Alpha-lactalbumin process (i)

Abstract

The present invention relates a process for the production of an aqueous solution enriched in alpha-lactalbumin from an aqueous proteinaceous composition, such as whey, comprising alpha-lactalbumin and at least one additional protein. The present invention further relates to process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition.

Description

Title of InventionAlpha-lactalbumin process (I)

[0001] This application claims priority from Australian Provisional Patent Application No. 2024901361 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 production of an aqueous solution enriched in alpha-lactalbumin from an aqueous proteinaceous composition, such as whey, comprising alpha-lactalbumin and at least one additional protein. The present invention further relates to process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition.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.

[0004] Alpha-lactalbumin is often produced on scale as it is a constituent of aqueous proteinaceous compositions derived from animals. Typically, those proteinaceous compositions, such as whey or reconstituted protein solutions, are derived from milk processing operations in the dairy industry and comprise additional proteins such as beta-lactoglobulin, immunoglobulins and lactoferrin. Often, acid or enzymes are used to produce proteinaceous compositions resulting in acidic or natural solutions.

[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 have previously been described. Other proteins present also have commercial value, such as immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

[0006] Previously described techniques for producing alpha-lactalbumin from such compositions have used a combination of an acidic pH and cationic exchange resin with the aim of separating particularity beta-lactoglobulin and alpha-lactalbumin from aqueous proteinaceous compositions. However, effective selective separation of alpha-lactalbumin from proteinaceous compositions remains a challenge.

[0007] The challenge of effective separation is due at least in part to poor separation of alpha-lactalbumin from other proteins, denaturing of alpha-lactalbumin due to incompatibility with the separation system and / or separation processes that require many steps and / or use of multiple solvent systems.

[0008] Accordingly, there remains an opportunity to develop alternative processes for the selective separation of alpha-lactalbumin from aqueous proteinaceous compositions that contain this protein.

[0009] 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 matters formed 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

[0010] 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 comprising alpha-lactalbumin and at least one additional protein.

[0011] 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 comprising alpha-lactalbumin and at least one additional protein. The process comprises contacting the aqueous proteinaceous composition with an anionic exchange resin, the pH during contacting of the aqueous proteinaceous composition with the anionic exchange resin can be maintained at a pH of about 6.0 to about 7.0 so that the at least one additional protein is at least partially selectively adsorbed onto the anionic exchange resin. Thus, providing the aqueous solution enriched in alpha-lactalbumin and an anionic exchange resin adsorbed with the at least one additional protein.

[0012] Without wishing to be bound by theory, the applicant has observed that the choice of an anionic exchange resin and specific pH range enables effective separation of alpha-lactalbumin from the at least one additional protein in proteinaceous compositions. The applicant has shown that at more acidic pHs below 6.0 the separation of alpha-lactalbumin is poor, for example poor separation with beta-lactoglobulin was observed. In addition, at more basic pHs such as above pH 7.0 the separation is also poor due at least in part to structural changes in alphalactalbumin, leading to degradation and / or denaturation. The choice of pH and anionic exchange resin enables the selective adsorption of the at least one additional protein onto the resin providing the aqueous solution enriched in alpha-lactalbumin in an efficient manner. The process is efficient in terms of step count and the number of solution systems used to obtain the aqueous solution enriched in alpha-lactalbumin.

[0013] In some embodiments the anionic exchange resin adsorbed with the at least one additional protein is separated from the aqueous solution enriched in alphalactalbumin. Separation may be conducted by filtration, centrifugation, decantation or use of a batch tank reactor.

[0014] In some embodiments the anionic exchange resin can be provided in an apparatus suitable for liquid flow therethrough. In some embodiments contacting aqueous proteinaceous composition occurs during passage of the aqueous proteinaceous composition through a chromatography apparatus as well as separation of the solution enriched in alpha-lactalbumin. Use of a chromatographyapparatus, such as a chromatography column is a convenient and efficient apparatus for conducting the process disclosed by the applicant.

[0015] In some embodiments any insoluble components of the aqueous proteinaceous composition are removed prior to contacting with the anionic exchange resin. Removal of insoluble components improves the contacting of the at least one additional protein with the anionic exchange resin enabling better selective adsorption of the at least one additional protein.

[0016] In some embodiments the aqueous solution enriched in alpha-lactalbumin can be lyophilised to produce isolated alpha-lactalbumin. Isolated solid alphalactalbumin is convenient for transport and distribution. Lyophilisation can be conduced by spray drying or freeze drying.

[0017] The present applicants have further conducted research aimed at developing a process for the isolation of alpha-lactalbumin from an aqueous proteinaceous composition comprising alpha-lactalbumin and at least one additional protein.

[0018] In a further aspect of the invention there is provided a process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition comprising alphalactalbumin and at least one additional protein. The process comprising, providing an anionic exchange resin in an apparatus suitable for liquid flow through, removal of any insoluble components of the aqueous proteinaceous composition prior to contacting with the anionic exchange resin, contacting the aqueous proteinaceous composition with the anionic exchange resin by eluting the aqueous proteinaceous composition through the apparatus suitable for liquid flow therethrough. The pH during contacting of the aqueous proteinaceous composition with the anionic exchange resin is maintained at a pH of about 6.0 to about 7.0 so that the at least one additional protein is at least partially selectively adsorbed onto the anionic exchange resin. Thus providing an aqueous solution enriched in alpha-lactalbumin separate from an anionic exchange resin adsorbed with the at least one additional protein, subjecting the aqueous solution enriched in alpha-lactalbumin to lyophilisation to provide the isolated alpha-lactalbumin. Lyophilisation can be conducted by spray drying or freeze drying.

[0019] Without wishing to be bound by theory, the applicant has observed that the choice of an anionic exchange resin and specific pH range enables effective separation of alpha-lactalbumin from the at least one additional protein in proteinaceous compositions. The disclosed process enables isolation of alphalactalbumin in an efficient manner. The process is efficient in terms of step count and the number of solutions systems used. In some embodiments, the process conveniently can be carried out using a chromatography column.

[0020] In some embodiments removal of any of the insoluble components present can be conducted by filtration. In some embodiments filtration can be conducted using a centrifugation for example. The choice of filter allows for removal of insoluble matter commonly found for example in dairy liquids such as lipid agglomerates which block interaction of the at least one additional protein with the anionic exchange resin.

[0021] In some embodiments passage of the aqueous proteinaceous composition can be conducted at a rate of about 0.3 to about 1 .4 column volumes per minute.

[0022] In some embodiments, the anionic exchange resin can be loaded with I Q- 70 column volumes of the proteinaceous composition.

[0023] In some embodiments the aqueous proteinaceous composition contains about 0.5 g / L to about 1 .5 g / L alpha-lactalbumin

[0024] In some embodiments the at least one additional protein can be eluted from the anionic exchange resin adsorbed with the at least one additional protein to provide an aqueous solution enriched in the at least one additional protein following eluting of the aqueous proteinaceous composition. By using the disclosed process, it can be possible to isolate alpha-lactalbumin as well as the at least one additional protein. By use of equipment suitable for liquid to flow therethrough, such as a chromatography column, this can be conducted conveniently in a step-wise manner.

[0025] In some embodiments, the aqueous proteinaceous composition can be adjusted to a pH of about 6.0 to about 7.0 prior to contacting with the anionic exchange resin. In some embodiments, adjusting of the pH of the aqueous proteinaceous is conducted using an effective amount of sodium hydroxide solutionand / or acetic acid. Some aqueous proteinaceous compositions inherently are within the pH range of 6.0 to 7.0, some however require adjustment to this pH which aids in selective adsorption of the at least one additional protein onto the anionic exchange resin. In some embodiments when inherently acidic aqueous proteinaceous compositions, such as whey, are used this pH adjustment may be necessary. In some embodiments the pH may not need adjusting because the aqueous proteinaceous compositions may inherently be in this range. A buffer can be included to maintain this pH range.

[0026] In some embodiments, the anionic exchange resin can be adjusted to an initial pH of about 6.0 to about 7.0 prior to contacting with the aqueous proteinaceous composition. In some embodiments, the pH of the anionic exchange resin can be adjusted using an effective amount of sodium hydroxide solution and then an effective amount of acetic acid. Adjusting the pH of the anionic exchange resin aids in selective adsorption of the at least one additional protein onto the anionic exchange resin.

[0027] In some embodiments, the at least one additional protein can be selected from is selected from the list consisting of beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

[0028] In some embodiments, the at least one additional protein can be 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.

[0029] In some embodiments, the at least one additional protein can be 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 least one additional protein can be bovine serum albumin and wherein the bovine serum albumin desorption solution can be 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 desorptionsolution can be an about 20 mS to an about 40 mS aqueous salt solution to provide a solution enriched in immunoglobulin.

[0030] In some embodiments, the order of eluting from the anionic exchange resin is sequential and as follows: glycomacropetide (caseinomacropeptide); bovine serum albumin; immunoglobulin; and then beta-lactoglobulin. The selective desorption of solutions enriched in the additional proteins can be subjected to additional processing to deliver isolated proteins. This enables an efficient process for isolating several proteins using in a single operation of the process disclosed.

[0031] In some embodiments the aqueous proteinaceous composition can be a dairy liquid. In some embodiments the diary liquid can be whey. For example a sweet whey, a salt whey or an acidic whey.

[0032] In some embodiments the aqueous proteinaceous composition can be subjected to defatting prior to contacting with the anionic exchange resin. In some embodiments defatting can be conducted by filtration.

[0033] In some embodiments the anionic exchange resin can be a high capacity quaternary amino group functionalised anion exchange resin. In some embodiments the quaternary amino group functionalised anion exchange resin can be a strong anionic cross-linked agarose with dextran surface extender, a strong anionic crosslinked agarose beads or a mixture thereof.

[0034] In some embodiments the aqueous solution enriched in alpha-lactalbumin can be subject to ultrafiltration. In some embodiments ultrafiltration can be conducted using a 50 kDa to about a 400 kDa filter.

[0035] In some embodiments the aqueous solution enriched in alpha-lactalbumin is subject to diafiltration. In some embodiments diafiltration can be conducted using about a 5 kDa to about a 50 kDa filter.

[0036] In some embodiments, ultrafiltration then diafiltration is performed sequentially.Brief Description of Drawings

[0037] Figure 1 is a chromatogram showing typical constituents of raw whey prior at pH5.5 to undergoing the process.

[0038] Figure 2 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following an embodiment of the process disclosed when conducted at a pH of 6.5.

[0039] Figure 3 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following an embodiment of the process disclosed when conducted at a pH of 7.0.

[0040] Figure 4 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following an embodiment of the process disclosed when conducted at a pH of 6.0.

[0041] Figure 5 is a chromatogram showing poor separation of alpha-lactalbumin following the process when conducted using the general procedure at a pH of 5.5.

[0042] Figure 6 is a chromatogram showing poor separation of alpha-lactalbumin following the process when conducted using the general procedure at a pH of 7.5.

[0043] Figure 7 is a chromatogram showing poor separation of alpha-lactalbumin following the process when conducted using the general procedure at a pH of 3.7 and using a cationic resin.

[0044] Figure 8 is a diagram showing a preferred embodiment of the process disclosed for isolation of alpha-lactalbumin from an aqueous proteinaceous composition.

[0045] Figure 9 chromatogram of the solution obtained by elution of the at least one additional protein (mostly beta-lactoglobulin) following the process when the pH maintained between about pH 6 to about pH 7.Detailed Description

[0046] 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.

[0047] As used herein the term “aqueous solution enriched” in alpha-lactalbumin or an additional protein relates to an aqueous solution produced by the process disclosed by the applicant. The relative abundance of alpha-lactalbumin or the additional protein is increased in relation to the any remaining proteins present as compared to the aqueous proteinaceous composition before the process is performed. Relative abundances can be measured by various analytical techniques, for example high-performance liquid chromatograph (HPLC).

[0048] As used herein “at least partially selectively adsorbed” relates to a preference under the disclosed process conditions for the adsorption of the at least additional protein into the resin from the aqueous proteinaceous composition over alpha-lactalbumin. The selective adsorption is reflected in the enrichment of alphalactalbumin found in the enriched in alpha-lactalbumin after contacting of the aqueous proteinaceous composition with the anionic exchange resin.

[0049] As used herein the “aqueous proteinaceous composition” relates to an any aqueous liquid comprising alpha-lactalbumin and at least one additional protein.

[0050] In some embodiments the aqueous proteinaceous composition may be a dairy liquid. In some embodiments the dairy liquid is derived from dairy animals such as a cow, a water buffalo, a goat, or a ewe. In some embodiments the dairy

[0051] liquid is milk, cream, buttermilk or whey. In some embodiments the dairy liquid can be raw, pasteurised or reconstituted.

[0052] In some embodiments the proteinaceous composition is whey derived from the processing of a milk produced by a cow. In some embodiments the dairy liquid has been subjected to processing. In some embodiments the proteinaceous composition is the product of an acidic process, for example cheese making, resulting in an acidic proteinaceous composition. In some embodiments the proteinaceous composition is the product of an enzymatic process, for example cheese making andcan result in composition having a pH closer to neutral. In some embodiments the proteinaceous composition is a whey produced as a filtrate following treatment and separation of an animal milk with an acid or an enzyme. In some embodiments the proteinaceous composition can be a reconstituted proteinaceous composition, for example reconstituted milk powder or whey powder.

[0053] 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 is selected from beta-lactoglobulin, bovine serum albumin, immunoglobulin A, immunoglobulin M, immunoglobulin, immunoglobulin C, lactoferrin, lactoperoxidase, glycomacropetide or mixtures thereof.

[0054] 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 Yeah (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.

[0055] As used herein “maintained at a pH of about 6.0 to about 7.0” relates to the pH of the anionic exchange resin or proteinaceous composition. In some embodiments the pH can be any one of, or any combination of, about 6.00, about 6.05 about 6.1 , about 6.15, about 6.2, about 6.25, about 6.3, about 6.35, about 6.4, about 6.44, about 6.5, about 6.55, about 6.6, about 6.65, about 6.7, about 6.75, about 6.8, about 6.85, about 6.9 about 6.95 or about 7.00. In some embodiments the pHcan be between about 6.1 to about 6.9. in some embodiments the pH is between about 6.2 to about 6.8. In some embodiments the pH is between about 6.3 to about 6.7. lin some embodiments the pH can be between about 6.4 to about 6.6. In some embodiments the pH can be about 6.0 to 6.5. In some embodiments the pH can be between about 6.1 to about 6.4. In some embodiments the pH can be between about 6.3 to about 6.4. In some embodiments the pH can be between about 6.5 to about 7.0. In some embodiments the pH can be between about 6.6 to about 6.9. In some embodiments the pH is between about 6.7 to about 6.8. To aid in maintaining the pH during the process, the pH of the anionic exchange resin and / or the pH proteinaceous composition can be adjusted prior to conducting the process as necessary.

[0056] Adjustment of the initial 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 it passes 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.

[0057] In some embodiments the anionic exchange resin pH can be adjusted as discussed above prior to contacting with the proteinaceous composition. In some embodiments the pH can be conducted using an effective amount of a nutritionally acceptable alkali and / or acid.

[0058] 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 an aqueous solution of sodium hydroxide and / or an effective amount of an aqueous solution of acetic acid 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 for example.

[0059] In some embodiments the proteinaceous composition pH can be adjusted as discussed above prior to contacting the anionic exchange resin. In some embodiments the pH of the proteinaceous composition can be adjusted using an effective amount of sodium hydroxide and / or acetic acid. In some embodiments an effective amount 1 M solution of sodium hydroxide and / or a 1 M 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 and trial and error. The skilled person will appreciate that other suitable acid / base pH adjustment systems may be used such as effective amounts potassium hydroxide and / or citric acid.

[0060] 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, arylsulfonic. In a similar vein, nutritionally acceptable acids alkalis can be selected from include NaOH, KOH, and the like.

[0061] 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 pH of either the proteinaceous composition or the anionic exchange resin. The pH may be adjusted to be lower, higher or to 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 product given that the proteinaceous composition is often derived from processing of animal products and may vary. 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.

[0062] In some embodiments the proteinaceous composition may already be within the range of pH of about 6.0 to about 7.0. In this case no adjustment may benecessary nevertheless an effective amount of acid / base may be added to increase the buffer capacity of the solution.

[0063] In some embodiments additional amounts of acid / base can be added during contacting the aqueous proteinaceous composition with an anionic exchange resin to maintain the pH of about 6.0 to about 7.0.

[0064] In some embodiments the anionic exchange resin adsorbed with the at least one additional protein is separated from the aqueous solution enriched in alphalactalbumin after contacting of the aqueous proteinaceous composition with the anionic exchange resin. Separation may be conducted by filtration, centrifugation or decantation. In some embodiments the resin may be washed with additional aqueous solution, the solution may be buffered as described above at a pH of about pH of about 6.0 to about 7.0.

[0065] 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 additional protein.

[0066] In some embodiments, the at least one additional protein can be glycomacropetide (caseinomacropeptide). In some embodiments the glycomacropetide desorption solution can be an about 1 mS to about 5 mS aqueous salt solution. In some embodiments the glycomacropetide desorption solution can be an about 2 mS to about 4 mS aqueous salt solution. Use of the glycomacropetide desorption can provide a solution enriched in glycomacropetide (caseinomacropeptide).

[0067] In some embodiments the at least one additional protein can be bovine serum albumin. In some the bovine serum albumin desorption solution can be an about 15 mS to an about 25 mS aqueous salt solution. In some the bovine serum albumin desorption solution can be 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.

[0068] In some embodiments, the at least one additional protein can be immunoglobulin. In some embodiments, the immunoglobulin desorption solution can be an about 20 mS to an about 40 mS aqueous salt solution. In some embodiments, the immunoglobulin desorption solution can be an about 25 mS to an about 35 mS aqueous. Use of the immunoglobulin desorption solution can provide a solution enriched in immunoglobulin.

[0069] In some embodiments, the at least one additional protein can be betalactoglobulin. In some embodiments, the beta-lactoglobulin desorption solution can be an about 30 mS to an about 100 mS aqueous salt solution. In some embodiments, the beta-lactoglobulin desorption solution is an about 50 mS to an about 100 mS. In some embodiments, the beta-lactoglobulin desorption solution is 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.

[0070] The skilled person will appreciate that in some embodiments, any one or combination of the above steps may be performed sequentially or simultaneously.

[0071] In some embodiments, the order of eluting from the anionic exchange resin adsorbed with the at least one additional protein can be sequential eluted as follows: glycomacropetide (caseinomacropeptide); alpha-lactalbumin; bovine serum albumin; immunoglobulin; and then beta-lactoglobulin.

[0072] In some embodiments, the at least one additional proteins are eluted from the anionic exchange resin adsorbed the at least one additional protein together using a desorption solution of about 100 mS to an about 120 mS.

[0073] 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.

[0074] In some embodiments the anionic exchange resin can be regenerated and reused after elution of the at least one additional protein.

[0075] 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 column or flow-chemistry equipment.

[0076] In some embodiments contacting of the aqueous proteinaceous composition occurs during passage of the aqueous proteinaceous composition through the chromatography column. In some embodiments the resin may be contacted with additional aqueous eluant following and / or during passage of the aqueous proteinaceous composition, the eluant may be adjusted as described above to a pH of about 6.0 to about 7.0.

[0077] In some embodiments contacting of the aqueous proteinaceous compositions with the anionic exchange resin can be conducted for about 0.1 to 30 minutes. In some embodiments contacting of the proteinaceous with the anionic exchange resin can be conducted for about 0.5 to 20 minutes. In some embodiments contacting of the proteinaceous with the anionic exchange resin can be conducted for about 1 .0 to 15 minutes. In some embodiments contacting of the proteinaceous with the anionic exchange resin can be conducted for about 2.0 to 10 minutes. In some embodiments contacting of the proteinaceous with the anionic exchange resin can be conducted for about 2.5 minutes.

[0078] In some embodiments, passage of the aqueous proteinaceous composition can be conducted at a rate of about 0.3 to about 1 .4 column volumes per minute. In some embodiments, passage of the aqueous proteinaceous composition can be conducted at a rate of about 0.5 to about 1 .2 column volumes per minute. In some embodiments, passage of the aqueous proteinaceous composition can be conducted at a rate of about 1 .0 column volumes per minute.

[0079] In some embodiments, the anionic exchange resin can be provided at a loading of about 10 column volumes to about 70 columns volumes of the aqueous proteinaceous composition. In some embodiments, the anionic exchange resin can be provided at a loading of about 20 column volumes to about 60 columns volumes of the aqueous proteinaceous composition. In some embodiments, the anionic exchange resin can be provided at a loading of about 30 column volumes to about 50 columns volumes of the aqueous proteinaceous composition. In some embodiments, the anionic exchange resin is provided at a loading of about 40.

[0080] In some embodiments, the aqueous proteinaceous composition can can contain about 0.75 g / L to about 1 .25 g / L alpha-lactalbumin. In some embodiments, the aqueous proteinaceous composition can contain about 1 .01 g / L alphalactalbumin.

[0081] 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.

[0082] 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.

[0083] In some embodiments the process can be conducted such that the aqueous elution of 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.

[0084] In some embodiments, any insoluble components of the aqueous proteinaceous composition are removed prior to contacting with the anionic exchange resin. Insoluble components may cause poor selective adsorption of the at least one additional protein onto the resin. Insoluble components may be lipid agglomerates, adventitious solid matter, proteinaceous agglomerates or mixtures thereof.

[0085] In some embodiments removal of lipid agglomerates also described as defatting is conducted by filtration. Filtration can be conducted using centrifugation andor decantation for example.

[0086] In some embodiments, the aqueous solution enriched in alpha-lactalbumin is lyophilised to produce isolated solid alpha-lactalbumin. In some embodiments, lyophilisation may be conducted by freeze drying or spray drying.

[0087] 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.

[0088] 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.

[0089] In some embodiments isolated alpha-lactalbumin, beta-lactoglobulin, immunoglobulin, lactoferrin or mixtures thereof are produced by the process hereinbefore described.

[0090] In some embodiments the aqueous solution enriched in alpha-lactalbumin contains about 0.25 g / L to about 1 .75 g / L alpha-lactalbumin. In some embodiments the aqueous solution enriched in alpha-lactalbumin contains about 0.50 g / L to about1 .50 g / L alpha-lactalbumin. In some embodiments the aqueous solution enriched in alpha-lactalbumin contains about 0.75 g / L to about 1.00 g / L alpha-lactalbumin.

[0091] In some embodiments the aqueous solution enriched in alpha-lactalbumin can contain 0 :1 :: beta-lactoglobulin : alpha lactalbumin as measured by HPLC. In some embodiments the amount of beta-lactoglobulin in the the aqueous solution enriched in alpha-lactalbumin is beyond detection limits. In some embodiments the aqueous solution enriched in alpha-lactalbumin can contain 0.01 :1 :: beta- lactoglobulin : alpha lactalbumin as measured by HPLC. In some embodiments the aqueous solution enriched in alpha-lactalbumin can contain 0.025 :1 :: beta- lactoglobulin : alpha lactalbumin as measured by HPLC. In some embodiments the aqueous solution enriched in alpha-lactalbumin can contain 0.05 :1 :: beta- lactoglobulin : alpha lactalbumin as measured by HPLC. In some embodiments the aqueous solution enriched in alpha-lactalbumin can contain 0.1 :1 :: beta-lactoglobulin : alpha lactalbumin as measured by HPLC.

[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 of about 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 8, a preferred embodiment of the applicant’s disclosed process is described. An aqueous proteinaceous composition is provided, preferably a dairy liquid and more preferably whey. This liquid is subject to a a process to remove insoluble matter such as agglomerates of lipids and / or other matter such as proteins. The removal of insoluble matter process is conducted preferably by filtration and more preferably by centrifugation. The pH of then filtereddairy liquid permeate is measured and adjusted if necessary to a pH of about 6.0 to about 7.0, preferably by use of an effective amount of a 1 M sodium hydroxide and / or 1 M acetic acid solution. A chromatography column is provided with anionic exchange resin preferably a high capacity strong anionic cross-linked agarose with dextran surface extender (also known as Capto Q) or a strong anionic cross-linked agarose beads (also known as QF) or a mixture thereof. The initial pH of the resin is adjusted if necessary to a pH of about 6.0 to about 7.0, preferably by use of an effective amount of a 1 M sodium hydroxide and / or 0.2% w / v acetic acid solution. The filtered dairy liquid is then eluted through the ion exchange column to provide an aqueous solution enriched in alpha-lactalbumin and an anionic exchange resin adsorbed with the at least one additional protein, by selective adsorption of the at least one additional protein onto the anionic exchange resin. The filtered dairy liquid permeate elutes through the column at a rate of 2 ml / min with a total of 30 column volumes. The aqueous solution enriched in alpha-lactalbumin is then subjected to ultrafiltration, preferable using a 100 kDa to about a 300 kDa filter to at least partially remove remaining small proteins and peptides such as bovine serum albumin, then diafiltration, preferable using about a 10 kDa to about a 40 kDa filter, to at least partially remove lactose, ashes and excess amount of water. Following ultrafiltration and / or diafiltration the utrafiltered and / or diafiltered aqueous solution enriched in alpha-lactalbumin is then subjected to lyophilization, preferably by freeze-drying or spray drying to provide isolated solid alpha-lactalbumin. The at least one additional protein can be eluted from the anionic exchange resin adsorbed with the at least one additional protein following passage of the aqueous proteinaceous composition by use of a desorption solution, for example a 100 mS sodium chloride solution to provide a solution enriched in the at least one additional protein, such as betalactoglobulin. The at least one additional protein can be subject to further process steps to recover, preferably, beta-lactoglobulin, immunoglobulins, glycomacropetide (caseinomacropeptide) and / or bovine serum albumin. In addition, a series of elution steps at specific salt conductivity (mS) can be conducted to selectively elute the additional proteins.Examples

[0094] General procedure

[0095] The following experiments were conducted according to the follow general procedure. High-capacity quaternary amine group-functionalised strong anion exchanger resin packed in a 10 ml radial column, and the column was connected to the Fast Protein Liquid Chromatography (FPLC) system.

[0096] Prior to the run, the resin bed was washed with an effective amount of 1 M NaOH and 0.1% acetic acid and rinsed with water to adjust the pH to be within the range of 6 to 7.

[0097] If the pH of the proteinaceous composition in this case whey, is not within the range of 6 to 7, adjust the pH of the to be within the range of 6 to 7 by using an effective amount of sodium hydroxide and / or acetic acid. In the present case the acid whey was adjusted from pH 5.5 to those pHs described below by addition of an effective amount of NaOH.

[0098] The proteinaceous composition was filtered before and / or after pH adjustment if required using a centrifuge to removed insoluble components such as lipid agglomerates.

[0099] The filtered proteinaceous composition of pH 6 to 7 was then contacted with anionic exchange resin (CaptoQ) by passing through the solution using a chromatography column at a rate of 2 ml / min with a total of 30 column volumes. This provided an aqueous solution enriched in alpha-lactalbumin and anionic exchange resin adsorbed with the at least one additional protein of alpha-lactalbumin.

[0100] The aqueous solution enriched in alpha-lactalbumin can then be filtered through the ultrafiltration membrane and / or a diafiltration membrane followed by lyophilization by spray drying or freeze drying resulting in an alpha-lactalbumin purity of around 90-95%.

[0101] Example 1 - selective adsorption of alpha lactalbumin from an aqueous proteinaceous composition to produce an aqueous solution enriched in alpha-lactalbumin

[0102] A series of experiments were conducted using whey as the aqueous proteinaceous composition with a pH of 5.5.

[0103] The general procedure was followed, adjusting the pH of as the aqueous proteinaceous composition and anionic resin to within the range of pH from 6.0 to 7.0. The results obtained are as follows.

[0104] Figure 1 is a chromatogram showing typical constituents of raw whey prior at pH of 5.5 to undergoing the process. Peaks on the chromatograph clearly show alpha-lactalbumin and at least one additional protein such as beta-lactoglobulin. The ratio in the untreated raw acidic whey is of 3.9 :1 :: beta-lactoglobulin : alpha lactalbumin.

[0105] Figure 2 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following the process when conducted using the general procedure at a pH of 6.5. This chromatograph shows the relative abundance of alphalactalbumin is greatly increased as comparted to at least one additional protein such as beta-lactoglobulin.

[0106] The table below shows the enrichment of alpha lactalbumin enabled by use of this process compared to beta-lactoglobulin.

[0107] Figure 3 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following the process when conducted using the general procedure at a pH of 6.0. This chromatograph shows the relative abundance of alphalactalbumin is greatly increased as comparted to at least one additional protein such as beta-globulin.

[0108] Figure 4 is a chromatogram showing the aqueous solution enriched in alpha-lactalbumin following the process when conducted using the general procedure at a pH of 7.0. This chromatograph shows the relative abundance of alphalactalbumin is greatly increased as comparted to at least one additional protein such as beta-globulin.

[0109] In conclusion, the applicant has shown by these experiments that by using an anionic exchange resin and maintaining the pH of between about 6.0 to 7.0, an aqueous solution enriched in alpha-lactalbumin is provided from an aqueous proteinaceous composition comprising alpha-lactalbumin and at least one additional protein by way of selective adsorption of the at least one additional protein onto the anionic exchange resin.

[0110] The measured conductivity of the whey sample and the aqueous solution enriched in alpha-lactalbumin samples was in the range of 7-8 mS.

[0111] The at least one additional protein was then eluted using an 80 mS aqueous solution of sodium hydroxide. As shown in Figure 9, a major constituent is beta-lactoglobulin.

[0112] Comparative example 1 - non-selective adsorption of alpha lactalbumin from an aqueous proteinaceous composition with a pH of less than 6.0

[0113] The above general procedure was conducted, without pH adjustment of the aqueous proteinaceous composition (acidic whey) from 5.5. Figure 5 shows that the separation is incomplete due to poor selective adsorption of at least one additional protein onto the anionic exchange resin.

[0114] Comparative example 2 - non-selective adsorption of alpha lactalbumin from an aqueous proteinaceous composition with a pH of more than 7.5

[0115] The above general procedure was conducted, with the pH of the whey adjusted to 7.5. Figure 6 shows that the separation is incomplete due to poor due to denaturing and / or degradations of proteins, including alpha-lactalbumin and betalactoglobulin as well as poor selective adsorption of at least one additional protein onto the anionic exchange resin.

[0116] Thus, from comparative examples, the applicant has shown that that the optimum range for selective adsorption of one additional protein onto the anionic exchange to produce a solution enrichened in alpha-lactalbumin is within the pH range of about 6.0 to about 7.0. In addition, below pH 6.0 and above 7.0 have been shown to be ineffectual.

[0117] Comparative example 3 - non-selective adsorption of alpha lactalbumin from a proteinaceous composition using a cationic exchange resin at a pH of less than 6

[0118] This example was performed according to the general procedure above, except where noted, and is broadly representative of the published method of WO 2018 / 149809 A1.

[0119] The general procedure was followed; however the radial column was packed with large cationic exchanger beads, and the initial pH of the column was adjusted using an effective amount of with acetic acid and / or sodium hydroxide. Subsequently, as described in the original study, the pH of the whey sample was adjusted to pH 3.7, and the adjusted whey was loaded onto the column.

[0120] According to the expected outcome, the beta in the column was anticipated to be replaced by the alpha. However, the results did not align with the stated expectations. Even in the flow-through sample, the beta and alpha components remained as close as in the raw sample (load sample). The displacement of beta with alpha was not observed in the replication experiment. The chromatograms shown in figure 7 and calculations below of alpha-lactalbumin and beta quantities are presented below.

[0121] The ratio of beta-lactoglobulin to alpha-lactalbumin of the solution collected after the process 1.0 : 2.5 :: beta-lactoglobulin : alpha-lactalbumin showing poorer selective adsorption of at least one additional protein onto the resin under these conditions and production of the solution enriched in alpha-lactalbumin.

[0122] In conclusion, the applicant found that use of a cationic exchange resin and an acidic pH 3.7 does not lead to a selective adsorption of one additional protein onto the anionic exchange to produce a solution enrichened in alpha-lactalbumin.

[0123] 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.

[0124] 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 valueand sub-range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0125] 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.

[0126] 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.

[0127] 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 found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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 any such 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 comprising alphalactalbumin and at least one additional protein, wherein the process comprises contacting the aqueous proteinaceous composition with an anionic exchange resin, wherein the pH during contacting of the aqueous proteinaceous composition with the anionic exchange resin is maintained at a pH of about 6.0 to about 7.0 so that the at least one additional protein is at least partially selectively adsorbed onto the anionic exchange resin, so providing the aqueous solution enriched in alphalactalbumin and an anionic exchange resin adsorbed with the at least one additional protein.

2. The process of claim 1 , wherein the anionic exchange resin adsorbed with the at least one additional protein is separated from the aqueous solution enriched in alpha-lactalbumin.

3. The process of any one of claims 1 to 2, wherein the anionic exchange resin is provided in an apparatus suitable for liquid flow therethrough.

4. The process of claim 3, wherein contacting of the aqueous proteinaceous composition occurs during passage of the aqueous proteinaceous composition through the apparatus suitable for liquid flow therethrough.

5. The process of claim 3 or claim 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 any water insoluble components of the aqueous proteinaceous composition are removed prior to contacting with the anionic exchange resin.

7. The process of any one of claims 1 to 6, wherein the aqueous solution enriched in alpha-lactalbumin is lyophilised to produce isolated alpha-lactalbumin.

8. A process for isolation of alpha-lactalbumin from an aqueous proteinaceous composition comprising alpha-lactalbumin and at least one additional protein, the process comprising: providing an anionic exchange resin in an apparatus suitable for liquid flowthrough, removal of any insoluble components of the aqueous proteinaceous composition prior to contacting with the anionic exchange resin, contacting the aqueous proteinaceous composition with the anionic exchange resin by passage of the aqueous proteinaceous composition through the apparatus suitable for liquid flow therethrough; wherein the pH during contacting of the aqueous proteinaceous composition with the anionic exchange resin is maintained at a pH of about 6.0 to about 7.0 so that the at least one additional protein is at least partially selectively adsorbed onto the anionic exchange resin, so providing an aqueous solution enriched in alphalactalbumin separated from an anionic exchange resin adsorbed with the at least one additional protein, subjecting the aqueous solution enriched in alpha-lactalbumin to lyophilisation to provide the isolated alpha-lactalbumin.

9. The process of claim 8, wherein the apparatus suitable for liquid flow therethrough is a chromatography apparatus.

10. The process of any one of claims 6 to 9, wherein removal of insoluble components is conducted by filtration.11 . The process of claim 10, wherein filtration is conducted using centrifugation or decantation.

12. The process of any one of claims 3 to 7 and 8 to 11 , wherein loading of the aqueous proteinaceous composition onto the resin in the apparatus suitable for liquidflow therethrough is conducted at a rate of about 0.3 to about 1 .4 column volumes per minute.

13. The process of any one of claims 3 to 7 and 8 to 12, wherein elution of the at least one additional protein through the apparatus suitable for liquid flow therethrough is conducted at a rate of about 0.15 to about 0.70 column volumes per minute.

14. The process of any one of claims 3 to 7 and 8 to 12, wherein the anionic exchange resin is provided in the apparatus suitable for liquid flow therethrough having the anionic exchange resin loaded with 10-70 column volumes of the proteinaceous composition.

15. The process of any one of claims 1 to 14, wherein the aqueous proteinaceous composition contains about 0.5 g / L to about 1 .5 g / L alpha-lactalbumin.

16. The process of any one of claims 1 to 15, wherein the aqueous proteinaceous composition is adjusted to a pH of about 6.0 to about 7.0 prior to contacting with the anionic exchange resin.

17. The process of any one of claims 1 to 16, wherein the anionic exchange resin is adjusted to an initial pH of about 6.0 to about 7.0 prior to contacting with the aqueous proteinaceous composition.

18. The process of claim 16 or claim 17, wherein adjusting of the pH is conducted using an effective amount of a nutritionally acceptable alkali and / or a nutritionally acceptable acid.

19. The process of claim 18, wherein the pH is adjusted using an effective amount of sodium hydroxide solution and then an effective amount of acetic acid.

20. The process of any one of claims 1 to 19, wherein the at least one additional protein is eluted using a desorption solution from the anionic exchange resin adsorbed with the at least one additional protein to provide an aqueous solutionenriched in the at least one additional protein following eluting of the aqueous proteinaceous composition.21 . The process to any one of claims 1 to 20 wherein the at least one additional protein is selected from is selected from the list consisting of beta-lactoglobulin, immunoglobulin, bovine serum albumin and glycomacropetide (caseinomacropeptide).

22. The process of claim 21 , wherein 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.

23. The process of any one of claims 21 or claim 22, wherein 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 least one 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.

24. The process of any one of claims 21 to 23, wherein the order of eluting from the anionic exchange reins is sequential and as follows: glycomacropetide (caseinomacropeptide); bovine serum albumin; immunoglobulin; and then beta- lactoglobulin.

25. The process of any one of claims 1 to 24, wherein the aqueous proteinaceous composition is a dairy liquid.

26. The process of claim 25, wherein the diary liquid is a whey.

27. The process of claim 26, wherein the whey is a sweet whey, acid whey or salt whey.

28. The process of any one of claims 1 to 27, wherein the aqueous proteinaceous composition is subjected to defatting prior to contacting with the anionic exchange resin.

29. The process of any one of claims 1 to 28, wherein the anionic exchange resin is a high-capacity quaternary amino group functionalised anion exchange resin.

30. The process of any one of claims 1 to 29, wherein the aqueous solution enriched in alpha-lactalbumin is subjected to ultrafiltration.31 . The process of claim 30, wherein ultrafiltration is conducted using a 50 kDa to about a 400 kDa filter.

32. The process of any one of claims 1 to 31 , wherein the aqueous solution enriched in alpha-lactalbumin is subject to diafiltration.

33. The process of claim 32, wherein diafiltration is conducted using about a 5 kDa to about a 50 kDa filter.

34. The process of any one of claims 30 to 32, wherein ultrafiltration is conducted sequentially and prior to diafiltration .

35. The process of any one of claims 1 to claim 34, wherein the aqueous solution enriched in alpha-lactalbumin contains about 0.25 to about 1 .75 g / L alpha-lactalbumin36. The process of any one of claims 1 to claim 35, wherein the aqueous solution enriched in alpha-lactalbumin contains about 0 : about 1 :: beta-lactoglobulin : alphalactalbumin as measured by HPLC.

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