Crystallization of proteins capable of crystallization under salt-soluble conditions using multiple protein feeds

Abstract

The present invention relates to an improved process for producing a product comprising a protein of interest (TP) capable of crystallizing under salt soluble conditions by crystallization of said TP. The present invention uses at least two different TP-containing protein feeds, which are mixed to produce a supersaturated protein solution in which TP crystallizes. One of the protein feeds must have a pH at least plus 0.1 to the pH (pHMCY, TP) providing the maximum crystallization yield of the TP, and one of the protein feeds must have a pH at most minus 0.1 to the pHMCY, TP.

Description

Technical Field

[0001] The present invention relates to an improved method for purifying a protein capable of crystallization under salting conditions (target protein, abbreviated as TP). Crystallization under salting conditions is usually possible only within a certain pH range. The present invention uses at least two different protein feeds containing TP, whose pH values ​​ensure that they are not oversaturated with respect to TP. The protein feeds containing TP are then mixed to provide a mixture having a pH that makes crystallization under salting conditions feasible and is supersaturated with respect to TP. The present invention requires the use of i) a pH higher than a pH (pH 50) that provides a maximum crystallization yield of TP; MCY,TP ) protein feed, referred to as "Type A feed", and ii) pH below pH MCY,TP The protein feed is called "Type B feed". Background Art

[0002] The concept of protein fractionation is well known in the art and has developed over the past several decades into a range of techniques for preparing products enriched in various protein species, each with specific properties and characteristics.

[0003] The isolation of β-lactoglobulin (BLG) from milk serum or whey is the subject of numerous publications and generally involves multiple separation steps and often chromatographic techniques to obtain a purified β-lactoglobulin product.

[0004] WO2018 / 115520 describes the industrial-scale separation of BLG from a protein solution by crystallization in a salting manner and at a pH range of 5 to 6. However, WO2018 / 115520 does not disclose the preparation of a supersaturated whey protein solution by mixing a type A protein feed and a type B protein feed containing BLG as defined in the present invention.

[0005] WO 2020 / 002422 describes the production of an alpha-lactalbumin-rich whey protein product by removing BLG from a whey protein solution on an industrial scale using BLG crystallization in a salting manner and at a pH in the range of 5 to 6. However, WO 2020 / 002422 does not disclose the preparation of a supersaturated whey protein solution by mixing a type A protein feed and a type B protein feed containing BLG as defined in the present invention.

[0006] de Jongh et al. (Mild Isolation Procedure Discloses New Protein Structural Properties of β-Lactoglobulin, J Dairy Sci., Vol. 84(3), 2001, pp. 562-571) describe the purification of BLG from freshly squeezed milk by low temperature acid coagulation of casein and by a combination of affinity chromatography (DEAE agarose gel) and gel permeation chromatography of the acid whey obtained. The BLG product obtained is said to contain 0.985 g of β-lactoglobulin per 1 g of protein.

[0007] Slack et al. (Journal of Food Processing and Preservation, Vol. 10, 1986, pp. 19-30) explored a different approach and prepared a BLG-rich precipitate by adjusting the pH of desalted acid whey and sweet whey to pH 4.65 and separating the formed precipitate by centrifugation and decantation. The precipitate pellet obtained was described as relatively insoluble and contained a significant amount of protein impurities in the additional BLG. No crystal formation was observed. It should be noted that the BLG precipitate that may be formed at pH 4.65 is not a BLG crystal.

[0008] Palmer (Crystalline Globulin from Cow's Milk, J. Biol. Chem., Vol. 104, 1934, pp. 359-372) reports a laborious and time-consuming process for producing protein crystals based on acid whey, which uses a variety of sequences of salt precipitation of unwanted proteins, pH adjustment and dialysis to remove other unwanted proteins. Finally, when a highly purified BLG solution is obtained, BLG crystallizes. The process lasts more than 12 days and requires the addition of toluene. Therefore, the procedure disclosed in Palmer is incompatible with safe food production and the product provided is clearly inedible.

[0009] Aschaffenburg et al. (Improved Method for the Preparation of Crystalline beta-Lactoglobulin and alpha-Lactalbumin from Cow's Milk, Bioch., Vol. 65, 1957, pp. 273-277) disclose a process improvement over the Palmer process that allows the preparation of beta-lactoglobulin crystals in a matter of days rather than weeks. However, the improved method still requires the removal of unwanted proteins prior to crystallization and also employs toluene for crystallization, which makes it incompatible with safe food production.

[0010] JP H10 218755A discloses the production of a cosmetic product containing a melanin production inhibitor, the cosmetic product comprising BLG as an active ingredient. The document also indicates that BLG can be separated, for example, by the following process: hydrochloric acid is added to milk to precipitate casein, followed by filtration to obtain whey. The pH of the whey is adjusted to 6.0, and a half-saturated amount of ammonium sulfate is added; the precipitated protein is removed by salting out, and the filtrate is recovered. The filtrate is saturated with ammonium sulfate and the precipitated protein is recovered. The recovered protein is dissolved in water again and dialyzed at pH 5.2 to separate the crystals, and β-lactoglobulin is prepared at a ratio of about 1.8 g per 1 L of whey. However, the general process steps of the proposed process described in JP H10 218755A are not sufficient to lead to the formation of BLG crystals. Therefore, the document does not contain a disclosure of the crystallization of BLG or the empowerment of BLG crystals.

[0011] US2790790 discloses a method for precipitating proteins from a solution, and more specifically, relates to fractional precipitation of relatively unconjugated proteins from an aqueous solution using sodium chloride as a precipitant. The process is shown to be useful for separating BLG by NaCl-induced precipitation at pH 3.6-3.8. In Example II of the document, it is shown that the NaCl-precipitate can be dialyzed in the usual manner to form crystalline β-lactoglobulin. However, US2790790 does not demonstrate that it is actually possible to form BLG crystals at pH 3.6-3.8, and does not contain the meaning of "the usual manner" of dialyzing the BLG precipitate. Therefore, the document does not contain a disclosure of the crystallization of BLG or the enabling of BLG crystals. Summary of the invention

[0012] It has been previously discovered that β-lactoglobulin (BLG) can be effectively isolated on an industrial scale by crystallization (see PCT application WO2018 / 115520). This crystallization method requires processing and handling of large amounts of supersaturated protein solutions.

[0013] The inventors subsequently discovered that large-scale implementation of WO2018 / 115520 may result in undesired crystallization of BLG prior to the intended crystallization of the process. This is a problem, for example, if BLG crystallization occurs in a membrane filtration unit or other equipment that is sensitive to the presence of particulate matter.

[0014] The inventors have also discovered that the separation of BLG by crystallization can be improved and made more robust by mixing two or more BLG-containing protein feeds that have no or only low levels of BLG supersaturation on their own, but provide a higher degree of supersaturation when combined, to prepare a supersaturated protein solution. The pH of one of these protein feeds should be at least 5.6 and more preferably at least 6.0, and the pH of the other should be at most 5.4 and more preferably at most 5.0. When combined, the two feeds provide a supersaturated BLG protein solution with a pH close to 5.5, which the inventors have found to be the optimal pH for the yield of BLG crystallization.

[0015] The step of combining the protein feeds is preferably performed by mixing the feeds and does not require the use of equipment that is sensitive to crystallization of the protein. Thus, the present invention reduces or even completely avoids the risk of early and unwanted BLG crystallization, which may lead to blockages and failures of the process equipment involved. The present invention is described in PCT / EP2022 / 053010.

[0016] The inventors have also realised that the techniques for protein crystallisation described in PCT / EP2022 / 053010 can be applied to other protein classes that can be crystallised under saline conditions, such as enzymes and immunoglobulins.

[0017] The schematic diagram of the formation of supersaturated protein solution is shown in Figure 1 wherein one or more Type B proteins are fed in an amount sufficient to provide an initial protein solution (having a pH less than pH MCY,TP , and preferably lower) with one or more Type A protein feeds (whose pH is above pH MCY,TP ) and the pH of the initial protein solution is at pH MCY,TP ±0.7 and it is supersaturated with respect to BLG.

[0018] The inventors have found that this method is very advantageous for large-scale crystallization of BLG, because separate type A and type B protein feeds can be produced without or with only a very limited risk of early (and therefore unwanted) BLG crystallization. However, once the type A protein feed and the type B feed are mixed, a supersaturated initial protein solution is formed.

[0019] Figure 2Schematically shows how the yield of BLG crystallization depends on the pH of the protein solution in which crystallization occurs. The inventors have found that the optimal pH is about 5.5. Therefore, by mixing the more acidic B-type protein feed with the less acidic A-type protein feed, an initial protein solution that is supersaturated with respect to BLG is obtained. If the A-type protein feed and / or the B-type protein feed are slightly supersaturated with respect to BLG, the initial protein solution must be even more supersaturated.

[0020] Guided by the disclosure of this application and common general knowledge, a skilled person can prepare a Type A protein feed and a Type B protein feed and mix them to provide an initial protein solution as described herein.

[0021] Therefore, one aspect of the present invention relates to a method for preparing a product, preferably an edible or pharmaceutically acceptable product, comprising a target protein (TP) capable of crystallizing under saline conditions, the method comprising the following steps:

[0022] a) preparing an initial protein solution comprising TP, said initial protein solution being supersaturated with respect to said TP and having a pH at which said TP can crystallize under saline conditions, preferably at a pH (pH 50) which provides a maximum crystallization yield of said TP; MCY,TP ) and most preferably within the range of + / - 0.7 pH units of the pH MCY,TP Within the range of + / -0.5 pH units,

[0023] b) crystallizing the TP in the supersaturated initial protein solution in a salting manner to obtain a solution containing TP crystals, and

[0024] c) preferably separating the TP crystals from the remaining liquid of said solution containing TP crystals,

[0025] wherein step a) involves preparing the initial protein solution by combining one or more type A protein feeds with one or more type B protein feeds,

[0026] And among them:

[0027] - "Type A protein feed" is defined as comprising the TP and having a pH greater than pH MCY,TP a protein feed at least 0.1 pH unit higher, and

[0028] - A "Type B protein feed" is defined as a feed comprising said TP and having a pH greater than pH MCY,TP Protein feed at least 0.1 pH unit lower,

[0029] And preferably, with the proviso that the TP is not β-lactoglobulin.

[0030] In the product obtained by the process, TP is preferably present in crystalline and / or isolated form.

[0031] In the context of the present invention, the terms "product", "product comprising TP" and "TP product" are used interchangeably and relate to the product of the process of the present invention.

[0032] The pH that provides the maximum crystallization yield of TP is abbreviated as “pH MCY,TP ", is determined as follows:

[0033] Determine the pH that provides the maximum crystallization yield of TP:

[0034] i) preparing a stock solution of TP having the same composition as the intended initial protein solution of step a) of the method of the present invention, but at a pH at the isoelectric point (pi) of TP. Determining the weight percentage of TP (including free TP, reversibly precipitated TP and crystallized TP) in the stock solution.

[0035] ii) A set of samples was prepared, each sample having a different pH, so that the pH range of the set of samples spanned 2-11 in steps of 0.1 pH units. The set of samples was prepared by measuring the same amount of stock solution into a glass beaker and adjusting its pH by adding the smallest possible weight of 0.5 M HCl (aq.) or 0.5 M NaOH (aq.) under stirring to reach the desired pH for each sample.

[0036] iii) adjusting the temperature of the sample to the lowest temperature used during step b) of the method of the present invention, but not below 0°C.

[0037] iv) Each sample was seeded with TP crystalline material in an amount such that the TP crystalline material comprised 1% w / w of the total weight of TP in the seeded sample.

[0038] v) The seeded sample is then stored for 24 hours under gentle stirring, maintaining the minimum temperature mentioned in step iii). Subsequently, the sample is centrifuged at 10000g for 60 minutes at the temperature of step iii); the supernatant and the precipitate are recovered separately. The precipitate of each sample is examined by microscopy using polarized light to check for the presence of TP crystalline material (which will be birefringent). If birefringent material is found in the precipitate, the corresponding supernatant is analyzed (preferably by HPLC) to determine its weight percentage of TP. Subsequently, the weight of TP in the recovered supernatant and the weight of TP in the seeded sample are calculated.

[0039] vi) The crystallization yield of each sample containing TP crystals was calculated as follows:

[0040]

[0041] vii) pH MCY,TP The pH of the sample that gave the highest crystallization yield was determined.

[0042] In the context of the present invention, the phrase "at pH MCY,TP "pH within the range of + / - x pH units" means that the pH is within (pH MCY,TP -x) to (pH MCY,TP +x). For example, if x = 0.5 and pH MCY,TP =5.7, the pH should be in the range of (5.7-0.5) to (5.7+0.5), that is, in the range of 5.2-6.2.

[0043] Another aspect of the present invention relates to the TP product obtainable by the process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Step a) of the process is shown, wherein a type A protein feed is mixed with a type B protein feed to prepare an initial protein solution.

[0045] Figure 2 is a schematic diagram of how the pH of Type A protein feed, Type B protein feed and the initial protein solution are positioned relative to the optimal pH for separation of BLG by crystallization. DETAILED DESCRIPTION

[0046] As mentioned above, one aspect of the present invention relates to a method for preparing a product, preferably an edible or pharmaceutically acceptable product, comprising a target protein (TP) capable of crystallizing under saline conditions, the method comprising the following steps:

[0047] a) preparing an initial protein solution comprising TP, said initial protein solution being supersaturated with respect to said TP and having a pH at which said TP can crystallize under saline conditions, preferably at a pH (pH 50) which provides a maximum crystallization yield of said TP; MCY,TP ) and most preferably within the range of + / - 0.7 pH units of the pH MCY,TP Within the range of + / -0.5 pH units,

[0048] b) crystallizing the TP in the supersaturated initial protein solution in a salting manner to obtain a solution containing TP crystals, and

[0049] c) preferably separating the TP crystals from the remaining liquid of the solution containing the TP crystals,

[0050] wherein step a) involves preparing the initial protein solution by combining one or more type A protein feeds with one or more type B protein feeds,

[0051] And among them:

[0052] - "Type A protein feed" is defined as comprising the TP and having a pH greater than pH MCY,TP A protein feed at least 0.1 pH unit higher, and

[0053] - A "Type B protein feed" is defined as a feed comprising said TP and having a pH greater than pH MCY,TP Protein feed at least 0.1 pH unit lower,

[0054] And preferably, with the proviso that said TP is not β-lactoglobulin.

[0055] In the context of the present invention, the term "edible product" relates to a product which is safe for human consumption and use as a food ingredient and does not contain problematic amounts of toxic ingredients such as toluene or other undesirable organic solvents.

[0056] In the context of the present invention, the term "crystal" refers to a solid material whose components (such as atoms, molecules or ions) are arranged in a highly ordered microstructure to form a lattice extending in all directions. TP crystals are protein crystals mainly containing TP that are arranged in a highly ordered microstructure to form a lattice extending in all directions. TP crystals can be, for example, monolithic or polycrystalline, and can be, for example, complete crystals, crystal fragments or combinations thereof. For example, when a complete crystal is subjected to mechanical shearing during processing, a crystal fragment is formed. Crystal fragments also have a highly ordered crystal microstructure, but may lack the uniform surface and / or uniform edge or corner of a complete crystal. For many examples of complete BLG crystals, see, for example, Figure 18 of WO2018115520 A1, and for examples of BLG crystal fragments, see Figure 13 of WO2018115520A1. In both cases, BLG crystals or crystal fragments can be visually identified as well-defined compact and coherent structures using optical microscopy. TP crystals or crystal fragments are usually at least partially transparent. Furthermore, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles as having a crystalline structure. Non-crystalline TP aggregates, on the other hand, appear to be poorly defined, opaque, and open or porous masses of irregular size.

[0057] In the context of the present invention, the term "crystallization" relates to the formation of protein crystals. Crystallization may, for example, occur spontaneously or be initiated by the addition of crystallization seeds.

[0058] The product preferably comprises TP in crystalline and / or isolated form. An edible product comprising TP in isolated form comprises at least 50% w / w TP relative to total solids. An edible product comprising TP in crystalline form comprises at least some TP crystals, and preferably a significant amount of TP crystals.

[0059] TP crystals can often be observed by microscopy and can even reach a size that makes them visible to the naked eye.

[0060] In the context of the present invention, a liquid that is "supersaturated" or "supersaturated with respect to TP" contains a concentration of dissolved TP that is higher than the saturation point of TP in the liquid under given physical and chemical conditions. The term "supersaturation" is well known in the art of crystallization (see, for example, Gérard Coquerela, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, pp. 2286-2300, No. 7, 2014), and supersaturation can be determined by many different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, supersaturation with respect to TP is determined by the following procedure.

[0061] Procedure for testing whether a liquid is supersaturated about TP under a specific set of conditions:

[0062] a) Transfer 50 ml of the liquid sample to be tested into a centrifuge tube (VWR catalog number 525-0402) with a height of 115 mm, an inner diameter of 25 mm and a capacity of 50 mL. Care should be taken to keep the sample and its subsequent fractions in the original physical and chemical conditions of the liquid during steps a) to h).

[0063] b) Immediately centrifuge the sample at 3000 g for 3.0 minutes with a maximum of 30 seconds acceleration and a maximum of 30 seconds deceleration.

[0064] c) Immediately after centrifugation, transfer as much supernatant as possible (without disturbing the pellet if one has formed) to a second centrifuge tube (of the same type as in step a).

[0065] d) Take a 0.05 mL subsample of the supernatant (subsample A).

[0066] e) 10 mg of TP crystals with a particle size of up to 200 microns (at least 98% pure TP relative to total solids) were added to a second centrifuge tube and the mixture was agitated.

[0067] f) Allow the second centrifuge tube to stand at the original temperature for 60 minutes.

[0068] g) Immediately after step f), the second centrifuge tube was centrifuged at 500 g for 10 minutes, and then another 0.05 mL supernatant subsample (subsample B) was taken.

[0069] h) Recover the centrifuged pellet of step g) (if any), resuspend it in milliQ water and immediately examine the suspension for the presence of crystals visible by microscopy.

[0070] i) Determine the concentration of TP in subsamples A and B and express the results as % TP w / w relative to the total weight of the subsamples. The TP concentration of subsample A is referred to as C TP,A , and the TP concentration of subsample B is called C TP,B .

[0071] j) If C TP,B Below C TP,A and crystals are observed in step i), then the liquid from which the sample of step a) was taken is supersaturated (under the specific conditions).

[0072] In the context of the present invention, the terms "liquid" and "solution" encompass compositions containing a combination of liquid and solid or semi-solid particles (such as, for example, protein crystals or other protein particles). Thus, a "liquid" or "solution" may be a suspension or even a slurry. However, "liquids" and "solutions" are preferably pumpable.

[0073] In the context of the present invention, a dried product (such as a powder) comprising "TP crystals" contains a product obtained by drying a suspension of TP crystals, and the crystal structure of the wet TP crystals may have been distorted during the drying process and may have lost at least part of its X-ray diffraction characteristics. Likewise, the terms "dried TP crystals" and "dried TP crystals" refer to particles obtained by drying wet TP crystals, and such dried particles themselves do not need to have a crystalline structure. However, the inventors have observed that when the dried TP crystals are resuspended in cold (4°C) desalted water in a weight ratio of 2 parts water to 1 part dried TP crystals, the TP crystals are rehydrated and recover essentially the same crystal structure (space group type and unit cell size) as before drying.

[0074] The analytical methods described in the context of PCT application WO 2018 / 115520 are equally applicable to the present invention and should be used to determine the parameters described herein.

[0075] In some preferred embodiments of the invention, the method is free of separation step c) and provides a product comprising both TP crystals and additional protein. If this method variant further comprises drying step f), it provides a dried product comprising TP crystals and additional protein. Preferably, the method contains steps a), b) and f) in direct sequence.

[0076] In some preferred embodiments of the present invention, the method further comprises a step d) of washing the TP crystals, such as the separated TP crystals obtained from step c).

[0077] In some preferred embodiments of the present invention, the method further comprises the step e) of recrystallizing the TP crystals, for example the TP crystals obtained from step c) or d).

[0078] The method may for example comprise or even consist of steps a), b), c), d) and e). Alternatively, the method may comprise or even consist of steps a), b), c) and e).

[0079] In some particularly preferred embodiments of the present invention, the method further comprises a step f) of drying the TP-containing composition derived from step b), c), d) or e).

[0080] The method may, for example, comprise or even consist of steps a), b) and f).

[0081] Alternatively, the method may comprise or even consist of steps a), b), c) and f).

[0082] Alternatively, the method may comprise or even consist of steps a), b), c), d) and f).

[0083] Alternatively, the method may comprise or even consist of steps a), b), c), d), e) and f).

[0084] The method of the invention preferably has the proviso that the TP is not β-lactoglobulin and / or has a sequence identity of at most 50% or even more preferably at most 26% with respect to bovine BLG.

[0085] In the context of the present invention, the term "BLG" or "β-lactoglobulin" pertains to BLG from mammalian species, e.g., in native and / or glycosylated form, and includes naturally occurring genetic variants. The term BLG also encompasses mammalian BLG produced by recombinant microorganisms. The term "BLG" or "β-lactoglobulin" as used herein excludes unfolded and aggregated BLG. β-lactoglobulin is a lipocalin and can bind to many hydrophobic molecules, suggesting a role in their transport. β-lactoglobulin has also been shown to be able to bind iron via siderophores and may therefore play a role in combating pathogens. After ingestion, BLG is able to shuttle complexed iron into human immune cells, thereby providing micronutrients to these cells and participating in immune tolerance.

[0086] The target protein preferably comprises a polypeptide sequence comprising at least 30 consecutive amino acid residues, more preferably at least 50 consecutive amino acid residues, even more preferably at least 100 consecutive amino acid residues and more preferably at least 150 consecutive amino acid residues.

[0087] Preferably, the TP is a globular protein, and preferably a globulin or a globin.

[0088] The term "TP" or "target protein" pertains to a native target protein that has not been denatured by heat or by other means.

[0089] In some preferred embodiments of the present invention, the TP is selected from the group consisting of enzymes, immunoglobulins, and hormones.

[0090] In some preferred embodiments of the invention, the TP is an enzyme, and preferably an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), or an isomerase (EC 5).

[0091] The "EC" number mentioned herein refers to the "enzyme commission number" used to classify enzymes according to their catalytic function.

[0092] In some preferred embodiments of the present invention, the TP is an oxidoreductase (EC 1), preferably one or more selected from the group consisting of laccase (EC 1.10.3.2), catalase (EC 1.11.1.6), glucose oxidase (EC 1.1.3.4), ligninase (EC 1.11.1.14) and peroxidase (EC 1.11.1).

[0093] In other preferred embodiments of the present invention, the TP is a transferase (EC2), preferably selected from acyltransferases (EC 2.3) or glycosyltransferases (EC 2.4).

[0094] In a further preferred embodiment of the present invention the TP is a hydrolase (EC 3).

[0095] In some embodiments where the TP has hydrolytic activity, the TP is an esterase (EC 3.1), preferably selected from the group consisting of a phytase (EC 3.1.3), a lipase (EC 3.1.1.3), a phosphatase (EC 3.1.3), a phytase and / or a nuclease (EC 3.1.11-3.1.31).

[0096] It is particularly preferred that the TP is a lipase (EC 3.1.1.3), a class of enzymes that may have important industrial applications. Examples of lipases that can be crystallized under saline conditions are found in, for example, US Pat. No. 5,719,048, which is incorporated herein by reference.

[0097] In other embodiments in which the TP has hydrolytic activity, the TP is a glycosylase (EC 3.2), preferably selected from α-amylase (EC 3.2.1.1), amyloglucosidase (EC 3.2.1.3 or EC 3.2.1.33), cellulase (EC 3.2.1.4), hemicellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), muramidase (EC 3.2.1.17), pullulanase (EC 3.2.1.41), pectinase (EC 3.2.1.67), β-galactosidase (EC 3.2.1.23), lactase (EC 3.2.1.108), glucoamylase (EC 3.2.1.20 or EC 3.2.1.3) and glucanase (EC 3.2.1.11).

[0098] In further embodiments where the TP has hydrolytic activity, the TP is a peptidase or protease (EC 3.4), for example selected from chymotrypsin (EC 3.4.21.1), trypsin, pepsin, rennet (EC 3.4.23.4) or alkaline protease.

[0099] Alternatively, the TP may be a lyase (EC 4), such as pectate lyase or alpha-acetolactate decarboxylase.

[0100] In other preferred embodiments, the TP is an isomerase (EC 5), such as glucose isomerase.

[0101] Alternatively, but also preferably, the TP may be a protein hormone, preferably human insulin (uniprot ID: P01308), a human insulin analogue, a human insulin agonist, human GLP-1, a human GLP-1 analogue or a human GLP-1 agonist.

[0102] In the context of the present invention, the term "analog" used with respect to a first protein hormone refers to a second protein having at least 90% sequence identity with respect to the first protein hormone and having the same or similar hormonal functionality as the first protein hormone. The determination of sequence identity is based on the mature protein and therefore ignores the signal peptide.

[0103] In the context of the present invention, term " sequence identity " relates to the dependency between two amino acid sequences.Sequence identity between two amino acid sequences uses the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) implemented in the Needle program of EMBOSS bag (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) (preferably 5.0.0 version or updated version) to determine.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (obtained using non-simplified option (nobrief option)) of the Needle marked as "longest identity" is used as identity percentage and is calculated as follows:

[0104] (Identical residues x 100) / (length of alignment - total number of gaps in the alignment).

[0105] Another type of useful TP is an immunoglobulin, and preferably a monoclonal immunoglobulin. The TP may be any type of immunoglobulin, however, immunoglobulin G (IgG) is currently preferred, and preferably in the form of a monoclonal IgG.

[0106] In some preferred embodiments of the invention, the TP is a bispecific IgG.

[0107] In other preferred embodiments of the present invention, the TP is an interferon, an interferon analog or an interferon agonist.

[0108] Useful examples of therapeutic proteins that are immunoglobulins or derived from immunoglobulins can be found in Lu et al.; Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https: / / doi.org / 10.1186 / s12929-019-0592-z, which is incorporated herein for all purposes.

[0109] TPs can be prepared by purification from natural sources, by fermentation in recombinant organisms, and / or by subsequent protein rearrangement.

[0110] The TP as such may be a recombinant protein, and for example a fusion protein, or a protein found in nature or in the human body.

[0111] It is particularly preferred that the TP is a therapeutic protein, i.e. a protein with a therapeutic effect. Available examples of TPs with a therapeutic effect are found in, for example, Lagassé et al. ("Recent advances in (therapeutic protein) drug development" [version 1; referees: 2 approved] F1000 Research 2017, 6 (F1000 Faculty Rev): 113 (doi: 10.12688 / f1000research.9970.1)) and / or Leader et al. ("Protein therapeutics: a summary and pharmacological classification"; Nature Reviews Drug Discovery; Vol. 7, pp. 21-39 (2008)), which are incorporated herein by reference.

[0112] Preferably, the TP is a therapeutic protein selected from the group consisting of: Muromonab-CD3, Abciximab, Rituximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Ibritumomab tiuxetan, Omalizumab, Cetuximab, Bevacizumab, Natalizumab, Panitumumab, Ranibizumab, Eculizumab, Certolizumab pegol), Ustekinumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Brentuximab vedotin, Pertuzumab, Trastuzumabemtansine), Raxibacumab, Obinutuzumab, Siltuximab, Ramucirumab, Vedolizumab, Blinatumomab, Nivolumab, Pembrolizumab, Idarucizumab, Necitumumab, Dinutuximab ), Secukinumab, Mepolizumab, Alirocumab, Evolocumab, Daratumumab, Elotuzumab, Ixekizumab, Reslizumab, Olaratumab, Bezlotoxumab, Atezolizumab, Obiltox aximab), Inotuzumabozogamicin, Brodalumab, Guselkumab, Dupilumab, Sarilumab, Avelumab, Ocrelizumab, Emicizumab, Benralizumab, Gemtuzumabozogamicin, Durvalumab rvalumab), Burosumab, Lanadelumab, Mogamulizumab, Erenumab, Galcanezumab, Tildrakizumab, Cemiplimab, Emapalumab, Fremanezumab, Ibalizumab, Moxetumomabpasudodox), Ravulizumab, Caplacizumab, Romosozumab, Risankizumab, Polatuzumab vedotin, Brolucizumab, and Crizanlizumab.

[0113] The skilled person knows how to quantify the amount of TP and will usually use a chromatography based (preferably HPLC) method in combination with a suitable assay method.

[0114] Step a) involves preparing an initial protein solution comprising TP, said initial protein solution being supersaturated with respect to said TP and having a pH at which said TP is able to crystallize under saline conditions, preferably at a pH that provides a maximum crystallization yield of said TP (pH MCY,TP ) and most preferably within the range of + / - 0.7 pH units of the pH MCY,TP within a range of + / - 0.5 pH units.

[0115] The initial protein solution of step a) is prepared by combining one or more type A protein feeds with one or more type B protein feeds, preferably by mixing one or more type A protein feeds with one or more type B protein feeds.

[0116] In the context of the present invention, a "Type A protein feed" is defined as a feed comprising TP and having a pH greater than pH MCY,TP The pH of the protein feed is at least 0.1 pH unit higher than the pH MCY,TP Any protein feed containing TP that is at least 0.1 pH unit higher and is used to prepare the initial protein solution is considered a Type A protein feed.

[0117] In the context of the present invention, a "Type B protein feed" is defined as a feed comprising TP and having a pH greater than pH MCY,TP The protein feed is at least 0.1 pH unit lower. Therefore, the pH is lower than the pH MCY,TP Any protein feed containing TP that was at least 0.1 pH unit lower and was used to prepare the initial protein solution was considered a Type B protein feed.

[0118] In the context of the present invention, the term "initial protein solution" pertains to a protein solution prepared by combining and preferably mixing one or more protein feeds of type A and one or more protein feeds of type B. The initial protein solution is a TP-containing solution which is supersaturated with respect to TP and in which the crystallization of step b) is initiated. If one or more of the protein feeds used already contain TP crystals, the initial protein solution is a solution having a pH of about pH 5. MCY,TP A first protein solution is prepared by combining, and preferably mixing, a Type A protein feed and a Type B protein feed, wherein the first protein solution is supersaturated with respect to TP and is preferably obtained by combining, and preferably mixing, a Type A protein feed and a Type B protein feed.

[0119] In the context of the present invention, the term "one or more type A protein feeds" means that the present invention can be implemented with a single type A protein feed or with several type A protein feeds, which are combined with a type B protein feed during preparation of the initial protein solution.

[0120] In the context of the present invention, the term "one or more type B protein feeds" means that the present invention can be implemented with a single type B protein feed or with several type B protein feeds, which are combined with a type A protein feed during preparation of the initial protein solution.

[0121] In the context of the present invention, the term "a Type A protein feed, if combined..." describes one or more Type A protein feeds based on the properties they would have if they were hypothetically mixed into a single Type A protein feed in the same amounts as used to prepare the initial protein solution. It is important to note that this term also encompasses the use of only a single Type A protein feed, in which case the properties relate to the single Type A protein feed. It is also important to note that this term does not require that the multiple Type A protein feeds are actually combined prior to mixing with the Type B protein feed(s).

[0122] In the context of the present invention, the term "a Type B protein feed, if combined..." describes one or more Type B protein feeds based on the properties they would have if they were hypothetically mixed into a single Type B protein feed in the same amounts as used to prepare the initial protein solution. It is important to note that this term also encompasses the use of only a single Type B protein feed, in which case the properties relate to the single Type B protein feed. It is also important to note that this term does not require that the multiple Type B protein feeds are actually combined prior to mixing with the Type A protein feed(s).

[0123] In addition to TP, the initial protein solution of step a) generally also comprises non-TP solids. In the context of the present invention, the term "non-TP solids" refers to solids, such as carbohydrates, minerals, lipids, peptides and other proteins except TP. The solids present in the initial protein solution have the characteristics of one or more protein sources for preparing the protein feed. The protein feed based on TP source prepared by fermentation generally contains impurities from the fermentation broth, such as residual nutrients and metabolic byproducts, such as other proteins, peptides and metabolic nutrients.

[0124] In addition to TP, the initial protein solution typically contains additional proteins. In the context of the present invention, the term "additional proteins" means proteins that are not TP. Additional proteins are typically protein impurities or cell debris from the fermentation broth, for example in the case where the TP has been produced by fermentation of a recombinant cell culture.

[0125] In some preferred embodiments of the invention, the initial protein solution of step a) comprises non-TP solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w and most preferably at least 10% w / w relative to total solids.

[0126] In some preferred embodiments of the invention, the initial protein solution of step a) comprises non-TP solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w and most preferably at least 40% w / w relative to total solids.

[0127] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises non-TP solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w relative to total solids.

[0128] In other preferred embodiments of the present invention, the initial protein solution of step a) comprises non-TP solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w and most preferably 0-5% w / w relative to total solids.

[0129] In a further preferred embodiment of the present invention the initial protein solution of step a) comprises non-TP solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w and most preferably 30-50% w / w relative to total solids.

[0130] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises at least 5% w / w of additional protein relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 10% w / w of additional protein relative to the total amount of protein. More preferably, the initial protein solution of step a) comprises at least 15% w / w of additional protein relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 20% w / w of additional protein relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise at least 30% w / w of additional protein relative to the total amount of protein.

[0131] In other preferred embodiments of the present invention, the initial protein solution of step a) comprises at least 35% w / w of additional protein relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at least 40% w / w of additional protein relative to the total amount of protein. More preferably, the initial protein solution of step a) may, for example, comprise at least 45% w / w of additional protein relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may comprise at least 50% w / w of additional protein relative to the total amount of protein.

[0132] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises additional protein in the range of 5-90% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise additional protein in the range of 10-80% w / w relative to the total amount of protein. The initial protein solution of step a) may, for example, comprise additional protein in the range of 20-70% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises additional protein in the range of 30-70% w / w relative to the total amount of protein.

[0133] As mentioned above, the inventors have found that TP can be crystallized without the use of organic solvents. This purification method can also be used to refine preparations containing other proteins that have been subjected to some TP purification and provides a simple method to further improve the purity of TP. Therefore, in some preferred embodiments of the present invention, the initial protein solution of step a) contains additional protein in the range of 1-20% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) may contain additional protein in the range of 2-15% w / w relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may, for example, contain additional protein in the range of 3-10% w / w relative to the total amount of protein.

[0134] The amounts and concentrations of TP and other proteins in the initial protein solution and protein feed all refer to dissolved protein and do not include precipitated or crystallized protein.

[0135] In the context of the present invention, the term "weight ratio" between component X and component Y means the weight ratio calculated by m X / m Y The value obtained, where m X is the amount (weight) of component X and m Y is the amount of component Y (by weight).

[0136] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises at least 1% w / w TP relative to the total amount of protein. Preferably, the initial protein solution of step a) comprises at least 2% w / w TP relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises at least 5% w / w TP relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at least 10% w / w TP relative to the total amount of protein.

[0137] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises at least 12% w / w TP relative to the total amount of protein. For example, the initial protein solution of step a) may comprise at least 15% w / w TP relative to the total amount of protein. The initial protein solution of step a) may, for example, comprise at least 20% w / w TP relative to the total amount of protein. Alternatively, the initial protein solution of step a) may comprise at least 30% w / w TP relative to the total amount of protein.

[0138] In other preferred embodiments of the present invention, the initial protein solution of step a) comprises at least 40% w / w TP relative to the total amount of protein. More preferably, the initial protein solution of step a) may comprise at least 45% w / w TP relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may, for example, comprise at least 50% w / w TP relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise at least 55% w / w TP relative to the total amount of protein.

[0139] In some particularly preferred embodiments of the present invention, the initial protein solution of step a) comprises at most 95% w / w TP relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 90% w / w TP relative to the total amount of protein. More preferably, the initial protein solution of step a) may, for example, comprise at most 85% w / w TP relative to the total amount of protein. Even more preferably, the initial protein solution of step a) may, for example, comprise at most 80% w / w TP relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 78% w / w TP relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise at most 75% w / w TP relative to the total amount of protein.

[0140] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises TP in the range of 1-95% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise TP in the range of 5-90% w / w relative to the total amount of protein. More preferably, the initial protein solution of step a) comprises TP in the range of 10-85% w / w relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises TP in the range of 10-80% w / w relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise TP in the range of 20-70% w / w relative to the total amount of protein.

[0141] In other preferred embodiments of the present invention, the initial protein solution of step a) comprises TP in the range of 10-95% w / w relative to the total amount of protein. Preferably, the initial protein solution of step a) may comprise TP in the range of 12-90% w / w relative to the total amount of protein. More preferably, the initial protein solution of step a) comprises TP in the range of 15-85% w / w relative to the total amount of protein. Even more preferably, the initial protein solution of step a) comprises TP in the range of 15-80% w / w relative to the total amount of protein. Most preferably, the initial protein solution of step a) may comprise TP in the range of 30-70% w / w relative to the total amount of protein.

[0142] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises TP in an amount of at least 0.4% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises TP in an amount of at least 1.0% w / w. More preferably, the initial protein solution comprises TP in an amount of at least 2.0% w / w. Even more preferably, the initial protein solution comprises TP in an amount of at least 4% w / w.

[0143] Higher concentrations of TP are even more preferred, and preferably, the initial protein solution comprises TP in an amount of at least 6% w / w. More preferably, the initial protein solution comprises TP in an amount of at least 10% w / w. Even more preferably, the initial protein solution comprises TP in an amount of at least 15% w / w.

[0144] In some preferred embodiments of the present invention, the initial protein solution of step a) comprises TP in an amount in the range of 0.4-40% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises TP in an amount in the range of 1-35% w / w. More preferably, the initial protein solution comprises TP in an amount in the range of 4-30% w / w. Even more preferably, the initial protein solution comprises TP in an amount in the range of 10-25% w / w.

[0145] In other preferred embodiments of the present invention, the initial protein solution of step a) comprises TP in an amount in the range of 1-45% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises TP in an amount in the range of 3-40% w / w. More preferably, the initial protein solution comprises TP in an amount in the range of 5-36% w / w. Even more preferably, the initial protein solution comprises TP in an amount in the range of 7-34% w / w.

[0146] In a further preferred embodiment of the present invention, the initial protein solution of step a) comprises TP in an amount in the range of 6-32% w / w relative to the weight of the initial protein solution. Preferably, the initial protein solution comprises TP in an amount in the range of 8-30% w / w. More preferably, the initial protein solution comprises TP in an amount in the range of 10-28% w / w. Even more preferably, the initial protein solution comprises TP in an amount in the range of 12-26% w / w.

[0147] Preferably, the initial protein solution is a desalted initial protein solution.

[0148] In this context, the term desalination means that the conductivity of the initial protein solution is at most 15 mS / cm, and preferably at most 10 mS / cm, and even more preferably at most 8 mS / cm. The UF permeate conductivity of the desalted initial protein solution is preferably at most 7 mS / cm, more preferably at most 4 mS / cm and even more preferably at most 1 mS / cm.

[0149] The terms “consists essentially of” or “consisting essentially of” mean that the claim or feature in question covers the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics of the claimed invention.

[0150] The proteins of the initial protein solution are preferably as close to their native state as possible and have preferably been subjected to only mild, non-denaturing heat treatment (if any at all in any case).

[0151] The process of the present invention is particularly advantageous for separating TP from a crude initial protein solution containing other solids besides TP.

[0152] The initial protein solution may for example contain carbohydrates, such as, for example, sucrose, glucose, lactose, oligosaccharides and / or hydrolysates of lactose (i.e., glucose and galactose). The initial protein solution may for example contain carbohydrates in the range of 0-40% w / w, such as in the range of 1-30% w / w or in the range of 2-20% w / w.

[0153] In some preferred embodiments of the invention, the initial protein solution contains at most 20% w / w carbohydrates, preferably at most 10% w / w carbohydrates, more preferably at most 5% w / w carbohydrates and even more preferably at most 2% w / w carbohydrates.

[0154] The initial protein solution may also contain lipids, for example in the form of triglycerides and / or other lipid types such as phospholipids.

[0155] In some embodiments of the invention, the initial protein solution of step a) comprises a total amount of lipids of at most 15% w / w relative to the total solids. Preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 10% w / w relative to the total solids. More preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 6% w / w relative to the total solids. Even more preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 1.0% w / w relative to the total solids. Most preferably, the initial protein solution of step a) comprises a total amount of lipids of at most 0.5% w / w relative to the total solids.

[0156] The total amount of protein in the initial protein solution is typically at least 1% w / w relative to the weight of the initial protein solution. Preferably, the total amount of protein in the initial protein solution is at least 5% w / w. More preferably, the total amount of protein in the initial protein solution is at least 10% w / w. Even more preferably, the total amount of protein in the initial protein solution is at least 15% w / w.

[0157] In some preferred embodiments of the present invention, the total amount of protein in the initial protein solution is in the range of 1-50% w / w. Preferably, the total amount of protein in the initial protein solution is in the range of 5-40% w / w. More preferably, the total amount of protein in the initial protein solution is in the range of 10-30% w / w. Even more preferably, the total amount of protein in the initial protein solution is in the range of 15-25% w / w.

[0158] The total solids of the initial protein solution are typically at least 1% w / w relative to the weight of the initial protein solution. Preferably, the total solids of the initial protein solution are at least 5% w / w. More preferably, the total solids of the initial protein solution are at least 10% w / w. Even more preferably, the total solids of the initial protein solution are at least 15% w / w.

[0159] In some preferred embodiments of the present invention, the total solids of the initial protein solution are in the range of 5-50% w / w. Preferably, the total solids of the initial protein solution are in the range of 8-40% w / w. More preferably, the total solids of the initial protein solution are in the range of 10-30% w / w. Even more preferably, the total solids of the initial protein solution are in the range of 15-25% w / w.

[0160] In other preferred embodiments of the present invention, the total solids of the initial protein solution are in the range of 8-50% w / w. Preferably, the total solids of the initial protein solution are in the range of 10-45% w / w. More preferably, the total solids of the initial protein solution are in the range of 12-40% w / w. Even more preferably, the total solids of the initial protein solution are in the range of 15-35% w / w.

[0161] The total amount of protein in the initial protein solution was determined according to Example 9.2 of WO2018 / 115520.

[0162] The initial protein solution of step a) is prepared by combining and preferably mixing one or more type A protein feeds with one or more type B protein feeds.

[0163] In a most preferred embodiment, the initial protein solution is prepared by combining and preferably mixing a single Type A protein feed with a single Type B protein feed. Crystallization is preferably initiated by the subsequent addition of a seed composition, preferably in liquid form or alternatively in powder form.

[0164] The initial protein solution may also contain a protein feed that is neither Form A nor Form B, for example, at a pH of MCY,TP Although it is possible to use a protein feed containing TP or a protein feed without TP, it is preferred to limit or even avoid the presence of such additional protein feeds unless they are used to optimize the process, for example by providing TP crystals or other seed material to initiate crystallization.

[0165] Preparation of the initial protein solution may involve the addition of other ingredients in addition to the protein feed, such as the addition of an acid or base to provide an initial protein solution with a suitable pH. However, it is preferred that the one or more Type A protein feeds and the one or more Type B protein feeds have been adjusted to provide the desired pH when combined, and thus limit or even avoid the addition of other ingredients.

[0166] The protein feed is typically an aqueous liquid and / or powder comprising TP and impurities. Examples of typical impurities are minerals, carbohydrates, other proteins, such as, for example, additional proteins.

[0167] Thus, one or more Type A protein feeds, one or more Type B protein feeds, and any additional ingredients may be combined in any order to provide an initial protein solution.

[0168] An initial protein solution is prepared by combining the protein feeds, preferably by mixing one or more Type A protein feeds with one or more Type B protein feeds, and optionally with any additional protein feeds or ingredients.

[0169] All protein feeds and any additional ingredients are provided with the appropriate chemical composition and are combined in amounts sufficient to provide an initial protein solution as described herein.

[0170] The present inventors have found that the maximum TP yield of TP crystallization is at about pH MCY,TP It is therefore preferred that the pH of the initial protein solution is closer to pH than MCY,TP :

[0171] - pH of the Type A protein feed in the case of pooling, or

[0172] - pH of the Type B protein feed in the case of pooling, or

[0173] - The pH of the Type A protein feed in case of pooling, and the pH of the Type B protein feed in case of pooling.

[0174] It is generally preferred to combine the protein feeds to provide an initial protein solution that is in the metastable region with respect to TP supersaturation, ie, in the supersaturation region where TP crystals can grow when seeding is used but crystallization does not initiate spontaneously.

[0175] In some preferred embodiments of the present invention:

[0176] - In the case of pooling, the pH of the Type A protein feed will be at least pH MCY,TP +0.1, more preferably at least pH MCY,TP+0.2, even more preferably at least pH MCY,TP +0.3 and most preferably at least pH MCY,TP +0.4, and

[0177] - In case of pooling, the pH of the Type B protein feed will be at most pH MCY,TP -0.1, more preferably up to pH MCY,TP -0.2, even more preferably up to pH MCY,TP -0.3 and most preferably up to pH MCY,TP -0.4.

[0178] In the case where only a single Type A protein feed is used, the phrase "when combined, will" refers to the single Type A protein feed itself. Similarly, in the case where only a single Type B protein feed is used, the phrase "when combined, will" refers to the single Type B protein feed itself.

[0179] Thus, the phrase "[a particular property] of the Type A protein feed, when mixed, will" means

[0180] - in the case of a single A-type protein feed, its [specified property], or

[0181] - In case of using two or more Type A protein feeds, in case of mixing, their [specific properties].

[0182] For example, it is stated that, in the case of mixing, the pH of the Type A protein feed will be at least pH MCY,TP +0.1 means:

[0183] - In case of using only a single A-type protein feed, its pH is at least pH MCY,TP +0.1

[0184] - In case two or more Type A protein feeds are used, the pH thereof, when mixed, is at least pH MCY,TP +0.1.

[0185] For example, in an embodiment where two Type A protein feeds are used, the phrase "upon combination, will" means that upon mixing the two Type A protein feeds, a particular property will be obtained.

[0186] Therefore, in some preferred embodiments of the present invention:

[0187] -About Type A protein feed:

[0188] - in the case of using only a single A-type protein feed, or

[0189] - In case two or more Type A protein feeds are used, the pH thereof, when mixed, is at least pH MCY,TP +0.1, more preferably at least pH MCY,TP +0.2, even more preferably at least pH MCY,TP +0.3 and most preferably at least pH MCY,TP +0.4, and

[0190] -About B-type protein feeding:

[0191] - in the case of using only a single B-type protein feed, or

[0192] - In case of using two or more B-type protein feeds, the pH thereof, in case of mixing, is at most pH MCY,TP -0.1, more preferably up to pH MCY,TP -0.2, even more preferably up to pH MCY,TP -0.3 and most preferably up to pH MCY,TP -0.4.

[0193] Preferably:

[0194] -About Type A protein feed:

[0195] - in the case of using only a single A-type protein feed, or

[0196] - In case two or more Type A protein feeds are used, the pH thereof, when mixed, is at least pH MCY,TP +0.5, more preferably at least pH MCY,TP +0.6, even more preferably at least pH MCY,TP +0.7 and most preferably at least pH MCY,TP +0.8, and

[0197] -About B-type protein feeding:

[0198] - in the case of using only a single B-type protein feed, or

[0199] - In case of using two or more B-type protein feeds, the pH thereof, in case of mixing, is at most pH MCY,TP -0.5, preferably at most pHMCY,TP -0.6, even more preferably up to pH MCY,TP -0.7 and most preferably up to pH MCY,TP -0.8.

[0200] In other preferred embodiments of the present invention:

[0201] -About Type A protein feed:

[0202] - in the case of using only a single A-type protein feed, or

[0203] - In case two or more Type A protein feeds are used, the pH thereof, when mixed, is at least pH MCY,TP +0.9, more preferably at least pH MCY,TP +1.0, even more preferably at least pH MCY,TP +1.2 and most preferably at least pH MCY,TP +1.5, and

[0204] -About B-type protein feeding:

[0205] - in the case of using only a single B-type protein feed, or

[0206] - In case of using two or more Type B protein feeds, the pH thereof, in case of mixing, is at most pH MCY,TP -0.9, preferably at most pHMCY,TP -1.0, even more preferably up to pH MCY,TP -1.2 and most preferably up to pH MCY,TP -1.5.

[0207] Preferably, with respect to the Type A protein feed:

[0208] - in the case of using only a single A-type protein feed, or

[0209] - In case of using two or more A-type protein feeds, in case of mixing,

[0210] Its pH range is pH MCY,TP +0.1 to pH MCY,TP +4.5, more preferably pH MCY,TP +0.3 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.5 to pH MCY,TP +1.5 and most preferably pH MCY,TP +0.6 to pH MCY,TP +1.0.

[0211] Alternatively, but equally preferably:

[0212] - in the case of using only a single A-type protein feed, or

[0213] - In case of using two or more A-type protein feeds, in case of mixing,

[0214] Its pH range is pH MCY,TP +0.5 to pH MCY,TP +2.5, more preferably pHMCY,TP +0.6 to pH MCY,TP +2.0, even more preferably pH MCY,TP +0.7 to pH MCY,TP +1.5 and most preferably pH MCY,TP +0.7 to pH MCY,TP +1.0.

[0215] In a further preferred embodiment of the present invention, with respect to the Type A protein feed:

[0216] - in the case of using only a single A-type protein feed, or

[0217] - In case of using two or more A-type protein feeds, in case of mixing,

[0218] Its pH range is pH MCY,TP +0.7 to pH MCY,TP +3.0, more preferably pH MCY,TP +0.8 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.9 to pH MCY,TP +2.0 and most preferably pH MCY,TP +1.0 to pH MCY,TP +1.5.

[0219] In some preferred embodiments of the present invention:

[0220] -About B-type protein feeding:

[0221] - in the case of using only a single B-type protein feed, or

[0222] - In case of using two or more B-type protein feeds, the pH range in case of mixing is pH MCY,TP -3.5 to pH MCY,TP -0.1, more preferably pH MCY,TP -2.5 to pH MCY,TP -0.3, even more preferably pH MCY,TP -1.5 to pH MCY,TP -0.5, and most preferably pH MCY,TP -1.0 to pH MCY,TP -0.6.

[0223] Preferably:

[0224] - in the case of using only a single B-type protein feed, or

[0225] - In case of using two or more B-type protein feeds, in case of mixing,

[0226] Its pH range is pH MCY,TP -1.5 to pH MCY,TP -0.5, more preferably pH MCY,TP -1.4 to pH MCY,TP -0.6, even more preferably pH MCY,TP -1.2 to pH MCY,TP -0.7, and most preferably pH MCY,TP -1.0 to pH MCY,TP -0.7.

[0227] Alternatively, but equally preferably, with respect to the Type B protein feed:

[0228] - in the case of using only a single B-type protein feed, or

[0229] - In case of using two or more B-type protein feeds, in case of mixing,

[0230] Its pH range is pH MCY,TP -2.0 to pH MCY,TP -0.7, more preferably pH MCY,TP -1.8 to pH MCY,TP -0.8, even more preferably pH MCY,TP -1.6 to pH MCY,TP -0.9, and most preferably pH MCY,TP -1.5 to pH MCY,TP -1.0.

[0231] In some preferred embodiments of the invention, at least one of the one or more Type A protein feeds is unsaturated with respect to TP, and more preferably all of the one or more Type A protein feeds are unsaturated with respect to TP.

[0232] In other preferred embodiments of the present invention, at least one of the one or more Type B protein feeds is unsaturated with respect to TP, and more preferably all of the one or more Type B protein feeds are unsaturated with respect to TP.

[0233] In a further preferred embodiment of the present invention, the type A or type B protein feed is fully saturated with respect to TP.

[0234] In some preferred embodiments of the present invention, at least one of the one or more type A protein feeds contains no TP crystals, and more preferably all of the one or more type A protein feeds contain no TP crystals.

[0235] In other preferred embodiments of the present invention, at least one of the one or more Type B protein feeds contains no TP crystals, and more preferably all of the one or more Type B protein feeds contain no TP crystals.

[0236] It should be noted that even if a feed contains no TP crystals, the feed may still be saturated or even supersaturated with respect to TP.

[0237] In a further preferred embodiment of the present invention, the type A or type B protein feed is entirely free of TP crystals.

[0238] In other preferred embodiments of the present invention, the Type A or Type B protein feed is entirely free of TP crystals.

[0239] In some preferred embodiments of the invention, at least one of the one or more Type A protein feeds is not oversaturated with respect to TP, and more preferably all of the one or more Type A protein feeds are not oversaturated with respect to TP.

[0240] In other preferred embodiments of the invention, at least one of the one or more Type B protein feeds is not oversaturated with respect to TP, and more preferably all of the one or more Type B protein feeds are not oversaturated with respect to TP.

[0241] In some preferred embodiments of the present invention, at least one of the one or more Type A protein feeds of step a) is saturated with respect to TP.

[0242] In some preferred embodiments of the present invention, at least one of the one or more Type B protein feeds of step a) is saturated with respect to TP.

[0243] In some preferred embodiments of the invention, at least one of the one or more Type A protein feeds is supersaturated with respect to TP, but is in a metastable region where spontaneous crystallization does not occur.

[0244] In other preferred embodiments of the invention, at least one of the one or more Type B protein feeds is supersaturated with respect to TP, but is in a metastable zone where spontaneous crystallization does not occur.

[0245] In some preferred embodiments of the present invention, the Type A and Type B protein feed free of TP crystals contributes at least 1% w / w, more preferably at least 10% w / w, even more preferably at least 20% w / w and most preferably at least 40% w / w of the TP content of the initial protein solution.

[0246] Preferably, the TP crystal-free Type A and Type B protein feed contributes at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w of the TP content of the initial protein solution.

[0247] In other preferred embodiments of the present invention, the type A and type B protein feed free of TP crystals contributes at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 96% w / w and most preferably at least 99% w / w of the TP content of the initial protein solution.

[0248] For example, it may be preferred that the Type A and Type B protein feed containing no TP crystals contributes 100% w / w of the TP content of the initial protein solution. This is typically the case, for example, if seed material is added after the initial protein solution is prepared.

[0249] The present inventors have found that it is generally desirable that at least some of the protein feed is not oversaturated with respect to TP, as this significantly reduces the risk of uncontrolled crystallization during production.

[0250] Thus, in some preferred embodiments of the present invention, the Type A and Type B protein feed which is not oversaturated with respect to TP contributes at least 1% w / w, more preferably at least 10% w / w, even more preferably at least 20% w / w and most preferably at least 40% w / w of the TP content of the initial protein solution.

[0251] Preferably, the type A and type B protein feed not oversaturated with respect to TP contributes at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w of the TP content of the initial protein solution.

[0252] In a further preferred embodiment of the present invention the type A and type B protein feed which is not oversaturated with respect to TP contributes at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 96% w / w and most preferably at least 99% w / w of the TP content of the initial protein solution.

[0253] It is generally preferred that the Type A and Type B protein feeds, which are not oversaturated with respect to TP, contribute about 100% w / w of the TP content of the initial protein solution.

[0254] It is preferred that Type A and Type B protein feeds be prepared, stored and used at temperatures that avoid protein denaturation and / or degradation.

[0255] In some preferred embodiments of the invention, the temperature of the Type A protein feed, when combined, is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

[0256] Therefore, in some preferred embodiments of the present invention:

[0257] -About Type A protein feed:

[0258] - in the case of using only a single A-type protein feed, or

[0259] - In case two or more type A protein feeds are used, their temperature, in case of mixing, is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

[0260] In other preferred embodiments of the present invention, the temperature of the Type A protein feed, in case of combination, is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C.

[0261] Therefore, in other preferred embodiments of the present invention:

[0262] -About Type A protein feed:

[0263] - in the case of using only a single A-type protein feed, or

[0264] - In case two or more type A protein feeds are used, their temperature, in case of mixing, is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C.

[0265] In some preferred embodiments of the invention, the temperature of the Type B protein feed, when combined, is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

[0266] Therefore, in some preferred embodiments of the present invention:

[0267] -About B-type protein feeding:

[0268] - in the case of using only a single B-type protein feed, or

[0269] - In case two or more Type B protein feeds are used, their temperature, in case of mixing, is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

[0270] In other preferred embodiments of the present invention, the temperature of the Type B protein feed, in case of combination, is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C.

[0271] Therefore, in other preferred embodiments of the present invention, with respect to the Type B protein feed:

[0272] - in the case of using only a single B-type protein feed, or

[0273] - In case of using two or more B-type protein feeds, in case of mixing,

[0274] Its temperature is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C.

[0275] The present inventors have found that initial protein solutions prepared from protein feeds having low conductivity generally provide higher TP crystallization yields.

[0276] In some preferred embodiments of the invention, the conductivity of the Type A protein feed, with mixing, is at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 4 mS / cm.

[0277] Therefore, in some preferred embodiments of the present invention:

[0278] -About Type A protein feed:

[0279] - in the case of using only a single A-type protein feed, or

[0280] - In case two or more type A protein feeds are used, their conductivity, in case of mixing, is at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 4 mS / cm.

[0281] In other preferred embodiments of the present invention, the conductivity of the Type A protein feed, in case of mixing, is at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm and most preferably at most 0.5 mS / cm.

[0282] Therefore, in other preferred embodiments of the present invention, with respect to the Type A protein feed:

[0283] - in the case of using only a single A-type protein feed, or

[0284] - In case of using two or more A-type protein feeds, in case of mixing,

[0285] Its conductivity is at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm and most preferably at most 0.5 mS / cm.

[0286] In some preferred embodiments of the invention, the conductivity of the Type B protein feed, with mixing, is at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 4 mS / cm.

[0287] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0288] - in the case of using only a single B-type protein feed, or

[0289] - In case of using two or more B-type protein feeds, in case of mixing,

[0290] Its conductivity is at most 10 mS / cm, more preferably at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 4 mS / cm.

[0291] In other preferred embodiments of the present invention, the conductivity of the Type B protein feed, in case of mixing, is at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm and most preferably at most 0.5 mS / cm.

[0292] Therefore, in other preferred embodiments of the present invention, with respect to the Type B protein feed:

[0293] - in the case of using only a single B-type protein feed, or

[0294] - In case of using two or more B-type protein feeds, in case of mixing,

[0295] Its conductivity is at most 3 mS / cm, more preferably at most 2 mS / cm, even more preferably at most 1 mS / cm and most preferably at most 0.5 mS / cm.

[0296] The inventors have also observed that if the ratio between the conductivity of the Type A protein feed and the Type B protein feed (expressed in mS / cm) and the total amount of protein (expressed in wt % of total protein relative to the total weight of the feed) is maintained at or below a certain threshold, a TP crystallization yield is obtained.

[0297] In some preferred embodiments of the present invention, the ratio between the conductivity of the Type A protein feed and the total amount of protein, in the case of mixing, will be at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12 and most preferably at most 0.10.

[0298] Therefore, in some preferred embodiments of the present invention:

[0299] -About Type A protein feed:

[0300] - in the case of using only a single A-type protein feed, or

[0301] - In case two or more type A protein feeds are used, the ratio between their conductivity and the total amount of protein, in case of mixing, is at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12 and most preferably at most 0.10.

[0302] In some preferred embodiments of the present invention, the ratio between the conductivity of the Type B protein feed and the total amount of protein, in the case of mixing, will be at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12 and most preferably at most 0.10.

[0303] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0304] - in the case of using only a single B-type protein feed, or

[0305] - In case of using two or more B-type protein feeds, in case of mixing,

[0306] The ratio between its conductivity and the total amount of protein is at most 0.3, more preferably at most 0.25, more preferably at most 0.20, more preferably at most 0.18, even more preferably at most 0.12 and most preferably at most 0.10.

[0307] In some preferred embodiments of the invention, the Type A protein feeds, when combined, will contain at least 1% w / w TP relative to the weight of the Type A protein feed combination, more preferably at least 3% w / w, even more preferably at least 5% w / w TP and most preferably at least 7% w / w TP relative to the weight of the Type A protein feed combination.

[0308] Thus, in some preferred embodiments of the invention, with respect to Type A protein feed:

[0309] - in the case of using only a single A-type protein feed, or

[0310] - In case of using two or more A-type protein feeds, in case of mixing,

[0311] It comprises at least 1% w / w TP relative to the total weight of the type A protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w TP and most preferably at least 7% w / w TP relative to the total weight of the type A protein feed.

[0312] In other preferred embodiments of the present invention, the Type A protein feeds, if combined, will contain 1-45% w / w TP relative to the weight of the Type A protein feed combination, more preferably 3-40% w / w, even more preferably 5-36% w / w TP and most preferably 7-34% w / w TP relative to the weight of the Type A protein feed combination.

[0313] Therefore, in other preferred embodiments of the present invention, with respect to the Type A protein feed:

[0314] - in the case of using only a single A-type protein feed, or

[0315] - In case of using two or more A-type protein feeds, in case of mixing,

[0316] It comprises 1-45% w / w TP relative to the total weight of the type A protein feed, more preferably 3-40% w / w, even more preferably 5-36% w / w TP and most preferably 7-34% w / w TP relative to the total weight of the type A protein feed.

[0317] In a further preferred embodiment of the present invention, the Type A protein feeds, when combined, comprise 6-32% w / w TP relative to the weight of the Type A protein feed combination, more preferably 8-30% w / w, even more preferably 10-28% w / w TP and most preferably 12-26% w / w TP relative to the weight of the Type A protein feed combination.

[0318] Therefore, in a further preferred embodiment of the present invention, with respect to the Type A protein feed:

[0319] - in the case of using only a single A-type protein feed, or

[0320] - In case of using two or more A-type protein feeds, in case of mixing,

[0321] It comprises 6-32% w / w TP relative to the total weight of the type A protein feed, more preferably 8-30% w / w, even more preferably 10-28% w / w TP and most preferably 12-26% w / w TP relative to the total weight of the type A protein feed.

[0322] In some preferred embodiments of the invention, the Type B protein feeds, if combined, will contain at least 1% w / w TP relative to the weight of the Type B protein feed combination, more preferably at least 3% w / w, even more preferably at least 5% w / w TP and most preferably at least 7% w / w TP relative to the weight of the Type B protein feed combination.

[0323] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0324] - in the case of using only a single B-type protein feed, or

[0325] - In case of using two or more B-type protein feeds, in case of mixing,

[0326] It comprises at least 1% w / w TP relative to the total weight of the Type B protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w TP, and most preferably at least 7% w / w TP relative to the total weight of the Type B protein feed.

[0327] In other preferred embodiments of the invention, the Type B protein feeds, if combined, will contain 1-45% w / w TP relative to the weight of the Type B protein feed combination, more preferably 3-40% w / w, even more preferably 5-36% w / w TP and most preferably 7-34% w / w TP relative to the weight of the Type B protein feed combination.

[0328] Therefore, in other preferred embodiments of the present invention, with respect to the Type B protein feed:

[0329] - in the case of using only a single B-type protein feed, or

[0330] - In case of using two or more B-type protein feeds, in case of mixing,

[0331] It comprises 1-45% w / w TP relative to the total weight of the Type B protein feed, more preferably 3-40% w / w, even more preferably 5-36% w / w TP and most preferably 7-34% w / w TP relative to the total weight of the Type B protein feed.

[0332] In a further preferred embodiment of the invention, the Type B protein feeds, if combined, will comprise 6-32% w / w TP relative to the weight of the Type B protein feed combination, more preferably 8-30% w / w, even more preferably 10-28% w / w TP and most preferably 12-26% w / w TP relative to the weight of the Type B protein feed combination.

[0333] Therefore, in a further preferred embodiment of the present invention, with respect to the Type B protein feed:

[0334] - in the case of using only a single B-type protein feed, or

[0335] - In case of using two or more B-type protein feeds, in case of mixing,

[0336] It comprises 6-32% w / w TP relative to the total weight of the Type B protein feed, more preferably 8-30% w / w, even more preferably 10-28% w / w TP and most preferably 12-26% w / w TP relative to the total weight of the Type B protein feed.

[0337] In some preferred embodiments of the invention, the Type A protein feed, in the case of mixing, will contain TP in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein.

[0338] Thus, in some preferred embodiments of the invention, with respect to Type A protein feed:

[0339] - in the case of using only a single A-type protein feed, or

[0340] - In case of using two or more A-type protein feeds, in case of mixing,

[0341] It comprises TP in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein.

[0342] In some preferred embodiments of the invention, the Type B protein feed, in the case of mixing, will comprise TP in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein.

[0343] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0344] - in the case of using only a single B-type protein feed, or

[0345] - In case of using two or more B-type protein feeds, in case of mixing,

[0346] It comprises TP in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein.

[0347] In some preferred embodiments of the invention, the Type A protein feed, if combined, will contain non-TP solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w and most preferably at least 10% w / w relative to the total solids.

[0348] Thus, in some preferred embodiments of the invention, with respect to Type A protein feed:

[0349] - in the case of using only a single A-type protein feed, or

[0350] - In case of using two or more A-type protein feeds, in case of mixing,

[0351] It is included in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w and most preferably at least 10% w / w of non-TP solids relative to total solids.

[0352] In other preferred embodiments of the present invention, the Type A protein feed, if mixed, will contain non-TP solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w and most preferably at least 40% w / w relative to the total solids.

[0353] Therefore, in other preferred embodiments of the present invention, with respect to the Type A protein feed:

[0354] - in the case of using only a single A-type protein feed, or

[0355] - In case of using two or more A-type protein feeds, in case of mixing,

[0356] It comprises at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w and most preferably at least 40% w / w non-TP solids relative to total solids.

[0357] In some preferred embodiments of the invention, the Type A protein feed, if combined, will contain non-TP solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w relative to total solids.

[0358] Thus, in some preferred embodiments of the invention, with respect to Type A protein feed:

[0359] - in the case of using only a single A-type protein feed, or

[0360] - In case of using two or more A-type protein feeds, in case of mixing,

[0361] It is included in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w of non-TP solids relative to total solids.

[0362] In some preferred embodiments of the present invention, the Type A protein feed, when mixed, will contain non-TP solids in an amount of 0-70% w / w, more preferably 0-60% w / w, even more preferably 0-50% w / w and most preferably 0-40% w / w relative to total solids.

[0363] Thus, in some preferred embodiments of the invention, with respect to Type A protein feed:

[0364] - in the case of using only a single A-type protein feed, or

[0365] - In case of using two or more A-type protein feeds, in case of mixing,

[0366] It is included in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w of non-TP solids relative to total solids.

[0367] In other preferred embodiments of the present invention, the Type A protein feed, if combined, will contain non-TP solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w and most preferably 0-5% w / w relative to total solids.

[0368] Therefore, in other preferred embodiments of the present invention, with respect to the Type A protein feed:

[0369] - in the case of using only a single A-type protein feed, or

[0370] - In case of using two or more A-type protein feeds, in case of mixing,

[0371] It is included in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w and most preferably 0-5% w / w of non-TP solids relative to the total solids.

[0372] In a further preferred embodiment of the invention, the Type A protein feed, if combined, will comprise non-TP solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w and most preferably 30-50% w / w relative to total solids.

[0373] Therefore, in a further preferred embodiment of the present invention, with respect to the Type A protein feed:

[0374] - in the case of using only a single A-type protein feed, or

[0375] - In case of using two or more A-type protein feeds, in case of mixing,

[0376] It is included in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w and most preferably 30-50% w / w of non-TP solids relative to total solids.

[0377] In some preferred embodiments of the invention, the Type B protein feed, if combined, will contain non-TP solids in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w and most preferably at least 10% w / w relative to the total solids.

[0378] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0379] - in the case of using only a single B-type protein feed, or

[0380] - In case of using two or more B-type protein feeds, in case of mixing,

[0381] It is included in an amount of at least 1% w / w, more preferably at least 2% w / w, even more preferably at least 5% w / w and most preferably at least 10% w / w of non-TP solids relative to total solids.

[0382] In other preferred embodiments of the present invention, the Type B protein feed, if combined, will contain non-TP solids in an amount of at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w and most preferably at least 40% w / w relative to the total solids.

[0383] Therefore, in other preferred embodiments of the present invention, with respect to the Type B protein feed:

[0384] - in the case of using only a single B-type protein feed, or

[0385] - In case of using two or more B-type protein feeds, in case of mixing,

[0386] It comprises at least 15% w / w, more preferably at least 20% w / w, even more preferably at least 30% w / w and most preferably at least 40% w / w non-TP solids relative to total solids.

[0387] In some preferred embodiments of the invention, the Type B protein feed, if combined, will contain non-TP solids in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w relative to total solids.

[0388] Thus, in some preferred embodiments of the invention, with respect to the Type B protein feed:

[0389] - in the case of using only a single B-type protein feed, or

[0390] - In case of using two or more B-type protein feeds, in case of mixing,

[0391] It is included in an amount of 0-70% w / w, more preferably 1-60% w / w, even more preferably 2-50% w / w and most preferably 3-40% w / w of non-TP solids relative to total solids.

[0392] In other preferred embodiments of the present invention, the Type B protein feed, if combined, will contain non-TP solids in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w and most preferably 0-5% w / w relative to the total solids.

[0393] Therefore, in other preferred embodiments of the present invention, with respect to the Type B protein feed:

[0394] - in the case of using only a single B-type protein feed, or

[0395] - In case of using two or more B-type protein feeds, in case of mixing,

[0396] It is included in an amount of 0-20% w / w, more preferably 0-15% w / w, even more preferably 0-10% w / w and most preferably 0-5% w / w of non-TP solids relative to the total solids.

[0397] In a further preferred embodiment of the invention, the Type B protein feed, if combined, will comprise non-TP solids in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w and most preferably 30-50% w / w relative to total solids.

[0398] Therefore, in a further preferred embodiment of the present invention, with respect to the Type B protein feed:

[0399] - in the case of using only a single B-type protein feed, or

[0400] - In case of using two or more B-type protein feeds, in case of mixing,

[0401] It is included in an amount of 15-70% w / w, more preferably 20-65% w / w, even more preferably 25-60% w / w and most preferably 30-50% w / w of non-TP solids relative to total solids.

[0402] In some embodiments of the invention, the protein feed has a low denatured protein content. Preferably, the protein feed, if combined, will have a protein denaturation level of at most 20%, more preferably at most 10%, even more preferably at most 5% and most preferably at most 2%.

[0403] In some preferred embodiments of the present invention, the initial protein solution comprises a total amount of type A protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution.

[0404] In other preferred embodiments of the present invention, the initial protein solution comprises a total amount of type A protein feed in the range of 5-95% w / w, more preferably 15-80% w / w, even more preferably 20-75% w / w and most preferably 35-70% w / w relative to the weight of the initial protein solution.

[0405] In some preferred embodiments of the present invention, the initial protein solution comprises a total amount of type B protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution.

[0406] In other preferred embodiments of the present invention, the initial protein solution comprises a total amount of type B protein feed in the range of 5-95% w / w, more preferably 15-80% w / w, even more preferably 20-75% w / w and most preferably 35-70% w / w relative to the weight of the initial protein solution.

[0407] In some preferred embodiments of the present invention, the initial protein solution comprises:

[0408] a total amount of type A protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, and

[0409] - A total amount of type B protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution.

[0410] In other preferred embodiments of the present invention, the initial protein solution comprises:

[0411] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP +0.3 to pH MCY,TP +3.5 of Type A protein feed, and

[0412] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP -3.0 to pH MCY,TP -0.3 of type B protein feed.

[0413] In a further preferred embodiment of the present invention, the initial protein solution comprises:

[0414] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP +0.5 to pH MCY,TP +2.5 of Type A protein feed, and

[0415] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP -2.5 to pH MCY,TP -0.5 of type B protein feed.

[0416] In an even further preferred embodiment of the present invention, the initial protein solution comprises:

[0417] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP +0.6 to pH MCY,TP +1.5 of Type A protein feed, and

[0418] - a total amount of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w of the pH relative to the weight of the initial protein solution MCY,TP -1.5 to pH MCY,TP -0.6 of type B protein feed.

[0419] In other preferred embodiments of the present invention, the initial protein solution comprises:

[0420] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution; MCY,TP +0.8 to pH MCY,TP +1.5 of Type A protein feed, and

[0421] - a total amount in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, at a pH of pH MCY,TP -1.5 to pH MCY,TP -0.8 of type B protein feed.

[0422] In some preferred embodiments of the invention, all of the protein feed is in liquid form.

[0423] In other preferred embodiments of the present invention, at least one of the protein feeds is in dry form.

[0424] In a further preferred embodiment of the present invention at least one of the protein feeds is in dry form, preferably in powder form, and at least one of the protein feeds is in liquid form.

[0425] The embodiments relating to the chemical composition of the initial protein solution apply equally to both Type A and Type B protein feeds, however typically at least one parameter of the protein feed is set to avoid supersaturation or at least spontaneous crystallization.

[0426] It is generally preferred that the supersaturation of the TP of the initial protein solution be higher than:

[0427] - in the case where only a single Type A protein feed is used, the supersaturation of said Type A protein feed, or

[0428] - In case two or more Type A protein feeds are used, the supersaturation of said Type A protein feeds, if combined.

[0429] Furthermore, it is generally preferred that the supersaturation of the TP of the initial protein solution be higher than:

[0430] - in the case where only a single Type B protein feed is used, the supersaturation of said Type B protein feed, or

[0431] - In case two or more Type B protein feeds are used, the supersaturation of said Type B protein feeds, if combined.

[0432] It is particularly preferred that the supersaturation of the TP of the initial protein solution is higher than both:

[0433] - in the case where only a single Type A protein feed is used, the supersaturation of said Type A protein feed, or

[0434] - in case two or more type A protein feeds are used, the supersaturation of said type A protein feeds in case of combination,

[0435] and both of the following:

[0436] - in the case where only a single Type B protein feed is used, the supersaturation of said Type B protein feed, or

[0437] - In case two or more Type B protein feeds are used, the supersaturation of said Type B protein feeds, if combined.

[0438] The supersaturation of the liquid is determined by seeding the liquid sample with TP crystals and quantifying the amount of additional TP crystal mass generated in the sample when the sample is kept under the same conditions (e.g., temperature, pressure, and humidity) for 24 hours. Quantification of the TP crystal mass is accomplished by centrifugation at 15,000 g for 5 minutes at the temperature of the liquid and subsequent quantification of the TP content in the obtained precipitate using assay 9.9 of WO 2018 / 115520A1. Supersaturation is the percentage of TP in the liquid sample that can be separated as a pellet.

[0439] In some preferred embodiments of the present invention, with respect to the Type A protein feed, in the case of using only a single Type A protein feed, or in the case of using two or more Type A protein feeds, in the case of mixing:

[0440] - its TP content is 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w BLG content, even more preferably 10-28% w / w and most preferably 12-26% w / w relative to the total weight of the type A protein feed,

[0441] - Its pH range is pH MCY,TP +0.1 to pH MCY,TP +3.5, more preferably pH MCY,TP +0.3 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.5 to pH MCY,TP+1.5 and most preferably pH MCY,TP +0.6 to pH MCY,TP +1.5,

[0442] - preferably, its temperature is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C,

[0443] - preferably, its TP content is in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein,

[0444] -It has one or more of the following:

[0445] - its conductivity is at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 3 mS / cm, and

[0446] - the ratio between its conductivity and the total amount of protein is at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0447] In some preferred embodiments of the present invention, with respect to the Type B protein feed, in the case of using only a single Type B protein feed, or in the case of using two or more Type B protein feeds, in the case of mixing:

[0448] - its TP content is 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w BLG content, even more preferably 10-28% w / w and most preferably 12-26% w / w relative to the total weight of the Type B protein feed,

[0449] - Its pH range is pH MCY,TP -3.5 to pH MCY,TP -0.1, more preferably pH MCY,TP -2.5 to pH MCY,TP -0.3, even more preferably pH MCY,TP -1.5 to pH MCY,TP -0.5 and most preferably pH MCY,TP -1.5 to pH MCY,TP -0.6,

[0450] - preferably, its temperature is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C,

[0451] - preferably, its TP content is in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein,

[0452] -It has one or more of the following:

[0453] - its conductivity is at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 3 mS / cm, and

[0454] - the ratio between its conductivity and the total amount of protein is at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0455] In a particularly preferred embodiment of the present invention,

[0456] - with respect to a Type A protein feed, in the case where only a single Type A protein feed is used, or

[0457] In the case of using two or more A-type protein feeds, in the case of mixing,

[0458] - its TP content is 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w BLG content, even more preferably 10-28% w / w and most preferably 12-26% w / w relative to the total weight of the type A protein feed,

[0459] - Its pH range is pH MCY,TP +0.1 to pH MCY,TP +3.5, more preferably pH MCY,TP +0.3 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.5 to pH MCY,TP +1.5 and most preferably pH MCY,TP +0.6 to pH MCY,TP +1.5,

[0460] - preferably, its temperature is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C,

[0461] - preferably, its TP content is in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein,

[0462] -It has one or more of the following:

[0463] - its conductivity is at most 7 mS / cm, even more preferably at most 5 mS / cm and most preferably at most 3 mS / cm, and

[0464] - the ratio between its conductivity and the total amount of protein is at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0465] and

[0466] - With respect to the B-type protein feed, in the case of using only a single B-type protein feed, or in the case of using two or more B-type protein feeds, in the case of mixing:

[0467] - its TP content is 4-32% w / w, more preferably 6-32% w / w, more preferably 8-30% w / w BLG content, even more preferably 10-28% w / w and most preferably 12-26% w / w relative to the total weight of the Type B protein feed,

[0468] - Its pH range is pH MCY,TP -3.5 to pH MCY,TP -0.1, more preferably pH MCY,TP -2.5 to pH MCY,TP -0.3, even more preferably pH MCY,TP -1.5 to pH MCY,TP -0.5 and most preferably pH MCY,TP -1.5 to pH MCY,TP -0.6,

[0469] - preferably, its temperature is at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C,

[0470] - preferably, its TP content is in an amount of at least 30% w / w, more preferably at least 40% w / w, even more preferably at least 50% w / w and most preferably at least 55% w / w relative to the total amount of protein,

[0471] -It has one or more of the following:

[0472] - its conductivity is at most 7 mS / cm, more preferably at most 5 mS / cm and most preferably at most 3 mS / cm, and

[0473] - the ratio between its conductivity and the total amount of protein is at most 0.3, more preferably at most 0.25, most preferably at most 0.20.

[0474] In some preferred embodiments of the invention, the protein feed is prepared by one or more of the following process steps:

[0475] - Adjust pH,

[0476] - Reduce conductivity

[0477] - Lower the temperature

[0478] - Increase protein concentration

[0479] - Add reagents to reduce water activity

[0480] -Change the ionic composition.

[0481] The present inventors have discovered that pressure driven membrane filtration is well suited for preparing liquid protein feeds.

[0482] In some preferred embodiments of the invention, at least one of the protein feeds is prepared by a process involving pressure driven membrane filtration of a protein source. Preferred pressure driven membrane filtrations include filtration by ultrafiltration, microfiltration, nanofiltration, and reverse osmosis. Ultrafiltration has been found to be particularly preferred for preparing protein feeds and can be used as the only type of pressure driven membrane filtration during preparation of the protein feed, or can be used in combination with other filtration methods.

[0483] Preparation of a protein feed often involves pH adjustment to provide a protein feed having a desired pH. Preferably, when preparing a Type A protein feed, the pH is adjusted to a pH of MCY,TP +0.5 to pH MCY,TP +1.5, and when preparing a Type B protein feed, adjust the pH to within the pH range of MCY,TP -1.5 to pH MCY,TP The pH of the mixture is preferably adjusted to a pH in the range of 0.1-0.5. Preferably, a food-acceptable acid and / or base is used to adjust the pH. Food-acceptable acids are particularly preferred, such as carboxylic acids. Examples of such acids include hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, maleic acid, tartaric acid, lactic acid, citric acid or gluconic acid and / or mixtures thereof.

[0484] In some preferred embodiments of the present invention, the pH is adjusted using a lactone (e.g., D-glucono-δ-lactone) which slowly hydrolyzes and simultaneously lowers the pH of the aqueous liquid containing it. The target pH after the hydrolysis of the lactone has been completed can be accurately calculated.

[0485] Useful examples of food-acceptable bases are, for example, hydroxide sources, such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, salts of food acids, such as, for example, trisodium citrate, and / or combinations thereof.

[0486] In other preferred embodiments of the present invention, by adding H + The pH can be adjusted by adding cation exchange materials in the form of beads / macroparticles. Bead / macroparticle cation exchange materials can be easily removed from the initial protein solution before or even after crystallization. +The use of cation exchange materials in the form of cation exchangers to adjust the pH is particularly advantageous in the present invention because the adjustment lowers the pH without adding negative counterions that significantly affect the conductivity of the protein feed.

[0487] In some preferred embodiments of the present invention, the preparation of the initial protein solution involves reducing the conductivity of the protein feed.

[0488] Unless otherwise stated, the conductivity values ​​mentioned herein have been normalized to 25°C.

[0489] The inventors have found that reducing the conductivity of the initial protein solution results in a higher yield of TP crystals. The minimum achievable conductivity of the initial protein solution depends on the composition of the protein fraction and the lipid fraction (if any). Some protein species contribute more to the conductivity than other protein species. Therefore, it is preferred to have the conductivity of the protein feed close to a level where the protein and the counterions of the protein are the main contributors to the conductivity. The reduction in conductivity generally involves removing at least some of the small free ions that are present in the liquid phase and are not tightly bound to the protein.

[0490] It is generally preferred that the conductivity of the protein feed is at most 10 mS / cm. In some preferred embodiments of the invention, the conductivity of the protein feed is at most 5 mS / cm. Preferably, the conductivity of the protein feed is at most 4 mS / cm.

[0491] Lower conductivities are even more preferred and result in higher TP crystal yields. Thus, the conductivity of the protein feed is preferably at most 3 mS / cm. In some preferred embodiments of the invention, the conductivity of the protein feed is at most 1 mS / cm. Preferably, the conductivity of the protein feed is at most 0.5 mS / cm.

[0492] The low conductivity of the protein feed is preferably obtained by dialysis or diafiltration. Diafiltration by ultrafiltration is particularly preferred because it allows washing out salts and small charged molecules while retaining protein. In some preferred embodiments of the present invention, the same UF unit is used for UF / diafiltration and subsequent protein feed concentration.

[0493] The inventors have seen evidence that the ratio between conductivity (expressed in mS / cm) and the total amount of protein in the initial protein solution (expressed in wt% total protein relative to the total weight of the initial protein solution) can advantageously be kept at or below a certain threshold to promote TP crystallization.

[0494] In some preferred embodiments of the present invention, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.3. Preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.25. Preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.20. More preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.18. Even more preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.12. Most preferably, the ratio between the conductivity and the total amount of protein in the protein feed is at most 0.10.

[0495] For example, it is preferred that the ratio between conductivity and the total amount of protein in the protein feed is about 0.07 or even lower.

[0496] The inventors have also found that the protein feed can be advantageously adjusted to obtain a UF permeate conductivity of up to 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520. When the term "conductivity" is used herein, it refers to the conductivity of the liquid in question. When the term "UF permeate conductivity" is used, it refers to the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520.

[0497] Preferably, the UF permeate conductivity of the protein feed is at most 7 mS / cm. More preferably, the UF permeate conductivity of the protein feed may be at most 5 mS / cm. Even more preferably, the UF permeate conductivity of the protein feed may be at most 3 mS / cm.

[0498] If a high yield of TP is to be obtained, even lower UF permeate conductivities may be used and are particularly preferred. Thus, preferably, the UF permeate conductivity of the protein feed is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the protein feed may be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the protein feed may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the protein feed may be at most 0.04 mS / cm.

[0499] For example, in case MilliQ water is used as diluent during diafiltration (the conductivity of MilliQ water is about 0.06 μS / cm), even lower UF permeate conductivity can be achieved. Thus, the UF permeate conductivity of the protein feed may be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the protein feed may be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the protein feed may be at most 0.0001 mS / cm.

[0500] In some preferred embodiments of the invention, preparation of the protein feed involves temperature reduction.

[0501] The temperature of the protein feed may be at most 30° C., preferably at most 20° C. and even more preferably at most 10° C. The inventors have found that even lower temperatures provide for higher supersaturation to be obtained when mixing the protein feed, therefore the temperature of the protein feed may, for example, be reduced to at most 5° C., preferably at most 2° C. and even more preferably at most 0° C. The temperature may even be below 0° C., however preferably the protein feed should remain pumpable, for example in the form of an ice slurry.

[0502] In some preferred embodiments of the invention, the protein feeds are an ice slurry before they are combined to prepare the initial protein solution.

[0503] Preparation of the protein feed preferably involves one or more protein concentration steps, such as ultrafiltration, nanofiltration, reverse osmosis and / or evaporation.

[0504] Ultrafiltration is particularly preferred as it allows for selective concentration of proteins.As mentioned above, ultrafiltration is preferably used for both diafiltration and concentration during preparation of the protein feed.

[0505] In some preferred embodiments of the invention, the TP concentration of the protein feed is below the level at which spontaneous crystallization of TP occurs.

[0506] In some preferred embodiments of the invention, preparation of the protein feed involves the addition of one or more water activity reducing agents.

[0507] Useful but non-limiting examples of such water activity reducing agents are polysaccharides and / or polyethylene glycol (PEG).

[0508] In some preferred embodiments of the invention, preparation of the protein feed involves ion exchange by addition of new ionic species, by dialysis or diafiltration.

[0509] In yet other preferred embodiments of the invention, the preparation of the protein feed involves reducing the conductivity, for example by diafiltration using a membrane that retains at least TP. Diafiltration by ultrafiltration is particularly preferred.

[0510] Any suitable protein source may be used to prepare the protein feed.In some preferred embodiments of the invention, the protein source is produced by fermenting one or more of mammalian cells, recombinant plant cells, recombinant mammalian cells and / or recombinant microorganisms capable of producing TP.

[0511] Typically, the protein feed is prepared by combining and preferably mixing two or more of the above methods.

[0512] In some preferred embodiments of the invention, the preparation of the protein feed involves subjecting the protein source to:

[0513] - pH adjustment to the desired pH, preferably at pH when preparing Type A protein feed MCY,TP +0.5 to pH MCY,TP +1.5, and when preparing a Type B protein feed, at pH MCY,TP -1.5 to pH MCY,TP -0.5 pH range, and

[0514] - Concentration by ultrafiltration, nanofiltration and / or reverse osmosis.

[0515] In a preferred embodiment of the invention, the preparation of the protein feed involves subjecting the protein source to:

[0516] - pH adjustment to the desired pH, preferably at pH when preparing Type A protein feed MCY,TP +0.5 to pH MCY,TP +1.5, and when preparing a Type B protein feed, at pH MCY,TP -1.5 to pH MCY,TP pH in the range of -0.5,

[0517] - concentration by ultrafiltration, nanofiltration and / or reverse osmosis,

[0518] - performing ultrafiltration / diafiltration to provide a protein feed with low conductivity, and

[0519] - Temperature regulation is performed to maintain the temperature of the liquid stream during processing at at most 20°C, more preferably at most 15°C, even more preferably at most 10°C and most preferably at most 5°C.

[0520] The inventors have also discovered that the TP yield of the process of the present invention can be increased by controlling the molar ratio between the sum of sodium + potassium and the sum of calcium and magnesium. Surprisingly, higher relative amounts of calcium and magnesium appear to increase the yield of TP and, therefore, the TP recovery efficiency of the process of the present invention.

[0521] In some preferred embodiments of the invention, the molar ratio between Na+K and Ca+Mg of the Type A protein feeds, if combined, will be at most 4. More preferably, the molar ratio between Na+K and Ca+Mg of the Type A protein feeds, if combined, will be at most 2. Even more preferably, the molar ratio between Na+K and Ca+Mg of the Type A protein feeds, if combined, will be at most 1.5, and even more preferably at most 1.0. Most preferably, the molar ratio between Na+K and Ca+Mg of the Type A protein feeds, if combined, will be at most 0.5, such as, for example, at most 0.2.

[0522] The molar ratio between Na+K and Ca+Mg is calculated as (m Na +m K ) / (m Ca +m Mg ), where m Na is the content of element Na in mol, m K is the content of element K in mol, m Ca is the content of element Ca in mol, and m Mg is the content of element Mg in mol.

[0523] In some preferred embodiments of the invention, the molar ratio between Na+K and Ca+Mg of the Type B protein feed, if combined, will be at most 4. More preferably, the molar ratio between Na+K and Ca+Mg of the Type B protein feed, if combined, will be at most 2. Even more preferably, the molar ratio between Na+K and Ca+Mg of the Type B protein feed, if combined, will be at most 1.5, and even more preferably at most 1.0. Most preferably, the molar ratio between Na+K and Ca+Mg of the Type A protein feed, if combined, will be at most 0.5, such as, for example, at most 0.2.

[0524] In some embodiments of the invention, the protein feed has a low denatured protein content. Preferably, the protein feed has a degree of protein denaturation of at most 20%, preferably at most 10%, more preferably at most 5% and most preferably at most 2%.

[0525] In step a), the initial protein solution is prepared to have a pH at which TP can crystallize under saline conditions, typically at pH MCY,TP The pH range is + / - 0.7 pH units.

[0526] All pH values ​​were measured using a pH glass electrode and normalized to 25°C.

[0527] More preferably, the pH of the initial protein solution is at pH MCY,TP within a range of + / - 0.5 pH units.

[0528] The inventors have observed that the maximum TP crystallization yield is at pH MCY,TP The pH of the initial protein solution can be advantageously selected at pH MCY,TP The pH of the initial protein solution is preferably at a pH of 1 to 5.5, but is moved closer to the optimal pH during crystallization, for example by adding edible acid or additional Type B protein feed or Type A protein feed. Thus, in some preferred embodiments of the invention, the pH of the initial protein solution is at a pH of MCY,TP -0.7 to pH MCY,TP Alternatively but preferably, the pH of the initial protein solution may be in the range of pH MCY,TP +0.2 to pH MCY,TP +0.7 range.

[0529] In other preferred embodiments of the present invention, the pH of the initial protein solution is at pH MCY,TP -0.5 to pH MCY,TP Alternatively but preferably, the pH of the initial protein solution may be in the range of pH MCY,TP +0.2 to pH MCY,TP +0.5 range.

[0530] The present inventors have found that it is advantageous to avoid spontaneous crystallization during crystallization, and that TP crystals formed by controlled crystallization in the metastable region have a more uniform particle size distribution and are more easily and efficiently separated from the mother liquor than TP crystals produced by spontaneous TP crystallization.

[0531] Therefore, in some preferred embodiments of the present invention, the initial protein solution and preferably the crystallizing protein solution are controlled to avoid spontaneous TP crystallization. For example, it is preferred that the initial protein solution is supersaturated with respect to TP, but is in a metastable region, meaning that spontaneous TP crystallization cannot occur.

[0532] The inventors have found that low conductivity of the initial protein solution results in a higher yield of TP crystals. The minimum achievable conductivity of the initial protein solution depends on the composition of the protein fraction and the lipid fraction (if any). Some protein species contribute more to the conductivity than other protein species. Therefore, it is preferred to have the conductivity of the protein feed close to a level where the protein and the counterions of the protein are the main contributors to the conductivity. The reduction in conductivity generally involves the removal of at least some of the small free ions that are present in the liquid phase and are not tightly bound to the protein.

[0533] It is generally preferred that the conductivity of the initial protein solution is at most 10 mS / cm. In some preferred embodiments of the invention, the conductivity of the initial protein solution is at most 5 mS / cm. Preferably, the conductivity of the initial protein solution is at most 4 mS / cm.

[0534] Lower conductivities are even more preferred and result in higher TP crystal yields. Thus, the conductivity of the initial protein solution is preferably at most 3 mS / cm. In some preferred embodiments of the invention, the conductivity of the initial protein solution is at most 1 mS / cm. Preferably, the conductivity of the initial protein solution is at most 0.5 mS / cm.

[0535] The inventors have seen evidence that the ratio between the conductivity of the initial protein solution (expressed in mS / cm) and the total amount of protein (expressed in wt% total protein relative to the total weight of the initial protein solution) can advantageously be kept at or below a certain threshold to promote TP crystallization.

[0536] In some preferred embodiments of the present invention, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.3. Preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.25. Preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.20. More preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.18. Even more preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.12. Most preferably, the ratio between the conductivity of the initial protein solution and the total amount of protein is at most 0.10.

[0537] For example, it is preferred that the ratio between the conductivity of the initial protein solution and the total amount of protein is about 0.07, or even lower.

[0538] The inventors have also found that the UF permeate conductivity of the initial protein solution can advantageously be at most 10 mS / cm. The UF permeate conductivity is a measure of the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520. When the term "conductivity" is used herein, it refers to the conductivity of the liquid in question. When the term "UF permeate conductivity" is used, it refers to the conductivity of the small molecule fraction of the liquid and is measured according to Example 9.10 of WO2018 / 115520.

[0539] Preferably, the UF permeate conductivity of the initial protein solution is at most 7 mS / cm. More preferably, the UF permeate conductivity of the initial protein solution may be at most 5 mS / cm. Even more preferably, the UF permeate conductivity of the initial protein solution may be at most 3 mS / cm.

[0540] If a high yield of TP is to be obtained, even lower UF permeate conductivities may be used and are particularly preferred. Thus, preferably, the UF permeate conductivity of the initial protein solution is at most 1.0 mS / cm. More preferably, the UF permeate conductivity of the initial protein solution may be at most 0.4 mS / cm. Even more preferably, the UF permeate conductivity of the initial protein solution may be at most 0.1 mS / cm. Most preferably, the UF permeate conductivity of the initial protein solution may be at most 0.04 mS / cm.

[0541] For example, in case MilliQ water is used as diluent during diafiltration (the conductivity of MilliQ water is about 0.06 μS / cm), even lower UF permeate conductivity can be achieved. Thus, the UF permeate conductivity of the initial protein solution may be at most 0.01 mS / cm. Alternatively, the UF permeate conductivity of the initial protein solution may be at most 0.001 mS / cm. Alternatively, the UF permeate conductivity of the initial protein solution may be at most 0.0001 mS / cm.

[0542] The present inventors have discovered that low temperatures increase TP crystallization yields.

[0543] In some preferred embodiments of the present invention, the temperature of the initial protein solution is at most 30° C., more preferably at most 20° C. and most preferably at most 10° C. Lower temperatures provide even higher supersaturation, therefore, the temperature of the initial protein solution is preferably at most 5° C., more preferably at most 2° C. and even more preferably at most 0° C. The temperature may even be below 0° C., however preferably the initial protein solution should remain pumpable, for example in the form of an ice slurry.

[0544] In some preferred embodiments of the present invention, the initial protein solution is an ice slurry when TP crystallization is initiated.Alternatively or additionally, the initial protein solution may be converted to or maintained as an ice slurry during the TP crystallization of step b).

[0545] It is particularly preferred that the initial protein solution is supersaturated with respect to TP by salting-in, so that TP can be crystallized from the initial protein solution in a salting-in manner.

[0546] In some embodiments of the present invention, the initial protein solution has a low content of denatured protein, especially in the case where the TP product of the present invention should also have a certain degree of protein denaturation. Preferably, the protein denaturation degree of the initial protein solution is at most 20%, preferably at most 10%, more preferably at most 5% and most preferably at most 2%.

[0547] Step b) of the method involves crystallizing at least some of the TPs of the supersaturated initial protein solution.

[0548] It is particularly preferred that the crystallization of step b) is carried out in a salting-in mode, i.e. in a liquid with low ionic strength and conductivity. This is in contrast to the salting-out mode in which a significant amount of salt is added to the solution to initiate crystallization.

[0549] The TP crystallization of step b) may, for example, involve one or more of the following:

[0550] - Wait for crystallization to occur,

[0551] - Adding crystallization seeds,

[0552] - further increase the supersaturation of TP, and / or

[0553] -Perform mechanical stimulation.

[0554] In some preferred embodiments of the invention, step b) involves adding crystallization seeds to the initial protein solution. The inventors have found that adding crystallization seeds allows control over when and where TP crystallization occurs to avoid sudden blockages and unexpected stops of process equipment during production. For example, it is often desirable to avoid initiating crystallization while concentrating the protein feed.

[0555] In principle, any seed material that initiates TP crystallization can be used. However, it is preferred to use hydrated TP crystals or dried TP crystals for seeding to avoid adding additional impurities to the initial protein solution.

[0556] The crystallization seeds may be in dry form or may form part of a suspension when added to the initial protein solution. It is currently preferred to add a suspension containing crystallization seeds (e.g. TP crystals) as it appears to provide a faster initiation of crystallization. Preferably, the pH of such a suspension containing crystallization seeds is in the range of 5-6 and the conductivity is at most 10 mS / cm.

[0557] In some embodiments of the invention, at least some of the crystallization seeds are located on a solid phase in contact with the initial protein solution.

[0558] The crystallization seeds preferably have a particle size smaller than the desired size of the TP crystals. The largest seeds can be removed by screening or other size classification processes to change the size of the crystallization seeds. Particle size reduction, such as by grinding, can also be employed prior to size classification.

[0559] In some embodiments of the invention, at least 90% w / w of the crystallization seeds have a particle size (measured by sieve analysis) in the range of 0.1-600 microns. For example, at least 90% w / w of the crystallization seeds may have a particle size in the range of 1-400 microns. Preferably, at least 90% w / w of the crystallization seeds may have a particle size in the range of 5-200 microns. More preferably, at least 90% w / w of the crystallization seeds may have a particle size in the range of 5-100 microns.

[0560] The particle size and dosage of the crystallization seeds can be tailored to provide optimal TP crystallization.

[0561] In some preferred embodiments of the invention, at least one of the one or more Type A or Type B protein feeds already contains TP crystals, in which case no further seeding is required, but it is generally preferred to ensure a sufficient amount of crystallization seeds.

[0562] In some preferred embodiments of the present invention, step b) involves further increasing the supersaturation of TP, preferably to a supersaturation at which TP crystallization begins immediately (i.e. within 20 minutes at most, preferably within 5 minutes at most). This is also called the nucleation zone, where crystallites spontaneously form and the crystallization process begins.

[0563] Supersaturation can be increased, for example, by one or more of the following:

[0564] - Further increase the protein concentration of the initial protein solution

[0565] - Further cooling of the initial protein solution

[0566] - Make the initial protein solution closer to the optimal pH for TP crystallization

[0567] -Further reduce the conductivity.

[0568] It is particularly preferred to further increase the protein concentration of the initial protein solution and / or bring the initial protein solution closer to the optimal pH (pH 50) for TP crystallization by adding additional Type A or Type B protein feed. MCY,TP ).

[0569] In some preferred embodiments of the invention, step b) involves waiting for TP crystals to form. This may take several hours and is typically used for an initial protein solution that is only slightly supersaturated with respect to TP and to which crystallization seeds have not yet been added.

[0570] In some preferred embodiments of the invention, providing the initial protein solution (step a) and TP crystallization (step b) are performed as two separate steps. However, if the initial protein solution already contains at least some TP crystals, step b) starts at the moment the initial protein solution has been prepared.

[0571] In some preferred embodiments of the invention, step b) involves additional adjustment of the crystallized protein solution, preferably to increase the supersaturation of TP, or at least maintain supersaturation. The additional adjustment preferably results in an increase in the yield of TP crystals or provides TP crystals of better quality, for example in the form of larger and more uniform crystal sizes.

[0572] Such additional adjustments may involve one or more of the following:

[0573] -Further increase the protein concentration of the crystallized protein solution

[0574] - Cooling the crystallized protein solution to even lower temperatures

[0575] - Make the crystallization protein solution closer to the optimal pH for TP crystallization

[0576] -Further reduce the conductivity of the crystallized protein solution.

[0577] In some preferred embodiments of the present invention, the crystallized protein solution is maintained in a metastable zone during step b) to avoid spontaneous formation of new crystallites.

[0578] It is particularly preferred to further increase the protein concentration of the crystallized protein solution and / or bring the crystallized protein solution closer to the optimal pH (pH 50) for TP crystallization by adding additional Type A or Type B protein feed. MCY,TP ). The pH adjustment is preferably performed slowly to avoid spontaneous crystallization or large local pH differences. A pH adjustment rate in the range of 0.05-0.5 pH units / hour has been found to be advantageous for controlled TP crystallization.

[0579] It is furthermore particularly preferred that during step b) the temperature of the crystallized protein solution is lowered to at least 2° C. below the temperature of the initial protein solution, more preferably at least 4° C. below, most preferably at least 6° C. below the temperature of the initial protein solution. However, it is preferred that the crystallized protein solution is not completely frozen.

[0580] The inventors have observed that the maximum TP crystallization yield is at pH MCY,TP and the pH of the initial protein solution may advantageously be selected outside the optimum but moved closer to the optimum pH during crystallization, for example by the addition of food acid or, more preferably, by additional feed of type B protein or feed of type A protein.

[0581] Therefore, in some preferred embodiments of the present invention, the pH of the initial protein solution is at pH MCY,TP -0.5 to pH MCY,TP -0.2 range or in pH MCY,TP+0.2 to pH MCY,TP In such preferred embodiments of the present invention, it is also preferred to adjust the pH of the crystallized protein solution so that the pH of the crystallized protein solution is closer to the pH of the initial protein solution. MCY,TP , preferably by adding additional Type A protein feed or additional Type B protein feed. It is particularly preferred that during crystallization the crystallizing protein solution is adjusted to a pH of about MCY,TP The additional type A protein feed or the additional type B protein feed used to adjust the crystallized protein solution preferably has an electrical conductivity that is equal to or lower than that of the crystallized protein solution. It is also preferred to combine the pH adjustment during crystallization described above with a temperature reduction of the crystallized protein solution, such that, for example, simultaneously or sequentially, the crystallized protein solution is cooled to a temperature that is lower than the temperature of the initial protein solution.

[0582] In some preferred embodiments of the present invention:

[0583] - The pH of the initial protein solution in step a) is at pH MCY,TP -0.5 to pH MCY,TP -0.2 range or in pH MCY,TP +0.2 to pH MCY,TP +0.5, and the temperature of the initial protein solution is in the range of 5-20°C,

[0584] - During step b), the pH of the crystallized protein solution is adjusted to pH MCY,TP -0.1 to pH MCY,TP +0.1 range, if the pH of the initial protein solution is within pH MCY,TP -0.5 to pH MCY,TP -0.2 range is preferably achieved by adding additional Type A protein feed, or if the pH of the initial protein solution is within a pH range of MCY,TP +0.2 to pH MCY,TP +0.5 is preferably achieved by adding additional Type B protein feed, and

[0585] - During step b), the temperature of the crystallized protein solution is lowered to a temperature which is at least 2° C. lower than the temperature of the initial protein solution.

[0586] The terms "additional protein feed type A" and "additional protein feed type B" relate to a composition containing TP which is added to an initial protein solution to initiate crystallization or to a crystallizing protein solution to at least change the pH and TP content of the crystallizing protein solution. Features and preferences described in the context of "protein feed type A" and "protein feed type B" are equally applicable to "additional protein feed type A" and "additional protein feed type B", respectively.

[0587] In some particularly preferred embodiments of the present invention, the method comprises a step c) of separating at least some of the TP crystals from the remaining liquid of the solution containing the TP crystals. This is particularly preferred when purification of the TP is desired.

[0588] Step c) may for example comprise separating the TP crystals to a solid content of at least 30% w / w. Preferably, step c) comprises separating the TP crystals to a solid content of at least 40% w / w. Even more preferably, step c) comprises separating the TP crystals to a solid content of at least 50% w / w.

[0589] The present inventors have found that a high solids content is advantageous for TP purification, since the aqueous fraction that adheres to the isolated TP crystals generally contains impurities that should be avoided. In addition, a high solids content reduces the energy consumption for converting the isolated TP crystals into a dry product, such as a powder, and increases the TP yield obtained from a drying unit with a given capacity.

[0590] In some preferred embodiments of the present invention, step c) comprises separating the TP crystals to a solid content of at least 60%. Preferably, step c) comprises separating the TP crystals to a solid content of at least 70%. Even more preferably, step c) comprises separating the TP crystals to a solid content of at least 80%.

[0591] In some preferred embodiments of the present invention, the separation in step c) involves one or more of the following operations:

[0592] - Centrifugation,

[0593] - decantation,

[0594] -filter,

[0595] -precipitation,

[0596] - A combination of the above.

[0597] These unit operations are well known and readily accomplished to those skilled in the art.Separation by filtration may, for example, involve the use of vacuum filtration, dynamic cross-flow filtration (DCF), filter presses or filter centrifuges.

[0598] Different filter pore sizes can be used depending on the desired results. Preferably, the filter allows native protein and small aggregates to pass through but retains TP crystals. The nominal pore size of the filter is preferably at least 0.1 microns. The nominal pore size of the filter can, for example, be at least 0.5 microns. Even more preferably, the nominal pore size of the filter can be at least 2 microns.

[0599] Filters with larger pore sizes can also be used, and indeed are preferred if primarily large crystals should be separated from the liquid containing TP crystals. In some embodiments of the invention, the nominal pore size of the filter is at least 5 microns. Preferably, the nominal pore size of the filter is at least 20 microns. Even more preferably, the pore size of the filter may be at least 40 microns.

[0600] The pore size of the filter may be, for example, in the range of 0.03-5000 microns, such as 0.1-5000 microns. Preferably, the pore size of the filter may be in the range of 0.5-1000 microns. Even more preferably, the pore size of the filter may be in the range of 5-800 microns, such as in the range of 10-500 microns or in the range of 50-500 microns.

[0601] In some preferred embodiments of the present invention, the pore size of the filter is in the range of 0.03-100 microns. Preferably, the pore size of the filter can be in the range of 0.1-50 microns. More preferably, the pore size of the filter can be in the range of 4-40 microns. Even more preferably, the pore size of the filter can be in the range of 5-30 microns, such as in the range of 10-20 microns.

[0602] The advantage of using filters with a pore size greater than 1 micron is that bacteria and other microorganisms are also at least partially removed during separation and optionally also during washing and / or recrystallization. Thus, the process according to the invention makes it possible to produce high-purity TP with a very low bacterial load while avoiding thermal damage to the protein.

[0603] Another advantage of using filters with pore sizes greater than 1 micron is that water removal and subsequent drying becomes easier and consumes less energy.

[0604] During preparation of the initial protein solution, the remaining liquid separated from the TP crystals can be recycled to the protein feed or feeds.

[0605] In some preferred embodiments of the present invention, step c) uses a filter centrifuge. In other preferred embodiments of the present invention, step c) uses a decanter centrifuge. The results (see Example 13 of WO2018 / 115520) show that the use of a filter centrifuge and / or a decanter centrifuge to separate TP crystals from the mother liquor provides a more robust process operation than, for example, vacuum filtration.

[0606] It is generally preferred to dry the formed filter cake with a drying gas to reduce the moisture content of the filter cake and preferably to allow the filter cake to be stripped from the filter. The use of a drying gas may form part of the separation step or, alternatively, may form a final drying step if the filter cake is converted directly to a dry TP product.

[0607] In some preferred embodiments of the present invention, step c) employs a DCF unit.

[0608] In some preferred embodiments of the invention, step c) is performed using a DCF unit equipped with a membrane capable of retaining TP crystals, the DCF permeate is recycled to form part of the initial protein solution or protein feed, and the DCF retentate may be recovered or returned to the crystallization tank. Preferably, the DCF permeate is treated, e.g. by ultrafiltration / diafiltration, to supersaturate it with respect to TP before mixing with the initial protein solution or protein feed.

[0609] Advantageously, these embodiments do not require the temperature of the liquid stream to be raised above 15°C and are therefore less susceptible to microbial contamination than process variants requiring higher temperatures. Another industrial advantage of these embodiments is that the supersaturation level is easy to control and can be maintained at a level where undesirable spontaneous crystallization does not occur. Therefore, the temperature of the liquid stream during these embodiments of the process is preferably at most 15°C, more preferably at most 12°C, even more preferably at most 10°C and most preferably at most 5°C.

[0610] These embodiments are illustrated in Example 10 of WO2018 / 115520 and can also be implemented with TP. These embodiments can be implemented as a batch process or a continuous process.

[0611] In some preferred embodiments of the present invention, the method comprises a step d) of washing the TP crystals, eg the TP crystals separated in c). The washing may consist of a single washing or multiple washing steps.

[0612] The washing of step d) preferably involves contacting the TP crystals with a washing liquid without completely dissolving the TP crystals, and subsequently separating the remaining TP crystals from the washing liquid.

[0613] The wash liquid is preferably selected to avoid complete dissolution of the TP crystals, and may, for example, comprise or even consist essentially of cold desalinated water, cold tap water, or cold reverse osmosis permeate.

[0614] The pH of the washing solution can be MCY,TP -0.7 to pH MCY,TP +0.7, more preferably in the pH range MCY,TP -0.5topH MCY,TP +0.5 and even more preferably in the pH range MCY,TP -0.4 to pH MCY,TP +0.4 range, such as in pH MCY,TP -0.3 to pH MCY,TP +0.3 range.

[0615] The conductivity of the wash liquor may be at most 0.1 mS / cm, preferably at most 0.02 mS / cm and even more preferably at most 0.005 mS / cm.

[0616] A washing liquid with an even lower conductivity may be used. For example, the conductivity of the washing liquid may be at most 1 μS / cm. Alternatively, the conductivity of the washing liquid may be at most 0.1 μS / cm, such as for example about 0.05 μS / cm.

[0617] The washing step is preferably performed at low temperature to limit dissolution of crystalline TP. The temperature of the washing liquid is preferably at most 30°C, more preferably at most 20°C and even more preferably at most 10°C.

[0618] The washing step may for example be performed at at most 5° C., more preferably at most 2° C., such as for example about 0° C. This temperature may be used as long as the washing liquid does not freeze at temperatures below 0° C., for example due to the presence of one or more freezing point depressants.

[0619] In some embodiments of the invention the wash liquor contains TP, for example in an amount of at least 1% w / w, preferably in an amount of at least 3% w / w, such as, for example, in an amount of 4% w / w.

[0620] The washing of step d) typically dissolves at most 80% w / w of the initial amount of TP crystals, preferably at most 50% w / w and even more preferably dissolves at most 20% w / w of the initial amount of TP crystals. Preferably, the washing of step d) dissolves at most 15% w / w of the initial amount of TP crystals, more preferably at most 10% w / w and even more preferably dissolves at most 5% w / w of the initial amount of TP crystals.

[0621] The weight ratio between the total amount of wash liquid and the initial amount of separated TP crystals is generally at least 1, preferably at least 2 and more preferably at least 5. For example, the weight ratio between the amount of wash liquid and the initial amount of separated TP crystals may be at least 10. Alternatively, the weight ratio between the total amount of wash liquid and the initial amount of separated TP crystals may be at least 20, such as, for example, at least 50 or at least 100.

[0622] The term "total amount of washing liquid" refers to the total amount of washing liquid used during the entire process.

[0623] In some preferred embodiments of the present invention, one or more washing procedures occur in the same filter arrangement as the TP crystal separation or in a similar filter arrangement. One or more procedures of washing liquid are added to the filter cake containing mainly TP crystals, the washing liquid is removed via the filter, and the remaining portion of the TP crystals remains in the filter cake.

[0624] In a particularly preferred embodiment of the present invention, the separation of step c) is performed using a filter that retains TP crystals. Subsequently, the filter cake is contacted with one or more washes that move through the filter cake and the filter. It is generally preferred that each wash is at most 10 times the volume of the filter cake, preferably at most 5 times the volume of the filter cake, more preferably at most 1 times the volume of the filter cake, even more preferably at most 0.5 times the volume of the filter cake, such as at most 0.2 times the volume of the filter cake. The volume of the filter cake includes both the solid and fluid (liquid and gas) of the filter cake. Preferably, the filter cake is washed at least 2 times, preferably at least 4 times, and even more preferably at least 6 times in this way.

[0625] The spent wash liquid from step d) can, for example, be recirculated to the protein feed or to the initial protein solution, where the washed out TP can be separated again.

[0626] The method may further comprise step e), which involves a recrystallization step. The recrystallization step may, for example, comprise:

[0627] - Dissolve the separated TP crystals in the recrystallization liquid,

[0628] - Adjust the recrystallization liquid to obtain supersaturation about TP,

[0629] - crystallizing TP in a supersaturated, conditioned recrystallization solution, and

[0630] - Separation of TP crystals from the remaining conditioned recrystallization solution.

[0631] Alternatively and generally more preferably, the separated TP crystals are converted into one or more Type A protein feeds and one or more Type B protein feeds, which are combined to form a new supersaturated protein solution as in step a), and TP is crystallized again in said new protein solution.

[0632] Step e) may comprise a single recrystallization procedure or multiple recrystallization procedures.

[0633] In some embodiments of the present invention, the TP crystals of step c) or d) are recrystallized at least 2 times. For example, the TP crystals may be recrystallized at least 3 times, such as at least 4 times.

[0634] The washing and recrystallization steps can be combined in any order and performed multiple times if desired.

[0635] The separated TP crystals of step c) can be subjected, for example, to the following process procedure:

[0636] - one or more washing steps (step d), followed by

[0637] - one or more recrystallization steps (step e).

[0638] Alternatively, the isolated TP crystals of step c) may be subjected to the following process procedure:

[0639] - one or more recrystallization steps (step e), followed by

[0640] - one or more washing steps (step d).

[0641] It is also possible to combine a plurality of washing and recrystallization steps, for example in the following order:

[0642] - one or more washing steps (step d),

[0643] - one or more recrystallization steps (step e),

[0644] - one or more washing steps (step d), and

[0645] - one or more recrystallization steps (step e).

[0646] Or for example in sequence:

[0647] - one or more recrystallization steps (step e),

[0648] - one or more washing steps (step d),

[0649] - one or more recrystallization steps (step e),

[0650] - one or more washing steps (step d).

[0651] In some embodiments of the invention, the method further involves subjecting the isolated TP to an additional TP enrichment step, for example based on chromatography or selective filtration. However, in other preferred embodiments of the invention, the method does not comprise an additional TP enrichment step after step b). The term "additional TP enrichment step" means a process step of enriching TP relative to the total amount of protein, which step is independent of crystallization of TP or treatment of TP crystals. An example of such an additional TP enrichment step is ion exchange chromatography. Washing of TP crystals and / or TP recrystallization are not considered "additional TP enrichment steps".

[0652] In some particularly preferred embodiments of the present invention, the method involves a drying step f), wherein the TP-containing composition originating from step b), c), d) or e) is converted into a dried composition.

[0653] In the context of the present invention, the term "dry" means that the composition or product under consideration contains at most 6% w / w of water, and preferably even less.

[0654] In the context of the present invention, the term "TP-containing composition" is used to describe the composition which is subjected to the drying of step f).

[0655] In the context of the present invention, a "TP-containing composition derived from step b), c), d) or e)" means a composition comprising at least some of the TP from step b), c), d) or e). In some preferred embodiments of the present invention, a "TP-containing composition derived from step b), c), d) or e)" is obtained directly from step b), c), d) or e). However, in other preferred embodiments of the present invention, a "TP-containing composition derived from step b), c), d) or e)" is the result of further processing of a composition obtained directly from step b), c), d) or e).

[0656] It is generally preferred that the TP-containing composition contains a significant amount of TP present in the composition obtained directly from step b), c), d) or e). In some preferred embodiments of the present invention, the TP-containing composition derived from step b), c), d) or e) comprises at least 50% w / w, preferably at least 70% and even more preferably at least 80% of the TP obtained from step b), c), d) or e).

[0657] Preferably, the TP-containing composition derived from step b), c), d) or e) comprises at least 85% w / w of TP obtained from step b), c), d) or e). More preferably, the TP-containing composition derived from step b), c), d) or e) comprises at least 90% w / w of TP obtained from step b), c), d) or e). Even more preferably, the TP-containing composition derived from step b), c), d) or e) comprises at least 95% w / w of TP obtained from step b), c), d) or e). Most preferably, the TP-containing composition derived from step b), c), d) or e) comprises at least 100% w / w of TP obtained from step b), c), d) or e).

[0658] In some preferred embodiments of the invention, the drying step involves one or more of spray drying, freeze drying, spin flash drying, spin drying and / or fluidized bed drying.

[0659] In some particularly preferred embodiments of the present invention, the drying step involves a TP-containing composition in which TP crystals have been dissolved, and wherein the resulting powder does not comprise TP crystals formed by step b) or by recrystallization prior to the drying step.

[0660] TP crystals can be dissolved, for example, by:

[0661] - Increase the temperature,

[0662] - increasing the conductivity, for example by adding one or more salts,

[0663] -Changed pH, e.g. out of range pH MCY,TP -0.5 to pH MCY,TP +0.5, more preferably out of range pH MCY,TP -0.7 to pH MCY,TP +0.7,

[0664] - reducing the TP concentration, for example by dilution,

[0665] - or a combination of the above.

[0666] Spray drying is the currently preferred method for drying TP-containing compositions free of TP crystals.

[0667] In other particularly preferred embodiments of the invention, the drying step involves a TP-containing composition still containing TP crystals, and wherein the resulting powder contains TP crystals. These embodiments are preferred in cases where the TP product should have a higher density than conventionally dried TP powder.

[0668] In some particularly preferred embodiments of the invention, the drying step involves a TP-containing composition that still contains TP crystals, and wherein the resulting powder contains TP crystals. These embodiments are preferred in cases where the TP product has a higher density than conventionally dried TP powder.

[0669] In some embodiments of the present invention, the temperature of the TP-containing composition containing TP crystals when reaching the outlet of the spray device (e.g., nozzle or atomizer) is at most 70° C., preferably at most 60° C., more preferably at most 50° C. In some preferred embodiments of the present invention, the temperature of the TP-containing composition containing TP crystals when reaching the outlet of the spray device is at most 40° C., preferably at most 30° C., more preferably at most 20° C., even more preferably at most 10° C. and most preferably at most 5° C.

[0670] The spray device of a spray dryer is a device, such as a nozzle or an atomizer, which converts the solution or suspension to be dried into droplets that enter the drying chamber of the spray dryer.

[0671] It is particularly preferred that the temperature of the TP-containing composition containing TP crystals when reaching the outlet of the spray device is in the range of 0-50°C, preferably in the range of 2-40°C, more preferably in the range of 4-35°C and most preferably in the range of 5-10°C.

[0672] In some preferred embodiments of the invention, the TP-containing composition has a TP crystallinity of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80% and most preferably at least 90%, such as preferably 97-100%. The TP-containing composition may be a TP isolate, e.g. containing TP in an amount greater than 90% w / w relative to total protein, or it may contain significant amounts of other proteins and thus contain TP in an amount of up to 90% w / w relative to total protein.

[0673] The gas inlet temperature of the spray dryer is preferably in the range of 140-220° C., more preferably in the range of 160-200° C. and even more preferably in the range of 170-190° C., such as, for example, preferably about 180° C. The outlet temperature of the gas from the spray dryer is preferably in the range of 50-95° C., more preferably in the range of 70-90° C. and even more preferably in the range of 80-88° C., such as, for example, preferably about 85° C. As a rule of thumb, it is said that the solid subjected to spray drying is heated to a temperature 10-15° C. lower than the gas outlet temperature.

[0674] In some preferred embodiments of the invention, the spray dryer is preferably in the range of 50-85°C, more preferably in the range of 60-80°C and even more preferably in the range of 65-75°C, such as like preferably about 70°C.

[0675] In some preferred embodiments of the invention, the TP-containing composition to be dried is mixed with a dried TP isolate to increase the solids content to a level at which the mixture can be dried by fluid bed drying. This is also known as backmixing and allows for very cost-effective drying of the TP product. These embodiments are particularly preferred for TP-containing compositions containing TP crystals.

[0676] An advantage of the process according to the invention is that the TP-containing composition to be dried can have a very high solids content before the drying step and therefore less water has to be removed and less energy is consumed in the drying operation.

[0677] In some preferred embodiments of the present invention, the solids content of the TP-containing composition derived from step b), c), d) or e) is at least 20% w / w. Preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is at least 30% w / w. More preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is at least 40% w / w. Even more preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is at least 50% w / w, such as, for example, at least 60% w / w.

[0678] In other preferred embodiments of the present invention, the solids content of the TP-containing composition derived from step b), c), d) or e) is in the range of 20-80% w / w. Preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is in the range of 30-70% w / w. More preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is in the range of 40-65% w / w. Even more preferably, the solids content of the TP-containing composition derived from step b), c), d) or e) is in the range of 50-65% w / w, such as like about 60% w / w.

[0679] The present inventors have found that the higher the crystallinity of the TP-containing composition, the less water is bound to the TP-containing composition and a higher total solids content of the TP-containing composition can be achieved before the drying step.

[0680] Thus, in some preferred embodiments of the present invention, the TP-containing composition has a TP crystallinity of at least 10% w / w. Preferably, the TP-containing composition has a TP crystallinity of at least 20% w / w. More preferably, the TP-containing composition has a TP crystallinity of at least 30% w / w. Even more preferably, the TP-containing composition has a TP crystallinity of at least 40% w / w.

[0681] Even higher crystallinity is generally preferred. Thus, in some preferred embodiments of the present invention, the TP-containing composition has a TP crystallinity of at least 50% w / w. Preferably, the TP-containing composition has a TP crystallinity of at least 60% w / w. More preferably, the TP-containing composition has a TP crystallinity of at least 70% w / w. Even more preferably, the TP-containing composition has a TP crystallinity of at least 80% w / w. Most preferably, the TP-containing composition has a TP crystallinity of at least 90% w / w, preferably at least 95% w / w, more preferably at least 97% w / w and even more preferably at least 99% w / w.

[0682] The present inventors have found that a decrease in water content tends to increase the crystallinity of the TP of the composition. Thus, under the same conditions, a composition with a high water:TP ratio (e.g., a suspension of 4% TP crystals in water) tends to have a lower TP crystallinity than a composition with a lower water:TP ratio (e.g., a filter cake or moist isolated crystals).

[0683] The method of the present invention can be operated using mild temperatures that do not destroy TP.

[0684] In some preferred embodiments of the invention, TP is not subjected to temperatures above 90° C. during the process. Preferably, TP is not subjected to temperatures above 80° C. during the process. Even more preferably, TP is not subjected to temperatures above 75° C. during the process. It should be noted that although spray drying typically employs temperatures in excess of 150° C., the short exposure time and simultaneous water evaporation means that the spray dried protein does not experience temperatures above 50-70° C.

[0685] The inventors have seen evidence that prolonged heating during the drying step reduces the amount of TP in crystalline form. In some preferred embodiments of the invention, the heat exposure during the drying step is kept low enough to provide a degree of TP denaturation of at most 10%, preferably at most 4%, more preferably at most 1%, even more preferably at most 0.4% and even more preferably at most 0.1%. Most preferably, the drying step does not result in detectable TP denaturation at all.

[0686] The degree of denaturation caused by the drying step is determined by determining the TP content (relative to the total solids) in the TP-containing composition to be dried in step f) (c 步骤f之前 ) and the TP content (relative to total solids) in the redissolved dry composition and calculated using the following formula:

[0687] Denaturation degree = ((c 步骤f之前 -c 步骤f之后 ) / c 步骤f之前 )×100%

[0688] The advantage of the process of the invention is that it is much faster than comparable TP crystallization processes of the prior art. The duration from initial adjustment of the protein feed to completion of the separation of step c may be at most 10 hours, preferably at most 4 hours, more preferably at most 2 hours and even more preferably at most 1 hour.

[0689] The TP product preferably contains TP in an amount of at least 30% w / w relative to the total amount of protein and total protein in an amount of at least 30% w / w relative to the total solids.

[0690] In some preferred embodiments of the present invention, the TP product has a TP crystallinity of at least 10% w / w. Preferably, the TP product has a TP crystallinity of at least 20% w / w. More preferably, the TP product has a TP crystallinity of at least 30% w / w. Even more preferably, the TP product has a TP crystallinity of at least 40% w / w.

[0691] Even higher crystallinity is generally preferred. Thus, in some preferred embodiments of the present invention, the TP product has a TP crystallinity of at least 50% w / w. Preferably, the TP product has a TP crystallinity of at least 60% w / w. More preferably, the TP product has a TP crystallinity of at least 70% w / w. Even more preferably, the TP product has a TP crystallinity of at least 80% w / w. Most preferably, the TP product has a TP crystallinity of at least 90% w / w, and preferably at least 95% w / w.

[0692] As outlined in Example 9.7 of WO2018 / 115520, but measuring TP instead of BLG, pH was measured at pH MCY,TP -0.5 to pH MCY,TP +0.5 TP crystallinity of the liquid. The TP crystallinity of the powdered material is measured as outlined in Example 9.8 of WO2018 / 115520, but measuring TP instead of BLG. If the product is a dry product but not in powder form, it must be converted to a powder, for example by grinding or milling, before being subjected to the method of Example 9.8 of WO2018 / 115520.

[0693] The TP product may, for example, contain up to 90% w / w TP relative to the total amount of protein, and a TP crystallinity of at least 10%. For example, the TP product may contain up to 80% w / w TP relative to the total amount of protein, and a TP crystallinity of at least 10%. The TP product may, for example, contain 30-70% w / w TP relative to the total amount of protein, and a TP crystallinity of at least 10%.

[0694] In other preferred embodiments of the present invention, the TP product comprises up to 90% w / w TP relative to the total amount of protein, and the TP crystallinity is at least 30%. Preferably, the TP product may comprise up to 80% w / w TP relative to the total amount of protein, and the TP crystallinity is at least 30%. Even more preferably, the TP product may comprise 30-70% w / w TP relative to the total amount of protein, and the TP crystallinity is at least 30%.

[0695] The inventors have found that the present invention allows the preparation of a TP product that is very low in phosphorus and other minerals, which is advantageous for patients suffering from kidney disease or reduced kidney function.

[0696] The TP product is preferably a low phosphorus product.

[0697] In the context of the present invention, term " low-phosphorus " relates to the product that phosphorus total content is 100mg phosphorus at the most per 100g protein, for example liquid, powder or another kind of food.Preferably, the total content of low-phosphorus product is 80mg phosphorus at the most per 100g protein.More preferably, the total content of low-phosphorus product can be 50mg phosphorus at the most per 100g protein.Even more preferably, the phosphorus total content of low-phosphorus composition can be 20mg phosphorus at the most per 100g protein.Even more preferably, the phosphorus total content of low-phosphorus composition can be 5mg phosphorus at the most per 100g protein.Low-phosphorus product according to the present invention can be used as food ingredient, for the production of food for the patient population of decreased renal function.

[0698] Thus, in some particularly preferred embodiments of the present invention, the TP product comprises at most 80 mg phosphorus per 100 g protein. Preferably, the TP product comprises at most 30 mg phosphorus per 100 g protein. More preferably, the TP product comprises at most 20 mg phosphorus per 100 g protein. Even more preferably, the TP product comprises at most 10 mg phosphorus per 100 g protein. Most preferably, the TP product comprises at most 5 mg phosphorus per 100 g protein.

[0699] The phosphorus content relates to the total amount of elemental phosphorus of the composition in question and is determined according to Example 9.5 of WO2018 / 115520.

[0700] In other preferred embodiments of the present invention, the TP product is a low mineral composition.

[0701] In the context of the present invention, the term "low in minerals" relates to a composition, such as a liquid, a powder or another food product, having at least one, preferably two and even more preferably all of the following:

[0702] - an ash content of at most 1.2% w / w relative to the total solids,

[0703] - a total calcium and magnesium content of up to 0.3% w / w relative to the total solids,

[0704] - a total sodium and potassium content of up to 0.10% w / w relative to the total solids,

[0705] - Total phosphorus content of not more than 100 mg phosphorus per 100 g protein.

[0706] Preferably, the low mineral product has at least one, preferably two or more and even more preferably all of the following:

[0707] - an ash content of at most 0.7% w / w relative to the total solids,

[0708] - a total calcium and magnesium content of up to 0.2% w / w relative to the total solids,

[0709] - a total sodium and potassium content of up to 0.08% w / w relative to the total solids,

[0710] - A total phosphorus content of not more than 80 mg phosphorus per 100 g protein.

[0711] Even more preferably, the low mineral product has at least one, preferably two or more and even more preferably all of the following:

[0712] - an ash content of at most 0.5% w / w relative to the total solids,

[0713] - a total calcium and magnesium content of up to 0.15% w / w relative to the total solids,

[0714] - a total sodium and potassium content of up to 0.06% w / w relative to the total solids,

[0715] - A total phosphorus content of not more than 50 mg phosphorus per 100 g protein.

[0716] It is particularly preferred that the low mineral product has the following:

[0717] - an ash content of at most 0.5% w / w relative to the total solids,

[0718] - a total calcium and magnesium content of up to 0.15% w / w relative to the total solids,

[0719] - a total sodium and potassium content of up to 0.06% w / w relative to the total solids,

[0720] - A total phosphorus content of not more than 50 mg phosphorus per 100 g protein.

[0721] In some preferred embodiments of the present invention, the TP product comprises a total amount of protein of at least 25% w / w relative to the total solids of the TP product. Preferably, the TP product comprises a total amount of protein of at least 50% w / w relative to the total solids of the TP product. More preferably, the TP product comprises a total amount of protein of at least 75% w / w relative to the total solids of the TP product. Even more preferably, the TP product comprises a total amount of protein of at least 90% w / w relative to the total solids of the TP product.

[0722] In some preferred embodiments of the present invention, the total amount of protein in the TP product is in the range of 25-100% w / w relative to the total solids. Preferably, the total amount of protein in the TP product is in the range of 50-100% w / w. More preferably, the total amount of protein in the TP product is in the range of 75-100% w / w relative to the total solids. Even more preferably, the total amount of protein in the TP product is in the range of 90-100% w / w relative to the total solids.

[0723] In some preferred embodiments of the present invention, the TP product comprises at least 75% w / w TP relative to the total amount of protein. Preferably, the TP product may comprise at least 90% w / w TP relative to the total amount of protein. More preferably, the TP product may comprise at least 95% w / w TP relative to the total amount of protein. Even more preferably, the TP product may comprise at least 97% w / w TP relative to the total amount of protein. Most preferably, the TP product comprises about 100% w / w TP relative to the total amount of protein.

[0724] In some preferred embodiments of the invention, the TP product contains at most 10% w / w carbohydrates, preferably at most 5% w / w carbohydrates, more preferably at most 1% w / w carbohydrates and even more preferably at most 0.1% w / w carbohydrates.

[0725] The TP product may also comprise lipids, for example in the form of triglycerides and / or other lipid types such as phospholipids.

[0726] In some embodiments of the invention, the TP product comprises a total amount of lipids of at most 1% w / w relative to the total solids. Preferably, the TP product comprises a total amount of lipids of at most 0.5% w / w relative to the total solids. More preferably, the TP product comprises a total amount of lipids of at most 0.1% w / w relative to the total solids. Even more preferably, the TP product comprises a total amount of lipids of at most 0.05% w / w relative to the total solids. Preferably, the TP product comprises a total amount of lipids of at most 0.01% w / w relative to the total solids.

[0727] In some preferred embodiments of the present invention, the TP product is a dry product, such as a powder. It is particularly preferred that the TP product is a spray-dried powder.

[0728] The inventors have seen evidence that TP products in powder form, where at least some of the TP is in crystalline form when dried, have a higher bulk density than comparable TP products that do not contain TP crystals.

[0729] Thus, in some preferred embodiments of the present invention, the bulk density of the TP product in powder form is at least 0.40 g / mL. Preferably, the bulk density of the TP product in powder form is at least 0.45 g / mL. More preferably, the bulk density of the TP product in powder form is at least 0.50 g / mL. Even more preferably, the bulk density of the TP product in powder form is at least 0.6 g / mL. The bulk density of the TP product in powder form may be, for example, at least 0.7 g / mL.

[0730] In some preferred embodiments of the present invention, the bulk density of the TP product in powder form is at least 0.45 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. More preferably, the bulk density of the TP product in powder form is at least 0.50 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the TP product in powder form is at least 0.6 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. The bulk density of the TP product in powder form may, for example, be at least 0.7 g / mL and comprise at least 70% w / w protein relative to the total weight of the product.

[0731] In other preferred embodiments of the present invention, the TP product in powder form has a bulk density of at least 0.45 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. More preferably, the TP product in powder form has a bulk density of at least 0.50 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. Even more preferably, the TP product in powder form has a bulk density of at least 0.6 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. The TP product in powder form may for example have a bulk density of at least 0.7 g / mL and comprise at least 80% w / w protein relative to the total weight of the product.

[0732] The bulk density of the TP product in powder form may for example be in the range of 0.40-1.5 g / mL and contain at least 80% w / w protein relative to the total weight of the product. Preferably, the bulk density of the powdered TP product is in the range of 0.45-1.0 g / mL and contains at least 80% w / w protein relative to the total weight of the product. More preferably, the bulk density of the powdered TP product may be in the range of 0.50-0.9 g / mL and contain at least 80% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the powdered TP product is in the range of 0.6-0.9 g / mL and contains at least 80% w / w protein relative to the total weight of the product. The bulk density of the powdered TP product may for example be in the range of 0.6-0.8 g / mL and contain at least 80% w / w protein relative to the total weight of the product.

[0733] The inventors have found that the high density powders of the present invention advantageously allow for more cost-effective powder packaging and logistics, as less packaging material is required per kg of powder and more powder (mass) can be transported by a given container or truck.

[0734] The bulk density of the TP product in powder form may for example be in the range of 0.40-1.5 g / mL. Preferably, the bulk density of the powdered TP product is in the range of 0.45-1.0 g / mL. More preferably, the bulk density of the powdered edible TP product may be in the range of 0.50-0.9 g / mL. Even more preferably, the bulk density of the powdered TP product is in the range of 0.6-0.9 g / mL. The bulk density of the powdered TP product may for example be in the range of 0.6-0.8 g / mL.

[0735] In other preferred embodiments of the present invention, the bulk density of the TP product in powder form is in the range of 0.50-1.5 g / mL. Preferably, the bulk density of the powdered TP product is in the range of 0.55-1.0 g / mL. More preferably, the bulk density of the powdered TP product may be in the range of 0.60-1.0 g / mL. Even more preferably, the bulk density of the powdered TP product is in the range of 0.65-1.0 g / mL. The bulk density of the powdered TP product may preferably be in the range of 0.70-1.0 g / mL.

[0736] The bulk density of the TP product in powder form may for example be in the range of 0.40-1.5 g / mL and contain at least 70% w / w protein relative to the total weight of the product. Preferably, the bulk density of the powdered TP product is in the range of 0.45-1.0 g / mL and contains at least 70% w / w protein relative to the total weight of the product. More preferably, the bulk density of the powdered TP product may be in the range of 0.50-0.9 g / mL and contain at least 70% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the powdered TP product is in the range of 0.6-0.9 g / mL and contains at least 70% w / w protein relative to the total weight of the product. The bulk density of the powdered TP product may for example be in the range of 0.6-0.8 g / mL and contain at least 70% w / w protein relative to the total weight of the product.

[0737] The bulk density of the TP product in powder form may for example be in the range of 0.40-1.5 g / mL and contain at least 80% w / w protein relative to the total weight of the product. Preferably, the bulk density of the powdered TP product is in the range of 0.45-1.0 g / mL and contains at least 80% w / w protein relative to the total weight of the product. More preferably, the bulk density of the powdered TP product may be in the range of 0.50-0.9 g / mL and contain at least 80% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the powdered TP product is in the range of 0.6-0.9 g / mL and contains at least 80% w / w protein relative to the total weight of the product. The bulk density of the powdered TP product may for example be in the range of 0.6-0.8 g / mL and contain at least 80% w / w protein relative to the total weight of the product.

[0738] In other preferred embodiments of the present invention, the bulk density of the TP product in powdered form is in the range of 0.50-1.5 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. Preferably, the bulk density of the powdered TP product is in the range of 0.55-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. More preferably, the bulk density of the powdered TP product may be in the range of 0.60-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the powdered TP product is in the range of 0.65-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the product. The bulk density of the powdered TP product may preferably be in the range of 0.70-1.0 g / mL and comprises at least 70% w / w protein relative to the total weight of the product.

[0739] In other preferred embodiments of the present invention, the bulk density of the TP product in powder form is in the range of 0.50-1.5 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. Preferably, the bulk density of the powdered TP product is in the range of 0.55-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. More preferably, the bulk density of the powdered TP product may be in the range of 0.60-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. Even more preferably, the bulk density of the powdered TP product is in the range of 0.65-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the product. The bulk density of the powdered TP product may preferably be in the range of 0.70-1.0 g / mL and comprises at least 80% w / w protein relative to the total weight of the product.

[0740] The bulk density of the powder was measured according to Example 9.3 of WO2018 / 115520.

[0741] In some preferred embodiments of the invention, the TP product is a liquid composition.The liquid TP product preferably comprises at least 20% w / w water, more preferably at least 30% w / w water and even more preferably at least 40% w / w water.

[0742] The liquid TP product may for example comprise water in the range of 20-90% w / w, more preferably in the range of 30-80% w / w and even more preferably at least 40% w / w of water.

[0743] The inventors have seen evidence that TP products according to the invention have a surprisingly low degree of protein denaturation, even though spray drying has been used to prepare the TP powder product.

[0744] Therefore, in some preferred embodiments of the present invention, the degree of protein denaturation of the TP product is at most 10%. Preferably, the degree of protein denaturation of the TP product is at most 5%. More preferably, the degree of protein denaturation of the TP product is at most 2%. Even more preferably, the degree of protein denaturation of the TP product is at most 1%. Even more preferably, the degree of protein denaturation of the TP product is at most 0.5%.

[0745] In some preferred embodiments of the present invention, the TP product is a dry powder, and preferably a spray-dried powder, and the degree of protein denaturation is at most 2%, and preferably at most 1.5%. More preferably, the degree of protein denaturation of the dry TP product, for example in the form of a spray-dried powder, is at most 1.0%. Even more preferably, the degree of protein denaturation of the dry TP product, for example in the form of a spray-dried powder, is at most 0.8%. Even more preferably, the degree of protein denaturation of the dry TP product, for example in the form of a spray-dried powder, is at most 0.5%.

[0746] In some preferred embodiments of the present invention, the TP product comprises:

[0747] - up to 6% w / w water

[0748] - At least 80% total protein relative to total solids

[0749] - at least 95% TP relative to total protein, and

[0750] The TP products:

[0751] - is a dry powder, and

[0752] - A bulk density of at least 0.50 g / mL, preferably at least 0.60 g / mL.

[0753] In other preferred embodiments of the present invention, the TP product comprises:

[0754] - up to 6% w / w water

[0755] - At least 80% total protein relative to total solids

[0756] - at least 95% TP relative to total protein, and

[0757] The TP products:

[0758] - It's a dry powder.

[0759] - the bulk density is at least 0.50 g / mL, and preferably at least 0.60 g / mL, and - the TP crystallinity is at least 20%, preferably at least 40%.

[0760] In another preferred embodiment of the present invention, the TP product comprises:

[0761] - up to 6% w / w water

[0762] - At least 80% total protein relative to total solids

[0763] - at least 95% TP relative to total protein, and

[0764] The TP products:

[0765] - It's a dry powder.

[0766] - the bulk density is at least 0.50 g / mL, and preferably at least 0.60 g / mL, and - the degree of protein denaturation is at most 2%, and preferably at most 1.0%.

[0767] In another preferred embodiment of the present invention, the TP product comprises:

[0768] - up to 6% w / w water

[0769] - at least 80% total protein relative to total solids,

[0770] - at least 95% TP relative to total protein,

[0771] - Up to 80 mg of phosphorus per 100 g of protein.

[0772] The TP products:

[0773] -It's a dry powder.

[0774] In a still preferred embodiment of the invention, the TP product comprises:

[0775] - up to 6% w / w water

[0776] - at least 90% total protein relative to total solids,

[0777] - at least 97% TP relative to total protein,

[0778] - Up to 50 mg of phosphorus per 100 g of protein.

[0779] The TP products:

[0780] -It's a dry powder.

[0781] In other preferred embodiments of the present invention, the TP product comprises:

[0782] - up to 6% w / w water

[0783] - at least 80% total protein relative to total solids, and preferably at least 90% total protein relative to total solids,

[0784] - 30-90% TP relative to total protein,

[0785] The TP products:

[0786] - is a dry powder, and

[0787] - TP crystallinity of at least 20%, preferably at least 40%.

[0788] In some preferred embodiments of the present invention, the TP product comprises:

[0789] - 20-80% w / w water, and preferably 20-60% w / w water,

[0790] - At least 80% total protein relative to total solids, and preferably at least 90% total protein

[0791] - at least 95% TP relative to total protein,

[0792] - Up to 80 mg of phosphorus per 100 g of protein.

[0793] The TP products:

[0794] - TP crystallinity of at least 20%, preferably at least 40, and

[0795] - Optionally, the degree of protein denaturation is at most 2%, and preferably at most 1.0%.

[0796] The liquid TP product according to these embodiments is particularly useful for preparing TP products in dry form, and is particularly suitable for spray drying and preparing high density TP protein powders having a normal concentration profile of other protein species but containing at least some of the TP in the form of dry TP crystals.

[0797] In other preferred embodiments of the present invention, the TP product comprises:

[0798] - 20-80% w / w water, and preferably 20-60% w / w water,

[0799] - at least 80% total protein relative to total solids, and preferably at least 90% total protein relative to total solids,

[0800] - 30-79% TP relative to total protein,

[0801] The TP products:

[0802] - TP crystallinity of at least 20%, preferably at least 40%.

[0803] Products according to these embodiments are particularly useful for preparing TP products in dry form, and are particularly suitable for spray drying and preparing high density TP protein powders having a normal concentration profile of other protein species but containing at least some of the TP in the form of dry TP crystals.

[0804] The process of the present invention is preferably performed at a temperature in the range of 1-65°C, preferably in the range of 2-50°C, more preferably in the range of 3-20°C, even more preferably in the range of 4-15°C.

[0805] Another aspect of the present invention relates to the TP product obtainable by the process of the present invention.

[0806] The present invention has been described above with reference to specific embodiments. However, within the scope of the present invention, other embodiments other than those described above are equally possible. Unless otherwise indicated, the various embodiments and aspects of the present invention may be combined in other ways than those described herein.

[0807] Example

[0808] Example 1: Beta-lactoglobulin (BLG) crystallization based on mixing of two WPI streams.

[0809] introduction:

[0810] During initial trials for large-scale implementation of the method of WO2018 / 115520, frequent spontaneous BLG crystallization events were observed in the ultrafiltration unit used to prepare the whey protein solution. Spontaneous BLG crystallization events led to production interruptions and time-consuming cleaning of the process units affected by the crystallization.

[0811] New method for BLG (described in PCT / EP2022 / 053010):

[0812] The inventors have subsequently hit upon the idea that instead of producing the supersaturated protein solution of WO2018 / 115520 from a single feed which could become supersaturated with respect to BLG under adverse conditions during preparation (e.g. in the presence of high local protein concentrations and / or sudden cooling), it would be more sensible to produce a supersaturated whey protein solution from two (or more) feeds, each having a pH such that it is significantly not supersaturated with respect to BLG (or is supersaturated to only a limited extent), and then mixing the two (or more) feeds to obtain the correct pH for BLG supersaturation, i.e. a pH close to pH 5.5.

[0813] This new method makes it possible to prepare protein feeds with very high protein contents without the risk of uncontrolled BLG crystallization. In addition, this method makes it possible to prepare the feed and subsequently the initial protein solution at a temperature significantly lower than the optimal temperature for microbial growth, which is advantageous because less heat treatment is required to reduce the microbial content of the final product to an acceptable level. The inventors have also found that the pH adjustment performed by the Type A and / or Type B protein feeds of the present invention only results in a very limited change in the electrical conductivity of the initial protein solution compared to the same pH change with conventional edible acids or edible bases. This is beneficial because a reduction in the electrical conductivity of the initial protein solution typically results in a higher BLG crystallization yield.

[0814] The novel method is also applicable to other protein species (invention):

[0815] The inventors have realized that the novel method disclosed in PCT / EP2022 / 053010 is also applicable to other protein species that can crystallize under salting conditions in addition to BLG, as long as the pH range is adjusted as described herein. The feasibility of the novel method is demonstrated in this example using BLG as a model protein that can crystallize under salting conditions, but can be applied to other protein species that can crystallize under salting conditions.

[0816] In this example, the protein feeds were based on whey protein isolate containing BLG, and the pH of the first protein feed (Batch One, Type A protein feed) was 6.2, and the pH of the second protein feed (Batch Two, Type B protein feed) was 4.9. Despite the high protein concentration of BLG, neither protein feed was supersaturated. However, by simply mixing the two feeds in appropriate amounts (to obtain a pH close to pH 5.5, which the inventors have found to be the pH that provides the maximum crystallization yield of BLG, pH MCY,BLG ), an initial protein solution supersaturated with respect to BLG is obtained, from which BLG can be crystallized.

[0817] process:

[0818] The whey raw material in this embodiment is a kind of lactose-free UF retentate derived from the sweet whey in the standard cheese production process, and the whey has been subjected to fat reduction treatment by Synder FR membrane before use. Two batches are produced from this raw material, and the feed pressure of 30 mill spacers and 1.5-3.0 bar is adjusted using Alfa Laval GR82PE membrane by ultrafiltration device. The product is demineralized using refined water as diafiltration medium, and the concentration of the feed is 18% TS (total solids) ± 5%. Continue to diafiltration at least until the conductivity in the retentate drops below 0.02mS / cm in the 20min period, when the TS in the retentate is stable (± 0.5% TS). The retentate is then concentrated to about 22% TS, and the feed composition of batch one and batch two is shown in Table 1.1 and Table 1.2.

[0819] The first batch (Batch One) was pH adjusted to pH 6.1 with food grade hydrochloric acid (HCl) before UF treatment at 10° C. to 12° C. The other batch (Batch Two) was pH adjusted to pH 4.9 with food grade hydrochloric acid (HCl) before UF treatment at 10° C. to 12° C.

[0820] Table 1.1: Data describing batch 1

[0821]

[0822] ALA = alpha-lactalbumin.

[0823] Table 1.2: Data describing Batch 2 *BDL = Below Detection Limit

[0824]

[0825] After UF treatment, the temperature of both batches was adjusted to 10°C and then portions of the batches were mixed together in five different ratios to produce protein solutions for crystallization, see Table 1.3 for details. Each mixture was seeded with 0.1% w / w seed material at 10°C and cooled to 5°C after seeding and agitated overnight.

[0826] To check whether batches one and two had been supersaturated, samples were taken from each of the two batches before mixing as described above and also seeded and stored overnight at 10° C. Visual inspection by microscope the next morning showed that no crystals were present after incubation, and it was therefore concluded that batches one and two were not supersaturated.

[0827] The seed material was produced by mixing together a small portion of batch one and batch two to achieve a final pH of 5.5 and then adding a small amount of dry BLG crystalline material. The seed solution was then placed on ice and stirred for one hour.

[0828] Samples of the protein solution were taken before seeding and after crystallization and centrifuged at 3000 g for 5 minutes, samples of the supernatant were analyzed by RP-HPLC as described in Example 1 of WO2018 / 115520, and the crystallization yield was calculated by the following formula:

[0829]

[0830] Where %BLG 结晶之前 is the BLG concentration in the sample measured by HPLC before seeding and crystallization, and %BLG 结晶之后 is the BLG concentration in the sample supernatant measured by HPLC after crystallization. Results:

[0831] As can be seen from the table below, the whey protein feed can be adjusted within a pH range with a lower supersaturation (or even no supersaturation) with respect to BLG, thereby reducing the risk of spontaneous crystallization in the UF device and increasing the maximum protein concentration that can be used without spontaneous BLG crystallization. The two feeds are then mixed to provide an initial protein solution with a high BLG supersaturation.

[0832] Table 1.3: Protein solution data.

[0833]

[0834] The inventors have recognized that the results of Example 1 indicate that if the pH relative to the protein as described herein MCY,TP By modifying the pH range of the feed, any protein that can be crystallized under saline conditions can be crystallized according to the method of Example 1.

[0835] Example 2: Crystallization based on a mixture of two WPI streams using commercially available WPI.

[0836] The whey materials used in this example were the commercially available Lacprodan DI-9213 and a second homemade pH 6.2 WPI.

[0837] pH 6.2WPI is made from the lactose-free UF retentate of sweet whey derived from the standard cheese production process, and the whey has been subjected to fat reduction treatment by Synder FR membrane before use. Before UF treatment at 10°C to 12°C, the pH of the raw material is adjusted to pH 6.2 with food-grade hydrochloric acid. A batch is produced from this raw material and adjusted by ultrafiltration device using AlfaLaval GR82PE membrane with 30 mill spacers and a feed pressure of 1.5-3.0 bar. Refined water is used as the diafiltration medium to desalinate the product, and the concentration of the feed is 18% TS ± 5%. Diafiltration is continued at least until the conductivity in the retentate drops below 0.02mS / cm in the 20min period, when the TS in the retentate is stable (± 0.5% TS). The retentate is then concentrated to about 22% TS, and the feed composition of batch one and batch two can be found in Table 2.1 and Table 2.2.

[0838] Batch 2 was made from Lacprodan DI-9213 in powder form.

[0839] The powder was conditioned by rehydrating it to a TS of 16.3 and stirring it at 10 °C until all the powder was dissolved.

[0840] Table 2.1: Characteristics of Batch 1

[0841]

[0842] Table 2.2: Data describing batch 2

[0843]

[0844]

[0845] After adjustment, two batches were processed as described in Example 1 (except that only two solutions were prepared) and analyzed.

[0846] The properties of the mixture can be found in Table 2.3.

[0847] result:

[0848] As can be seen from the table below, the whey protein feed can be adjusted within a pH range where it is not oversaturated with respect to BLG, making it possible to reduce the risk of spontaneous crystallization and increase the protein concentration level without the risk of crystallization in the UF plant, and then mix the two together to obtain a high supersaturation. It can be seen that higher conductivity affects the yield as expected.

[0849] Table 2.3: Protein solution data.

[0850]

[0851] The inventors have recognized that the results of Example 2 indicate that if the pH relative to the protein as described herein MCY,TP By modifying the pH range of the feed, any protein that can be crystallized under saline conditions can be crystallized according to the method of Example 2.

[0852] Example 3: Crystallization based on mixing of two WPI streams (pH 4.0 and 6.1).

[0853] The whey raw material in this embodiment is a kind of lactose-free UF retentate derived from the sweet whey in the standard cheese production process, and the whey has been subjected to fat reduction treatment by Synder FR membrane before use. Two batches are produced from this raw material, and by ultrafiltration device, Alfa Laval GR82PE membrane is used to adjust with 30 mill spacers and 1.5-3.0 bar feed pressure. Refined water is used as diafiltration medium to desalinate the product, and the concentration of the feed is 18% TS ± 5%. Continue diafiltration at least until the conductivity in the retentate drops below 0.02mS / cm in the 20min period, when the TS in the retentate is stable (± 0.5% TS). The retentate is then concentrated to about 22% TS, and the feed composition of batch one and batch two is shown in Table 3.1 and Table 3.2.

[0854] One batch (Batch One) was pH adjusted to pH 6.1 with food grade hydrochloric acid (HCl) prior to UF treatment at 10° C. to 12° C. The other batch (Batch Two) was pH adjusted to pH 4.0 with food grade hydrochloric acid (HCl) prior to UF treatment at 10° C. to 12° C.

[0855] Table 3.1: Data describing batch 1

[0856]

[0857] Table 3.2: Data describing Batch 2 *BDL = Below Detection Limit

[0858]

[0859] After adjustment, two batches were processed as described in Example 1 of WO2018 / 115520 (except that only four solutions were prepared) and analyzed.

[0860] The properties of the mixture can be found in Table 3.3.

[0861] result:

[0862] As can be seen from the table below, the whey protein feed can be adjusted within a pH range where it has a low supersaturation with respect to BLG, making it possible to reduce the risk of spontaneous crystallization and increase the protein concentration level without the risk of crystallization in the UF plant, and then mix the two together to obtain a high supersaturation.

[0863] Table 3.3: Protein solution data.

[0864]

[0865] The inventors have recognized that the results of Example 3 indicate that if the pH relative to the protein as described herein MCY,TP By modifying the pH range of the feed, any protein that can be crystallized under saline conditions can be crystallized according to the method of Example 3.

[0866] Example 4: Crystal size distribution

[0867] The whey raw material in this embodiment is a kind of lactose-free UF retentate derived from the sweet whey in the standard cheese production process, and the whey has been subjected to fat reduction treatment by Synder FR membrane before use. Two batches are produced from this raw material for each crystallization, and the feed pressure of 30 mill spacers and 1.5-3.0 bar is regulated by ultrafiltration device using Alfa Laval GR82PE membrane. The product is desalted using refined water as the diafiltration medium, and the concentration of the feed is 18% TS ± 5%. Continue to diafiltration at least until the conductivity in the retentate drops below 0.02mS / cm in the 20min period, when the TS in the retentate is stable (± 0.5% TS). The retentate is then concentrated to about 22% TS, and the feed composition of batch one and batch two can be seen in the table below.

[0868] One batch (Batch One) was pH adjusted to pH about 6.1 with food grade hydrochloric acid (HCl) prior to UF treatment at 10° C. to 12° C. The other batch (Batch Two) was pH adjusted to pH 4.8 with food grade hydrochloric acid (HCl) prior to UF treatment at 10° C. to 12° C.

[0869] Crystallization was carried out in a 300 L crystallization tank and 0.1% w / w seed material was used. The seed material was produced as in Example 1.

[0870] The first crystallization

[0871] Table 4.1: Data describing batch 1

[0872]

[0873] Table 4.2: Data describing Batch 2 *BDL = Below Detection Limit

[0874]

[0875] After adjustment, the two batches were mixed together to obtain a final pH of 5.50, the mixture was seeded and rapidly cooled to 5°C and left overnight to crystallize. Slurry samples were analyzed to calculate yield as described in Example 1. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0876] Second crystallization

[0877] Table 4.3: Data describing batch 1

[0878]

[0879] Table 4.4: Data describing Batch 2 *BDL = below detection limit

[0880]

[0881]

[0882] After adjustment, the two batches were mixed together to obtain a final pH of 5.45, the mixture was seeded and rapidly cooled to 5°C and left overnight to crystallize. Slurry samples were analyzed to calculate yield as described in Example 1. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0883] The third crystallization

[0884] Table 4.5: Data describing batch 1

[0885]

[0886] Table 4.6: Data describing Batch 2 *BDL = below detection limit

[0887]

[0888]

[0889] After the two batches were mixed together to obtain a pH of 5.80, the mixture was seeded and incubated for 1 hour and then adjusted to a final pH of 5.45 by further addition of batch two over 2 hours and then rapidly cooled to 5°C and left overnight to crystallize. Slurry samples were analyzed to calculate yield as described in Example 1. The particle size distribution of the crystal slurry was analyzed by Malvern particle size characterization.

[0890] result:

[0891] The crystallization results are shown in Table 4.7 and as can be seen, despite the use of different raw materials and a different crystallization process, the crystal size distribution is similar.

[0892] Table 4.7: Summary of crystallization results

[0893] crystallization BLG yield (%) Dx(10) Dx(50) Dx(90) 1 73 80 167 263 2 75 58 153 253 3 71 57 127 248

[0894] The inventors have recognized that the results of Example 4 indicate that if the pH relative to the protein as described herein MCY,TP By modifying the pH range of the feed, any protein that can be crystallized under saline conditions can be crystallized according to the method of Example 4.

[0895] Example 5

[0896] Examples 1-4 of US 5,719,048 can be modified according to the present invention as follows.

[0897] Example 1 of US5,719,048:

[0898] The diafiltrate of Example 1 of US5,719,048 was divided into two batches and pH adjusted, and further diafiltrated to prepare:

[0899] - Type A feed having a pH of 5.1, a conductivity of 3 mS / cm and containing 13% w / w lipase, and

[0900] - Feed type B having a pH of 3.9, a conductivity of 3 mS / cm and containing 13% w / w lipase.

[0901] The initial protein solution of step a) of the present invention is prepared by mixing a type A feed with a type B feed in an amount sufficient to provide a pH of 4.5 in the mixture.

[0902] Example 2 of US5,719,048:

[0903] The diafiltrate of Example 2 of US5,719,048 was divided into two batches and pH adjusted, and further diafiltrated to prepare:

[0904] - Type A feed having a pH of 4.9, a conductivity of 3 mS / cm and containing 11% w / w lipase, and

[0905] - Feed type B having a pH of 3.9, a conductivity of 3 mS / cm and containing 11% w / w lipase.

[0906] The initial protein solution of step a) of the present invention is prepared by mixing Type A feed with Type B feed in an amount sufficient to provide a pH of 4.3 in the mixture.

[0907] Example 3 of US5,719,048:

[0908] The diafiltrate of Example 3 of US5,719,048 was divided into two batches and pH adjusted, and further diafiltrated to prepare:

[0909] - Type A feed having a pH of 5.6, a conductivity of 3 mS / cm and containing 10% w / w lipase, and

[0910] - Feed type B having a pH of 4.4, a conductivity of 3 mS / cm and containing 10% w / w lipase.

[0911] The initial protein solution of step a) of the present invention is prepared by mixing Type A feed with Type B feed in an amount sufficient to provide a pH of 5.0 in the mixture.

[0912] Example 4 of US5,719,048:

[0913] The diafiltrate of Example 4 of US5,719,048 was divided into two batches and pH adjusted, and further diafiltrated to prepare:

[0914] - Type A feed having a pH of 4.9, a conductivity of 3 mS / cm and containing 10% w / w lipase, and

[0915] - Feed type B having a pH of 3.9, a conductivity of 3 mS / cm and containing 10% w / w lipase.

[0916] The initial protein solution of step a) of the present invention is prepared by mixing Type A feed with Type B feed in an amount sufficient to provide a pH of 4.3 in the mixture.

Claims

1. A method for preparing a product, preferably an edible or pharmaceutically acceptable product, comprising a target protein (TP) capable of crystallizing under saline conditions, the method comprising the following steps: a) preparing an initial protein solution comprising the TP, the initial protein solution being supersaturated with respect to the TP and having a pH at which the TP can crystallize under saline conditions, b) crystallizing the TP in the supersaturated initial protein solution by salting to obtain a solution containing TP crystals, and c) preferably separating the TP crystals from the remaining liquid of said solution containing TP crystals, wherein step a) involves preparing the initial protein solution by combining one or more type A protein feeds with one or more type B protein feeds, And among them: - "Type A protein feed" is defined as comprising the TP and having a specific pH MCY,TP a protein feed having a pH at least 0.1 pH unit higher, and - "Type B protein feed" is defined as comprising said TP and having a specific pH MCY,TP A protein feed having a pH at least 0.1 pH unit lower, And preferably, with the proviso that said TP is not β-lactoglobulin.

2. The method according to claim 1, wherein: -About Type A protein feed: - in the case of using only a single A-type protein feed, or - In case two or more A-type protein feeds are used, and in case of mixing, the pH thereof is at least pH MCY,TP +0.1, more preferably at least pH MCY,TP +0.2, even more preferably at least pH MCY,TP +0.3 and most preferably at least pH MCY,TP +0.4, and -About B-type protein feeding: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH is at most pH MCY,TP -0.1, more preferably up to pH MCY,TP -0.2, even more preferably up to pH MCY,TP -0.3 and most preferably up to pH MCY,TP -0.

4.

3. A method according to any one of the preceding claims, wherein: -About Type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its pH is at least pH MCY,TP +0.5, more preferably at least pH MCY,TP +0.6, even more preferably at least pH MCY,TP +0.7 and most preferably at least pH MCY,TP +0.8, and -About B-type protein feeding: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH is at most pH MCY,TP -0.5, preferably at most pHMCY,TP -0.6, even more preferably up to pH MCY,TP -0.7 and most preferably up to pH MCY,TP -0.

8.

4. A method according to any one of the preceding claims, wherein: -About Type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its pH is at least pH MCY,TP +0.9, more preferably at least pH MCY,TP +1.0, even more preferably at least pH MCY,TP +1.2 and most preferably at least pH MCY,TP +1.5, and -About B-type protein feeding: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH is at most pH MCY,TP -0.9, preferably at most pHMCY,TP -1.0, even more preferably up to pH MCY,TP -1.2 and most preferably up to pH MCY,TP -1.

5.

5. The process according to any one of the preceding claims, wherein with respect to the type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP +0.1 to pH MCY,TP +4.5, more preferably pH MCY,TP +0.3 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.5 to pH MCY,TP +1.5 and most preferably pH MCY,TP +0.6 to pH MCY,TP +1.

0.

6. The process according to any one of the preceding claims, wherein with respect to the type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP +0.5 to pH MCY,TP +2.5, more preferably pH MCY,TP +0.6 to pH MCY,TP +2.0, even more preferably pH MCY,TP +0.7 to pH MCY,TP +1.5 and most preferably pH MCY,TP +0.7 to pH MCY,TP +1.

0.

7. The process according to any one of the preceding claims, wherein with respect to the type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP +0.7 to pH MCY,TP +3.0, more preferably pH MCY,TP +0.8 to pH MCY,TP +2.5, even more preferably pH MCY,TP +0.9 to pH MCY,TP +2.0 and most preferably pH MCY,TP +1.0 to pH MCY,TP +1.

5.

8. The process according to any one of the preceding claims, wherein with respect to the Type B protein feed: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP -3.5 to pH MCY,TP -0.1, more preferably pH MCY,TP -2.5 to pH MCY,TP -0.3, even more preferably pH MCY,TP -1.5 to pH MCY,TP -0.5, and most preferably pH MCY,TP -1.0 to pH MCY,TP -0.

6.

9. The process according to any one of the preceding claims, wherein with respect to the Type B protein feed: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP -1.5 to pH MCY,TP -0.5, more preferably pH MCY,TP -1.4 to pH MCY,TP -0.6, even more preferably pH MCY,TP -1.2 to pH MCY,TP -0.7, and most preferably pH MCY,TP -1.0 to pH MCY,TP -0.

7.

10. The process according to any one of the preceding claims, wherein with respect to the Type B protein feed: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its pH range is pH MCY,TP -2.0 to pH MCY,TP -0.7, more preferably pH MCY,TP -1.8 to pH MCY,TP -0.8, even more preferably pH MCY,TP -1.6 to pH MCY,TP -0.9, and most preferably pH MCY,TP -1.5 to pH MCY,TP -1.

0.

11. The method according to any one of the preceding claims, wherein at least one of the one or more A-type protein feeds does not contain TP crystals, more preferably all of the one or more A-type protein feeds do not contain TP crystals, and / or At least one of the one or more Type B protein feeds does not contain TP crystals, and more preferably all of the one or more Type B protein feeds do not contain TP crystals.

12. The process according to any one of the preceding claims, wherein at least one of the one or more Type A protein feeds is not oversaturated with respect to TP, and more preferably all of the one or more Type A protein feeds are not oversaturated with respect to TP, and / or Wherein at least one of the one or more Type B protein feeds is not oversaturated with respect to TP, and more preferably all of the one or more types are not oversaturated with respect to TP.

13. The process according to any one of the preceding claims, wherein the type A protein feed and the type B protein feed free of TP crystals contribute at least 1% w / w, more preferably at least 10% w / w, even more preferably at least 20% w / w and most preferably at least 40% w / w of the TP content of the initial protein solution.

14. The process according to any one of the preceding claims, wherein the type A protein feed and the type B protein feed free of TP crystals contribute at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w of the TP content of the initial protein solution.

15. The method according to any one of the preceding claims, wherein with respect to the type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, Its temperature is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

16. The process according to any one of the preceding claims, wherein with respect to the Type B protein feed: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, Its temperature is at most 60°C, more preferably at most 50°C, even more preferably at most 40°C and most preferably at most 30°C.

17. The method according to any one of the preceding claims, wherein with respect to the type A protein feed: - in the case of using only a single A-type protein feed, or - In case of using two or more A-type protein feeds, and in case of mixing, It comprises at least 1% w / w TP relative to the total weight of the type A protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w TP and most preferably at least 7% w / w TP relative to the total weight of the type A protein feed.

18. The process according to any one of the preceding claims, wherein with respect to the Type B protein feed: - in the case of using only a single B-type protein feed, or - In case of using two or more B-type protein feeds, and in case of mixing, It comprises at least 1% w / w TP relative to the total weight of the Type B protein feed, more preferably at least 3% w / w, even more preferably at least 5% w / w TP, and most preferably at least 7% w / w TP relative to the total weight of the Type B protein feed.

19. The method according to any one of the preceding claims, wherein the initial protein solution comprises a total amount of type A protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution.

20. The method according to any one of the preceding claims, wherein the initial protein solution comprises a total amount of Type B protein feed in the range of 1-99% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution.

21. The method according to any one of the preceding claims, wherein the initial protein solution comprises: a total amount of type A protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said type A protein feed having a pH MCY,TP +0.3 to pH MCY,TP pH +2.5, and a total amount of Type B protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said Type B protein feed having a pH MCY,TP -2.5 to pH MCY,TP - pH 0.

3.

22. The method according to any one of the preceding claims, wherein the initial protein solution comprises: a total amount of type A protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said type A protein feed having a pH MCY,TP +0.5 to pH MCY,TP pH +2.5, and a total amount of Type B protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said Type B protein feed having a pH MCY,TP -2.5 to pH MCY,TP - pH of 0.

5.

23. The method according to any one of the preceding claims, wherein the initial protein solution comprises: a total amount of type A protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said type A protein feed having a pH MCY,TP +0.6 to pH MCY,TP pH of +1.0, and a total amount of Type B protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said Type B protein feed having a pH MCY,TP -1.0 to pH MCY,TP - pH 0.

6.

24. The method according to any one of the preceding claims, wherein the initial protein solution comprises: a total amount of type A protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said type A protein feed having a pH MCY,TP +0.8 to pH MCY,TP pH +1.5, and a total amount of Type B protein feed in the range of 5-90% w / w, more preferably 10-80% w / w, even more preferably 20-70% w / w and most preferably 30-60% w / w relative to the weight of the initial protein solution, said Type B protein feed having a pH MCY,TP -1.5 to pH MCY,TP - pH of 0.

8.

25. The process according to any one of the preceding claims, wherein protein feed type A and protein feed type B contribute at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w of the protein content of the initial protein solution.

26. The process according to any one of the preceding claims, wherein protein feed type A and protein feed type B contribute at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 96% w / w and most preferably at least 99% w / w of the protein content of the initial protein solution.

27. The process according to any one of the preceding claims, wherein protein feed type A and protein feed type B contribute at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w of the solids content of the initial protein solution.

28. The process according to any one of the preceding claims, wherein protein feed type A and protein feed type B contribute at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 96% w / w and most preferably at least 99% w / w of the solids content of the initial protein solution.

29. The process according to any one of the preceding claims, wherein type A protein feed and type B protein feed contribute at least 50% w / w, more preferably at least 70% w / w, even more preferably at least 80% w / w and most preferably at least 90% w / w by weight of the initial protein solution.

30. The process according to any one of the preceding claims, wherein protein feed type A and protein feed type B contribute at least 92% w / w, more preferably at least 94% w / w, even more preferably at least 96% w / w and most preferably at least 99% w / w by weight of the initial protein solution.

31. The method of any one of the preceding claims, wherein the supersaturation of the TP of the initial protein solution is greater than both: - in the case where only a single Type A protein feed is used, the supersaturation of said Type A protein feed, or - in case two or more type A protein feeds are used, the supersaturation of said type A protein feeds in case they are combined, and both of the following: - in the case where only a single Type B protein feed is used, the supersaturation of said Type B protein feed, or - In case two or more Type B protein feeds are used, the supersaturation of said Type B protein feeds in case they are combined.

32. The method according to any one of the preceding claims, wherein the pH of the initial protein solution is at a pH (pH MCY,TP ) and most preferably within the range of + / - 0.7 pH units of the pH MCY,TP within a range of + / - 0.5 pH units.

33. The method according to any one of the preceding claims, wherein the TP is a globular protein, and preferably a globulin or a globin.

34. The method according to any one of the preceding claims, wherein the TP is selected from the group consisting of an enzyme, an immunoglobulin, a hormone.

35. The method according to any one of the preceding claims, wherein the TP is an enzyme, preferably an oxidoreductase (EC1 ), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4) or an isomerase (EC 5).

36. The method according to any of the preceding claims, wherein the TP is an oxidoreductase (EC 1), preferably selected from one or more of the following: laccase (EC 1.10.3.2), catalase (EC 1.11.1.6), glucose oxidase (EC 1.1.3.4), ligninase (EC 1.11.1.14) and peroxidase (EC 1.11.1).

37. The method of any one of the preceding claims, wherein the TP is a hydrolase (EC 3).

38. The method according to any one of the preceding claims, wherein the TP is an esterase (EC 3.1), preferably selected from phytase (EC 3.1.3), lipase (EC number 3.1.1.3), phosphatase (EC 3.1.3), phytase and / or nuclease (EC 3.1.11-3.1.31).

39. The method according to any one of the preceding claims, wherein the TP is a glycosylase (EC 3.2), preferably selected from α-amylase (EC 3.2.1.1), amyloglucosidase (EC 3.2.1.3 or EC 3.2.1.33), cellulase (EC 3.2.1.4), hemicellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), muramidase (EC 3.2.1.17), pullulanase (EC 3.2.1.41), pectinase (EC 3.2.1.67), β-galactosidase (EC 3.2.1.23), lactase (EC 3.2.1.108), glucoamylase (EC 3.2.1.20 or EC 3.2.1.3) and glucanase (EC 3.2.1.11).

40. The method according to any one of the preceding claims, wherein the TP is a peptidase or a protease (EC 3.4), for example selected from chymotrypsin (EC 3.4.21.1), trypsin, pepsin, rennet (EC 3.4.23.4) or an alkaline protease.

41. The method of any one of the preceding claims, wherein the TP is a lyase (EC 4), such as pectate lyase or alpha-acetolactate decarboxylase.

42. The method of any one of the preceding claims, wherein the TP is an isomerase (EC 5), such as glucose isomerase.

43. The method according to any of the preceding claims, wherein the TP is a hormone, preferably human insulin (uniprot ID: P01308), a human insulin analogue, a human insulin agonist, human GLP-1, a human GLP-1 analogue or a human GLP-1 agonist.

44. The method according to any one of the preceding claims, wherein the TP is an immunoglobulin, and preferably a monoclonal immunoglobulin.

45. The method according to any one of the preceding claims, wherein the TP is immunoglobulin G (IgG), and preferably monoclonal IgG.

46. ​​The method of any one of the preceding claims, wherein the TP is a bispecific IgG.

47. The method of any of the preceding claims, wherein the TP is an interferon, an interferon analog, or an interferon agonist.

48. The method of any one of claims 30-32, wherein the TP is a therapeutic protein selected from the group consisting of muromonab-CD3, abciximab, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab , eculizumab, becelimumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, vembrolizumab, pertuzumab, emtansuzumab, raxicumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, belintuximab, nivolumab, pembrolizumab, idarucizumab, necituzumab monoclonal antibody, datuximab, secukinumab, mepolizumab, alirocumab, eloumab, datumumab, elotuzumab, ixekizumab, reslizumab, olaratumumab, belotuzumab, atezolizumab, otuximab, omagaituzumab, bodalumab, guselkumab, dupilumab, salizumab, avelumab, ocrelizumab, emicizumab, benrelizumab monoclonal antibody, ogamicin, durvalumab, brolucizumab, ranarumab, moglizumab, efenazumab, ganavir, tetracycline, cemiplizumab, imatinib, remanezumab, ibalizumab, mosetuzumab, ravulizumab, calicitumomab, romuzumab, risankizumab, vepotuzumab, brolucizumab, and rizotinib.

49. The method according to any one of the preceding claims, wherein the TP is prepared by purification from a natural source, by fermentation of a recombinant organism and / or by subsequent protein rearrangement.

50. The method of any preceding claim, wherein the TP is a therapeutic protein.

51. A TP product obtainable by a process according to one or more of claims 1-50.

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