WO2018164937A1 - Process for crystallizing 2'-fucosyllactose and related compositions - Google Patents
Process for crystallizing 2'-fucosyllactose and related compositions Download PDFInfo
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- WO2018164937A1 WO2018164937A1 PCT/US2018/020431 US2018020431W WO2018164937A1 WO 2018164937 A1 WO2018164937 A1 WO 2018164937A1 US 2018020431 W US2018020431 W US 2018020431W WO 2018164937 A1 WO2018164937 A1 WO 2018164937A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
Definitions
- This specification relates to a process for crystallizing an oligosaccharide, particularly a human milk oligosaccharide, and, more particularly, 2'-fucosyllactose ("2'-FL").
- This specification also relates to compositions (e.g., crystalline products) produced using such a process.
- Human milk oligosaccharides are important for nutrition and therapeutics.
- One of the most important human milk oligosaccharides is 2'-fucosyllactose (also known as 2'-0- fucosyllactose or "2'-FL").
- This oligosaccharide is the most abundant oligosaccharide found in human breast milk, and is believed to have several beneficial biological roles, including prebiotic, antibacterial, antiviral, immune system enhancing and brain development enhancing effects.
- Such benefits make 2'-FL a potentially attractive ingredient to be included in food, dietary supplements and medicines, and particularly infant formula.
- development of economically feasible processes for large scale production of 2'-FL continues to be a challenge.
- DiFL difucosyllactose
- monosaccharides amino acids, polypeptides, proteins, monovalent and divalent salts, organic acids, nucleic acids, etc.
- Special equipment is often required to recover the solvent after the crystallization. In some instances, there are solvent losses associated with such solvent recovery. For some solvents, special equipment is also needed due to explosion or other safety reasons. And use of solvents also can be problem if residual solvent remains in the crystalline product. Even trace levels of some organic solvents may have an adverse effect on taste, odor, color and/or food safety.
- this specification generally discloses a process for crystallizing 2'-FL, as well as compositions produced using such a process.
- this specification discloses, in part, a process for making crystalline 2'-FL from an aqueous starting solution comprising 2'-FL and at least one other carbohydrate.
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-FL, and then precipitating a 2'-FL crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of greater than 60°C.
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2'-FL crystal.
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-fucosyllactose, and then precipitating a 2'- fucosyllactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40°C.
- the starting solution has a dry solids content with a 2'-fucosyllactose concentration of from 70 to 95% (wherein the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography).
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2'-fucosyllactose crystal.
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-fucosyllactose, and then precipitating a 2'- fucosyllactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40°C.
- the starting solution has a dry solids content with a 2'-fucosyllactose concentration of less than about 98% (wherein the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography).
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2'-fucosyllactose crystal.
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-fucosyllactose, and then precipitating a 2'- fucosyllactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40°C.
- the starting solution has a dry solids content with a 2' -fucosyllactose concentration of less than about 95% (wherein the percentage corresponds to a normalized peak area concentration obtained using high performance liquid chromatography).
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2' -fucosyllactose crystal.
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-fucosyllactose, and then precipitating a 2'- fucosyllactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40°C.
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2' -fucosyllactose crystal.
- the crystalline 2' -fucosyllactose product has a melting point of from about 230 to about 239°C (as determined with a l°C/min heating rating using the European
- the process comprises concentrating the starting solution to a supersaturated state with respect to 2'-fucosyllactose, and then precipitating a 2'- fucosyllactose crystal from the supersaturated solution while subjecting the supersaturated solution to a temperature of at least 40°C.
- the supersaturated solution comprises no greater than 1% (by weight) organic solvent during the precipitation of the 2' -fucosyllactose crystal.
- the crystalline 2'-fucosyllactose product exhibits an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ⁇ 0.20, 13.65 ⁇ 0.20 and 18.32 ⁇ 0.20 2 ⁇ angles.
- This specification also discloses, in part, a process for making amorphous 2'-FL.
- the process comprises making crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the crystalline 2'-FL in a solvent (e.g., water) to form a purified 2'-FL solution, and precipitating amorphous 2'-FL from the purified 2'-FL solution.
- a solvent e.g., water
- This specification also discloses, in part, a processes for making a food, dietary supplement or medicine.
- the process for making the food, dietary supplement or medicine comprises making crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), and then mixing the crystalline 2'-FL with one or more ingredients suitable for the food, dietary supplement or medicine.
- the process for making the food, dietary supplement or medicine comprises making crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the crystalline 2'-FL in a solvent (e.g., water), and mixing the dissolved 2'-FL with one or more ingredients suitable for the food, dietary supplement or medicine.
- a solvent e.g., water
- the process for making the food, dietary supplement or medicine comprises making amorphous 2'-FL as described above (or elsewhere in this specification), and then mixing the amorphous 2'-FL with one or more ingredients suitable for the food, dietary supplement or medicine.
- the process for making the food, dietary supplement or medicine comprises making amorphous 2'-FL as described above (or elsewhere in this specification), dissolving the amorphous 2'-FL in a solvent (e.g., water), and mixing the dissolved 2'-FL with one or more ingredients suitable for the food, dietary supplement or medicine.
- a solvent e.g., water
- This specification also discloses, in part, a processes for making infant formula.
- the process for making infant formula comprises making crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), and then mixing the crystalline 2'-FL with one or more infant formula ingredients.
- the process for making infant formula comprises making crystalline 2'-FL according to the crystallization process described above (or elsewhere in this specification), dissolving the crystalline 2'-FL in a solvent (e.g., water), and mixing the dissolved 2'-FL with one or more infant formula ingredients.
- a solvent e.g., water
- the process for making an infant formula comprises making amorphous 2'-FL as described above (or elsewhere in this specification), and then mixing the amorphous 2'-FL with one or more infant formula ingredients.
- the process for making an infant formula comprises making amorphous 2'-FL as described above (or elsewhere in this specification), dissolving the amorphous 2'-FL in a solvent (e.g., water), and mixing the dissolved 2'-FL with one or more infant formula ingredients.
- a solvent e.g., water
- This specification also discloses, in part, a crystalline 2'-FL product obtained from a crystallization process described above (or elsewhere in this specification).
- This specification also discloses, in part, amorphous 2'-FL obtained from a process described above (or elsewhere in this specification) for making amorphous 2'-FL.
- This specification also discloses, in part, a food, dietary supplement or medicine prepared by a process for making a food, dietary supplement or medicine described above (or elsewhere in this specification).
- This specification also discloses, in part, an infant formulation obtained from a process for making an infant formula described above (or elsewhere in this specification).
- This specification relates to a process for crystallizing 2'-FL from a 2'-FL- containing solution.
- this process comprises bringing a 2'-FL-containing solution to a supersaturated state with respect to 2'-FL, and then crystallizing the 2'-FL from the solution by evaporation until a crystallization mass is obtained which has a crystal yield of at least about 1% with respect to 2'-FL.
- the solution from which the 2'-FL is crystallized comprises a natural source of 2'-FL, such as, for example, an animal milk (e.g., human milk) or a composition derived from animal milk.
- the solution from which the 2'- FL is crystallized is produced by a chemical synthesis.
- the 2'-FL- containing solution is obtained from a microbial fermentation process.
- the 2'-FL source may be a fermentation broth obtained by microbial fermentation using a recombinant microorganism, such as bacteria or yeast.
- the microorganism is a yeast.
- the microorganism is a bacteria.
- the microorganism is Escherichia coli.
- the fermentation occurs in a chemically defined medium.
- the source of 2'-FL may be subjected to one or more purification processes.
- the 2'-FL source e.g., fermentation broth
- the 2'-FL source is subjected to a centrifugation, sedimentation or one or more other process to remove cell biomass.
- the 2'-FL source e.g.,
- the fermentation broth is subjected to ultrafiltration. This can be helpful, for example, to remove cells and large biomolecules, such as proteins, nucleic acids and lipopolysaccharides.
- the 2'-FL source e.g., fermentation broth
- nanofiltration This can be helpful, for example, to concentrate the 2'-FL by reducing water while also removing minerals and various small biomolecules.
- the 2'-FL source e.g., fermentation broth
- a chromatographic separation such as gel-filtration chromatography.
- the 2'-FL source e.g., fermentation broth
- microfiltration is subjected to microfiltration. This can be helpful, for example, to remove microbiological contamination.
- the 2'-FL source e.g., fermentation broth
- the 2'-FL source is subjected to cation exchange, anion exchange, ion exchange resin, mixed bed ion exchange and/or electrodialysis. These can be helpful, for example, to remove small charged molecules, salts and trace metals.
- the 2'-FL source e.g., fermentation broth
- the 2'-FL source is subjected to a de-coloration, by, for example, contacting it with powdered activated carbon or charcoal filtration.
- the 2'-FL source e.g., fermentation broth
- evaporation This can be helpful, for example, to concentrate the 2'-FL by removing water.
- the 2'-FL source e.g., fermentation broth
- the 2'-FL source is subjected to a combination of two or more of the above purification steps before the crystallization.
- Such embodiments may include use of the steps in various orders, as well as repeating various steps at different points in the process.
- the 2'-FL source is a fermentation broth, which is subjected to ultrafiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a supersaturated state for the crystallization.
- the 2'-FL source is a fermentation broth, which is subjected to centrifugation, ultrafiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a supersaturated state for the crystallization.
- the 2'-FL source is a fermentation broth, which is subjected to ultrafiltration, nanofiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a supersaturated state for the crystallization.
- the 2'-FL source is a fermentation broth, which is subjected to centrifugation, ultrafiltration, nanofiltration, cation exchange, anion exchange, mixed bed ion exchange and powdered activated carbon before the solution is brought to a supersaturated state for the crystallization.
- the 2'-FL source is a fermentation broth, which is subjected to ultrafiltration, nanofiltration, optional microfiltration, optional ion removal (e.g., ion exchange resin and/or electrodialysis), optional pre-concentration (e.g., evaporation or nanofiltration), decoloration (e.g., charcoal filtration), microfiltration, and optional nanofiltration before the solution is brought to a supersaturated state for the
- the 2'-FL concentration (or "purity") in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than 99%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than 98%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 91%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 90%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 85%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 80%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is less than about 75%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 50%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 55%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 60%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 65%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 70%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps from the pre-crystallization purification steps is at least about 75%). In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 80%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 85%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 90%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is at least about 95%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 60 to about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 60 to about 90%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre- crystallization purification steps is from about 65 to about 95%. In some embodiments, the 2'- FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 65 to about 90%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 70 to about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 70 to about 90%). In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 70 to about 85%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 70 to about 80%.
- the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 70 to about 75%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 75 to about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 80 to about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 85 to about 95%. In some embodiments, the 2'-FL concentration in the dry solids content of the syrup resulting from the pre-crystallization purification steps is from about 90 to about 95%.
- the 2'-FL is crystallized from a solution comprising 2'-FL and at least one other carbohydrate.
- the solution may contain, for example, a complex mixture of lactose, oligosaccharides in addition to the 2'-FL (e.g., difucosyllactose ("DiFL")), monosaccharides, amino acids, polypeptides, proteins, monovalent and divalent salts, organic acids, nucleic acids, etc.
- the total concentration of carbohydrates other than 2'-FL in the solution is at least about 1%.
- the total concentration of carbohydrates other than 2'-FL in the solution is at least about 2%. In some embodiments, for example, the total concentration of carbohydrates other than 2'-FL in the solution is at least about 5%. In some embodiments, the concentration of DiFL in the solution is at least about 1%. In some embodiments, the concentration of DiFL in the solution is at least about 2%. In some embodiments, the concentration of DiFL in the solution is at least about 5%. In some embodiments, the concentration of DiFL in the solution is at least about 7%. In some embodiments, the concentration of DiFL in the solution is from about 1 to about 20%. In some embodiments, the concentration of DiFL in the solution is from about 2 to about 20%.
- the concentration of DiFL in the solution is from about 2 to about 18%. In some embodiments, the concentration of DiFL in the solution is from about 5 to about 18%. In some embodiments, the concentration of DiFL in the solution is from about 2 to about 10%. In some embodiments, the concentration of DiFL in the solution is from about 5 to about 10%. In some embodiments, the concentration of DiFL in the solution is from about 10 to about 18%. In some embodiments, the concentration of lactose in the solution is no greater than about 0.1%. In some embodiments, the concentration of lactose in the solution is at least about 0.1%. In some embodiments, the concentration of lactose in the solution is at least about 2%.
- the concentration of lactose in the solution is at least about 5%. In some embodiments, the concentration of lactose in the solution is from about 0.1 to about 15%. In some embodiments, the concentration of lactose in the solution is from about 0.1 to about 0.5%. In some embodiments, the concentration of lactose in the solution is from about 1 to about 12%. In some embodiments, the concentration of lactose in the solution is from about 2 to about 12%. In some embodiments, the concentration of lactose in the solution is from about 5 to about 12%. In some embodiments, the concentration of lactose in the solution is from about 2 to about 15%. In some embodiments, the concentration of lactose in the solution is from about 5 to about 15%. These percentages correspond to normalized peak area concentrations (or "purities") obtained using HPLC.
- the solution is brought to a 2'-FL supersaturation of greater than about 1.0. In some embodiments, the solution is brought to a 2'-FL supersaturation of from about 1.05 to about 1.8. In some embodiments, the solution is brought to a 2'-FL supersaturation of from about 1.1 to about 1.7. In some embodiments, the solution is brought to a 2'-FL supersaturation of from about 1.1 to about 1.5. In some embodiments, the solution is brought to a 2'-FL supersaturation of from about 1.2 to about 1.7. In some embodiments, the solution is brought to a 2'-FL supersaturation of from about 1.3 to about 1.7.
- the solution is brought to a 2'-FL supersaturation of from about 1.3 to about 1.5.
- the terms “supersaturation” and “supersaturated” correspond to a dimensionless ratio of the measured 2'-FL content to the solubility of 2'-FL, the ratio being calculated from the following equation:
- the solution is brought to a supersaturated state by increasing the dry solids content to at least about 50% (by weight). In some such embodiments, the dry solids content is increased to at least about 55% (by weight). In some such embodiments, the dry solids content is increased to at least about 60% (by weight). In some such embodiments, the dry solids content is increased to at least about 65% (by weight). In some such embodiments, the dry solids content is increased to no greater than about 80% (by weight). In some such embodiments, the dry solids content is increased to no greater than about 78%) (by weight). In some such embodiments, the dry solids content is increased to no greater than about 75% (by weight).
- the dry solids content is increased to from about 50 to about 80%> (by weight). In some embodiments, the dry solids content is increased to from about 55 to about 78% (by weight). In some embodiments, the dry solids content is increased to from about 60 to about 75% (by weight). In some embodiments, the dry solids content is increased to from about 65 to about 75% (by weight). In some such
- the dry solids content is increased to about 60% (by weight).
- the solution is brought to a supersaturated state with respect to 2'-FL by evaporation.
- the evaporation (or at least a portion thereof) is carried out at atmospheric pressure.
- the evaporation (or at least a portion thereof) is carried out at a lower pressure.
- the evaporation (or at least a portion thereof) is carried out at a pressure of no greater than about 250 mbar.
- the evaporation (or at least a portion thereof) is carried out at a pressure of from about 120 to about 250 mbar.
- the evaporation (or at least a portion thereof) is carried out at a pressure of from about 130 to about 220 mbar. In some embodiments, the evaporation (or at least a portion thereof) is carried out at a pressure of from about 150 to about 210 mbar
- the temperature at which the evaporation is carried out will depend on, for example, the pressure.
- the temperature of the evaporation is the boiling temperature of the solution at the pressure.
- the evaporation temperature is at least about 40°C. In some embodiments, the evaporation temperature is at least about 45°C. In some embodiments, the evaporation temperature is at least about 50°C. In some embodiments, the evaporation temperature is at least about 55°C. In some embodiments, the evaporation temperature is at least about 60°C. In some embodiments, the evaporation temperature is greater than 60°C. In some embodiments, the evaporation temperature is no greater than about 100°C.
- the evaporation temperature is no greater than about 90°C. In some embodiments, the evaporation temperature is no greater than about 80°C. In some embodiments, the evaporation temperature is no greater than about 75°C. In some embodiments, the evaporation temperature is no greater than about 70°C (while temperatures greater than about 70°C can be advantageous for the evaporation, use of such temperatures can cause undesirable product degradation and/or color formation). In some embodiments, the evaporation temperature is from about 45 to about 80°C. In some embodiments, the evaporation temperature is from about 50 to about 80°C. In some embodiments, the evaporation temperature is from about 55 to about 80°C.
- the evaporation temperature is from about 60 to about 80°C. In some embodiments, the evaporation temperature is greater than 60 to no greater than about 80°C. In some embodiments, the evaporation temperature is from about 60 to about 75°C. In some embodiments, the evaporation temperature is greater than 60 to no greater than about 75°C. In some embodiments, the evaporation temperature is from about 65 to about 75°C. In some embodiments, the evaporation temperature is from about 40 to about 70°C. In some embodiments, the evaporation temperature is from about 45 to about 70°C. In some embodiments, the evaporation temperature is from about 50 to about 70°C. In some embodiments,
- the evaporation temperature is from about 55 to about 70°C. In some embodiments, the evaporation temperature is from about 55 to about 70°C.
- the evaporation temperature is from about 60 to about 70°C. In some embodiments, the evaporation temperature is from about 60 to about 70°C.
- the evaporation temperature is greater than 60 to no greater than about 70°C.
- seeding is used to initiate formation of 2'-FL crystals from the supersaturated solution.
- seeding is effected by adding seed crystals to the supersaturated solution.
- the seed crystals comprise a particulate 2'-FL powder.
- the seed crystals comprise anhydrous 2'-FL seed crystals.
- Seed crystals can made by various processes, including, for example, those discussed in this specification.
- the dry seeds are milled to get smaller particle size.
- the seed crystals can be used also in suspension form.
- the desired amount of seed crystals may depend on, for example, the size of the seed crystals.
- the amount of dry seed crystals used is from about 0.001 to about 1% (by weight) of particulate 2'-FL, based on the 2'-FL of the crystallization mass.
- a suspension of seed crystals is used, and the amount of suspension seed crystals is from about 1 to about 30% (by weight) of particulate 2'-FL, based on the 2'-FL of the crystallization mass.
- crystallization is initiated without adding 2'-FL seed crystals to the supersaturated solution.
- seeding is effected using spontaneous seeding or ultrawave seeding.
- initiation of crystallization is carried out when a suitable supersaturation has been achieved.
- initiation of crystallization is carried out when the 2'-FL supersaturation is greater than about 1.0.
- initiation of crystallization is carried out when the 2'-FL supersaturation is from about 1.05 to about 1.8.
- initiation of crystallization is carried out when the 2'-FL supersaturation is from about 1.1 to about 1.7.
- initiation of crystallization is carried out when the 2'-FL supersaturation is from about 1.1 to about 1.5. In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the 2'-FL supersaturation is from about 1.2 to about 1.7. In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the 2'-FL supersaturation is from 1.3 to about 1.7. In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the 2'-FL supersaturation is from about 1.3 to about 1.5.
- initiation of crystallization is carried out when the dry solids content of the syrup at least about 60% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup at least about 65% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is no greater than about 80% (by weight). In some embodiments, initiation of
- crystallization e.g., addition of seed crystals
- initiation of the dry solids content of the syrup is no greater than about 75% (by weight).
- crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is from about 60 to about 80% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is from about 65 to about 80% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is from about 65 to about 77% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is from about 65 to about 75% (by weight).
- initiation of crystallization is carried out when the dry solids content of the syrup is from about 65 to about 70% (by weight). In some embodiments, initiation of crystallization (e.g., addition of seed crystals) is carried out when the dry solids content of the syrup is from about 70 to about 76% (by weight).
- the crystallization may generally be carried out at the boiling point of the solution.
- At least a portion (or all) of the crystallization is carried out at atmospheric pressure.
- At least a portion of the crystallization is carried out at a pressure that is less than atmospheric pressure. In some such embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of no greater than about 250 mbar. In some embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of from about 50 to about 250 mbar. In some embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of from about 50 to about 120 mbar. In some embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of from about 120 to about 250 mbar.
- the crystallization (or at least a portion thereof) is carried out at a pressure of from about 130 to about 220 mbar. In some embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of from about 150 to about 210 mbar. In some embodiments, the crystallization (or at least a portion thereof) is carried out at a pressure of from about 170 to about 205 mbar.
- At least about 50% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, at least about 75% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, at least about 90% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, at least about 95% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, at least about 98% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, at least about 99% of the crystallization is carried out at a sub-atmospheric pressure range discussed above. In some embodiments, the entire
- crystallization is carried out at a sub-atmospheric pressure range discussed above.
- At least a portion of the crystallization is carried out at a the boiling temperature of the solution at the pressure. In some embodiments, the temperature during the crystallization does not decrease to below about 20°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at least about 40°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at least about 45°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of at least about 50°C. In some embodiments at least a portion of the crystallization is carried out at a temperature of at least about 55°C.
- At least a portion of the crystallization is carried out at a temperature of at least about 60°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of greater than 60°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of no greater than about 100°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of no greater than about 90°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of no greater than about 80°C. In some
- At least a portion of the crystallization is carried out at a temperature of no greater than about 75°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of no greater than about 70°C (while temperatures greater than about 70°C can be advantageous for crystallization, use of such temperatures can cause undesirable product degradation and/or color formation). In some embodiments, at least a portion of the
- crystallization is carried out at a temperature of from about 45 to about 80°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of from about 50 to about 80°C. In some embodiments at least a portion of the crystallization is carried out at a temperature of from about 55 to about 80°C. In some embodiments at least a portion of the crystallization is carried out at a temperature of from about 60 to about 80°C. In some embodiments at least a portion of the crystallization is carried out at a temperature of greater than 60 and no greater than about 80°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of from about 60 to about 75°C.
- At least a portion of the crystallization is carried out at a temperature of greater than 60 and no greater than about 75°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of from about 65 to about 75°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of about from 40 to about 70°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of about from 45 to about 70°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of from about 50 to about 70°C.
- At least a portion of the crystallization is carried out at a temperature of from about 55 to about 70°C. In some embodiments, at least a portion of the crystallization is carried out at a temperature of from about 60 to about 70°C. In some embodiments at least a portion of the crystallization is carried out at a temperature of greater than 60 and no greater than about 70°C.
- At least about 50% of the crystallization is carried out at a temperature range discussed above. In some embodiments, at least about 75% of the
- crystallization is carried out at a temperature range discussed above. In some embodiments, at least about 90% of the crystallization is carried out at a temperature range discussed above. In some embodiments, at least about 95% of the crystallization is carried out at a temperature range discussed above. In some embodiments, at least about 98% of the crystallization is carried out at a temperature range discussed above. In some embodiments, at least about 99% of the crystallization is carried out at a temperature range discussed above. In some embodiments, the entire crystallization is carried out at a temperature range discussed above.
- the pH of the crystallization is from about 4 to about 11. In some embodiments, the pH during at least a portion of the crystallization is from about 4 to about 8. In some embodiments, the pH during at least a portion of the crystallization is from about 5 to about 7. In some embodiments, the pH during at least a portion of the crystallization is from about 5 to about 6. In some embodiments, the pH during all the crystallization is no greater than about 7. In some embodiments, the pH during all the crystallization is from about 5 to about 7. In some embodiments, the pH during all the crystallization is from about 5 to about 6.
- the crystal mass is typically mechanically mixed.
- the mixing is optimally arranged to maximize homogeneity throughout the entire volume of the crystallization mass. This generally provides for desirable heat and mass transfer and small supersaturation gradients throughout whole crystallization volume. In some embodiments, this is achieved using traditional rotating mixer blades. In general, the intensity of the mixing is moderated to prevent excess of fines formation.
- the crystal suspension is generally subjected to boiling and evaporation until a sufficient degree of crystallization (yield, reduction in 2'-FL purity of the mother liquor and/or crystal size) has been achieved.
- the crystallization by evaporation is continued for from about 1 to about 50 hr. In some embodiments, the crystallization by evaporation is continued for from about 2 to about 40 hr. In some
- the crystallization by evaporation is continued for from about 2 to about 30 hr. In some embodiments, the crystallization by evaporation is continued for from about 2 to about 20 hr. In some embodiments, the crystallization by evaporation is continued for from about 2 to about 15 hr. In some embodiments, the crystallization by evaporation is continued for from about 2 to about 5 hr.
- the target yield is the maximum crystallization yield that allows for effective crystal separation, washing and drying. More specifically, low crystal yields are typically undesirable because they create losses in manufacturing capacity and efficiency. However, increases in crystal yield cause the viscosity of the product suspension to increase. A high viscosity, in turn, can create difficulty (and, in some instances, even prevent) crystal separation. Thus, in some embodiments, the target yield is the maximum yield corresponding to a viscosity allowing for cost-effective separation.
- a crystallization mass is obtained which has a crystal yield of from about 1 to about 80% with respect to 2'-FL. In some embodiments, a crystallization mass is obtained which has a crystal yield of from about 30 to about 80% with respect to 2'-FL. In some embodiments, a crystallization mass is obtained which has a crystal yield of from about 50 to about 80%) with respect to 2'-FL. In some embodiments, a crystallization mass is obtained which has a crystal yield of from about 55 to about 75% with respect to 2'-FL. In some embodiments, a crystallization mass is obtained which has a crystal yield of from about 60 to about 75%) with respect to 2'-FL.
- the crystal yields above refer to yields immediately after boiling.
- the crystal size (i.e., the largest dimension of a crystal) of the main crystal population is at least about 1 ⁇ . In some embodiments, the crystal size of the main crystal population is at least about 5 ⁇ . In some embodiments, the crystal size of the main crystal population is at least about 10 ⁇ . In some embodiments, the crystal size of the main crystal population is at least about 20 ⁇ . In some embodiments, the crystal size of the main crystal population is at least about 30 ⁇ . In some embodiments, the crystal size of the main crystal population is no greater than about 10000 ⁇ . In some embodiments, the crystal size of the main crystal population is no greater than about 5000 ⁇ . In some embodiments, the crystal size of the main crystal population is no greater than about 1000 ⁇ . In some embodiments, the crystal size of the main crystal population is no greater than about 500 ⁇ . In some
- the crystal size of the main crystal population is no greater than about 300 ⁇ . In some embodiments, the crystal size of the main crystal population is no greater than about 200 ⁇ . In some embodiments, the crystal size of the main crystal population ranges from about 1 to about 10000 ⁇ . In some embodiments, the crystal size of the main crystal population ranges from about 10 to about 500 ⁇ . In some embodiments, the crystal size of the main crystal population ranges from about 30 to about 200 ⁇ . In some embodiments, the crystal size of the main crystal population ranges from about 30 to about 100 ⁇ . In some embodiments, the crystal size of the main crystal population ranges from about 100 to about 200 ⁇ . As used in this specification, the term "main crystal population" is a visual judgement of a microscopic image from a crystallization mass. Outlier crystals that are either abnormally small or abnormally large are ignored.
- additional feed liquid is added to the 2'-FL solution simultaneously with the crystallization by evaporation to increase the level of the 2'-FL solution in the crystallizer and/or to increase the dry substance content of the 2'-FL solution.
- the additional feed liquid is added continuously.
- the additional feed liquid is added batchwise.
- the 2'-FL-containing solution is cooled simultaneously with the crystallization.
- the crystallization comprises a combined boiling- and-cooling process.
- the temperature may, for example, be decreased to a temperature of from about 10 to about 20°C less than the seeding point temperature.
- the cooling rate is from about l°C/hr to about 5°C/hr.
- the crystal mass is mixed before allowing the mass to cool.
- the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 40 to about 75°C.
- the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 40 to about 70°C.
- the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 45 to about 70°C.
- the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 50 to about 70°C.
- the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 55 to about 70°C. In some embodiments, the mixing is carried out under, for example, atmospheric pressure, at a temperature of from about 60 to about 70°C. In some embodiments, the mixing is carried out under, for example, atmospheric pressure, at a temperature of greater than 60 and no greater than about 70°C. In some embodiments, the mixing is continued for from about 0.5 to about 30 hr. [69] In some embodiments, when the crystallization by evaporation is terminated, the temperature of the crystallization mass is decreased to room temperature. In some embodiments, when the crystallization by evaporation is terminated, the temperature of the crystallization mass is decreased to about 20°C. In some embodiments, a longer cooling period is used when the - FL content is low.
- the cooling is conducted over a period of up to about 3 days. In some embodiments, the cooling is conducted over a period of from about 1 to about 3 days. In some embodiments, the cooling is conducted over a period of from about 1 to about 30 hr.
- the cooling rate is from about l°C/h to about 5°C/h.
- the apparent viscosity of the crystallization mass during the crystallization by evaporation is from about 3 to about 200 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization by evaporation is from about 5 to about 200 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization by evaporation is from about 5 to about 100 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization by evaporation is from about 50 to about 100 Pa-s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization by evaporation is from about 5 to about 50 Pa- s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization mass during the crystallization by evaporation is from about 5 to about 50 Pa- s. In some embodiments, the apparent viscosity of the crystallization mass during the crystallization mass
- crystallization by evaporation is from about 5 to about 30 Pa-s.
- a viscosity in these ranges is generally suitable for effective separation of the crystallized material. If the crystallization mass becomes too viscous for effective separation, the viscosity of the crystallization mass may be decreased by, for example, decreasing the supersaturation of the crystallization mass. The supersaturation of the crystallization mass may be decreased by, for example, raising the temperature and/or diluting the crystallization mass with water or a 2'-FL-containing solution.
- the crystals can be separated by, for example, centrifugation, filtration, decantation etc. In some embodiments, separation of the crystals comprises centrifugation.
- the crystals are typically dried by, for example, contacting them with hot air. In some embodiments, the crystals are washed following separation. In some embodiments, the crystals are washed with water and then dried by, for example, contacting them with hot air. In some embodiments, the water content of the resulting crystal fraction is less than about 1% (by weight). In some embodiments, the water content of the resulting crystal fraction is less than about 0.5% (by weight). Water content may be measured by weight loss on drying or by Karl Fisher titration.
- solid-liquid-separation is performed on the crystalline product to remove micro-impurities, such as traces of protein and DNA, amino acids, carbohydrate impurities, trace elements, etc.
- the 2'-FL content of the resulting crystal fraction of this process is at least about 85%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 90%. In some embodiments, the 2' ⁇ -FL content of the resulting crystal fraction is at least about 91%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 92%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 93%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 94%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 95%.
- the - -FL content of the resulting crystal fraction is at least about 96%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 97%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 98%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 99%. In some embodiments, the - -FL content of the resulting crystal fraction is at least about 99.5%. In some embodiments, the 2'-FL content of the resulting crystal fraction is at least about 99.9%. These percentages correspond to normalized peak area concentrations or purities obtained using UPLC.
- the melting point of the resulting crystals of this process is from about from about 230 to about 239°C. In some embodiments, the melting point of the resulting crystals of this process is from about from about 230 to about 232°C. In some embodiments, the melting point of the resulting crystals of this process is from about from about 230 to about 231°C. In some embodiments, the melting point of the resulting crystals of this process is from about from about 232 to about 239°C. In some embodiments, the melting point of the resulting crystals of this process is from about from about 232 to about 238°C. In some embodiments, the melting point of the resulting crystals of this process is from about from about 233 to about 238°C.
- the melting point of the resulting crystals of this process is from about from about 234 to about 237°C. In some embodiments, the melting point of the resulting crystals of this process is less than 236°C. In some embodiments, the melting point of the resulting crystals of this process is at least about 233°C and less than 236°C. In some embodiments, the melting point of the resulting crystals of this process is at least about 234°C and less than 236°C. In some embodiments, the melting point of the resulting crystals of this process is from about 233°C to 235°C. The above melting points correspond to a melting point determined with a l°C/min heating rate using the European Pharmacopoeia capillary melting point method.
- the resulting crystals exhibit an X-ray powder diffraction spectra discussed in International Patent Appl. Publ. No. WO201 1/150939 for the crystalline form cited in WO201 1/150939 as "crystalline 2'-0-fucosyllactose polymorph II.”
- the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ⁇ 0.20, 13.65 ⁇ 0.20 and 18.32 ⁇ 0.20 2 ⁇ angles. In some embodiments, the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ⁇ 0.20, 13.65 ⁇ 0.20, 18.32 ⁇ 0.20 and 21.70 ⁇ 0.20 2 ⁇ angles. In some embodiments, the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at
- the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ⁇ 0.20, 13.65 ⁇ 0.20, 18.32 ⁇ 0.20, 21.7Ot0.20, 15.22 ⁇ 0.20 and 20.63 ⁇ 0.20 2 ⁇ angles. In some embodiments, the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at 16.98 ⁇ 0.20, 13.65 ⁇ 0.20, 18.32 ⁇ 0.20, 21.7Ot0.20, 15.22 ⁇ 0.20 and 20.63 ⁇ 0.20 2 ⁇ angles. In some embodiments, the resulting crystals exhibit an X-ray powder diffraction reflection, based on a measurement using CuKa radiation, at
- recrystallization is performed one or more times to increase 2'-FL purity. Recrystallization may be carried out by, for example, dissolving the 2'-FL crystals in water (typically deionized water), bringing the resulting solution to a supersaturated state with respect to 2'-FL (via, for example, evaporation), and crystallizing using the crystallization-by-evaporation methodology described above.
- water typically deionized water
- bringing the resulting solution to a supersaturated state with respect to 2'-FL via, for example, evaporation
- crystallizing using the crystallization-by-evaporation methodology described above.
- yield is increased by performing crystallization of the mother liquor produced by the initial crystallization.
- Such a crystallization may be carried out by, for example, bringing the mother liquor to a supersaturated state with respect to 2'-FL (via, for example, evaporation), and crystallizing using the crystallization-by-evaporation
- the 2'-FL crystalline product of the process described in this specification is incorporated into a food (e.g., human or pet food), dietary supplement or medicine.
- the crystalline product is incorporated into a human baby food (e.g., infant formula).
- the 2'-FL is mixed with other ingredients of the food, dietary supplement or medicine.
- the 2'-FL may be mixed with other infant formula ingredients such as, for example, nonfat milk, a carbohydrate source (e.g., lactose), a protein source (e.g., whey protein concentrate), a fat source (e.g., high oleic safflower oil), vitamins, minerals, etc.
- a carbohydrate source e.g., lactose
- a protein source e.g., whey protein concentrate
- a fat source e.g., high oleic safflower oil
- the 2'-FL concentration in the infant formula is approximately the same concentration as the 2'-FL concentration generally present in human breast milk.
- this process may be used to produce an anhydrous crystalline product.
- the moisture content of the dried 2'-FL crystal product as measured by Karl Fischer titration, is less than 1.0% (by weight). In some such embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.9% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.8% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.7% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.6% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.5% (by weight).
- the moisture content of the dried 2'-FL crystal product is less than 0.4% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.3% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.2% (by weight). In some embodiments, the moisture content of the dried 2'-FL crystal product is less than 0.1% (by weight). In some embodiments, no moisture is detected in the dried 2'-FL crystal product, as measured by Karl Fischer titration.
- the 2'-FL crystalline product of the process described in this specification is converted into a different physical form for an intended application.
- the crystalline product is dissolved or suspended in water or other solvent to form a solution or suspension, which, in turn, is incorporated into (or otherwise used in the manufacture of) a food (e.g., infant formula), dietary or medicine.
- the crystalline product is dissolved in water or other solvent to form a solution, which is subsequently spray dried to form an amorphous 2'-FL composition, which, in turn, is incorporated into (or otherwise used in the manufacture of) a food (e.g., infant formula), dietary or medicine.
- the amount of the 2'-FL is an amount that is effective to convey a benefit, such as a prebiotic, antibacterial, antiviral, immune system enhancing and/or brain development enhancing effect.
- no alcohol e.g., methanol, ethanol, etc.
- no alcohol is added to the solution from which the 2'-FL is crystallized.
- no alcohol is added while the solution is being brought to supersaturation with respect to 2'-FL.
- no alcohol is added while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL.
- no alcohol is added while 2'-FL crystallization is occurring.
- the total concentration of any alcohol present in the solution while the solution is being brought to supersaturation with respect to 2'-FL is no greater than about 1% (by weight).
- the total concentration of any alcohol present in the solution while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL is no greater than 1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while 2'-FL crystallization is occurring is no greater than about 1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while the solution is being brought to supersaturation with respect to 2'-FL is no greater than about 0.1% (by weight). In some embodiments, the total concentration of any alcohol present in the solution while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL is no greater than 0.1% (by weight).
- the total concentration of any alcohol present in the solution while 2'-FL crystallization is occurring is no greater than about 0.1% (by weight).
- the solution consists essentially of no alcohol while the solution is being brought to supersaturation with respect to 2'-FL.
- the solution consists essentially of no alcohol while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL.
- the solution consists essentially of no alcohol while 2'-FL crystallization is occurring.
- the phrase "consists essentially of no alcohol” means the amount of alcohol present in the solution (if any is present at all) is insufficient to materially affect the crystallization.
- no organic acid solvent e.g., acetic acid
- no organic acid solvent is added to the solution from which the 2'-FL is crystallized.
- no organic acid solvent is added while the solution is being brought to supersaturation with respect to 2'-FL.
- no organic acid solvent is added while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL.
- no organic acid solvent is added while 2'-FL crystallization is occurring.
- the total concentration of any organic acid solvent present in the solution while the solution is being brought to supersaturation with respect to 2'-FL is no greater than about 1% (by weight).
- the total concentration of any organic acid solvent present in the solution while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL is no greater than 1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while 2'-FL crystallization is occurring is no greater than about 1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while the solution is being brought to supersaturation with respect to 2'- FL is no greater than about 0.1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL is no greater than 0.1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while 2'-FL crystallization is occurring is no greater than about 0.1% (by weight). In some embodiments, the total concentration of any organic acid solvent present in the solution while 2'-FL crystallization is occurring is no greater than about 0.1% (
- the solution consists essentially of no organic acid solvent while the solution is being brought to supersaturation with respect to 2'-FL. In some embodiments, the solution consists essentially of no organic acid solvent while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL. In some embodiments, the solution consists essentially of no organic acid solvent while 2'-FL crystallization is occurring.
- the phrase "consists essentially of no organic acid solvent” means the amount of organic acid solvent present in the solution (if any is present at all) is insufficient to materially affect the
- no organic solvent is added to the solution from which the 2'-FL is crystallized. In some embodiments, no organic solvent is added while the solution is being brought to supersaturation with respect to 2'-FL. In some embodiments, no organic solvent is added while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL. In some embodiments, no organic solvent is added while 2'-FL crystallization is occurring. In some embodiments, the total concentration of any organic solvent present in the solution while the solution is being brought to supersaturation with respect to 2'-FL is no greater than about 1% (by weight).
- the total concentration of any organic solvent present in the solution while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL is no greater than 1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while 2'-FL crystallization is occurring is no greater than about 1% (by weight). In some embodiments, the total concentration of any organic solvent present in the solution while the solution is being brought to
- the solution consists essentially of no organic solvent while the solution is being brought to supersaturation with respect to 2'-FL.
- the solution consists essentially of no organic solvent while evaporation is being used to bring the solution to supersaturation with respect to 2'-FL.
- the solution consists essentially of no organic solvent while 2'-FL crystallization is occurring.
- the phrase "consists essentially of no organic solvent” means the amount of organic solvent present in the solution (if any is present at all) is insufficient to materially affect the
- a value identified by "[1]” refers to normalized peak area purity or concentration obtained using HPLC (CarboPac PA100, Thermo Fisher Scientific, Waltham, MA USA). This is a percentage of peak area relative to the total area of peaks.
- a value identified by "[2]” refers to 2'-FL yield calculated from the purity values.
- a value identified by “[3]” refers to dry solid content ("DS") as measured by
- a value identified by "[4]” refers to dry solid content calculated from a mass balance based on the initial dry solid content measured by using Karl Fischer titration and the amount of water evaporated or added.
- a value identified by "[5]” refers to moisture content measured by weight loss on drying.
- a value identified by "[6]” refers to a crystal size measurement (i.e., measurement of the largest dimension of a crystal) made from a microscopic image. Outlier crystals that are either abnormally small or abnormally large are ignored.
- a value identified by "[7]” refers to a sugar color under the International Commission for Uniform Process of Sugar Analysis (“ICUMSA”) sugar color grading system.
- a value identified by "[8]” refers to a supersaturation with respect to 2'-FL defined as a ratio between 2'-FL concentration in solution (g / 100 g water) and solubility of 2'- FL in water (g / 100 g water) at the same temperature.
- a value identified by "[9]" is the melting point measured with a l°C/min heating rate using the European Pharmacopoeia capillary melting point method.
- a value identified by “[10]” refers to normalized peak area purity or
- HPLC high performance liquid chromatography
- the crystal mass was centrifuged with a batch-wise centrifuge having 22.5 cm basket diameter. For this centrifugation, 343 g of crystal mass was loaded onto the centrifuge at the acceleration stage at a 500 rpm rotation speed. Wash water (75 mL) was added at a 2000 rpm rotation speed, and then centrifugation was continued for 5 min at a rotation speed of 3500 rpm. The resulting centrifuged crystals (85 g) were dried in a heating chamber at 50°C for 3.4 hr. The moisture content after drying was 0.8% [3].
- Crystals prepared in accordance with the process illustrated here were used in Example 2. Before being used, the crystals were washed with ethanol, dried and then grinded in a porcelain mill to break the crystal structure and provide irregular shaped particles to initiate secondary nucleation. The washed crystals had a 2'-FL purity of 94.0% [1].
- Example 1 at a temperature of 55°C.
- the seeded syrup was stirred while being maintained at 55°C. Ethanol was added in two portions: the first portion (610 g) was added 0.9 hr after seeding within 0.3 hr, and the second portion (221 g) was added 2.3 hr after seeding.
- the crystallizing solution was then cooled from 55°C to 15°C within 17 hr while stirring. After cooling to 15°C, the stirring was continued at 15°C for 0.9 hr.
- the resulting crystal mass (1416 g) was centrifuged with 100 mL wash water (batchwise centrifuge, basket diameter 22.5 cm, 3500 rpm, 5 min). A total of 557 g of crystal cake was obtained. Of this, 241 g were dried in a heating chamber at 60°C for 2.3 hr. The moisture content after drying was 0.8 % [3].
- Crystals prepared in accordance with this process were, in turn, used as seed crystals in Example 3 below. Before being used, the crystals were washed with ethanol, dried and then grinded in a porcelain mill. The washed crystals had a 2'-FL purity of 91.6% [1].
- the crystal mass was diluted to a DS of 79.5% [4] by adding deionized water, and then centrifuged with a batch-wise centrifuge having 22.5 cm basket diameter. Here, 693 g of crystal mass was loaded onto the standing centrifuge. Wash water (50 mL) was then added during the acceleration stage at the rotation speed of 2000 rpm. The centrifugation time was continued for 5 min at a rotation speed of 3500 rpm. The 2'-FL centrifugation yield was 54% [2]. The mother liquor 2'-FL purity was 79.5% [1], and the moisture content of the non-dried cake 5.7% [5].
- Crystal cake from the centrifugation (274 g) was dried in a heating chamber at 60°C for 2 hr and then cooled in a desiccator.
- the dried crystals had a 2'-FL purity of 100.0%) [1], melting point of 236.8 - 237.5°C [9], color corresponding to 22 ICUMSA [7] and moisture content of 0.3% [3].
- centrifugation of a small sample of the mass without washing (Hettich Rotanta 460R centrifuge, 2500 rpm, 10 min) provided a crystalline cake having a 2'-FL purity of 95.7% [1] and a mother liquor having a 2'-FL purity of 51.5%) [1]. This corresponds to a 76 % 2'-FL yield [2].
- the crystal size of the main crystal population was 40-100 ⁇ [6].
- the crystal mass (165 g) was centrifuged with a batch-wise centrifuge without washing (basket diameter 22.5 cm, 3500 rpm, 3 min). The centrifugation 2'-FL yield was 76% [2].
- the centrifuged and dried (60°C, 2.4 hr) crystals had a 2'-FL purity of 93.2% [1] with a color of 157 ICUMSA [7] and moisture content of 0.5% [3].
- the mother liquor 2'-FL purity was 53.9% [1] with a color of 609 ICUMSA [7].
- Example 6 2'-FL Crystallization from Centrifugation Mother Liquor of Example 5
- the syrup was diluted to a DS of 72.6% [4] and kept at 67°C without vacuum for 1 hr. Boiling of the syrup was then continued at a temperature of 65-67°C and pressure of 200 mbar for 5.2 hr while simultaneously adding small portions of deionized water (around 500 g in total) to the mass.
- the crystal mass (371 g) was centrifuged with 30 mL wash water (batchwise centrifuge, basket diameter 22.5 cm, 3500 rpm, 5 min).
- the 2'-FL centrifugation yield was 54% [2].
- the 2'-FL purity of the centrifuged and dried (60°C, 2 hr) crystals was 93.4% [1].
- the color was 100 ICUMSA [7] and the moisture content was 0.9% [3].
- the mother liquor 2'-FL purity was 65.4% [1] and the color was 287 ICUMSA [7].
- the seeded syrup was evaporated to a DS of 80.4% [3] within 5.3 hr in three steps such that, in each step, the syrup was first boiled under vacuum for 0.9 - 1.2 hr (67 - 72°C and 190 - 200 mbar), followed by mixing at atmospheric pressure for 0.6 - 0.9 hr (67 - 72°C). The resulting crystal mass was moved to a 6 L mixing tank and kept at 69°C overnight (19 hr). Progression of crystallization was monitored by centrifuging (Hettich Rotanta 460R centrifuge, 2500 rpm, 10 min) small samples of the mass without washing. The results are summarized in Table 1.
- the moisture content of the combined, non-dried cake was 4.4% [5].
- the 2'- FL purity of the centrifuged and dried (60°C, 2 hr) crystals was 100.0%) [1].
- the color was 462 ICUMSA [7] and the moisture content was 0.9%> [3].
- the crystal size of the main crystal population was 30-100 ⁇ [6].
- the DS of the resulting crystal mass was 80.8%> [3]. Centrifugation of a small sample of the mass without washing (Hettich Rotanta 460R centrifuge, 2500 rpm, 10 min) provided a crystalline cake 2'-FL having a purity of 96.5% [1] and a mother liquor 2'-FL having a purity of 76.7% [1]. This corresponds to a 60% 2'-FL yield [2].
- the crystal mass was kept in rotating Rotavapor bottle at a temperature of 68°C overnight (18 hr). After 23 hr from seeding, centrifugation of a small sample of the mass without washing (Hettich Rotanta 460R centrifuge, 2500 rpm, 10 min) provided a crystalline cake having a 2'-FL purity of 97.3% [1] and a mother liquor 2'-FL purity of 69.7% [1]. This corresponds to a 71% 2'-FL yield [2].
- the crystal size of the main crystal population was 30- 80 ⁇ [6].
- Centrifugation of a small sample of the mass without washing (Hettich Rotanta 460R centrifuge, 2500 rpm, 10 min) provided a crystalline cake having a 2'-FL purity of 95.7% [1] and a mother liquor 2'-FL purity of 61.8% [1]. This corresponds to 71% 2'-FL yield [2].
- the moisture content of the combined, non-dried cake was 3.4% [5].
- the 2'-FL purity of the centrifuged and dried (60°C, 2 hr) crystals was 100.0% [1].
- the color was 35 ICUMSA [7] and the moisture content was 0.2% [3].
- the syrup for seeding mass was seeded with 0.5 g of 2'-FL dry seed (crystals from Example 3) at a temperature of 68°C (supersaturation of 1.32 [8]).
- the seeded syrup was boiled at a temperature of 65-68°C and pressure of 190 mbar for 5 hr.
- the obtained seeding mass which had a DS of 84.5%> [3], was kept in a rotating Rotavapor bottle at a temperature of 55°C overnight (19 hr).
- the crystal mass was centrifuged in 4 batches with wash water in an amount equal to 70-87 mL/kg mass DS (batchwise centrifuge, basket diameter 22.5 cm, 3500 rpm, 10 min).
- the average 2'-FL centrifugation yield was 55%> [2].
- the average 2'-FL purity of the centrifuged and dried (18 hr at 40°C and 1 hr at 60°C) crystals was 97.9% [1].
- the color was 204 ICUMSA [7] and the moisture content was 0.8%> [3].
- the mother liquor 2'-FL purity was 59.4% [1] and the color was 1409 ICUMSA [7].
- the crystal mass was centrifuged with wash water in an amount equal to 71 mL/kg mass DS (batchwise centrifuge, basket diameter 22.5 cm, 3500 rpm, 5 min). The moisture content of the combined, non-dried cake was 5.8% [5]. The 2'-FL purity of the centrifuged and dried (21 hr at 40°C and 1 hr at 60°C) crystals was 100.0%) [1]. The color was 142 ICUMSA [7] and the moisture content was 0.1% [3].
- the crystal mass was centrifuged with wash water in an amount equal to 75 mL/kg mass DS (batchwise centrifuge, basket diameter 22.5 cm, 3500 rpm, 5 min).
- the moisture content of the combined, non-dried cake was 4.6%> [5].
- the 2'-FL purity of the centrifuged and dried (19 hr at 40°C and 1 hr at 60°C) crystals was 100.0% [1].
- the melting point was 233.2 - 234.1°C [9]
- the color was 47 ICUMSA [7]
- the moisture content was 0.1% [3].
- the resulting crystal mass was kept mixing at 75 °C for about 6 hr, and then centrifuged with wash water in an amount equal to about 60 mL/kg mass DS.
- the average 2'-FL centrifugation yield was 17.2%, and the average 2'-FL purity of the centrifuged crystals was 97.4% [10].
- the drying process lasted for 457 minutes.
- Moisture content (by weight) 2.12-2.21%
Abstract
Description
Claims
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CN201880029649.2A CN110621685A (en) | 2017-03-06 | 2018-03-01 | Methods and related compositions for crystallizing 2' -fucosyllactose |
AU2018230564A AU2018230564A1 (en) | 2017-03-06 | 2018-03-01 | Process for crystallizing 2'-fucosyllactose and related compositions |
BR112019018410A BR112019018410A2 (en) | 2017-03-06 | 2018-03-01 | process for crystallizing 2'-fucosyl-lactose and related compositions |
EP18764563.5A EP3596095A4 (en) | 2017-03-06 | 2018-03-01 | Process for crystallizing 2'-fucosyllactose and related compositions |
SG11201907911PA SG11201907911PA (en) | 2017-03-06 | 2018-03-01 | Process for crystallizing 2'-fucosyllactose and related compositions |
JP2019548667A JP2020509073A (en) | 2017-03-06 | 2018-03-01 | Process for crystallizing 2'-fucosyllactose and related compositions |
KR1020197028768A KR20190126350A (en) | 2017-03-06 | 2018-03-01 | 2'-fucosyllactose crystallization method and related composition |
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Cited By (9)
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WO2020079114A1 (en) | 2018-10-18 | 2020-04-23 | Basf Se | Method for obtaining crystalline 2'-fucosyllactose |
EP3686210A1 (en) | 2019-01-24 | 2020-07-29 | DuPont Nutrition Biosciences ApS | Process for purifying a human milk oligosaccharide and related compositions |
EP3822282A1 (en) | 2019-11-18 | 2021-05-19 | DuPont Nutrition Biosciences ApS | Process for removing an antifoam agent from a solution comprising a human milk oligosaccharide and related compositions |
WO2022072333A1 (en) | 2020-09-29 | 2022-04-07 | Dupont Nutrition Biosciences Aps | Process for purifying a human milk oligosaccharide and related compositions |
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