecule(s) and/or ion(s) that have been separated or purified from a cell extract, plant extract, bodily fluid, biomolecule synthesis mixture, fermentation broth, or food/beverage-derived liquids (including but not limited to milk and whey) or the like, as described herein. The composition may include, for example, but not limited to, one or more polypeptide(s), one or more carbohydrate(s), one or more lipid(s), and/or one or more polynucleotide(s), and may optionally further include one or more ion(s). In some embodiments, the composition may be a solution in which one or more biomolecule(s) and/or ion(s) are more concentrated than in the solution from which they were derived. In some embodiments, one or more biomolecule(s) in the composition is (are) in a native (e.g., biologically active) conformation. In some embodiments, the solution in which one or more biomolecule(s) and/or ion(s) are present, passes through the electrochemical cell from one outlet to the other without fouling the membrane which is capable of water-splitting. In some embodiments, at least a portion of the liquid may be removed from a solution that contains the separated or purified biomolecule(s) (e.g., by drying (e.g., spray drying), lyophilization, or the like).

[0176] In some embodiments, the composition includes one specific biomolecule that has been separated or purified from a cell extract, plant extract, bodily fluid, biomolecule synthesis mixture, fermentation broth, or food/beverage-derived liquids (including but not limited to milk and whey) or the like, as described herein. The composition may include, for example, but not limited to, a polypeptide such as lactoferrin as described in Example 4. In some embodiments, the bound and recovered specific polypeptide may be more concentrated than in the solution from which it was derived. In some embodiments, the polypeptide is in a native (e.g., biologically active) conformation. In some embodiments, at least a portion of the liquid may be removed from a solution that contains the separated or purified polypeptide (e.g., by drying (e.g., spray drying), lyophilization, or the like).

[0177] In some embodiments, the composition includes one or more biomolecule(s) that have been recovered in the flow through (e.g., biomolecule(s) that did not bind to the water-splitting ion exchange membrane) in a method as described herein. The composition may include, for example, but not limited to, one or more polypeptide(s), and/or one or more carbohydrate(s), and/or one or more lipid(s), and/or one or more polynucleotide(s). In some embodiments, one or more biomolecule(s) in the composition is (are) in a native (e.g., biologically active) conformation. In some embodiments, at least a portion of the liquid may be removed from a solution that contains the biomolecule(s) recovered in the flow through (e.g., by drying (e.g., spray) drying, lyophilization, or the like).

[0178] In some embodiments, the composition is a solution (e.g., a cell extract, plant extract, bodily fluid, biomolecule synthesis mixture, fermentation broth, or food/beverage-derived liquids (including but not limited to milk and whey) etc.) from which at least a portion of one or more biomolecule(s) and/or ion(s) have been removed by passage through an electrochemical cell, in a method as described herein. For example, the composition may be a solution from which, for example, but not limited to, one or more polypeptide(s), and/or one or more carbohydrate(s), and/or one or more lipid(s), and/or one or more polynucleotide(s) have been removed.

[0179] In one embodiment, the composition includes one or more polypeptide(s) separated from a milk solution (e.g., whey and whey derivatives including, but not limited to, demineralized whey, or ultrafiltered retentate (UFR)). Polypeptide(s) separated from whey or derivatives of whey may include, but are not limited to, lactoferrin, lactoperoxidases, immunoglobulins, bovine serum albumin, beta lactoglobulin, alpha lactalbumin, and/or glycomacropeptide. In some embodiments, one or more of the polypeptide(s) is more concentrated than in the solution (e.g., whey or a derivative of whey such as but not limited to demineralized whey, or UFR),) from which they were derived. In some embodiments, one or more of the polypeptide(s) is in a native (e.g., biologically active) conformation.

[0180] In some embodiments, the composition includes one or more ion(s) that have been separated or removed from a solution that contains one or more biomolecule(s) and one or more ion(s), including, but not limited to, food and/or beverage-derived liquids (including but not limited to milk and whey) as described herein. The composition may include, for example, but not limited to, at least some portion of ion(s), and/or at least some portion of one or more polypeptide(s), and/or at least some portion of carbohydrate(s), and/or at least some portion of lipid(s). In some embodiments, the composition may be a solution in which one or more ions(s) are more concentrated than in the solution from which they were derived. In some embodiments, one or more ions(s) in the composition is (are) in their natural form and ratios (e.g., ions present in whey, known as milk minerals). In some embodiments, at least a portion of the liquid may be removed from a solution that contains the separated or removed ion(s) (e.g., by heat and evaporation, drying (e.g., spray drying), lyophilization, or the like).

[0181] In some embodiments, the composition includes one or more biomolecule(s) that have been recovered in the flow through as a demineralized solution (e.g., biomolecule(s) that did not bind to the water-splitting ion exchange membrane) in a method as described herein. The composition may include, for example, but not limited to, one or more polypeptide(s), and/or one or more carbohydrate(s), and/or one or more lipid(s), and/or one or more polynucleotide(s). In some embodiments, one or more biomolecule(s) recovered in the flow through as a demineralized solution, in the composition is (are) in a native (e.g., biologically active) conformation. In some embodiments, at least a portion of the liquid may be removed from a solution that contains the biomolecule(s) recovered in the flow through as a demineralized solution (e.g., by drying (e.g., spray) drying, lyophilization, or the like).

[0182] In some embodiments, the composition is a solution (e.g., a cell extract, plant extract, bodily fluid, biomolecule synthesis mixture, fermentation broth, or food/beverage-derived liquids (including but not limited to milk and whey) etc.) from which at least a portion of one or more ions(s) have been removed (or demineralized) by passage through an electrochemical cell, in a method as described herein.

[0183] In one embodiment, the composition is a food and/or beverage solution, for example, a milk solution (e.g., whey and whey derivatives including but not limited to, demineralized whey, UFR and deproteinized whey/UFP) from which at least a portion of one or more polypeptide(s) have been separated by passage through an electrochemical cell, in a method as described herein. In an embodiment, whey polypeptide(s), at least a portion of which may have been purified, include, but are not limited to, lactoferrin, lactoperoxidases, immunoglobulins, bovine serum albumin, beta lactoglobulin, alpha lactalbumin, and/or glycomacropeptide. In some embodiments, the composition from which at least a portion of one or more polypeptide(s) have been separated, includes carbohydrate(s) (e.g., lactose) and/or lipid(s).

[0184] In one embodiment the composition of a solution containing one or more biomolecule(s) and one or more ion(s) comprises, consists of, or consists essentially of whey that has previously been treated by a prior method, including but not limited to microfiltration, ultrafiltration, nanofiltration, reverse osmosis, electrodialysis, ion exchange or diafiltration.

[0185] In one embodiment, the composition of a product is lactose or deproteinized whey powder comprising >80% lactose or deproteinized whey that has been separated from the milk minerals of whey (demineralized).

[0186] In one embodiment, the composition of a product is a milk mineral powder comprising cationic and anionic milk minerals separated from other components in whey (or derivatives of whey such as but not limited to, deproteinized whey).

[0187] In one embodiment, the composition of a product is a demineralized whey (or a demineralized derivative of whey such as, but not limited to, ultrafiltration retentate or permeate) powder comprising cationic and/or anionic polypeptides, and/or lactose/and/or fat that have been separated from milk minerals of whey.

[0188] In one embodiment, the composition of a product is a protein powder comprising cationic and/or anionic polypeptides of whey, which have been separated from milk minerals and/or fat and/or lactose.

[0189] In one embodiment, the composition of a product is a specific protein powder comprising a cationic or anionic polypeptide of whey, which has been separated from other components in whey due to targeted specific binding, for example, as described in Example 4.

[0190] The following examples are intended to illustrate, but not limit, the invention.

EXAMPLES

[0191] Example 1

Procedure

[0192] An experiment was performed to determine the recovery and reproducibility of using an electrochemical system with a bipolar water-splitting ion exchange membrane for demineralization of whey and whey derivatives (e.g., UFP and UFR). A commercially available LINX? 140T TDS Cartridge system, available from Pionetics Corp., was used. This system includes two cartridges, each with a spiral bound textured bipolar sheet that contains a cation exchange layer abutting an anion exchange layer.

[0193] Pasteurized liquid whey was obtained from Petaluma Creamery, as was the ultrafiltration retentate (or UFR, containing concentrated protein, fat, ions and at least a portion of lactose after processing through an ultrafiltration system). The ultrafiltration permeate (or UFP, containing fat, ions, concentrated lactose and at least a portion of the whey proteins after processing through an ultrafiltration system), was also processed as deproteinized whey.

[0194] Samples of whey, UF retentate (concentrated whey protein and ions) and UF permeate (deproteinized whey/concentrated lactose and ions) were passaged through the LINX? 140T TDS Cartridge system with an electric current applied (voltage set at 300V). The operating parameters were 100-120 Vac, 50/60 Hz, 6 A, and flow rate >0.5 L/min. All samples were diluted with distilled water prior to use, at a dilution factor of approximately 1:5 (sample: water), to lower the conductivity of the solutions to approximately 2000 ?S/cm (to stay within the operating range of the current scale of the LINX? 140T TDS Cartridge system. Approximately 10 L of each dilute sample (whey, UF retentate and UF permeate respectively) were passaged through LINX? 140T TDS Cartridge system under an electric current, separating at least a portion of ions that bound to the water-splitting membrane, from the other components in the solutions (biomolecules), that did not bind to the water-splitting membrane and flowed through the cartridge as a demineralized solution. Bound ions from each sample were recovered by regenerating the membrane (reversing the polarity of the electrodes), using distilled water to produce a regenerated solution that contained the ions (milk minerals).

[0195] All solutions (diluted whey/UFR/UFP samples, deionized solutions and regenerated solutions), were analyzed for presence and quantity of ions, as measured by a digital conductivity meter (HM Digital COM-100 Waterproof Combo Meter for EC, TDS and Temperature).

[0196] Each dilute sample (whey, UF retentate or UF permeate) was treated in a continuous alternating process between the two cells of the LINX? 140T TDS Cartridge system. Each of the two cells treated the dilute sample volume (10 L) simultaneously during the demineralization phase after which, water (at pH 8.0) was used as the regeneration solution during the regeneration phase. Regeneration began with the first cell providing 2 L of demineralized water to the second cell as a solution for its regeneration process (i.e., the release of ions bound to the second cell). Next, the second cell, in turn, provided 2 L of demineralized water to the first cell as a solution for its regeneration process (i.e., the release bound ions bound to the first cell). After both cells regenerated, the cycle was complete and the system was then ready to treat the next sample.

[0197] Both the regenerated sample and the flow through sample collected from each experiment were analyzed quantitatively for either protein concentration or lactose concentration (depending on the source solution) as determined by spectrophotometry, and presence of ions as determined by a conductivity measurement.

Quantitative Analysis

[0198] Protein concentration was determined using the Quick Start? Bradford protein assay. The assay uses a dye reagent (Coomassie Brilliant Blue G-250), at 1?concentration. The dye exists in three forms; cationic (red), neutral (green) and anionic (blue). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form. However, when the dye binds to protein it is converted to a stable unprotonated blue form which is detected using a spectrophotometer at 595 nm. A standard curve was prepared using seven pre-diluted standards at the following concentrations (0.125, 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 mg/ml bovine serum albumin) and was used to calculate the concentration of protein in each of the samples.

[0199] Lactose content was determined using the EnzyChrom? Lactose Assay Kit from Bioassays systems. This kit uses specific enzyme-coupled reactions in which lactose is cleaved into glucose and galactose molecules. The resulting galactose forms a colored product which can be read using a spectrophotometer at 570 nm. The color intensity is directly proportional to the lactose concentration in the sample.

[0200] Results are shown in Table 1.

[0201] Over 80% of the ions (milk minerals), regardless of charge, size or valence were bound and removed from each of the dilute feed streams (whey, UF retentate and UF permeate) (Table 1). The milk minerals (ions) which were bound to the membrane, were recovered during the regeneration step (by reversing the polarity of the electrodes), and efficiently removed from the feed streams producing demineralized whey, UF retentate or UF permeate respectively. The milk minerals (ions) that were bound to the membrane were recovered in water (without the use of any chemicals) during regeneration and hence could have been lyophilized or spray dried to produce natural milk minerals in their naturally occurring ratios. The resulting demineralized solutions from each of the samples still retained the majority of biomolecules originally present (proteins and/or lactose) which could have been further processed or separated using methods described herein.

[0202] Example 2

[0203] Procedure

[0204] An experiment was performed to determine the recovery and reproducibility of using an electrochemical system with a bipolar water-splitting ion exchange membrane for purification of biomolecules such as proteins from whey. A commercially available LINX? 140T Cartridge system, available from Pionetics Corp., was used. This system includes two cartridges, each with a spiral bound textured bipolar sheet that contains a cation exchange layer abutting an anion exchange layer.

[0205] Whey was produced by heating 4 gallons of 1% milk to about 65? C. Approximately ? cup of dilute distilled vinegar was added per gallon of milk to separate the curd (coagulated casein protein) from the whey. The mixture was further separated by running the entire mixture through a fine cheesecloth. The whey was immediately cooled on ice until further processing.

[0206] Samples of dilute liquid whey were passaged through the LINX? 140T TDS Cartridge system with an electric current applied (voltage set at 300V). The operating parameters were 100-120 Vac, 50/60 Hz, 6 A, and flow rate <0.5 L/min. Whey was diluted with distilled water prior to use, at a dilution factor of approximately 1:15, to lower the conductivity of the whey solution to closely match the conductivity of hard, unfiltered tap water (approximately 1000 ?S/cm). Dilution to lower the conductivity of the whey was performed to artificially represent a demineralized whey solution in which, protein binding would be favored over ion binding. Demineralization can be achieved by either removal of ions as described in Example 1 (or any other known demineralization method previously described) or dilution with water. Once whey is sufficiently demineralized, it can be further processed using methods described herein to extract and purify biomolecules such as proteins.

[0207] Two consecutive samples of dilute whey (approximately 21 L each) at a protein concentration of approximately 2.5 g/L (about 50 g total protein) were processed through the LINX? 140T TDS Cartridge system under sufficient electric current, to separate biomolecules that bound to the membrane (bound biomolecules) from those that did not bind and flowed through the cartridge (flow through biomolecules). Bound biomolecules were recovered by regenerating the system (reversing the polarity of the electrodes), with the same dilute whey as the original feed solution to produce a regenerated solution that contained the previously bound biomolecules (recovered bound biomolecules, such as recovered bound protein).

[0208] Each batch of dilute whey was processed in a continuous alternating process between the two cells of the LINX? 140T TDS Cartridge system. Each of the two cells processed the 21 L of dilute whey simultaneously during the separation phase after which, the cells entered regeneration phase. The first cell provided 2 L of deproteinized flow through material to the second cell as a solution for its regeneration process (i.e., to release biomolecules bound to the second cell). Subsequently, the second cell continued to provide 2 L of deproteinized flow through material to the first cell as a solution for its regeneration process (i.e., to release bound biomolecules bound to the first cell). After both cells regenerated, the cycle was complete and the system was then ready to treat the next batch. At the end of each cycle, approximately 6.5 L of regenerated solution was collected, which contained the concentrated bound biomolecules (proteins). Both samples containing the recovered bound biomolecules and the flow through biomolecules were analyzed quantitatively for protein concentration, qualitatively for protein content and size, and protein identification/biological state using polyacrylamide gel electrophoresis banding patterns.

Quantitative Analysis

[0209] Protein concentration was determined by absorbance at 280 nm (A.sub.280). Virtually all proteins exhibit a strong UV absorbance maximum near 280 nm. This characteristic absorbance is due almost entirely to the absorbance by the aromatic rings in the side chains of the amino acids tryptophan and tyrosine. The crude sample (undiluted whey) was diluted 1:100 due to the high initial concentration of whey. The regenerated and flow through samples were diluted 1:10. Sample dilutions were prepared in duplicate and A.sub.280 readings were determined. The average protein concentration of each sample was calculated by multiplying the dilution factor by the average absorption value and then dividing by 1.4, which is the molar extinction coefficient of mammalian immunoglobulin. The results are shown in Table 2. About 5 times more concentrated protein was recovered in the regenerated stream than in the flow through.

TABLE-US-00001 TABLE 2 Volume Average Total % of Protein protein Yield sample Concen- in each of collected tration sample total Sample (L) (g/L) (grams) protein Crude extract 38.11 (undiluted whey) Dilute Whey 21 2.54 52.5 100% (1:15 dilution) Regenerated 6.5 5.00 32.5 62% Protein Sample #1 Regenerated 6.5 5.31 34.5 65% Protein Sample #2 Flow Through 14.5 1.23 17.8 34% Sample #1

[0210] Yield of bound and extracted protein was calculated as a percentage of total protein processed per cycle. An average of 64% of all whey proteins processed using the LINX 140T TDS Cartridge system were bound and extracted during the regeneration phase. Fat content was determined in the regenerated samples from batches 1 and 2, and flow through sample from batch number 1, using the method described in Forcato (2005) J Dairy Sci 88(2):478-481. Lipids present in milk can be determined by UV spectrophotometry based upon the property of fatty acids to absorb UV light proportional to their concentrations. A standard curve of milk fat and UV absorbance at 208 nm was established in order to calculate fat content in each of the samples. 30 ?L of each sample was added to 1.5 mL of pure ethanol and chilled at ?20? C. for 2 hours to precipitate the protein. The samples were then centrifuged at 13,000 rpm for 15 minutes and the supernatant removed for absorbance measurement at 208 nm. The results are shown in Table 3. The regenerated whey samples contained an average of 120 g/ml fat and the flow through sample contained approximately 375 g/ml. The results are shown in Table 3. About 3 times more fat was recovered in the flow through sample than in the regenerated samples. Since the bound biomolecules in this experiment were eluted during the regeneration phase using dilute whey (identical solution to the feed solution), the presence of fat in the regenerated solution can be accounted for due to carry through. If the bound biomolecules were to be eluted during regeneration phase using an aqueous or buffered solution, the amount of fat in the regenerated solution would be greatly reduced.

TABLE-US-00002 TABLE 3 Standard curve calculation Average (y = A208, Fat Average x = ?g/mL content Sample A.sub.208 Milk fat) (?g/mL) Crude sample 0.4309 y = 0.0004x 1077.25 (undiluted whey) Regenerated 0.0509 y = 0.0004x 127.25 Sample #1 Recovered 0.0449 y = 0.0004x 112.50 Sample #2 Flow Through 0.150 y = 0.0004x 375.00 Sample #1

[0211] Lactose content was determined in the regenerated samples from batch numbers 1 and 2, and flow through solution from batch number 1. The presence and characterization of lactose was determined by HPLC-RI method (by ion exclusion HPLC). 500 ?L of each sample was filtered through 3K MWCO (Molecular Weight Cut off) centrifugal filter units at 14000 rpm for 10 minutes. Filtered extracts of these samples were analyzed by Ion Exclusion HPLC using an organic acid analysis column. 5 mg/mL lactose was used as a reference standard and duplicate injections of both reference standards and samples were made. Qualitative identification of the lactose in the samples was by comparison of retention times with reference standard and quantitative identification of lactose in samples was by extrapolation with the reference standard area. The regenerated whey samples contained an average of 25% w/w lactose and the flow through sample contained 75% w/w lactose. The results are shown in Table 4. About 4 times more lactose was recovered in the flow through sample than in the regenerated sample. Since the bound biomolecules were eluted the during regeneration phase using dilute whey (identical solution to the feed solution), the presence of lactose in the regenerated stream can be accounted for due to carry through. If the bound biomolecules were to be eluted during regeneration phase using an aqueous or buffered solution, the amount of lactose in the regenerated samples would be greatly reduced.

TABLE-US-00003 TABLE 4 Total lactose Approximate Concen- Volume per percentage of tration per sample sample total lactose Sample ID (mg/mL) (L) (g) per cycle Crude sample 59.78 (undiluted whey) Diluted whey 4.76 21 99.9 100% Regenerated Sample #1 4.35 6.5 28 25% Regenerated Sample #2 4.25 6.5 27.6 25% Flow through Sample #1 5.15 14.5 74.6 75%

Qualitative Analysis

[0212] The samples described in Table 4 were examined by polyacrylamide gel electrophoresis (PAGE) (4-20% acrylamide), with or without prior denaturation of proteins. For reference, one lane included size standards and another lane included 2 mg/ml bovine serum albumin (BSA). For the denaturing gel, fully reducing conditions were employed where samples were pre-treated with ?-mercaptoethanol (reduces disulfide bonds) and heat treated to fully denature the proteins prior to loading the gel. For the non-denaturing gel, non-reducing conditions were employed where the samples were not treated with any additional chemicals or heat prior to gel loading to retain proteins in native (non-denatured) configurations. Both sets of samples were run with sodium dodecyl sulfate (SDS) in the buffer (providing uniform negative charge). Proteins were stained with Coomassie Blue for visualization. By visual inspection, the protein banding patterns in both recovered protein and flow through samples were essentially identical in each sample, both under reducing and non-reducing conditions. This indicates that major proteins in whey were recovered in native (e.g., monomeric) form after processing with the LINX? 140T TDS Cartridge system. Since the same proteins were also recovered in the flow through although in lower amounts (concluded by visual inspection of banding patterns on SDS gels) the amount of sample run through the system may have contained more protein than available sites on the ion exchange membranes. This may be alleviated by controlling flow rates and appropriate dilutions of the incoming whey stream.

[0213] Example 3

Procedure

[0214] An experiment was performed to determine the reliability and reproducibility of using an electrochemical system with a bipolar water-splitting ion exchange membrane for controlled demineralization of wine. A commercially available LINX? 140T TDS Cartridge system, available from Pionetics Corp., was used. This system includes two cartridges, each with a spiral bound textured bipolar sheet that contains a cation exchange layer abutting an anion exchange layer.

[0215] A commercially available cask wine (Franzia-chardonnay) was obtained from Safeway. Samples of wine were passaged through the LINX? 140TTDS Cartridge system with an electric current applied. The conductivity of the wine was approximately 2500 ?S/cm the wine was at pH 3.7. The operating parameters were 100-120 Vac, 50/60 Hz, 6 A. The voltage and flow rates were varied; 300V and flow rate <0.5 L/min for Max voltage conditions; 65V and flow rate of >0.5 L/min for Min voltage conditions. Approximately 5 L of wine was processed through the LINX? 140T TDS Cartridge system under an electric current, separating ions that bound to the membrane from other components in the solutions that did not bind and flowed through the cartridge as a demineralized solution. Bound ions were recovered by regenerating the membrane (by reversing the polarity of the electrodes), with distilled water to produce a regenerated solution that contained the ions.

[0216] All samples were analyzed for presence and quantity of ions as a measure of conductance measured by a digital conductivity meter (HM Digital COM-100 Waterproof Combo Meter for EC, TDS and Temperature).

[0217] Each wine sample was treated in a continuous alternating process between the two cells of the LINX? 140T TDS Cartridge system. Each of the two cells treated the wine simultaneously during the demineralization phase after which, water (at pH 8.0) was used as the fluid for the regeneration phase. Regeneration began with the first cell providing 2 L of demineralized water to the second cell as a solution for its regeneration process (i.e., to release ions bound to the second cell). Next, the second cell provided 2 L of demineralized water to the first cell as a solution for its regeneration process (i.e., to release bound ions bound to the first cell). After both cells regenerated, the cycle was complete and the system was then ready for the next cycle.

Quantitative Analysis

[0218] Both the regenerated and the flow through samples were collected from each batch and were analyzed quantitatively for the presence of ions as determined by a conductivity measurement. Results are shown in Table 5.

[0219] A range between 60 and 90% of the ions, regardless of charge or valence, were removed from each of the wine samples treated respectively, depending on the set voltage applied across the membranes during deionization (Table 5). By varying the voltage across the membrane and controlling the flow rate, the degree of demineralization (amount of ions bound and removed) was able to be controlled. The ions were recovered during the regeneration phase and efficiently removed from the original sample (wine) producing varying degrees of demineralized wine.

Example 4

[0220] An electrochemical system with a bipolar water-splitting ion exchange membrane is used for purification of a specific biomolecule such as a cationic or anionic protein from whey, such as lactoferrin. Lactoferrin is bound to the membrane by using its inherent affinity to bind Fe.sup.3+ ions. A commercially available LINX? 140T Cartridge system, available from Pionetics Corp., is used. This system includes two cartridges, each with a spiral bound textured bipolar sheet that contains a cation exchange layer abutting an anion exchange layer.

[0221] Effective binding and separation of a single protein such as lactoferrin is achieved in a two-step process as described herein. Regardless of the composition of the cation and anion materials used to extrude the bipolar membranes that capable of water-splitting, ions on the membrane (either on the cation or the anion layer or both), such as Fe.sup.3+ present in an aqueous solution such as, but not limited to Iron (III) sulfate (or ferric sulfate with the formula Fe.sub.2(SO.sub.4).sub.3), may be replaced during the first few minutes of the deionization phase. Since multivalent metals have a very high affinity for the cation and anion exchange layers on the membrane, the surfaces of the membrane will be replaced with Fe.sup.3+ ions. Once the membranes are charged with Fe.sup.3+ ions, demineralized whey (or a derivative of whey such as UFR), can be passaged through the cell as a high flow rate. During this phase, lactoferrin (which is the only whey protein that has an affinity for binding iron) binds to the membrane while the other proteins and biomolecules present in the solution pass through as unbound molecules. Since lactoferrin is present in whey at very low concentrations (<1% of the total proteins), a large volume of whey ((or a derivative of whey such as UFR), can be treated before the system would require regeneration using polarity reversal of the electrodes.

[0222] Detection of lactose presence and quantitation in all samples (original feed sample, regenerated sample and the flow through sample) is carried out using commercially available Bovine Lactoferrin Elisa kits. The assay uses affinity purified anti-bovine lactoferrin antibodies for solid phase (micro titer wells) immobilization and horseradish peroxidase (HRP) conjugated anti-bovine lactoferrin antibodies for detection.

[0223] Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention, which is delineated in the appended claims. Therefore, the description should not be construed as limiting the scope of the invention.

[0224] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.