Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons

Definitions

• the present invention pertains to a method for isolating osteopontin from a milk- derived feed, e.g. a feed based on milk serum, sweet whey, or acid whey.
• the present method involves the use of a relatively high protein concentration and a narrow window of pH and specific conductance of the milk- derived feed, which surprisingly has proven to provide a very efficient isolation of osteopontin from chemically complex feeds.
• Osteopontin is an acidic, highly phosphorylated, sialic acid rich, calcium binding protein. Osteopontin contains approx. 28 moles of bound phosphate per mol osteopontin and binds approx. 50 moles of Ca per mole osteopontin.
• Osteopontin is a multifunctional bioactive protein that is implicated in numerous biological processes, such as bone remodelling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk.
• OPN is involved in the urinary tract's defence against the formation of renal stones because OPN can inhibit growth and aggregation of calcium oxalate monohydrate crystals.
• the biological role of OPN in milk is not clear; however, several functions could be hypothesized. Osteopontin has been reported to be involved in mammary gland development and differentiation, and high levels of OPN expression have been observed in the mammary gland in early lactation.
• osteopontin is typically purified from bone or milk and it is typically present in bovine milk in a concentration of 20 mg/L.
• osteopontin is a serum protein but may also to some extent associate with the casein micelles depending on the Ca 2+ level.
• Acid whey is the preferred raw material for industrial production of osteopontin. When acid whey is formed osteopontin is thought to leave the casein micelles as Ca 2+ leaks out into the serum phase. This aspect makes acid whey a straightforward source of osteopontin.
• sweet whey has a slightly lower osteopontin content.
• sweet whey contains caseino macropeptide (CMP) from enzymatic cleavage of the kappa-casein.
• CMP caseino macropeptide
• CMP has many biochemical resemblances with osteopontin - both are small, flexible, acidic, phosphorylated glycoproteins.
• CMP and osteopontin is believed to be quite similar in their binding to ion exchange resins, which will pose a problem in purifying osteopontin from a CMP-containing raw material.
• Another aspect is the likely degradation of osteopontin by proteolytic enzymes used for cheese making.
• WO 02/28413 Al describes a method of producing an osteopontin-containing composition from feedstocks, such as milk and acid whey, by means of low pH anion exchange.
• feedstocks such as milk and acid whey
• cGMP is a type of CMP which is known to bind to anion exchangers and which would inhibit the binding of osteopontin.
• WO 01/149741 A2 describes a process wherein osteopontin is purified from a milk material by mixing the milk material with soluble calcium and adjusting the pH of the mixture to selectively precipitate the other protein components of whey while keeping osteopontin in solution.
• osteopontin can be isolated by anion exchange in high yield and high purity from complex milk-derived feeds despite the presence of competing proteins of the feed.
• the present inventors have found that the specific conductance of the feed is particularly important for the yield and purity of osteopontin and have identified an optimum window of the specific conductance for the isolation of osteopontin from milk derived feeds.
• an aspect of the invention relates to a method for isolating osteopontin from a milk-derived feed, the method comprising the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a pH in the range of pH 3.6-6.5 at 25 degrees C and a specific conductance in the range of 4-10 mS/cm at 25 degrees C, b) subjecting said milk-derived feed to anion exchange chromatography, which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• An aspect of the invention may for example relate to a method for isolating osteopontin from a milk-derived feed, the method comprising the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a pH in the range of pH 3.6-6.5 at 25 degrees C and a specific conductance in the range of 4-10 mS/cm at 25 degrees C, and wherein the milk-derived feed comprises a total amount of protein in the range of 50-250 g/L milk-derived feed, b) subjecting said milk-derived feed to anion exchange chromatography, which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• the present method allows for a more cost-efficient use of the anion exchange medium, and a higher yield of osteopontin per kg anion exchange medium.
• the "specific conductance" (sometimes referred to as the "specific conductivity") of an aqueous solution is a measure of the ability of the solution to conduct electricity.
• the specific conductance may e.g. be determined by measuring the AC resistance of the solution between two electrodes and the result is typically given in the unit miliSiemens per cm (mS/cm).
• the measurement of specific conductance may for example be measured according to the EPA (the US Environmental Protection Agency) Method No. 120.1.
• Yet an aspect of the invention relates to the osteopontin-containing composition obtained by the present method.
• a further aspect of the invention pertains to an osteopontin-containing composition as such.
• an aspect of the invention pertains to a method for isolating osteopontin from a milk-derived feed, the method comprising the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a pH in the range of pH 3.6-6.5 at 25 degrees C and a specific conductance in the range of 4-10 mS/cm at 25 degrees C, and the wherein milk-derived feed comprises a total amount of protein in the range of 50-250 g/L milk-derived feed, b) subjecting said milk-derived feed to anion exchange chromatography, which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• step c) of the method is omitted and in this case the method comprises the steps a), b), and d).
• the term "isolating osteopontin” relates to enrichment of osteopontin to a weight percent of at least 70% (w/w), preferably at least 80% (w/w), and even more preferably at least 90% (w/w) relative to the total weight of protein recovered from the anion exchange medium during step d).
• Isolating osteopontin may for example involve the enrichment of osteopontin to a weight percent of at least 95% (w/w), such as at least 97% (w/w) relative to the total weight of protein recovered from the anion exchange medium during step d).
• the method is particularly useful for improving the selective enrichment of osteopontin from a milk-derived feed comprising additional protein having an isoelectric point (pi) ⁇ 5.0, for example milk-derived feeds which contain CMP.
• selective enrichment should be understood as increasing the molar ratio between osteopontin and the total amount of other proteins of the milk- derived feed.
• the isoelectric point of a protein is preferably determined by isoelectric focusing at 25 degrees C.
• milk-derived feed pertains to the liquid feed which contacts the anion exchange medium.
• the milk-derived feed is derived from milk from one or more mammalian source(s), e.g. milk from human, cow, sheep, goat, buffalo, camel, llama, horse and/or deer.
• the milk-derived feed is derived from bovine milk.
• milk-derived feed In the context of the present invention, the terms “milk-derived feed”, “sweet whey-derived feed”, “acid whey-derived feed”, and “milk serum-derived feed” relate to feeds wherein at least 50% (w/w) of the total protein originates from milk, sweet whey, acid whey or milk serum, respectively. In some preferred embodiments of the invention at least 90% (w/w), and preferably substantially all, of the total protein of the milk-derived feed originates from a milk, sweet whey, acid whey or milk serum.
• milk relates to the liquid obtained from the mammary glands of mammals during lactation. The term “milk” should be interpreted broadly and covers both the raw milk, i.e.
• Whey is a collective term referring to the watery by-product which is produced during the manufacture of cheese or casein from milk.
• sweet whey relates to whey, which is obtained during rennet-based coagulation of milk, which for example takes place during the production of yellow cheese.
• the term "acid whey” relates to whey, which is obtained during chemical or biological acidification of milk, which for example takes place during the production of cottage cheese or quark, or in the production of casein/caseinates.
• milk serum which also is known as “serum whey”, “native whey” or “lactoserum”, pertains to milk from which milk fat and casein micelles have been removed.
• milk serum typically contains some free casein species, which have dissociated from the native casein micelles before the micelles were removed.
• Milk serum may for example be produced by microfiltering a skimmed milk through a filter or membrane having a pore size of approx. 0.1 micrometer and collecting the resulting permeate as the milk serum. Milk serum may be produced according to Evans et a/.
• the milk, sweet whey, acid whey or milk serum may have been subjected to several process steps to prepare the milk-derived feed.
• Processing of milk or milk-related products typically involves one or more heat treatment processes, such as a pasteurisation (e.g. 72 degrees C for 15 sec) or a high pasteurisation (e.g. 85 degrees C for 20 sec).
• the processing may involve one or more filtration step(s).
• Microfiltration may be used to remove microorganisms, or alternatively to concentrate casein.
• Ultrafiltration or nanofiltration may e.g. be used to concentrate whey protein or milk serum protein.
• the processing may involve one or more
• centrifugation step(s) e.g. for separating fat from skimmed milk and/or separating microorganisms from the milk.
• the processing may involve one or more evaporation step(s) for removing water and thus concentrating dry matter such at proteins and/or minerals.
• the processing may also comprise one or more pH adjustment(s). Acidification may e.g. be used to coagulate casein and pH adjustment may furthermore be important when the processing involves ion exchange chromatography.
• the milk-derived feed comprises one more process stream(s) from the production of other milk protein fractions, such as filtered, heat treated whey or alpha-lactalbumin- and/or beta-lactoglobulin- depleted whey.
• the milk-derived feed comprises, or even essentially consists of, a milk serum-derived feed.
• the milk-derived feed may for example be a milk serum, and preferably a milk serum protein concentrate, e.g. in the form of the retentate obtained by ultrafiltration of milk serum.
• the content of osteopontin in the milk-derived feed depends on the specific feed type.
• the milk-derived feed comprises osteopontin in an amount in the range of 0.01-20% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise osteopontin in an amount in the range of 0.05-5% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may e.g. comprise osteopontin in an amount in the range of 0.1-2% (w/w).
• the milk-derived feed may comprise osteopontin in an amount in the range of 0.1-1% (w/w) relative to the total amount of protein of the milk-derived feed.
• a product, component, method, or method step which is stated to "essentially consist of" one or more subcomponents or one or more activities consists of the one or more specifically mentioned sub-components or the one or more specifically mentioned activities but may also include one or more additional unnamed sub-components or activities which do not materially affect the basic and novel characteristic(s) of the present invention.
• the present method is particularly effective for isolating osteopontin from complex feeds, i.e. feeds which contain molecular entities that interfere with the isolation of osteopontin, such as proteins having a pi lower than 5.0.
• proteins having a pi lower than 5.0 have been found to compete with osteopontin for the functional groups of the anion exchange medium.
• proteins having a pi ⁇ 5.0 are alpha lactalbumin, proteose peptone- 3, proteose peptone-5, and proteose peptone-8, and casein-derived peptides such as caseino macropeptide and/or caseino phosphopeptides.
• the additional protein may comprise one or more proteins from the group consisting of alpha lactalbumin, proteose peptone-3, proteose peptone-5, and proteose peptone-8, casein-derived peptide, caseino macropeptide, caseino phosphopeptide, and combinations thereof.
• Free alpha-casein and free beta-casein are other example of proteins having a pi value ⁇ 5.0.
• the milk-derived feed comprises an amount of additional protein having a pi ⁇ 5.0 of at least 0.1% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk- derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 of at least 0.5% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 of at least 2% (w/w) relative to the total amount of protein of the milk-derived feed.
• the additional protein having a pi ⁇ 5.0 does not include osteopontin but typically includes one or more other proteins having a pi in the specified range.
• the milk-derived feed comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 0.1-50% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk- derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 0.5-40% (w/w) relative to the total amount of protein of the milk- derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 2-25% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may e.g. comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 0.1-20% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 0.3-15% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 0.5-10% (w/w) relative to the total amount of protein of the milk- derived feed.
• the milk-derived feed comprises an amount of additional protein having a pi ⁇ 5.0 in the range of 1-50% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 10-45% (w/w) relative to the total amount of protein of the milk- derived feed.
• the milk-derived feed may comprise an amount of additional protein having a pi ⁇ 5.0 in the range of 15-40% (w/w) relative to the total amount of protein of the milk-derived feed.
• the additional protein having a pi ⁇ 5.0 has a pi ⁇ 4.5, and preferably a pi ⁇ 4.0.
• proteins encompasses both large aggregates of polypeptides, single polypeptide chains and peptides such as di- or tri-peptides. Chemically, proteins are polymers comprising different and/or identical amino acids linked by so-called peptide bonds.
• the milk-derived feed furthermore comprises caseino macropeptide (CMP).
• CMP caseino macropeptide
• the term "CMP” or "caseino macropeptide” pertains to a small protein, which is released from kappa-casein upon exposure to rennet enzymes. CMP encompasses both glycosylated variants and a non- glycosylated variant of the protein. The glycosylated variants of the protein is sometimes referred to as caseino glycomacropeptide (cGMP).
• cGMP caseino glycomacropeptide
• the milk-derived feed comprises, or even essentially consists of, sweet whey-derived feed.
• the milk-derived feed may for example be sweet whey, and preferably sweet whey protein concentrate, e.g. in the form of the retentate obtained by ultrafiltration of sweet whey.
• the milk-derived feed may comprise, or even essentially consists of, an acid whey-derived feed.
• the milk-derived feed may for example be an acid whey, and preferably an acid whey protein concentrate, e.g. in the form of the retentate obtained by ultrafiltration of acid whey.
• milk serum protein
• the concentrate pertains to an aqueous composition which contains at least 80% (w/w) of the total protein which was present in original milk serum, sweet whey, or acid whey, respectively, and which has a total protein content of at least 25% (w/w) relative to the dry weight of the aqueous composition.
• the milk-derived feed is not an acid whey-derived feed.
• the milk-derived feed e.g. a sweet whey-derived feed
• the milk-derived feed comprises CMP in an amount of at least 1% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk- derived feed e.g. a sweet whey-derived feed
• the milk-derived feed e.g. a sweet whey- derived feed
• the milk-derived feed comprises CMP in an amount in the range of 1-40% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk- derived feed e.g. a sweet whey-derived feed
• the present inventors have observed that, surprisingly, a precipitate is formed during the present process when the feed is based on milk serum or a concentrate thereof.
• the precipitate causes problems during the anion exchange process and reduces its robustness.
• the inventors have investigated the precipitate and found indications that it contains precipitated casein species, which were present in dissolved form such as free beta-casein or free alpha-casein.
• free beta-casein or “free alpha-casein” pertains beta-casein molecules or alpha-casein molecules which are not bound to the native casein micelles of milk products.
• Such free beta-casein or free alpha-casein includes dissolved single molecules of beta-casein or alpha- casein or small aggregates of alpha-casein and/or beta-casein.
• Single molecules of beta-casein are for example known to form small beta-casein micelles, which also is an example of free beta-casein.
• the milk-derived feed furthermore comprises free alpha-casein and free beta-casein. Free alpha-casein and free beta-casein are typically present in milk-serum derived feed.
• step a) of the method involves removing precipitate from the acidified liquid which is to form the milk- derived feed. Such removal may e.g. involve centrifugation or filtration of the acidified liquid.
• the milk-derived feed may therefore have a pH in the range of 5.0-6.5, and preferably in the range of pH 5.0-6.0.
• a feed/process temperature above 15 degrees C, such as 20-40 degrees C.
• dissolved beta-casein forms small beta-casein micelles, not to be confused with native casein micelles of milk, and these beta-casein micelles seem to interfere less with the anion exchange process than single beta- casein molecules.
• the temperature of the milk-derived feed and anion exchange material during step b) may therefore be in the range of 15-40 degrees C, and preferably in the range of 20-38 degrees C.
• the milk-derived feed may e.g. comprise a total amount of free alpha-casein and free beta-casein of at least 0.5% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise a total amount of free alpha-casein and free beta-casein of at least 2% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may e.g. comprise a total amount of free alpha-casein and free beta-casein of at least 10% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises a total amount of free alpha-casein and free beta-casein in an amount in the range of 0.5-40% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise a total amount of free alpha- casein and free beta-casein in an amount in the range of 2-20% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may e.g. comprise a total amount of free alpha-casein and free beta-casein in an amount in the range of 5-15% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises alpha- lactalbumin in an amount of at least 1% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise alpha-lactalbumin in an amount of at least 10% (w/w) relative to the total amount of protein of the milk-derived feed, preferably at least 20% (w/w), and even more preferably at least 30% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises alpha- lactalbumin in an amount in the range of 1-50% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise alpha-lactalbumin in an amount in the range of 5-40% (w/w) relative to the total amount of protein of the milk-derived feed, preferably in the range of 10-35% (w/w), and even more preferably in the range of 12-30% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises beta- lactoglobulin in an amount of at least 1% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise beta-lactoglobulin in an amount of at least 15% (w/w) relative to the total amount of protein of the milk-derived feed, preferably at least 30% (w/w), and even more preferably at least 40% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises beta- lactoglobulin in an amount in the range of 1-70% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed may comprise beta-lactoglobulin in an amount in the range of 10-65% (w/w) relative to the total amount of protein of the milk-derived feed, preferably in the range of 20-60% (w/w), and even more preferably in the range of 35-55% (w/w) relative to the total amount of protein of the milk-derived feed.
• Caseins are known to precipitate at pH-values at or below about 4.6.
• milk-derived feed having a pH in the range of about 3.6 - 4.6 has a relatively low concentration of casein, such as at most 1% (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises at most 0.1% casein (w/w) relative to the total amount of protein of the milk-derived feed. It may even be preferred that the milk-derived feed comprises at most 0.01% casein (w/w) relative to the total amount of protein of the milk-derived feed.
• the milk-derived feed comprises a total amount of protein in the range of 6-250 g/L milk-derived feed.
• the milk-derived feed may comprise a total amount of protein in the range of 50- 150 g/L milk-derived feed. It may even be preferred that the milk- derived feed comprises a total amount of protein in the range of 75-125 g/L milk- derived feed.
• the milk-derived feed may comprise a total amount of protein in the range of 50-250 g/L milk-derived feed.
• the milk-derived feed may comprise a total amount of protein in the range of 50-150 g/L milk-derived feed. It may even be preferred that the milk-derived feed comprises a total amount of protein in the range of 75-150 g/L milk-derived feed.
• the milk-derived feed may comprise a total amount of protein in the range of 75-250 g/L milk-derived feed.
• the milk-derived feed comprises a total amount of protein of at least 6 g/L milk-derived feed.
• the milk-derived feed may comprise a total amount of protein of at least 50 g/L milk- derived feed. It may even be preferred that the milk-derived feed comprises a total amount of protein of at least 75 g/L milk-derived feed.
• the milk-derived feed has a pH in the range of pH 3.8-6.0 at 25 degrees C.
• the pH of the milk-derived feed at 25 degrees C is in the range of pH 4.0-5.5.
• the pH of the milk-derived feed at 25 degrees C is in the range of pH 4.2-5.0, such as e.g. pH 4.3-4.5.
• the desired specific conductance of the milk-derived feed may for example be obtained by:
• the milk-derived feed has a specific conductance in the range of 4.5-9.0 mS/cm at 25 degrees C.
• the specific conductance of the milk-derived feed may be in the range of 5.0-8.0 mS/cm at 25 degrees C.
• it may be even more preferable that the specific conductance of the milk-derived feed is in the range of 5.5-7.0 mS/cm at 25 degrees C.
• the milk-derived feed may for example have a specific conductance in the range of 4-7 mS/cm at 25 degrees C.
• the milk-derived feed may have a specific conductance in the range of 7-10 mS/cm at 25 degrees C.
• the milk-derived feed may have a specific conductance in the range of 5-8 mS/cm at 25 degrees C.
• the milk-derived feed has a specific conductance in the range of 4-7 mS/cm at 25 degrees C and a pH in the range of pH 3.6-5.0 at 25 degrees C.
• the milk-derived feed has a specific conductance in the range of 7-10 mS/cm at 25 degrees C and a pH in the range of pH 5.0-6.5 at 25 degrees C.
• the milk-derived feed has a specific conductance in the range of 5-8 mS/cm at 25 degrees C and a pH in the range of pH 4.0-5.5 at 25 degrees C.
• the milk-derived feed may have a specific conductance in the range of 5-6 mS/cm at 25 degrees C and a pH in the range of pH 4.2-5.0 at 25 degrees C.
• the milk-derived feed has a pH in the range of 3.6 to 6.5 and
• the milk-derived feed may have a pH in the range of 3.6 to 5.0 and a specific conductance of
• the milk-derived feed may have a pH in the range of 5.0 to 6.5 and a specific conductance of
• the anion exchange medium comprises a solid phase and one or more cationic groups.
• At least some of the cationic groups are attached to the surface of the solid phase and/or to the surface of pores which are accessible through the surface of the solid phase.
• the solid phase of the anion exchange medium comprises one or more components selected from the group consisting of a plurality of particles, a filter, and a membrane.
• the solid phase may for example comprise, or even essentially consists of, polysaccharide. Cross-linked polysaccharides are particularly preferred. Examples of useful polysaccharides are cellulose, agarose, and/or dextran.
• the solid phase may comprise, or even essentially consists of, a non-carbohydrate polymer. Examples of useful non-carbohydrate polymers are methacrylate, polystyrene, and/or styrene-divinylbenzene.
• the cationic groups comprises, or even essentially consists of, amino groups.
• Tertiary amino groups are particularly preferred and result in quaternary ammonium groups under appropriate pH conditions.
• Quaternary ammonium groups provide strong anion exchange characteristics to the anion exchange medium.
• the cationic groups may comprise one or more primary or secondary amino groups.
• a substantial amount of primary or secondary amino groups typically provides the anion exchange medium with weak anion exchange characteristics.
• the optimal protein load per cycle depends on the design of the anion exchange chromatography system and the characteristics of the anion exchange medium.
• the process conditions during the anion exchange chromatography, including pressure, flow rate, etc., depend on the actual process implementation, the used equipment and the used anion exchange medium.
• the temperature of the milk-derived feed during step b) is typically sufficiently low to avoid microbial growth and heat damaging of the protein and the anion exchange medium but sufficiently high to provide an acceptable viscosity.
• the temperature of the milk-derived feed during step b) is in the range of 2-40 degrees C.
• the temperature of the milk-derived feed during step b) is in the range of 4-20 degrees C, and even more preferably in the range of 6-12 degrees C.
• the method of the invention comprises a step c) of washing the anion exchange medium with a washing solution after contacting it with the milk-derived feed.
• Useful washing solutions are typically pH neutral or weak acidic aqueous solutions capable of removing loosely bound molecules from the anion exchange medium.
• the method of the present invention does not contain step c).
• Step d) of the present invention involves recovering the osteopontin bound to the anion exchange medium.
• the recovery is typically performed by contacting the anion exchange medium with an eluent and collecting the resulting eluate, i.e. the eluent plus the molecules released from the anion exchange medium.
• the eluent is an aqueous solution having an ion strength and/or pH sufficient to release bound osteopontin from the anion exchange medium.
• Examples of useful eluents are pH-neutral, 1.0 M aqueous solutions of salts such as NaCI, CaCI 2 , KCI, MgCI 2 , or a combination thereof.
• the recovered composition may be subjected to additional process steps e.g. for demineralising and concentrating the composition, and subsequently transforming it into a powder.
• the recovered composition is furthermore subjected to one or more of the process step(s) selected from the group consisting of concentration, diafiltration, evaporation of solvent, spray- drying, and substitution of protein-bound cations.
• the recovered composition may be subjected to a concentration step.
• the recovered composition may be subjected to a diafiltration step.
• the recovered composition may be subjected to an evaporation step.
• the recovered composition may be subjected to a spray-drying step.
• the recovered composition is subjected to the following steps: i) concentrating, e.g. by ultrafiltration,
• cation replacement by contacting the aqueous composition of step ii) or iii) with a water soluble calcium salt, e.g. CaCI 2 ,
• the present method may both be implemented as a batch process or a semi- batch-process.
• the semi-batch process may for example be implemented by operating a first and a second anion exchange column, and performing step b) on the first anion exchange column while performing steps c) and/or d) on the second anion exchange column, and vice versa.
• the present method for isolating osteopontin from a milk-derived feed comprises the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a pH in the range of pH 3.6-6.5 at 25 degrees C and a specific conductance in the range of 4-10 mS/cm at 25 degrees C, and wherein the milk-derived feed comprises a total amount of protein in the range of 50-250 g/L milk-derived feed, and wherein the milk-derived feed furthermore comprises additional protein having an isoelectric point (pi) ⁇ 5.0.
• anion exchange chromatography which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• the present method for isolating osteopontin from a milk-derived feed comprises the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a the milk-derived feed has a pH in the range of 3.6 to 6.5 and
• milk-derived feed comprises a total amount of protein in the range of 50-250 g/L milk-derived feed
• anion exchange chromatography which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• the present method for isolating osteopontin from a milk-derived feed comprises the steps of: a) providing a milk-derived feed comprising osteopontin, said milk- derived feed having a the milk-derived feed has a pH in the range of 3.6 to 6.5 and
• milk-derived feed comprises a total amount of protein in the range of 50-250 g/L milk-derived feed, and wherein the milk-derived feed furthermore comprises additional protein having an isoelectric point (pi) ⁇ 5.0, b) subjecting said milk-derived feed to anion exchange chromatography, which includes contacting the milk-derived feed with an anion exchange medium, c) optionally, washing the anion exchange medium, and d) recovering the protein bound to the anion exchange medium, thereby obtaining a composition comprising isolated osteopontin.
• an aspect of the invention relates to an osteopontin-containing composition obtainable by the method described herein.
• Example 1 Enrichment of osteopontin from controlled conductance sweet whey concentrate
• Samples of the raw material was diluted to a final protein concentration of 10 % w/v, hereafter pH (pH 3.0 - 5.7, at 4.7 mS/cm) and specific conductance (2.3 - 10.3 mS/cm at pH 4.3) were adjusted by addition of HCI and NaCI, respectively. Unbound proteins were washed out of the column by 3 bed volumes of 100 mM NaCI, pH 5.0 solution. Subsequently, the bound proteins were eluted by passage of 1 M NaCI, pH 5.0 solution through the column until no more protein was detected in the eluate.
• pH pH 3.0 - 5.7, at 4.7 mS/cm
• specific conductance 2.3 - 10.3 mS/cm at pH 4.3
• osteopontin OPN
• CMP CMP
• the sample was filtered through a 0.22 pm filter and subjected to HPLC with column MonoQ HR 5/5 (1ml), Pharmacia and detection at 280 nm.
• the concentration of the sample was calculated by the external standard method (comparison with peak area of standard with known OPN content). It is a prerequisite for this analytical procedure that the samples comprise relatively pure OPN, e.g. as determined by SDS-PAGE (Laemmli, 1970), due to the low specificity of the method.
• Buffer A lOmM NaCI, 20mM Tris HCL, pH 8.0
• Buffer B 0.8 M NaCI, 20mM Tris HCI, pH 8.0
• a standard calibration curve was made from 5 standards in the concentration range 1-10 mg/ml of OPN standard in buffer A. All standards were filtered by 0.22 ⁇ filters before loading onto the column. Sampling and pretreatment:
• Samples for analysis were diluted with Milli Q water, HPLC grade, if they were out of range of the standard calibration curve. Dilution was in some instances also necessary to enable binding of OPN to the anion exchange resin if much NaCI from the eluent was present. An amount equivalent to 25 pL of 1-10 mg/mL OPN was injected for analysis. Samples were filtered through 0.22 pm filters before injection to HPLC.
• HPLC conditions Flow 1 ml/min, injection volume 25 pL, gradient: 0-3 min 0% B, 3-17 min 0-60% B, 17-30 min 60-100% B, 30-33 min 100% B, 33-34 min 100- 0% B, 34-40 min 0% B.
• the concentration of OPN in each sample was calculated by reference to the standard curve and by observing the employed dilutions.
• the purity was calculated by the ratio between OPN and total protein, using the OPN specific Jones factor of 7.17 as most or all protein determined by Kjeldahl digestion in the current context was OPN. Quantification of major whey proteins by HPLC
• TSKgeBOOOPWxl (7.8 mm x 30 cm) connected in series with attached precolumn PWxl (6 mm 4 cm)(Tosohass, Japan).
• the standard solution for calibration of the system consisted of: 225 mg CMP; 225 mg alpha-lactalbumin and 50 mg ⁇ -lactoglobulin dissolved in 500.0 ml 0.02 M phosphate buffer 7.5.
• Pretreatment of samples Powder samples were dissolved in phosphate buffer at 1 mg/ml and left over night for solubilisation. Alternatively, liquid samples were diluted with phosphate buffer to obtain a content of approx. 0.1% protein. If the protein content was below 0.1%, the sample was measured undiluted. All samples were filtered through 0.22 pm filters before injection to the column.
• the first series of experiments with varying the pH value of the applied feed show that the yield of OPN (mg OPN from 70 g total protein in the feed) increases as the pH increases from pH 3 to approx. pH 4.3.
• the high yield is maintained when the pH is increased beyond pH 4.3.
• the purity of OPN in the obtained OPN preparations is very high at low pH values, but when the pH is increased above approx. pH 4.5 the purity slowly declines but is still acceptable until approx. pH 6.5.
• the best yield of high purity OPN is obtained in the pH window pH 3.8-5.5, and particularly in the range pH 4.3-4.5.
• Example 2 Enrichment of osteopontin from controlled conductance acid whey concentrate The following purification experiment was conducted on FPLC equipment similar to Example 1. The raw material, however, was acid whey concentrate having a total protein content of 10% (w/w) and approx. 70 g of total protein was loaded on the column. The concentrated acid whey was diluted to different degrees giving rise to specific conductivities in the range from 2.5 to 10 mS/cm. OPN and total protein was measured in the feed and the eluate by the methods described in Example 1. The pH was adjusted to 4.3 by addition of HCI and the raw material applied to the anion exchange column. Unbound proteins were washed out of the column by 3 bed volumes of 0.1 M NaCI and the bound proteins were subsequently eluted by passage of 1 M NaCI solution through the column until no more protein was detected in the eluate.
• the data in Figure 3 shows an enrichment of OPN from acid whey by the described procedure.
• Example 1 where sweet whey was used as raw material, it is again observed that low specific conductance in the feed increases the yield on the sacrifice of purity of the product.
• acid whey derived feeds do not contain large quantities of CMP the effect is not as detrimental to the enrichment of OPN as in the case where the feed is derived from sweet whey.
• Acid whey derived feed does contain minor components, which binding can be minimised by the same parameter settings as employed to avoid binding of CMP.
• the narrow optimum range of OPN isolation identified in Example 1 applies to the OPN isolation from acid whey as well.
• milk serum can be used as raw material for OPN production by the disclosed method.
• Milk serum was produced by microfiltration (filter pore size of approx. 0.1 micron) of skimmed milk at 24 degrees so as to remove micellar casein. Samples of milk serum were thereafter adjusted to pH 4.3 or pH 4.7 by HCI and diafiltered to obtain a specific
• OPN was enriched from the modified milk serum samples by anion exchange at approx. 5 degrees C as described in example 1.
• Milk serum is produced by microfiltration of skimmed milk so as to remove caseins, whereas sweet and acid whey are casein depleted by precipitation of caseins by action of rennet or acid, respectively.
• sweet and acid whey are casein depleted by precipitation of caseins by action of rennet or acid, respectively.
• all three categories of milk derived feeds are essentially free of native casein micelles, which would interfere with anion exchange chromatographic procedures due to precipitation and flocculation of caseins.
• This problem can solved by filtering the milk derived feeds prior to anion exchange using e.g. a microfilter.
• the anion exchange step may be performed at a pH where precipitation is limited or even absent, e.g. in the range of pH 5.0-6.5.
• a feed/process temperature above 15 degrees C, such as 20-40 degrees C.
• dissolved beta-casein forms small beta-casein micelles, not to be confused with native casein micelles, and these beta-casein micelles interfere less with the anion exchange process than single beta-casein molecules.
• Example 1 The following purification experiment was conducted on FPLC equipment similar to Example 1.
• the raw material was sweet whey isolate and approx. 1 g of total protein was loaded on the column. Before loading to the column the sweet whey was concentrated by ultrafiltration, diafiltered after addition of an equal volume of water and finally diluted again by an equal volume of water. The resulting specific conductance was 2.1 mS/cm.
• CMP and total protein was measured in feed and eluate by the methods described in Example 1. The amount of OPN could not be measured due to interfering proteins in both feed and eluate.
• OPN data in Table 1 is estimated from known yields in experiments from Example 1.
• the pH was adjusted to 4.3 by addition of HCI and the raw material applied to the anion exchange column. Unbound proteins were washed out of the column by 3 bed volumes of water and the bound proteins were hereafter eluted by passage of 1 M NaCI solution through the column until no more protein was detected in the eluate.
• Example 5 Comparison and conclusion (comparing Example 1 with Example 4)
• Example 1 is much more specific for enriching OPN than the method of Example 4.
• much more protein can be loaded to the column per cycle and the yield of OPN per cycle is much higher with the method of Example 1.
• This method results in practically pure OPN, whereas CMP dominates the product of Example 4.

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