Alpha-lactalbumin process (II)

Abstract

The present invention relates to a process for the or the production of an aqueous solution enriched in alpha-lactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid.

Description

Title of InventionAlpha-lactalbumin process (II)

[0001] This application claims priority from Australian Provisional Patent Application No. 2024901362 filed on 10 May 2024, the contents of which are to be taken as incorporated herein by this reference.Technical Field

[0002] The present invention relates to a process for the isolation of alphalactalbumin from a permeate of a microfiltered dairy liquid.Background of Invention

[0003] Alpha-lactalbumin is a globular protein that regulates production of lactose in most animals. The primary structure of alpha-lactalbumin typically consists of 123 amino acids including four disulfide bridges with a molecular weight of approximately 14 kDa. This protein has a number of uses such as in nutrition, particularly for newborn children. Additionally, this protein can be used in various therapeutic formulations. Alpha-lactalbumin undergoes structural changes associated with pH changes, for example under acidic conditions the protein may release calcium ions and undergo beta-sheet unfolding.

[0004] Alpha-lactalbumin is often produced on scale as it is a constituent of proteinaceous compositions derived directly or indirectly from animals, such as milk, whey and isolated protein powder. Often, those compositions have undergone processing operations in the dairy industry, for example cheese making operations. Such proteinaceous compositions may contain additional proteins such as betalactoglobulin, immunoglobulins and lactoferrin.

[0005] Beta-lactoglobulin is often a prevalent protein constituent along with alphalactalbumin. Beta-lactoglobulin is a protein of 162 residues having a size of 18.4 kDa. Although beta-lactoglobulin has uses in forming gels and for improving the bioavailability of micronutrients for example, its properties as a human allergen havepreviously been described. Other proteins found in the permeate of microfiltered dairy liquids also have commercial value, such as immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

[0006] Processing of proteinaceous compositions derived from animals, such as milk, has conventionally been conducted by use of acids or enzymes including rennet. More recent processing techniques have been developed that do not rely on the addition of chemical or biological entities but rely instead on physical processes. An example of this is conducted during the production of micellar casein concentrate (MCC) from milk. Because no acid or enzymes are used in the production of this product, often the proteins in the resultant proteinaceous compositions (whey) are less likely to suffer any structural degradation leading to a more desirable product.

[0007] Production of MCC and related products is achieved primarily through microfiltration by use of a semipermeable membrane having a pore size typically in the range of about 0.1 to 1 .0 pm. Typical production of such products is by microfiltration of a dairy product, such as skim milk. Passage of the dairy product through a microfiltration membranate results in a microfiltered permeate and a microfiltered retentate. The permeate comprises serum proteins such as alphalactalbumin and beta-lactoglobulin. The retentate comprises casein-enriched solutions that are often referred to as micellar casein concentrate. This retentate can be further processed into powders for example by spray drying. The retentate products are of commercial value, for example in the food industry.

[0008] The permeate can be understood as a waste stream although it nevertheless contains useful products such as alpha-lactalbumin. The purification of alpha-lactalbumin and other proteins from such waste streams remains a challenge.

[0009] Accordingly, there remains an opportunity to develop alternative processes for the isolation of alpha-lactalbumin and other proteins from aqueous proteinaceous compositions obtained from the permeate of microfiltration of dairy liquids.

[0010] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these mattersformed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.Summary of Invention

[0011] The present applicants have conducted research aimed at developing a process for the production of an aqueous solution enriched in alpha-lactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid.

[0012] Accordingly in one aspect of the invention there is provided a process for the production of an aqueous solution enriched in alpha-lactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid. The aqueous proteinaceous composition comprises alpha-lactalbumin, and at least one additional protein and the proteinaceous composition solution has a pH of about 6.3 to about pH 7.0. The process comprises the steps of providing an anionic exchange resin and adjusting the anionic exchange resin to an initial pH of about the isoelectric point of alpha-lactalbumin. Contacting the anionic exchange resin having the initial pH of about the isoelectric point of alpha-lactalbumin with the aqueous proteinaceous composition so that the alpha-lactalbumin and the at least one additional protein are at least partially selectively adsorbed onto the anionic exchange resin. Thus, providing an aqueous raffinate and an anionic exchange resin adsorbed with the alpha-lactalbumin the at least one additional protein. Separating of the anionic exchange resin adsorbed with the alpha-lactalbumin the at least one additional protein from the aqueous raffinate followed by eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein, by use of an alpha-lactalbumin desorption solution provides a solution enriched in alpha-lactalbumin.

[0013] Without wishing to be bound by theory, the applicant has observed that the purification of alpha-lactalbumin as well as additional proteins such as betalactoglobulin from proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid can be achieved without altering the inherent pH, for example by acidification or by enzymatic means, thus assisting in preserving thenative structures of the proteins. The pH of the permeate tends to be in the range of about about of such solutions tends to be in the range of about 6.3 to about pH 7.0. Isolating alpha-lactalbumin and additional proteins from such solutions has been found to be effective by adjusting the initial pH of the anionic exchange resin close to the isoelectric point of alpha-lactalbumin in order to achieve effective separation from beta-lactoglobulin other additional proteins.

[0014] In some embodiments, the anionic exchange resin is provided in an apparatus suitable for liquid flow therethrough. This provides a convenient means for conducing the process.

[0015] In some embodiments, contacting of the anionic exchange resin with the aqueous proteinaceous composition solution occurs during passage through apparatus suitable for liquid flow therethrough as well as separation from the aqueous raffinate.

[0016] Accordingly in one aspect of the invention there is provided a process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid. The aqueous proteinaceous composition comprises alpha-lactalbumin and at least one additional protein and wherein the proteinaceous composition solution has a pH of about 6.3 to about pH 7.0. The process comprises the steps of providing an anionic exchange resin in an apparatus suitable for liquid flow therethrough and adjusting the anionic exchange resin to an initial pH of about the isoelectric point of alpha-lactalbumin. Contacting the anionic exchange resin having the initial pH of about the isoelectric point of alphalactalbumin with the aqueous proteinaceous composition can occur during passage through apparatus suitable for liquid flow therethrough so that the alpha-lactalbumin and at least one additional protein are at least partially selectively adsorbed onto the anionic exchange resin. Thus providing an aqueous raffinate separated from an anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and by use of an alpha-lactalbumin desorption solution to provide a solution enriched in alpha-lactalbumin. The aqueous solution enriched inalpha-lactalbumin can then be subjected to lyophilisation to provide the isolated alpha-lactalbumin.

[0017] In some embodiments the apparatus suitable for liquid flow therethrough is a chromatography apparatus.

[0018] In some embodiments, the permeate of microfiltration has been subjected to filtration with a semipermeable membrane pore size of about 0.1 pm to about 1 .0 pm. Without wishing to be bound by theory, use of microfiltration may not cause the same level of degradation to proteins that may be found with chemical and / or enzymatic treatments.

[0019] In some embodiments the alpha-lactalbumin desorption solution is an about 1 mS to about 15 mS salt solution. Use of this solution can enable selective desorption of alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein.

[0020] In some embodiments, the process further comprises eluting of the at least one additional protein from the anionic exchange resin adsorbed with the alphalactalbumin and at least one additional protein by use of an at least one additional protein desorption solution to provide a solution enrichened in the at least one additional protein.

[0021] In some embodiments, the at least one additional protein is selected from the list consisting of beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

[0022] In some embodiments, the at least one additional protein is beta- lactoglobulin and the beta-lactoglobulin desorption solution is an about 30 mS to an about 100 mS aqueous salt solution to provide a solution enriched in beta- lactoglobulin.

[0023] In some embodiments, the at least one additional protein is glycomacropetide (caseinomacropeptide) and wherein the glycomacropetide desorption solution is an about 2 mS to 4 mS aqueous salt solution to provide a solution enriched in glycomacropetide (caseinomacropeptide); wherein the at leastone additional protein is bovine serum albumin and wherein the bovine serum albumin desorption solution is an about 15 mS to an about 25 mS aqueous salt solution to provide a solution enriched in bovine serum albumin; or wherein the at least one additional protein is immunoglobulin and the immunoglobulin desorption solution is an about 20 mS to an about 40 mS aqueous salt solution to provide a solution enriched in immunoglobulin.

[0024] In some embodiments, the order of eluting from the anionic exchange reins is sequential and as follows: glycomacropetide (caseinomacropeptide); alphalactalbumin; bovine serum albumin; immunoglobulin; and then beta-lactoglobulin.

[0025] In some embodiments, the salt is sodium chloride.

[0026] In some embodiments, the initial pH of the anionic exchange resin following adjustment is about pH 4.0 to about pH 5.0.

[0027] Without wishing to be bound by theory, the inventor has surprisingly found that by carefully adjusting the pH of an anionic exchange resin prior to elution with an aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid (preferably MCC whey derived from cow’s milk), alpha lactalbumin can be adsorbed onto the anionic exchange resin. The combination of use of the aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid that has not been enzymatically or chemically treated, in combination with the pH adjusted anionic exchange resin can deliver alpha-lactalbumin as well as additional proteins in high purity and in an efficient manner. The inventor has shown that when the anionic exchange is adjusted to a pH greater than the isoelectric point of alpha-lactalbumin that adsorption of alpha-lactalbumin is poor, specifically at a pH greater than pH 5. In addition, without wishing to be bound by theory, conducing the process when the anionic exchange is adjusted to a pH lower than the anionic exchange is adjusted to a pH lower than the isoelectric point of alpha-lactalbumin (lower than pH 4 for example) is likely to lead to denaturing and / or degrading of the proteins such as alpha-lactalbumin.

[0028] In some embodiments, adjusting of the anionic exchange resin is conducted using an effective amount of a nutritionally acceptable alkali and an effective amount of a nutritionally acceptable acid sequentially or simultaneously.

[0029] In some embodiments, adjusting of the anionic exchange resin is conducted using about 4 to about 8 column volumes of a nutritionally acceptable alkali and about 4 to about 8 column volumes of an aqueous nutritionally acceptable acid solution.

[0030] In some embodiments, the nutritionally acceptable alkali is sodium hydroxide and the nutritionally acceptable acid is acetic acid.

[0031] In some embodiments, adjusting of the anionic exchange resin is conducted using about 0.5 M to about a 1 .5 M solution sodium hydroxide solution and then about 0.1 to about 0.3 w / v aqueous acetic acid solution.

[0032] In some embodiments, adjusting of the anionic exchange resin is conducted using the following steps: washing with 1 column volume of water; 6 column volumes of a about 0.5M to about 1 M aqueous solution of sodium hydroxide; the anionic exchange resin; washing with 1 column volume of water; 6 column volumes of 0.2% w / w acetic acid solution; then washing with 5 column volumes water.

[0033] In some embodiments, adjusting of the anionic exchange resin is conducted using a clean-in-place (CIP) protocol.

[0034] In some embodiments, passage of the aqueous proteinaceous composition solution is at a rate about 0.3 CV / minute to about 1 .4 CV / minute.

[0035] In some embodiments, the solution enriched in beta-lactoglobulin is subjected to lyophilization to provide isolated beta-lactoglobulin.

[0036] In some embodiments, the dairy liquid is a milk, or a cream.

[0037] In some embodiments, the aqueous solution enriched in alpha-lactalbumin is subjected to an ultrafiltration.

[0038] In some embodiments, the ultrafiltration is conducted using a 50 kDa to about a 400 kDa filter.

[0039] In some embodiments, the aqueous solution enriched in alpha-lactalbumin is subject to a diafiltration.

[0040] In some embodiments, the diafiltration is conducted using about a 5 kDa to about a 50 kDa filter.

[0041] In some embodiments, the ultrafiltration is conducted sequentially and prior to the diafiltration.

[0042] In some embodiments, the ultrafiltration and the diafiltration are conducted prior to subjecting the aqueous solution enriched in alpha-lactalbumin to lyophilisation.

[0043] In some embodiments, the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein is washed with water prior to eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alphalactalbumin and at least one additional protein.Brief Description of Drawings

[0044] Figure 1 is a chromatogram showing typical constituents of an aqueous proteinaceous composition obtained as a permeate of a microfiltered dairy liquid prior to undergoing the process.

[0045] Figure 2 is a chromatogram showing the solution enriched in alphalactalbumin following embodiment of the process using about a 14 mS elution salt solution.

[0046] Figure 3 is a chromatogram showing the solution enriched in the at least one additional protein, in this case beta-lactoglobuin following embodiment of the process using about a 100 mS elution salt solution.

[0047] Figure 4 is a diagram showing a preferred embodiment of the process disclosed for the production of an enriched solution of alpha-lactalbumin and a soluionenriched in beta-lactoglobulin from a dairy liquid obtained as a permeate of a microfiltered dairy liquid.

[0048] Figure 5 is a chromatogram showing the showing typical constituents of an aqueous proteinaceous composition obtained as a permeate of a microfiltered dairy liquid prior to undergoing the process used in the comparative example.

[0049] Figure 6 is a chromatogram showing that following passing of the aqueous proteinaceous composition, the aqueous raffinate primarily comprises alphalactalbumin when the pH of the anionic exchange resin is adjusted not to the isoelectric point of alpha-lactalbumin, greater than pH 5 and in the comparative example between about pH 5.3 to pH 5.5. Therefore showing that alpha-lactalbumin is not adsorbed onto the anionic exchange resin under these conditions.

[0050] Figure 7 is a chromatogram showing that following passing of the aqueous proteinaceous composition, subsequent washing the anionic exchange resin with water primarily delivers alpha-lactalbumin. Therefore showing that alpha-lactalbumin is not adsorbed onto the anionic exchange resin under these conditions.

[0051] Figure 8 is a chromatogram showing that following washing with water, a protein desorption solution of 10 mS sodium chloride primarily delivers beta- lactoglobulin, therefore showing that alpha-lactalbumin is not selectively adsorbed onto the anionic exchange resin under these conditions.Detailed Description

[0052] In this specification a number of terms are used that are well known to a skilled addressee. Nevertheless, for the purposes of clarity a number of terms will be defined.

[0053] As used herein the term “aqueous solution enriched in” relates to an aqueous solution produced by the process disclosed by the applicant. The relative abundance of the relevant protein, for example alpha-lactalbumin or beta- lactoglobulin, is increased in relation any remain proteins as compared to the aqueous proteinaceous composition before the process is performed. Relativeabundances can be measured by various analytical techniques, for example high- performance liquid chromatograph (HPLC).

[0054] As used herein the aqueous proteinaceous composition can be obtained from “a dairy liquid”. In some embodiments the dairy liquid is derived from a dairy animal such as a cow, a water buffalo, a goat, or an ewe. In some embodiments the dairy liquid can be a milk or a cream. In some embodiments the dairy liquid can be raw, pasteurised or reconstituted. In some embodiments the dairy liquid has not been subjected to processing by use of an acid or an enzyme.

[0055] In some embodiments the aqueous proteinaceous composition derived from a permeate of microfiltration of a dairy liquid can have a pH in the range of about pH 6.3 to about pH 7.0. In some embodiments the pH of the aqueous proteinaceous composition can be any one of, or any combination of, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9, about pH 7.0. In some embodiments the pH of the aqueous proteinaceous composition can be about pH 6.4 to about pH 6.7. In some embodiments the pH of the aqueous proteinaceous composition can be about pH 6.5 to about pH 6.8. In some embodiments the dairy liquid not been subjected to processing by use of an acid or an enzyme therefore the pH of permeate can be close to the inherent pH of the permeate and / or diary liquid.

[0056] As used here the aqueous proteinaceous composition can be obtained as a “permeate of microfiltration”. The skilled person will appreciate that processing of dairy liquids can be conducted by physical means such as by microfiltration. The microfiltration results in a permeate and retentate. The retentate comprises material that does not pass through the membrane, for example micellar casein concentrate (MCC). The permeate contains material that does pass through and comprises proteins such as alpha-lactalbumin and beta-lactoglobulin as well as additional proteins.

[0057] The permeate of the proteinaceous composition can undergo further processing, for example, by use of further filtration steps and / or freeze drying to produce a powder. The powdered can be reconstituted before being subject to the process disclosed herein.

[0058] In some embodiments the pore size of a membrane for the microfiltration process can be about 0.1 pm to about 1 .0 pm. In some embodiments the pore size of a membrane for the microfiltration process can be any one of, or any combination of, 0.1 pm, about 0.25 pm, about 0.5 pm, about 1 .0 pm.

[0059] As used herein “at least partially selectively adsorbed” relates to a preference under the disclosed process conditions for the adsorption of the alphalactalbumin and the at least one additional protein into the resin from the aqueous proteinaceous composition and leading to an aqueous "raffinate”. The skilled person will appreciate that a raffinate can be understood as a solution that has had a protein or proteins at least partially removed. The raffinate can comprise lactose and other extraneous matter.

[0060] As used herein “at least one additional protein” refers another protein that would not be understood to the skilled person as alpha-lactalbumin. In some embodiments the at least one additional protein is of dairy origin. In some embodiments the at least one additional protein can be selected from bovine serum albumin, immunoglobulin A, immunoglobulin M, immunoglobulin, immunoglobulin C, lactoferrin, lactoperoxidase, glycomacropetide or mixtures thereof.

[0061] As used herein “anionic exchange resin” relates to a water insoluble resin functionalised with anionic groups. In some embodiments the resin is a high capacity quaternary amino group functionalised anionic exchange resin. In some embodiments the quaternary amino group functionalised anionic exchange resin is a anionic cross-linked agarose with dextran surface extender (also known as Capto Q) or a anionic Q Sepharose fast flow resin (also known as QF) or a mixture thereof. QFF is anionic exchange chromatography resin with a quaternary amine (Q) functional group [-CH2-N+(CH3)3] attached to Sepharose(crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed) strong anionic exchange resin. The Q group serves as a "strong anion exchanger" meaning that it is completely ionized over a broad pH range. The initial counterion in QFF resin is (SO4)2-. Capto Q resin comprises a rigid agarose matrix with attached quaternary amine (Q) functional groups. Similarly to QFF It is engineered for, selectivity, and flow properties, having a high binding capacity for proteins.

[0062] As used here “isoelectric point of alpha-lactalbumin” is the pH at which a molecule carries no net electrical charge. The skilled person will appreciate that the isoelectric point may be affected by the environment alpha-lactalbumin is contained in. For example, the aqueous proteinaceous composition is a biological product and may vary depending on batch due to seasonal changes and animal behavior. The composition may further comprise salts, short peptides, hormones, lipids et cetera that may affect the pH of the isoelectric point of alpha-lactalbumin.

[0063] In some embodiments the pH of the isoelectric point of alpha-lactalbumin can be about pH 4.0 to about pH 5.0. In some embodiments the isoelectric point of alpha-lactalbumin can be any one of, or any combination of about pH 4.0, about pH 4.1 , about pH 4.2 pH, about 4.3, pH about 4.4, pH about 4.5, pH about 4.6, about pH 4.7, pH about 4.8, pH 4.9, about pH 5.0. In some embodiments the pH of the isoelectric point of alpha-lactalbumin can be about pH 4.1 to about pH 4.9. In some embodiments the pH of the isoelectric point of alpha-lactalbumin can be about pH 4.2 to about pH 4.8. In some embodiments the pH of the isoelectric point of alphalactalbumin can be about pH 4.3 to about pH 4.7. In some embodiments the pH of the isoelectric point of alpha-lactalbumin can be less than about pH 5.0.

[0064] In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of about pH 4.0 to about pH 5.0. In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of any one of, or any combination of about pH 4.0, about pH 4.1 , about pH 4.2 pH, about 4.3, pH about 4.4, pH about 4.5, pH about 4.6, about pH 4.7, pH about 4.8, pH 4.9, about pH 5.0. In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of about pH 4.1 to about pH 4.9. In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of about pH 4.2 to about pH 4.8. In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of about pH 4.3 to about pH 4.7. In some embodiments the pH of the anionic exchange resin can be adjusted to an initial pH of less than about pH 5.0.

[0065] Adjustment of the pH of the anionic exchange resin resin can be measured by use of a pH probe. For example, when using a fast protein liquid chromatography a pH sensor gives the live pH of the system by measuring the pH of the solution as itpasses through the anionic exchange resin. The pH of the solution that has passed through the resin is used to ascertain the pH of the resin. Once the pH of the solution has stabilized it is assumed that the pH of the resin has been adjusted to the pH measured. The skilled person will appreciate that the pH may change during operation of the process. The “initial” pH refers to the pH observed before contacting of the proteinaceous composition.

[0066] The term "nutritionally acceptable" refers to an acid or an alkali that the relevant protein, for example alpha-lactalbumin, counterion or adduct retains the desired biological activity of the relevant protein and includes acid addition salts and alkali addition salts, adducts or entrained material. Nutritionally acceptable acids can be an inorganic acid or an organic acid. Examples of such inorganic acids can be selected from hydrochloric, sulfuric, and phosphoric acid. Suitable organic acids can be selected from citric, acetic, propanoic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic or arylsulfonic. In a similar vein, nutritionally acceptable acids alkalis can be selected from include NaOH, KOH, and the like.

[0067] An "effective amount" refers to at least an amount effective, at amounts necessary, to achieve the desired result. For example, the desired result may be adjustment of the anionic exchange resin to a relevant pH. The pH may be adjusted to be lower, higher or increase buffer capacity. An effective amount can be provided in one or more applications. The effective amount may vary according to the desired result and according to the particular batch of anionic exchange resin. Typically, the effective amount will fall within a relatively broad range. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity.

[0068] In some embodiments the anionic exchange resin adsorbed with the alphalactalbumin and the at least one additional protein can be separated from the aqueous raffinate after contacting of the aqueous proteinaceous composition with the anionic exchange resin. Separation may be conducted by filtration, by centrifugation, decantation or by use of a batch tank reactor.

[0069] In some embodiments the anionic exchange resin can be provided in an apparatus suitable for liquid flow therethrough. In some embodiments the anionic exchange resin is provided in a chromatography apparatus, such as achromatography column, or flow-chemistry equipment. In some embodiments contacting of the aqueous proteinaceous composition occurs during elution of the aqueous proteinaceous composition through the chromatography column.

[0070] In some embodiments eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and by use of an alpha-lactalbumin desorption solution to provide a solution enriched in alpha-lactalbumin. In some embodiments the alpha-lactalbumin desorption solution comprises about 1 mS to about 15 mS salt solution. In some embodiment the alpha-lactalbumin desorption solution comprises about 10 mS to about 15 mS salt solution. In some embodiments the alpha-lactalbumin desorption solution comprises about a 15 mS or about a 14 mS salt solution. In some embodiments the salt comprises sodium chloride. In some embodiments the alpha-lactalbumin desorption solution comprises about 7 mS to about 12 mS salt solution. In some embodiments the alpha-lactalbumin desorption solution comprises about 8 mS to about 11 mS salt solution. In some embodiments the alpha-lactalbumin desorption solution comprises about 9 mS to about 10 mS salt solution.

[0071] In some embodiments, the at least one additional protein is selected from the list consisting of beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide). The skilled person will appreciate that varying amounts of the additional proteins can be present in the permeate of a microfiltered diary liquid. This is due to seasonal changes, dietary variations, and the extent to which the diary liquid has been processed. The process can be tailored for selective desorption of the desired additional protein by choice of desorption solution and order of desorption as necessary. The conductivity (mS) of the aqueous salt desorption solution is adjusted so as to enable selective deposition of the protein.

[0072] In some embodiments, the at least one additional protein can be glycomacropetide (caseinomacropeptide). In some embodiments the glycomacropetide desorption solution comprises an about 1 mS to about 5 mS aqueous salt solution. In some embodiments the glycomacropetide desorption solution comprises an about 2 mS to about 4 mS aqueous salt solution. Use of theglycomacropetide desorption can provide a solution enriched in glycomacropetide (caseinomacropeptide).

[0073] In some embodiments the at least one additional protein can be bovine serum albumin. In some the bovine serum albumin desorption solution comprises an about 15 mS to an about 25 mS aqueous salt solution. In some the bovine serum albumin desorption solution comprises an about 16 mS to an about 18 mS aqueous salt solution. Use of the bovine serum albumin desorption solution can provide a solution enriched in bovine serum albumin.

[0074] In some embodiments, the at least one additional protein is immunoglobulin. In some embodiments, the immunoglobulin desorption solution comprises an about 20 mS to an about 40 mS aqueous salt solution. In some embodiments, the immunoglobulin desorption solution comprises an about 25 mS to an about 35 mS aqueous. Use of the immunoglobulin desorption solution can provide a solution enriched in immunoglobulin.

[0075] In some embodiments, the at least one additional protein is betalactoglobulin. In some embodiments, the beta-lactoglobulin desorption solution comprises an about 30 mS to an about 100 mS aqueous salt solution. In some embodiments, the beta-lactoglobulin desorption solution comprises an about 50 mS to an about 100 mS. In some embodiments, the beta-lactoglobulin desorption solution comprises an about 60 mS to an about 80 mS aqueous salt solution. Use of the beta- lactoglobulin desorption solution provides a solution enriched in beta-lactoglobulin.

[0076] The skilled person will appreciate that in some embodiments, any one or combination of the above steps may be performed sequentially or simultaneously. In some embodiments, eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein, by use of an alpha-lactalbumin desorption solution to provide a solution enriched in alphalactalbumin is performed first.

[0077] In some embodiments, eluting beta-lactoglobulin from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein, by use of an beta-lactoglobulin desorption solution to provide a solutionenriched in beta-lactoglobulin is performed after eluting alpha-lactalbumin from the anionic exchange resin.

[0078] In some embodiments, the order of eluting from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein can be sequential and can be as follows: glycomacropetide (caseinomacropeptide); alphalactalbumin; bovine serum albumin; immunoglobulin; and then beta-lactoglobulin.

[0079] In some embodiments, the at least one additional proteins are eluted from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein together using a desorption solution of about 100 mS to an about 120 mS.

[0080] The solutions enrichened in beta-lactoglobulin, immunoglobulin, bovine serum albumin and / or glycomacropetide (caseinomacropeptide) can be subjected to further processing steps such as, ultrafiltration, diafiltration and / or lyophilization (for example by spray drying) to provide isolated proteins.

[0081] In some embodiments the pH of the anionic exchange resin is adjusted using an effective amount of a nutritionally acceptable alkali and / or a nutritionally acceptable acid. In some embodiments an effective amount of a solution of sodium hydroxide and / or an effective amount of a solution of acetic acid can be used. The skilled person will appreciate that the effective amount can be monitored by use of a pH probe. The nutritionally acceptable alkali and a nutritionally acceptable acid can be added sequentially in any order.

[0082] In some embodiments adjusting of the anionic exchange resin can be conducted using about 4 to about 8 column volumes of an aqueous nutritionally acceptable alkali solution and then 4 to about 8 column volumes of an aqueous nutritionally acceptable acid solution. In some embodiments adjusting of the anionic exchange resin can be conducted using about 5 to about 7 column volumes of an aqueous nutritionally acceptable alkali solution and then 5 to about 7 column volumes of an aqueous nutritionally acceptable acid solution. In some embodiments adjusting of the anionic exchange resin can be conducted using about 6 of an aqueousnutritionally acceptable alkali solution and then about 6 column volumes of an nutritionally acceptable acetic acid solution.

[0083] In some embodiments adjusting of the anionic exchange resin is conducted using about 0.5 M to about 1 .5 M solution sodium hydroxide solution and then about 0.1 % w / v to about 0.3 % w / v aqueous acetic acid solution. In some embodiments adjusting of the anionic exchange resin is conducted using about 1 .0 M solution sodium hydroxide solution and then about 0.2 % w / v aqueous acetic acid solution.

[0084] In some embodiment adjusting of the anionic exchange resin is conducted using the following steps: washing with 1 column volume of water; 6 column volumes of a 1 M aqueous solution of sodium hydroxide; the anionic exchange resin; washing with 1 column volume of water; 6 column volumes of 0.2% w / w acetic acid solution; then washing with 5 column volumes water.

[0085] In some embodiments adjusting of the anionic exchange resin is conducted using a clean-in-place (CIP) protocol, particularly when a fast protein liquid chromatography system is used.

[0086] In some embodiments the feed rate of the aqueous proteinaceous composition can be about 0.3 CV / minute to about 1 .4 CV / minute. In some embodiments the feed rate of the aqueous proteinaceous composition can be about 0.5 to about 1 ,2CV / minute. In some embodiments the feed rate of the aqueous proteinaceous composition can be about 1 .0 CV / minute.

[0087] In some embodiments the process can be conducted with a 500 L, 400 L, 380 L, 370 L, 360 L, 200 L or 100 L column volume. In some embodiments, the aqueous proteinaceous composition can be loaded at a rate of about 0.6 to about 1 .3 column volumes per minute. In some embodiments, the aqueous proteinaceous composition can be loaded at a rate of about 0.7 to about 1 .2 column volumes per minute. In some embodiments, the aqueous proteinaceous composition can be loaded at a rate of about 0.8 to about 1 .1 column volumes per minute. In some embodiments, the aqueous proteinaceous composition can be loaded at a rate of about 0.9 column volumes per minute.

[0088] In some embodiments the process can be conducted such that the aqueous proteinaceous composition can be loaded at a loading flow rate of about 400 mL / minute, about 380 mL / minute, about 370 mL / minute, about 360 mL / minute or 350 mL / minute.

[0089] In some embodiments the process can be conducted such that the aqueous elution of the alpha-lactalbumin or the at least one additional protein can be conducted at a rate of about 220 mL / minute, about 210 mL / minute, about 200, mL / minute, about 190 m / minute, about 180 mL / minute, or about 170 mL / minute. In some embodiments the aqueous solution enriched in alpha-lactalbumin is subject to ultrafiltration. In some embodiments ultrafiltration is conducted using a 50 kDa to about a 400 kDa filter. In some embodiments ultrafiltration is conducted using a 75 kDa to about a 350 kDa filter. In some embodiments ultrafiltration is conducted using a 100 kDa to about a 300 kDa filter. Ultrafiltration aims to at least partially remove remaining small proteins and peptides such as bovine serum albumin.

[0090] In some embodiments the aqueous solution enriched in alpha-lactalbumin is subject to diafiltration. In some embodiments the diafiltration is conducted using about a 5 kDa to about a 50 kDa filter. In some embodiments the diafiltration is conducted using about a 10 kDa to about a 40 kDa filter. Diafiltration aims to at least partially remove lactose, ashes and excess amount of water.

[0091] In some embodiments, the aqueous solution enriched in alpha-lactalbumin or beta-lactoglobulin is lyophilised to produce isolated alpha-lactalbumin or betalactoglobulin. In some embodiments, lyophilisation may be conducted by spray drying or freeze drying.

[0092] In some embodiments the aqueous solution enriched in alpha-lactalbumin is at a purity of about at least 60%. In some embodiments the aqueous solution enriched in alpha-lactalbumin is at a purity of about at least 70%. In some embodiments the aqueous solution enriched in alpha-lactalbumin is at a purity of about at least 80%. In some embodiments the aqueous solution enriched in alphalactalbumin is at a purity of about at least 85%. In some embodiments the aqueous solution enriched in alpha-lactalbumin is at a purity of about at least 90%. In some embodiments the aqueous solution enriched in alpha-lactalbumin is at a purity ofabout at least 95%. In some embodiments the aqueous solution enriched in alphalactalbumin is at a purity of about at least 99%. Purity of the solution may be measured by analytical techniques such as HPLC.

[0093] With reference to figure 5, a preferred embodiment of the applicant’s disclosed process is described. A aqueous proteinaceous composition obtained from a permeate of microfiltration of a dairy liquid comprising alpha-lactalbumin and at least one additional protein (for example beta-lactoglobulin) is provided, an anionic exchange resin, QFF resin, is provided in an apparatus suitable for liquid flow therethrough, such as a fast protein liquid chromatography system, the pH of the anionic exchange resin is adjusted to an initial pH of about the isoelectric point, preferably lower than about pH 5 or from about pH 4.5 to about pH 5, of alphalactalbumin by washing with 1 column volume of water; 6 column volumes of a 1 M aqueous solution of sodium hydroxide; the anionic exchange resin; washing with 1 column volume of water; 6 column volumes of an acetic acid solution; then washing with 5 column volumes water. The anionic exchange resin is then contacted with the aqueous proteinaceous composition during passage through apparatus suitable for liquid flow therethrough at a rate of 0.11 CV / minute, so that the alpha-lactalbumin and the at least one additional protein are at least partially selectively adsorbed onto the anionic exchange resin. This provides an aqueous raffinate comprising lactose for example, and an anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein. Eluting of the alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin can be conducted by use of a 14 mS salt solution to provide a solution enriched in alpha-lactalbumin. The aqueous solution enriched in alpha-lactalbumin can then subjected to an ultrafiltration, using a 200 kDa filtration membrane to provide an ultrafiltered retentate. The ultrafiltered retentate can then subject to a diafiltration using a 5 kDa filtration membrane to provide a diafiltered retentate. The applicant observed that alpha-lactalbumin was present in this ultrafiltered permeate was present in this diafiltered retentate in a about 93 % to 96 % as measured by HPLC. This solution can then be subjected to lyophilisation to provide the isolated alpha-lactalbumin, for example by spray drying. The at least one additional protein can be eluted using, for example, a 100 mS salt solution to provide a solution enriched in beta-lactoglobulin.

[0094] In a further embodiments, the applicant discloses a process for the production of an aqueous solution enriched in a protein, wherein the protein selected from the list consisting of alpha-lactalbumin, beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide) from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid, wherein the proteinaceous composition solution having a pH of about 6.3 to about pH 7.0, the process comprising: providing an anionic exchange resin and adjusting the anionic exchange resin to an initial pH of about the isoelectric point of alpha-lactalbumin, preferably lower than about pH 5 or from about pH 4.5 to about pH 5, contacting the anionic exchange resin with the aqueous proteinaceous composition so the protein is selectively adsorbed onto the anionic exchange resin, so providing an aqueous raffinate and an anionic exchange resin adsorbed with the protein, separating the anionic exchange resin adsorbed with the protein from the aqueous raffinate, selectivity eluting the protein by use of an aqueous salt desorption solution to provide the solution enriched in the protein. The conductivity (mS) of the aqueous salt desorption solution is adjusted so as to enable selective deposition of the protein.Examples

[0095] Example 1 - production of an aqueous solution enriched in alphalactalbumin from an aqueous proteinaceous composition obtained from microfiltration of a dairy liquid

[0096] The experiment utilised a strong anion exchanger resin packed in a 700 ml radial column, which was connected to a Fast Protein Liquid Chromatography (FPLC) system. A HPLC trace the aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid (cow’s milk) solution prior to treatment is shown in figure 1 .

[0097] Initial anionic exchange resin adjustment

[0098] The resin bed underwent adjustment using the following clean-in-place procedure: washing with water (1 column volumes), addition of 1 M aqueous solution of NaOH (6 column volumes), washing with water (1 column volume), addition ofacetic acid 0.2% w / w (6 column volumes), and water (5 column volumes). This adjusted the pH of the column to about the isoelectric point of alpha-lactalbumin, to a pH of about 4.6.

[0099] Contacting the anionic exchange resin with the aqueous proteinaceous composition

[0100] Subsequently, the aqueous proteinaceous composition solution was passed through the column at a rate of 80 ml / min for a total of 10 column volumes. Thus, the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein.

[0101] The anionic exchange resin was then washed with water.

[0102] Eluting alpha-lactalbumin and the at least one additional protein from the anionic exchange resin

[0103] Alpha-lactalbumin was then eluted using a 10 mS NaCI solution to provide an enriched aqueous solution of alpha-lactalbumin. The eluate was collected and characterised using HPLC as shown in figure 2.

[0104] Following the 10 mS elution, the at least one additional protein, the majority being beta-lactoglobulin, and some additional proteins were eluted with a 100 mS sodium chloride solution. This eluate was also collected and characterised analytically. The representative isolated protein concentrations are presented in the table below.

[0105] The applicant observed by HPLC that the enriched aqueous solution of alpha-lactalbumin contains about 86 % to about 87% as measured by HPLC.

[0106] The aqueous solution enriched in alpha-lactalbumin can then subjected to an ultrafiltration, using a 200 kDa filtration membrane to provide an ultrafiltered retentate. The ultrafiltered retentate can then subject to a diafiltration using a 5 kDa filtration membrane to provide a diafiltered retentate. The applicant observed that alpha-lactalbumin was present in this ultrafiltered retentate was present in this diafiltered retentate in a about 93 % to 96 % as measured by HPLC.

[0107] Comparative Example 1 - use of an initial anionic exchange resin adjustment with a pH greater than the isoelectric point of alpha-lactalbumin

[0108] This comparative example was conducted using utilised a strong anion exchanger resin packed in a 700 ml radial column, which was connected to a Fast Protein Liquid Chromatography (FPLC) system. A HPLC trace of the aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid (cow’s milk) solution prior to treatment is shown in figure 5.

[0109] In this comparative example the the anionic exchange was adjusted to an initial pH that was not about the isoelectric point of alpha-lactalbumin with the aqueous proteinaceous composition. The pHs examined were from about pH 5.3 to about pH 5.5.

[0110] The aqueous proteinaceous composition was then contacted with the pH adjusted resin in accordance with the claimed invention. Figure 6 shows following elution, the raffinate conducted under these conditions delivers primarily alphalactalbumin thus showing that by not adjusting the column to the claimed conditions does not lead to adsorption of alpha-lactalbumin onto the resin.

[0111] Following elution, the resin was then washed with water as shown in Figure 7. This wash primarily contained alpha-lactalbumin again showing that this pH outside of the claimed invention does not lead to adsorption of alpha-lactalbumin onto the resin.

[0112] Following the water wash, the resin was then contacted with a 10 mS sodium chloride solution to desorb any protein that had become adsorbed onto theresin. As shown in Figure 8, following this solution primarily contained betalactoglobulin.

[0113] Thus the inventor has surprisingly found that by carefully adjusting the pH of the resin prior to elution with an aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid (preferably MCC whey derived from cow’s milk), alpha-lactalbumin can be adsorbed selectively onto resin. The combination of use of the aqueous proteinaceous composition obtained as a permeate from microfiltration of a dairy liquid that has not been enzymatically or chemically treated, in combination with the pH adjusted resin can deliver alphalactalbumin in high purity and in an efficient manner. Additional proteins can then also be selectively desorbed by using desorption solutions of specific mS concentrations.

[0114] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0115] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub-range is explicitly recited. The statement “about X% to Y%” has the same meaning as “about X% to about Y%,” unless indicated otherwise.

[0116] The term “about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of + / - 10% or less, + / - 5% or less, + / - 1 % or less, or + / - 0.1 % or less of and from the numerical value or range recited or claimed.

[0117] It is also to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0118] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter foundthroughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0119] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0120] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0121] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0122] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications.The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0123] Future patent applications may be filed in Australia or overseas on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and / or by claiming a continuation status. It is to be understood that the following provisional claims are provided by way of example only and are not intended to limit the scope of what may be claimed in anysuch future application. Furthermore, the claims should not be considered to limit the understanding of (or exclude other understandings of) the invention inherent in the present disclosure. Features may be added to or omitted from the provisional claims at a later date, so as to further define the invention.

Claims

1 . A process for the production of an aqueous solution enriched in alphalactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid, wherein the aqueous proteinaceous composition comprises alpha-lactalbumin, and at least one additional protein and wherein the proteinaceous composition solution has a pH of about 6.3 to about pH 7.0, the process comprising: providing an anionic exchange resin and adjusting the anionic exchange resin to an initial pH of about the isoelectric point of alpha-lactalbumin, contacting the anionic exchange resin having the initial pH of about the isoelectric point of alpha-lactalbumin with the aqueous proteinaceous composition so that the alpha-lactalbumin and the at least one additional protein are at least partially selectively adsorbed onto the anionic exchange resin, so providing an aqueous raffinate and an anionic exchange resin adsorbed with the alpha-lactalbumin the at least one additional protein, separating the anionic exchange resin adsorbed with the alpha-lactalbumin the at least one additional protein from the aqueous raffinate, eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and the at least one additional protein, by use of an alphalactalbumin desorption solution to provide the solution enriched in alpha-lactalbumin.

2. The process of claim 1 , wherein the anionic exchange resin is provided in an apparatus suitable for liquid flow therethrough.

3. The process of claim 1 or claim 2, wherein contacting of the anionic exchange resin with the aqueous proteinaceous composition solution occurs during passage through apparatus suitable for liquid flow therethrough.

4. A process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition obtained as a permeate of microfiltration of a dairy liquid,wherein the aqueous proteinaceous composition comprises alpha-lactalbumin, and at least one additional protein and wherein the proteinaceous composition solution has a pH of about 6.3 to about pH 7.0, the process comprising: providing an anionic exchange resin in an apparatus suitable for liquid flow therethrough and adjusting the anionic exchange resin to an initial pH of about the isoelectric point of alpha-lactalbumin, contacting the anionic exchange resin having the initial pH of about the isoelectric point of alpha-lactalbumin with the aqueous proteinaceous composition during passage through apparatus suitable for liquid flow, therethrough so that the alpha-lactalbumin and at least one additional protein are at least partially selectively adsorbed onto the anionic exchange resin, so providing an aqueous raffinate separated from an anionic exchange resin adsorbed with the alphalactalbumin and at least one additional protein, eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein by use of an alpha-lactalbumin desorption solution to provide a solution enriched in alpha-lactalbumin, subjecting the aqueous solution enriched in alpha-lactalbumin to lyophilisation to provide the isolated alpha-lactalbumin.

5. The process of any one of claims 1 to 4, wherein the apparatus suitable for liquid flow therethrough is a chromatography apparatus.

6. The process of any one of claims 1 to 5, wherein the permeate of microfiltration has been subjected to filtration with a semipermeable membrane pore size of about 0.1 pm to about 1 .0 pm.

7. The process of any one of claims 1 to 6, wherein the alpha-lactalbumin desorption solution comprises an about 5 mS to about 15 mS salt solution.

8. The process of any one of claims 1 to 7, further comprising eluting of the at least one additional protein from the anionic exchange resin adsorbed with the alphalactalbumin and the at least one additional protein by use of an at least one additionalprotein desorption solution to provide a solution enrichened in the at least one additional protein.

9. The process of claim 8, wherein the at least one additional protein is selected from the list consisting of beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

10. The process of claim 9, wherein the at least one additional protein is beta- lactoglobulin and the beta-lactoglobulin desorption solution comprises an about 30 mS to an about 100 mS aqueous salt solution to provide a solution enriched in beta- lactoglobulin.11 . The process of any one of claims 8 to 10, wherein the at least one additional protein is glycomacropetide (caseinomacropeptide) and wherein the glycomacropetide desorption solution comprises an about 2 mS to 4 mS aqueous salt solution to provide a solution enriched in glycomacropetide (caseinomacropeptide); wherein the at least one additional protein is bovine serum albumin and wherein the bovine serum albumin desorption solution comprises an about 15 mS to an about 25 mS aqueous salt solution to provide a solution enriched in bovine serum albumin; or wherein the at least one additional protein is immunoglobulin and the immunoglobulin desorption solution comprises an about 20 mS to an about 40 mS aqueous salt solution to provide a solution enriched in immunoglobulin.

12. The process of any one of claims 8 to 11 , wherein the order of eluting from the anionic exchange reins is sequential and as follows: glycomacropetide (caseinomacropeptide); alpha-lactalbumin; bovine serum albumin; immunoglobulin; and then beta-lactoglobulin.

13. The process of any one of claims 8 to claim 12, wherein the salt comprises sodium chloride.

14. The process of any one of claims 1 to 13, wherein the initial pH of the anionic exchange resin having the initial pH of about the isoelectric point of alpha-lactalbumin following adjustment is about pH 4.0 to about pH 5.0.

15. The process of any one of claims 1 to 14, wherein adjusting of the anionic exchange resin is conducted using an effective amount of a nutritionally acceptable alkali and an effective amount of a nutritionally acceptable acid sequentially or simultaneously.

16. The process of any one of claims 1 to 15, wherein adjusting of the anionic exchange resin is conducted using about 4 to about 8 column volumes of a nutritionally acceptable alkali and about 4 to about 8 column volumes of an aqueous nutritionally acceptable acid solution.

17. The process of claim 15 or claim 16, wherein the nutritionally acceptable alkali is sodium hydroxide and the nutritionally acceptable acid is acetic acid.

18. The process of claim 17, wherein adjusting of the anionic exchange resin is conducted using about 0.5 M to about a 1 .5 M solution sodium hydroxide solution and then about 0.1 % w / v to about 0.3 w / v aqueous acetic acid solution.

19. The process of any one of claims 1 to 18, wherein adjusting of the anionic exchange resin having the initial pH of about the isoelectric point of alpha-lactalbumin is conducted using the following steps: washing with 1 column volume of water;6 column volumes of a about 0.5 M to about 1 .5 M aqueous solution of sodium hydroxide; the anionic exchange resin; washing with 1 column volume of water;6 column volumes of 0.2% w / w acetic acid solution; then washing with 5 column volumes water.

20. The process of any one of claims 1 to 19, adjusting of the anionic exchange resin is conducted using a clean-in-place (CIP) protocol.21 . The process of any one of claims 2 to 17, wherein passage of the aqueous proteinaceous composition solution through the apparatus suitable for liquid flow therethrough is at a rate about 0.3 CV / minute to about 1 .4 CV / minute.

22. The process of any one of claims 1 to 21 , wherein the dairy liquid is a milk, a cream or a reconstituted milk product.

23. The process of any one of claims 1 to 22, wherein the aqueous solution enriched in alpha-lactalbumin is subjected to an ultrafiltration.

24. The process of claim 23, wherein the ultrafiltration is conducted using a 50 kDa to about a 400 kDa filter.

25. The process of any one of claims 1 to 22, wherein the aqueous solution enriched in alpha-lactalbumin is subject to a diafiltration.

26. The process of claim 25, wherein the diafiltration is conducted using about a 5 kDa to about a 50 kDa filter.

27. The process of any one of claims 24 to 26, wherein the ultrafiltration is conducted sequentially and prior to the diafiltration.

28. The process of any one of claims 24 to 27, wherein the ultrafiltration and the diafiltration are conducted prior to subjecting the aqueous solution enriched in alphalactalbumin to lyophilisation.

29. The process of any one of claims 1 to 28, wherein the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein is washed with water prior to eluting alpha-lactalbumin from the anionic exchange resin adsorbed with the alpha-lactalbumin and at least one additional protein.

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