Crystallization of 2’-FL

Abstract

The present invention relates to a method for selectively crystallizing 2'-FL from an aqueous solution containing 2'-FL and one or more other fucosylated carbohydrates by adding acetic acid to the solution.

Description

Technical Field

[0001] The present invention relates to a method for crystallizing 2'-O-fucosyllactose (2'-FL) from an aqueous solution, in particular an aqueous solution obtained from the biotechnological production of 2'-FL, using acetic acid. Background Art

[0002] Crystallization or recrystallization is one of the simplest and cheapest methods for separating a product from a reaction mixture, separating it from contaminants / impurities, and obtaining a pure substance. Separation or purification using crystallization makes the entire technical process more robust and cost-effective, and thus has advantages.

[0003] 2'-FL can exist in different crystalline forms, see for example WO 2011 / 150939 (polymorphs I and II), WO 2014 / 009921 (polymorphs A, B, and C), or WO 2014 / 069625. In this regard, the crystallization of 2'-FL can advantageously form part of its production, especially during its purification and separation from the aqueous medium in which it is produced. WO 2014 / 086373 discloses the crystallization of 2'-FL from a freeze-dried powder derived from an aqueous fermentation broth using methanol. WO 2015 / 188834 discloses a method for selectively crystallizing polymorph II of 2'-FL from an aqueous solution containing 2'-FL and fucosylated carbohydrates other than 2'-FL (preferably DFL) by adding one or more C1-C4 alcohols (preferably methanol) to the solution. WO2016 / 095924 discloses an alternative method for selectively crystallizing polymorph II of 2'-FL from an aqueous solution containing 2'-FL and one or more other fucosylated carbohydrates by adding acetic acid to the solution. The latter method provides crystalline 2'-FL that is particularly suitable for dietary / food / infant formula applications. WO 2018 / 164937 claims a method for preparing crystalline 2'-FL by evaporation crystallization from a supersaturated aqueous solution containing no more than 1% (by weight) of an organic solvent.

[0004] For crystalline 2'-FL, there is still a need for an improved crystallization method that consumes a smaller amount of antisolvent and / or produces crystals with improved properties, such as a stable crystal morphology, better filterability, lower levels of moisture / volatiles, faster powder drying, and / or better dry powder physical and rheological properties, while maintaining or even exceeding the good yields and purities provided by prior art methods. Summary of the Invention

[0005] A first aspect of the present invention relates to a method for crystallizing 2'-FL (advantageously polymorph II of 2'-FL), which comprises:

[0006] 1) Provide an aqueous solution or syrup containing 2’-FL,

[0007] 2) Add a suspension of crystalline 2’-FL (advantageously polymorph II of 2’-FL) in acetic acid to the aqueous solution or syrup containing 2’-FL, thereby producing a slurry,

[0008] 3) Add acetic acid to the slurry to obtain a crystalline substance, and

[0009] 4) Filter the crystalline 2’-FL, advantageously polymorph II of 2’-FL, from the crystalline substance.

[0010] A second aspect of the invention relates to crystalline 2’-FL, advantageously polymorph II of 2’-FL, which is obtained or obtainable according to the first aspect of the invention.

[0011] A third aspect of the invention relates to crystalline 2’-FL, advantageously polymorph II of 2’-FL, which has the following determinations determined by a combination of quantitative measurements: 2’-FL: at least 97%, DFL: less than 2%, acetic acid: less than 0.5%.

[0012] A fourth aspect of the invention relates to a nutritional or pharmaceutical composition comprising crystalline 2’-FL according to the second or third aspect of the invention, advantageously polymorph II of 2’-FL.

[0013] A fifth aspect of the invention relates to the use of crystalline 2’-FL (advantageously polymorph II of 2’-FL) according to the second or third aspect of the invention in the preparation of a food composition or a food supplement.

[0014] A sixth aspect of the invention relates to the use of crystalline 2’-FL (advantageously polymorph II of 2’-FL) according to the second or third aspect of the invention as a food or a food supplement. Detailed Description

[0015] For the crystallization of 2'-FL polymorph II, it is preferred to crystallize from an aqueous solution obtained from the microbial fermentation production of 2'-FL after one or more purification steps of the fermentation medium, and the aqueous solution contains, in addition to 2'-FL, by-products of one or more other fucosylated carbohydrate types, such as difucosyllactose (DFL) for example. The solvent-antisolvent type crystallization method from an aqueous acetic acid solution (see WO 2016 / 095924) has proven to be technically advantageous and can provide crystalline 2'-FL with good purity, which is suitable and approved for use in nutritional compositions and infant formulas. The crystalline 2'-FL polymorph II obtainable by the method disclosed in WO 2016 / 095924 usually contains a residual acetic acid content of less than 1%, preferably not more than 0.5%, which does not represent any safety or health issues. However, in order to obtain crystalline 2'-FL with high yield and good purity, cca. 5-7 liters of acetic acid are used per kilogram of 2'-FL in the aqueous solution during the crystallization process.

[0016] The inventors have strived to reduce the amount of acetic acid used for crystallization while maintaining the good crystallization yield and purity of 2'-FL provided by the original method, and surprisingly found that the crystals thus produced have beneficial properties and crystal quality.

[0017] Accordingly, there is provided a method for crystallizing 2'-FL (advantageously 2'-FL polymorph II), which comprises:

[0018] 1) providing an aqueous solution or syrup containing 2'-FL,

[0019] 2) adding a suspension of crystalline 2'-FL (advantageously 2'-FL polymorph II) in acetic acid to the aqueous solution or syrup containing 2'-FL, thereby producing a slurry,

[0020] 3) adding acetic acid to the slurry to obtain a crystalline mass, and

[0021] 4) filtering crystalline 2'-FL, advantageously 2'-FL polymorph II, from the crystalline mass.

[0022] 2’-FL Polymorph II refers to the crystalline variant of 2’-FL disclosed in WO 2011 / 150939, which, based on measurements using CuKα radiation, contains X-ray powder diffraction reflections at 16.98±0.20, 13.65±0.20 and 18.32±0.20 2θ angles, more preferably at 16.98±0.20, 13.65±0.20, 18.32±0.20 and 21.70±0.20 2θ angles, even more preferably at 16.98±0.20, 13.65±0.20, 18.32±0.20, 21.70±0.20 and 15.22±0.20 2θ angles, most preferably at 16.98±0.20, 13.65±0.20, 18.32±0.20, 21.70±0.20, 15.22±0.20 and 20.63±0.20 2θ angles, especially at 16.98±0.20, 13.65±0.20, 18.32±0.20, 21.70±0.20, 15.22±0.20, 20.63±0.20 and 11.94±0.20 2θ angles.

[0023] The above method provides crystalline 2’-FL, preferably 2’-FL Polymorph II, which has beneficial characteristics compared to the 2’-FL crystalline materials known in the prior art. In addition, the claimed crystallization method is characterized by a lower consumption of anti-solvent (acetic acid) (meaning a more economical process), while the crystallization yield and crystal purity (usually measured by HPLC) are not reduced.

[0024] The first step of the method is to provide an aqueous solution or syrup containing 2’-FL. The aqueous solution or syrup does not contain organic solvents and is preferably homogeneous. If it is an aqueous solution, it does not need to be a supersaturated solution of 2’-FL. A supersaturated solution of 2’-FL is a solution that contains more than the maximum amount of 2’-FL that can be dissolved at a given temperature. The “maximum amount of 2’-FL that can be dissolved at a given temperature” is actually the solubility of 2’-FL, which is a thermochemical property referring to the ability of 2’-FL to dissolve in a solvent. It is measured based on the maximum amount of solute dissolved in the solvent at equilibrium (here in water). The resulting solution is called a saturated solution. The solubility of 2’-FL in water can be measured, for example, as disclosed in Example 15 of WO 2018 / 164937, which is incorporated herein by reference. The concentration of 2’-FL in the aqueous solution or syrup is at least 40 wt%, preferably at least 45 wt%, more preferably at least 50 wt%, even more preferably at least 53 wt%, such as 53 - 68 wt%, 55 - 61 wt%, 58 - 63 wt% or 57 - 61 wt% (determined by HPLC).

[0025] In one embodiment, an aqueous solution or syrup containing 2’-FL is provided at an elevated temperature (above room temperature) or heated to an elevated temperature before the next step. The elevated temperature is at least 35 °C, preferably at least 45 °C, for example about 45 - 55 °C or 50 ± 2 °C.

[0026] The aqueous solution or syrup containing 2’-FL disclosed above may contain one or more other carbohydrates or carbohydrate derivatives, depending on how the 2’-FL was previously produced. 2’-FL can be prepared by total chemical synthesis from simpler carbohydrate precursors, which synthesis includes the chemical fucosylation of a suitably protected lactose acceptor; such methods are disclosed, for example, in WO 2010 / 070616, WO 2010 / 115934, WO 2010 / 115935, WO 2014 / 009921, WO2015 / 032413, WO 2016 / 038192, WO 2017 / 134176 or WO 2017 / 153452. In addition to 2’-FL, the carbohydrate derivatives contaminating 2’-FL are typically intermediate derivatives in the disclosed 2’-FL synthetic routes, especially the final intermediate prior to complete deprotection to 2’-FL. Such a final intermediate is a fully or partially protected 2’-FL derivative, where the protecting group can be an acyl group (mainly acetyl or benzoyl), an optionally substituted benzyl group, an acetal or ketal (mainly isopropylidene or benzylidene) and / or a silyl group. In the fermentative production of 2’-FL (see, for example, Drouillard et al. Angew.Chem.Int.Ed. 45, 1778 (2006), WO 2012 / 097950, WO 2012 / 112777, WO 2015 / 032412, WO 2015 / 188834, WO 2016 / 095924), typical carbohydrate by-products may contain fucosylated carbohydrates other than 2’-FL and / or lactose and / or lactulose and / or fucose and / or glucose and / or sucrose and / or galactose and / or FLU (2’-O-fucosyllactose) and / or FFL (Fuc(α1-2)Fuc(α1-2)Gal(β1-4)Glc). The fucosylated carbohydrates other than 2’-FL can be any other monofucosyllactose formed during fermentation due to lack, defect or impaired fucosylation other than α-1,2-fucosylation on the galactose moiety of lactose (e.g., one that results in 3-O-fucosyllactose, 3-FL), or fucose migration on 2’-FL under culture conditions or post-fermentation manipulations, or hydrolysis of fucose from a polyfucosylated (preferably difucosylated) lactose; or can be a polyfucosylated (preferably difucosylated) lactose formed due to over-fucosylation of lactose under culture conditions. The difucosylated lactose is preferably 2,2’-di-O-fucosyllactose or 2’,3-di-O-fucosyllactose (DFL), especially DFL as a characteristic by-product formed in the fermentative production of 2’-FL.Other carbohydrate contaminants that may form during fermentation or during purification / separation steps after fermentation are FLU or lactulose (by rearrangement), fucose, glucose, galactose or lactose (due to hydrolysis of the product and intermediates), glucose, lactose or sucrose (as unconsumed isolates or raw materials added during fermentation).

[0027] As disclosed in the experimental section, the total solids content of the aqueous solution or syrup according to step 1), measured with a refractometer, is at least 57 °Bx (Brix), for example 57 - 80, 60 - 80, 65 - 80, 70 - 80, 65 - 75 or 70 - 75 °Bx. It refers to any solute dissolved in the aqueous solution, i.e., it includes substances other than 2’-FL and its accompanying carbohydrate derivatives as disclosed above, such as salts.

[0028] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 67 - 77 °Bx and a dissolved 2’-FL of 52 - 62 wt%, as estimated by HPLC.

[0029] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 70 - 75 °Bx and a dissolved 2’-FL of 56 - 62 wt%, as estimated by HPLC.

[0030] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 67 - 71 °Bx and a dissolved 2’-FL of 52 - 56 wt%, as estimated by HPLC.

[0031] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 67 - 77 °Bx and a dissolved 2’-FL of 62 - 70 wt%, for example 64 - 68 wt%, as estimated by HPLC.

[0032] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 70 - 75 °Bx and a dissolved 2’-FL of 62 - 70 wt%, for example 64 - 68 wt%, as estimated by HPLC.

[0033] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL has a total solids content of 67 - 71 °Bx and a dissolved 2’-FL of 62 - 70 wt%, for example 64 - 68 wt%, as estimated by HPLC.

[0034] In one embodiment of step 1), the aqueous solution or syrup containing 2’-FL contains DFL and optionally lactose as accompanying carbohydrates.

[0035] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL, where the weight ratio of 2'-FL:DFL is greater than 2, preferably greater than 4, more preferably greater than 6, especially between 8 and 13.

[0036] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and lactose, where the weight ratio of 2'-FL:lactose is 10 - 110, and the weight ratio of DFL:lactose is 1 - 10.

[0037] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and lactose, where the weight ratio of 2'-FL:DFL is greater than 6, preferably 8 - 13, the weight ratio of 2'-FL:lactose is 10 - 110, and the weight ratio of DFL:lactose is 1 - 10.

[0038] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and lactose, where the weight ratio of 2'-FL:DFL is greater than 13, preferably greater than 25, such as greater than 50, and the weight ratio of DFL:lactose is less than 1.

[0039] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 75 °Bx and a dissolved 2'-FL of 52 - 62 wt%, as estimated by HPLC, and where the weight ratio of 2'-FL:DFL is 8 - 13.

[0040] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70 - 75 °Bx and a dissolved 2'-FL of 56 - 62 wt%, as estimated by HPLC, where the weight ratio of 2'-FL:DFL is greater than 6, preferably 8 - 13, the weight ratio of 2'-FL:lactose is 10 - 110, and the weight ratio of DFL:lactose is 1 - 10.

[0041] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 71 °Bx and a dissolved 2'-FL of 52 - 56 wt%, as estimated by HPLC, where the weight ratio of 2'-FL:DFL is greater than 6, preferably 8 - 13, the weight ratio of 2'-FL:lactose is 20 - 50, and the weight ratio of DFL:lactose is 2 - 6.

[0042] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67-77 °Bx and 62-70 wt%, for example 64-68 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, and the DFL:lactose weight ratio is less than 1.

[0043] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70-75 °Bx and 62-70 wt%, for example 64-68 wt% of dissolved 2'-FL as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, and the DFL:lactose weight ratio is less than 1.

[0044] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67-71 °Bx and 62-70 wt%, for example 64-68 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, and the DFL:lactose weight ratio is less than 1.

[0045] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67-75 °Bx and 52-62 wt% of dissolved 2'-FL, as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0046] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70-75 °Bx and 56-62 wt% of dissolved 2'-FL, as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0047] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67-71 °Bx and 52-56 wt% of dissolved 2'-FL, as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0048] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67-77 °Bx and 62-70 wt%, for example 64-68 wt% of dissolved 2'-FL, as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0049] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70 - 75 °Bx and 62 - 70 wt%, for example 64 - 68 wt% of dissolved 2'-FL as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0050] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 71 °Bx and 62 - 70 wt%, for example 64 - 68 wt% of dissolved 2'-FL, as estimated by HPLC, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0051] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and optionally lactose as accompanying carbohydrates, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0052] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL, wherein the 2'-FL:DFL weight ratio is greater than 2, preferably greater than 4, more preferably greater than 6, especially between 8 - 13, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0053] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and lactose, wherein the 2'-FL:lactose weight ratio is 10 - 110, the DFL:lactose weight ratio is 1 - 10, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0054] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL contains DFL and lactose, wherein the weight ratio of 2'-FL:DFL is greater than 6, preferably 8 - 13, the 2'-FL:lactose weight ratio is 10 - 110, the DFL:lactose weight ratio is 1 - 10, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0055] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 75 °Bx and 52 - 62 wt% of dissolved 2'-FL as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is 8 - 13, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0056] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70 - 75 °Bx and 56 - 62 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 6, preferably 8 - 13, the 2'-FL:lactose weight ratio is 10 - 110, the DFL:lactose weight ratio is 1 - 10, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0057] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 71 °Bx and 52 - 56 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 6, preferably 8 - 13, the 2'-FL:lactose weight ratio is 20 - 50, the DFL:lactose weight ratio is 2 - 6, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0058] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 77 °Bx and 62 - 70 wt%, for example 64 - 68 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, the DFL:lactose weight ratio is less than 1, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0059] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 70 - 75 °Bx and 62 - 70 wt%, for example 64 - 68 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, the DFL:lactose weight ratio is less than 1, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0060] In one embodiment of step 1), the aqueous solution or syrup containing 2'-FL has a total solids content of 67 - 71 °Bx and 62 - 70 wt%, for example 64 - 68 wt% of dissolved 2'-FL, as estimated by HPLC, wherein the 2'-FL:DFL weight ratio is greater than 13, preferably greater than 25, for example greater than 50, the DFL:lactose weight ratio is less than 1, and is provided at at least 45 °C, preferably at 50 ± 2 °C.

[0061] In the second step, to initiate crystallization, at an elevated temperature, preferably at the same temperature as in step 1), and preferably with continuous stirring, a suspension of crystalline 2'-FL (preferably 2'-FL polymorph II) in acetic acid is added to the aqueous solution or syrup containing 2'-FL according to step 1) to form a slurry. After adding the suspension of crystalline 2'-FL (preferably 2'-FL polymorph II) in acetic acid, the amount of acetic acid in the slurry should be greater than 1 wt%, preferably greater than 1.5 wt%, more preferably greater than 2 wt%, such as between 2 to 4 wt% or 2 to 3 wt%. The suspension of crystalline 2'-FL (preferably 2'-FL polymorph II) in acetic acid to be added to the aqueous solution or syrup containing 2'-FL provided in step 1) is about 10 - 30 wt% (i.e., 100 g of the suspension contains about 10 - 30 g of crystalline 2'-FL, preferably 2'-FL polymorph II). In addition, the amount of seed crystals is suitable for forming the slurry. Preferably, relative to the total solid content of the aqueous solution or syrup provided in step 1), the amount of seed crystals is at least 0.5 wt%, such as 0.5 - 2 wt%, preferably 0.9 - 1.2 wt%. The acetic acid applicable to prepare the above suspension is glacial acetic acid.

[0062] Generally, the 2'-FL / acetic acid suspension is added immediately to the aqueous solution or syrup containing 2'-FL, preferably with stirring. After seeding, the mixture is stirred at the same temperature for several hours, such as 1 - 8 or 3 - 7 hours, preferably 4 - 6 hours, such as about 5 hours, to form a slurry.

[0063] In the above step of initiating crystallization, no vacuum or reduced pressure is applied.

[0064] It is advantageous to use the 2'-FL / acetic acid suspension to seed the aqueous solution or syrup containing 2'-FL because it represents a different nucleation mechanism, as opposed to, for example, the evaporation crystallization or simple addition of seed crystals disclosed in WO 2018 / 164937, thus affecting the morphology of the resulting crystalline material.

[0065] In one embodiment of step 2), the amount of 2'-FL for seeding is 0.5 - 2 wt% of the solid content of the aqueous solution or syrup provided in step 1), the 2'-FL / acetic acid suspension is 10 - 30 wt%, and the amount of acetic acid in the slurry after adding the suspension is 2 - 3 wt%.

[0066] In one embodiment of step 2), the amount of 2'-FL for seeding is 0.9 - 1.2 wt% of the solid content of the aqueous solution or syrup provided in step 1), the 2'-FL / acetic acid suspension is 10 - 30 wt%, and the amount of acetic acid in the slurry after adding the suspension is 2 - 3 wt%.

[0067] In one embodiment of step 2), the amount of 2’-FL inoculated is 0.5-2 wt% of the solid content of the aqueous solution or syrup provided in step 1), the 2’-FL / acetic acid suspension is 24±2 wt%, and the amount of acetic acid in the slurry after adding the suspension is 2-3 wt%.

[0068] In one embodiment of step 2), the amount of 2’-FL inoculated is 0.9-1.2 wt% of the solid content of the aqueous solution or syrup provided in step 1), the 2’-FL / acetic acid suspension is 24±2 wt%, and the amount of acetic acid in the slurry after adding the suspension is 2-3 wt%.

[0069] In one embodiment of step 2), the stirring in the case of adding the 2’-FL / acetic acid suspension and / or the stirring of the slurry after adding the 2’-FL / acetic acid suspension is carried out at 150-300 rpm.

[0070] In one embodiment of step 2), after adding the 2’-FL / acetic acid suspension, the slurry is stirred at the same temperature as that for the previous inoculation for at least 2-5 hours.

[0071] In the next step 3), after step 2), pure acetic acid is added to the slurry obtained above under continuous stirring. In one embodiment, the temperature at which the pure acetic acid is added to the slurry is the same as the temperature in the previous step. In other embodiments, the temperature is lower than the temperature in step 2). In a preferred embodiment, the slurry is first cooled, usually within about 5-60 minutes, or allowed to cool itself to the desired temperature, then pure acetic acid is added, and this temperature is maintained throughout the addition of pure acetic acid. This temperature is preferably about 30-40 °C, such as about 35±2 °C. Calculate the amount of acetic acid added in step 3) such that the weight fraction of acetic acid to water in the crystal suspension at the end of adding acetic acid in step 3) is 4-6, preferably 4.6-6 or 4.6-5.5. In the weight fraction of acetic acid to water, the amount of acetic acid should include the acetic acid added in step 3) and the acetic acid constituting the 2’-FL / acetic acid suspension used for inoculation in step 2). The water content of the suspension at the end of step 3) is actually the amount of water in the aqueous solution or syrup containing 2’-FL provided in step 1), and can be calculated as follows: water wt% = 100 - Brix [the amount of the aqueous solution or syrup containing 2’-FL provided in step 1].

[0072] Pure acetic acid can be continuously added in step 3) at a given addition rate, or in several portions, preferably in equal portions. The calculated amount of acetic acid is added relatively slowly, within several hours, but not less than 3 hours, such as not less than 5, 7 or 9 hours, preferably not less than 10 hours, more preferably 12±2 hours. At the end of the addition of acetic acid, the slurry becomes a crystalline substance.

[0073] In the above step of adding acetic acid, no vacuum or reduced pressure is applied.

[0074] Optionally, after adding pure acetic acid according to step 3) above and obtaining the crystalline substance of 2’-FL, if necessary, the final temperature is reduced to between 0 °C and room temperature within several hours, preferably at about 20 - 25 °C. At this final temperature, the crystalline substance of 2’-FL is stirred for several hours, such as at least 2 or 3 hours, such as 5 ± 1 hour. In the step of equilibration of the crystalline substance of 2’-FL, no vacuum or reduced pressure is applied.

[0075] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over at least 3 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4 - 6.

[0076] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over at least 10 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4 - 6.

[0077] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over 12 ± 2 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4 - 6.

[0078] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over at least 3 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4.6 - 5.5.

[0079] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over at least 10 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4.6 - 5.5.

[0080] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) over 12 ± 2 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4.6 - 5.5.

[0081] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C over at least 3 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4 - 6.

[0082] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C over at least 10 hours to achieve a weight fraction of acetic acid to water in the crystalline substance of 4 - 6.

[0083] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C for 12 ± 2 hours to achieve a weight fraction of acetic acid to water in the crystalline material of 4 - 6.

[0084] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C for at least 3 hours to achieve a weight fraction of acetic acid to water in the crystalline material of 4.6 - 5.5.

[0085] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C for at least 10 hours to achieve a weight fraction of acetic acid to water in the crystalline material of 4.6 - 5.5.

[0086] In one embodiment of step 3), acetic acid is added to the slurry obtained in step 2) at 35 ± 2 °C for 12 ± 2 hours to achieve a weight fraction of acetic acid to water in the crystalline material of 4.6 - 5.5.

[0087] In step 3), any of the specific embodiments disclosed above optionally follow:

[0088] - Cooling the obtained crystalline material to 0 - 25 °C, preferably 20 - 25 °C,

[0089] - Stirring the obtained crystalline material for at least 2 - 3 hours, preferably 5 ± 1 hour, or

[0090] - Cooling the obtained crystalline material to 0 - 25 °C, preferably 20 - 25 °C, and then stirring it for at least 2 - 3 hours, preferably 5 ± 1 hour.

[0091] Then, in step 4), the 2’-FL crystalline material obtained in step 3) or the above optionally equilibrated crystalline material is filtered. The crystalline 2’-FL is separated from the mother liquor by conventional methods, such as dead-end filtration, centrifugation, or decantation at a temperature between 0 °C and room temperature, preferably at about 20 - 25 °C. In some embodiments, the separated 2’-FL crystals, preferably those of 2’-FL polymorph II, are washed with glacial acetic acid. Then the optionally washed crystals are dried by conventional methods.

[0092] The crystalline 2'-FL obtained or obtainable by the method of the present invention, preferably 2'-FL polymorph II, has a purity of at least 92%, preferably at least 95%, more preferably at least 98% (determined by HPLC), and / or, compared to 2'-FL, the content of DFL is less than 3% by weight, preferably less than 2%, and / or the acetic acid content is less than 1%, preferably less than 0.5%, and / or the water content is less than 0.5%, preferably less than 0.25% or 0.1%. The crystalline 2'-FL obtained or obtainable by the method of the present invention, preferably 2'-FL polymorph II, has the following determinations determined by a combination of quantitative measurements: 2'-FL: at least 97%, DFL: less than 2%, acetic acid: less than 0.5%; or 2'-FL: at least 97%, DFL: less than 2%, acetic acid: less than 0.5% and water: less than 0.1%.

[0093] The above method provides crystalline 2'-FL, preferably 2'-FL polymorph II, which has at least one of the following beneficial characteristics not possessed by the crystalline 2'-FL obtainable by WO 2016 / 095924, as demonstrated by comparative tests of the examples disclosed in the further detailed description:

[0094] - Due to the larger crystal size (evaluated according to the particle size distribution) and the crystal morphology stable to mechanical shear stress, there is less dust,

[0095] - Better filterability results in a shorter filtration cycle,

[0096] - A lower volatile content (such as measured by loss on drying, LoD) means a shorter drying period. Preferably, the LoD value is reduced by at least 25%, more preferably at least 40%, even more preferably at least 50%,

[0097] - The content of residual solvents (i.e., AcOH and water) after drying is lower,

[0098] - Better flowability, as indicated by the Carr index and Hausner ratio,

[0099] - An increase in bulk and tapped density, and / or

[0100] - A higher crystallization yield.

[0101] In addition, compared to the prior art method according to 2016 / 095924, the above method consumes significantly less (at least 25% less, preferably at least 35% less, for example about 40, 45 or 50% less) AcOH.

[0102] Due to the significant improvement in powder properties, downstream operations can be significantly facilitated, such as the direct formulation process, such as dry blending.

[0103] Furthermore, the crystallization method claimed above is superior to the method of evaporative crystallization from water disclosed, for example, in WO 2018 / 164937, because there is essentially no need to provide a supersaturated aqueous solution or syrup containing 2’-FL from which 2’-FL is crystallized, and / or the obtained crystals exhibit better performance in terms of filterability.

[0104] This crystallization method is highly suitable for selectively crystallizing 2’-FL from an aqueous solution by treating the aqueous solution with acetic acid as described above. The aqueous solution is preferably obtained from a fermentation broth and contains 2’-FL and at least one fucosylated carbohydrate other than 2’-FL, particularly DFL, and optionally other carbohydrate contaminants. This selective crystallization provides high-purity 2’-FL in one step, and it is generally possible to achieve crystallization of batches of at least 100 g of 2’-FL, such as at least 1 kg, or at least 100 kg, or even at least 1 ton of 2’-FL, although the concentration range of contaminating saccharide compounds in such aqueous solutions is wide. In this regard, 2’-FL can be crystallized with a yield of at least 70%, preferably at least 75%, more preferably at least 80%, such as at least 85%.

[0105] The aqueous solution or syrup containing 2’-FL from which 2’-FL is crystallized according to the present invention is preferably a processed / purified fermentation broth, and the fermentation provides 2’-FL by culturing genetically modified cells. The fermentation is preferably carried out as follows.

[0106] Exogenously added lactose is internalized by genetically modified cells from the culture medium as a receptor and is converted to 2’-FL in a reaction including enzymatic fucosylation. In one embodiment, the internalization can occur by a passive transport mechanism, during which lactose diffuses passively through the plasma membrane of the cell. The flow is guided by the concentration difference of lactose in the extracellular and intracellular spaces, and lactose should flow from a place with a higher concentration to a lower concentration region to tend to equilibrium. In another embodiment, lactose can be internalized in the cell by means of an active transport mechanism, during which lactose diffuses through the plasma membrane of the cell under the influence of a transport protein or permease in the cell. Lactose permease (LacY) is specific for lactose. Thus, lactose can be easily internalized by cells expressing the LacY permease (such cells are also referred to as LacY in this article) +Phenotypic cells) take up and accumulate in the cells before being fucosylated (see, for example, WO 01 / 04341, Fort et al. J. Chem. Soc., Chem. Commun. 2558 (2005), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), WO 2012 / 112777, WO 2015 / 036138). Preferably, the cells expressing the lacY gene encoding lactose permease lack the enzymes capable of degrading the internalized lactose. Preferably, due to the inactivation or deletion of the endogenous lacZ gene, the cells lack β1,4-galactosidase activity (such cells are also referred to herein as LacZ - phenotypic cells) or at least have reduced β1,4-galactosidase activity, see, for example, Escherichia coli (E. coli) with low galactosidase activity according to WO 2012 / 112777.

[0107] In a preferred embodiment, the internalization of lactose occurs via an active transport mechanism mediated by the lactose permease of the cell (more preferably LacY).

[0108] After being internalized into the cells, lactose is fucosylated by a fucosyltransferase expressed by the corresponding heterologous gene or nucleic acid sequence, which is introduced into the cells by known techniques such as by integrating it into the chromosome of the cell or using an expression vector. The fucosyltransferase necessary for the production of 2'-FL is α-1,2-fucosyltransferase. The corresponding donor GDP-Fuc that provides the fucose residue for fucosylation can be generated by the action of enzymes (ManB, ManC, Gmd, and WcaG) involved in the GDP-Fuc de novo biosynthesis pathway in the cells, starting from simple carbon sources such as glycerol, fructose, or glucose, in a stepwise reaction sequence. Alternatively, genetically modified cells can utilize recycled fucose, which is phosphorylated by a kinase and then converted to GDP-Fuc by a pyrophosphorylase (see, for example, WO 2010 / 070104).

[0109] 2’-FL can be produced by genetically modified microorganisms according to, for example, Drouillard et al., Angew. Chem. Int. Ed. 45, 1778 (2006), WO 01 / 04341, WO 2010 / 070104, WO 2010 / 142305, WO 2012 / 112777, WO 2015 / 032412, WO 2015 / 036138, WO 2015 / 197082, WO 2017 / 101958, WO 2017 / 188684, US 2017 / 0152538, WO 2018 / 077892, WO 2018 / 194411 or WO 2019 / 008133.

[0110] In a preferred embodiment, the genetically modified microorganism is Escherichia coli.

[0111] Thus, in a preferred embodiment, the production process comprises the following steps:

[0112] a) Providing genetically modified Escherichia coli cells with a LacY + phenotype or a LacZ - phenotype, wherein the cells comprise: + - an α-1,2-fucosyltransferase, and

[0113] - one or more genes encoding the GDP-Fuc biosynthetic pathway, and

[0114]

[0115] b) Culturing the genetically modified Escherichia coli cells with a LacY + phenotype or a LacZ - phenotype in the presence of exogenous lactose and a suitable carbon source, thereby producing a fermentation broth comprising 2’-FL. +

[0116] The Escherichia coli strain preferably has only one recombinant glycosyltransferase-encoding gene, which is an α-1,2-fucosyltransferase, more preferably an α-1,2-fucosyltransferase encoded by the futC gene from Helicobacter pylori.

[0117]

[0118] The fermentation broth thus produced contains 2’-FL both in the production cells and in the culture medium. To harvest intracellular 2’-FL and thereby increase the product titer, the above method may further comprise an optional step c) of disrupting or permeabilizing the cells, for example by heating.

[0118] The fermentation broth containing neutral HMO may be accompanied by other carbohydrates. Usually, another carbohydrate is lactose, which is used as a receptor during fermentation and is not converted. Although the amount of lactose can be significantly reduced before the fermentation broth undergoes the separation / purification steps disclosed below (such as those disclosed in WO 2012 / 112777 or WO 2015 / 036138), it is not necessary to do so. In one embodiment, the purification method is applicable to separating 2'-FL accompanied by carbohydrates from non-carbohydrate contaminants, and the relative proportion of carbohydrates remains substantially unchanged during the claimed process. However, another embodiment of the claimed method is suitable for purifying neutral HMO by separating neutral HMO from carbohydrates and non-carbohydrate contaminants, thereby providing 2'-FL in a substantially pure form.

[0119] Therefore, the carbohydrates accompanying 2'-FL fermentation production are mainly lactose and DFL (Fucα1-2Galβ1-4[Fucα1-3]Glc) as over-fucosylated 2'-FL, which has biological properties similar to 2'-FL. In addition, there may be other non-HMO carbohydrate contaminants in the fermentation broth. These are usually lactulose and its glycosylated derivatives. When lactose is heat-sterilized before and / or during fermentation, lactose can form lactulose by rearrangement. Since lactulose is also internalized by cells, it can be glycosylated similarly to lactose in concurrent biotransformation reactions. However, the amount of lactulose and its glycosylated derivatives does not exceed two-tenths of the weight percent of the total dry solids of the culture broth after biomass separation (a couple of tenth weight %).

[0120] Remove / separate non-carbohydrate particles and substances from the fermentation broth. This process can include a conventional demineralization step in which minerals, salts, and other charged molecules are extracted from the fermentation broth containing 2'-FL and other carbohydrates. Conventional ion exchange resins can be used for demineralization, such as passing the fermentation broth through a + cation exchange resin in the H-form and an anion exchange resin in the free base form. The cation exchange resin is preferably a strong exchanger, and the anion exchange resin can be a weak exchanger or a strong exchanger. In addition to removing salts and charged molecules from the fermentation broth, the ion exchange resins can physically adsorb proteins, DNA, and chromatosomes / caramel bodies that may optionally remain in the fermentation broth after the previous purification steps. Alternatively, demineralization can be carried out by conventional electrodialysis or a conventional membrane filtration / filtration system using an appropriate particle size cut-off. Then the solution obtained in any of the above ways can be concentrated by a conventional evaporation step or a conventional nanofiltration step.

[0121] In addition, the above method for removing / separating non-carbohydrate particles and substances from carbohydrates may further include conventional charcoal treatment to remove chromophores and optionally water-soluble biomolecules (such as nucleic acids, peptides, proteins, amino acids, exopolysaccharides, and lipids) left over from previous purification steps. The affinity of charcoal for carbohydrates in an aqueous medium is weaker than its affinity for certain water-soluble lipophilic contaminants (such as proteins and amino acids containing lipophilic moieties, lipids, and colored aromatic bodies). Therefore, carbohydrates free of lipophilic contaminants on the charcoal can be easily washed off the charcoal with (distilled) water. In addition, the above method may further include a conventional clarification step for removing cells, cell debris, and proteins after fermentation, preferably before the above charcoal treatment. Clarification can be carried out in a conventional manner, such as by precipitation in a centrifuge to produce a clarified or partially clarified supernatant. Alternatively, the fermentation broth can be ultrafiltered in a conventional manner to remove high-molecular-weight components. The semi-permeable membrane used for ultrafiltering the 2'-FL fermentation broth may suitably have a cut-off of 5-50 kDa, preferably 10-25 kDa, more preferably about 15 kDa. Depending on the characteristics of the fermentation broth to be clarified, a combination of higher and lower cut-off membranes within the above given range can be used (in this order). Optionally, nanofiltration can be carried out after centrifugation or ultrafiltration, during which the aqueous solution containing 2'-FL and accompanying carbohydrates is concentrated in a conventional manner before treatment with charcoal. In this nanofiltration step, the pore size of its membrane can ensure retention of 2'-FL with a molecular weight of 488; thus, a membrane with a cut-off of 200-300 Da can generally be used. Alternatively, if the UF permeate contains a relatively high amount of lactose, the MWCO of the membrane suitable for nanofiltration is 600-3500 Da, ensuring retention of 2'-FL and allowing at least a portion of lactose to pass through the membrane, and the active (top) layer of the membrane is composed of polyamide, where the MgSO4 rejection factor on the membrane is about 20-90%, preferably 50-90%. In the case of relatively high lactose rejection (about 90%), it may be necessary to subsequently diafilter with pure water to bring all or at least most of the lactose into the permeate. The higher the lactose rejection, the more diafiltration water is required for effective separation. This nanofiltration membrane should be tight for 2'-FL so that 2'-FL is effectively retained. Preferably, the rejection of 2'-FL is greater than 95%, more preferably 97%, and even more preferably 99%. Membranes with a MWCO greater than 3500 Da are expected to allow more or a significant amount of 2'-FL to pass through the membrane, thus showing a reduction in 2'-FL retention and are therefore not suitable. Preferably, the rejection of lactose is not greater than 80-90%. If the lactose rejection becomes 90 ± 1-2%, the 2'-FL rejection should preferably be about 99% or higher to achieve a practically satisfactory separation. These requirements are simultaneously met when the membrane is relatively loose for MgSO4, i.e., its rejection is about 50-90%.In this regard, the above-specified membrane is tight for 2’-FL, while loose for monosaccharides and lactose as well as MgSO4. Therefore, lactose (a precursor for preparing 2’-FL by fermentation) can be efficiently separated from 2’-FL by nanofiltration, and additionally most divalent ions also pass through to the permeate. Preferably, the rejection factor of the membrane for NaCl is lower than that for MgSO4. At about 20 - 30% NaCl rejection, a significant reduction of all monovalent salts in the retentate can also be achieved. The active layer or top layer of the above nanofiltration membrane is preferably composed of polyamide, more preferably the polyamide membrane is a polyamide constructed with phenylenediamine or piperazine building blocks as the amine, and even more preferably piperazine (also known as piperazinyl polyamide). An example of a suitable piperazinyl polyamide TFC membrane is. UA60.

[0122] Example

[0123] General:

[0124] The term "Brix" refers to degrees Brix, i.e., the sugar content of an aqueous solution (grams of sugar in 100 g of solution). In this regard, the Brix of the 2’-FL solution in this application refers to the total carbohydrate content of the solution, including 2’-FL and its accompanying carbohydrates, and thus actually represents the total dissolved solids (TDS). Brix is measured at room temperature using a calibrated refractometer. The Brix measurement value is verified by measuring the remaining moisture in the solution using the Karl-Fischer titration method.

[0125] HPLC: The impurity concentration is analyzed by HPLC on an apHeraNH2 polymer (250 mm × 4.6 mm; 5 μm), using 72 v / v% acetonitrile (ACN) at a flow rate of 1.1 ml / min and 25 °C, with a charged aerosol detector (CAD). The concentration of 2’-FL is measured by HPLC on a TSKgel Amide-80 (150 mm × 4.6 mm, particle size: 3 μm), using 64 v / v% acetonitrile at a flow rate of 1.1 ml / min and 25 °C, with a refractive index detector at 37 °C.

[0126] The water content of the dry crystalline powder is measured by the Karl-Fischer titration method. The residual AcOH content is measured using Megazyme K-ACETRM 07 / 12.

[0127] Powder X-ray diffraction studies are carried out using a Philips PW 1830 / PW1050 instrument in transmission geometry, monochromatized using CuKα radiation through a graphite monochromator. Based on the wavelength, the D-spacing is calculated from the 2θ values. As a general rule, the error rate of the 2θ values is Based on their diffraction patterns, all crystalline 2’-FL samples produced according to the following examples proved to be polymorph II as disclosed in WO 2011 / 150939.

[0128] Fermentation and purification: Using LacZ - , LacY + A genetically modified Escherichia coli strain with a LacZ, LacY phenotype is used to generate a fermentation broth containing 2’-FL by fermentation, wherein the strain contains a recombinant gene encoding an α1,2-fucosyltransferase and genes encoding the biosynthetic pathway of GDP-fucose, and the α1,2-fucosyltransferase is capable of transferring the fucose of GDP-fucose to internalized lactose. Fermentation is carried out by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, for example, according to WO 2015 / 197082 or WO 2016 / 095924, thereby producing 2’-FL, accompanied by DFL (≈5 - 14%, relative to 2’-FL) and unreacted lactose (≈0.8 - 10%, relative to 2’-FL) as the main carbohydrate impurities in the fermentation broth. The fermentation broth is treated as follows: ultrafiltration, nanofiltration, decolorization with activated carbon, and ion exchange treatment with strong acidic (H + ) resin and weak basic resin.

[0129] Example 1

[0130] The treated aqueous fermentation broth containing 2’-FL, DFL, and lactose is concentrated to 73.0 °Bx (238.3 g; containing 57.4 wt% of 2’-FL, 1.6 wt% of lactose, and 6.4 wt% of DFL), and then added to a 1-liter crystallizer. The syrup is heated to 50 °C while stirring at 150 rpm. Crystallization of the preheated clear syrup is initiated by loading 2’-FL polymorph II crystals relative to 1 wt% of the total solids according to the Brix. The seed crystals are loaded after being suspended in acetic acid at 24.1 wt%. The slurry is stirred for 5 hours and then cooled to 35 °C. Then acetic acid is added to the slurry over 12 hours to achieve a weight fraction of acetic acid to water of 4.69. At the end of the acetic acid feed, the crystalline material is cooled to 25 °C while stirring for another 5 hours.

[0131] Collect a slurry sample (75 ml) and perform pressure filtration at 1 bar. Collect another slurry sample (75 ml), perform pressure filtration at 1 bar, and wash the filter cake with acetic acid (28 g). Dry the washed and unwashed solid wet filter cakes overnight at 65 °C and 50 mbar, yielding drying losses (LoD) of 17.3% and 23.2%, respectively. Determine the concentrations of 2’-FL and other impurities in the mother liquor and the dried solids by HPLC. The unwashed dried crystalline material contains 95.3% of 2’-FL and yields 81.5% of crystalline 2’-FL polymorph II, while the purity of the washed filter cake increases to 97.0%.

[0132] Example 2

[0133] Follow the protocol of Example 1 with the following parameters (parameters not specifically mentioned are the same as in Example 1):

[0134] The concentrated syrup is 71.0 °Bx (166.8 g; containing 60.9 wt% of 2’-FL, 0.6 wt% of lactose, and 4.9 wt% of DFL);

[0135] The weight fraction of acetic acid to water at the end of acetic acid addition is 5.58.

[0136] Dry the unwashed solid wet filter cake overnight at 65 °C and 50 mbar, yielding a drying loss (LoD) of 24.7%. The unwashed dried crystalline material contains 98.1% of 2’-FL and yields 86.2% of crystalline 2’-FL polymorph II.

[0137] Example 3

[0138] Follow the protocol of Example 1 with the following parameters (parameters not specifically mentioned are the same as in Example 1):

[0139] The concentrated syrup is 74.5 °Bx (148.3 g; containing 57.9 wt% of 2’-FL, 4.4 wt% of lactose, and 5.4 wt% of DFL);

[0140] Heat the syrup to 35 °C and inoculate at 35 °C;

[0141] The weight fraction of acetic acid to water at the end of acetic acid addition is 4.70.

[0142] Dry the washed and unwashed solid wet filter cakes overnight at 65 °C and 50 mbar, yielding drying losses (LoD) of 16.1% and 22.5%, respectively. The unwashed dried crystalline material contains 95.7% of 2’-FL and yields 81.0% of crystalline 2’-FL polymorph II, while the purity of the washed filter cake increases to 99.9%.

[0143] Example 4

[0144] Following the protocol of Example 1, the parameters are as follows (parameters not specifically mentioned are the same as in Example 1):

[0145] The concentrated syrup was 69.0 °Bx (161.4 g; containing 54.3 wt% 2'-FL, 1.4 wt% lactose, and 6.0 wt% DFL);

[0146] The syrup was heated to 35 °C and inoculated at 35 °C while stirring at 300 rpm;

[0147] The slurry was stirred for 2 h after inoculation;

[0148] Acetic acid was added over 15 h;

[0149] The weight fraction of acetic acid to water at the end of acetic acid addition was 4.68.

[0150] The unwashed solid wet filter cake was dried overnight at 65 °C and 50 mbar, yielding a drying loss (LoD) of 48.5%. The unwashed dried crystalline material contained 92.8% 2'-FL and yielded 84.9% of the crystalline 2'-FL polymorph II.

[0151] Example 5 (Comparative experiment)

[0152] Samples (75 ml) of the crystallization slurry were collected separately from Examples 1 - 3 above and from the prior art (Example 2 according to WO 2016 / 095924, except that the procedure was carried out at 35 °C instead of room temperature and the concentrated syrup was 62 °Bx (140.2 g; containing 48.8 wt% 2'-FL, 1.3 wt% lactose, and 5.4 wt% DFL)) after crystallization was completed, and pressure filtration was carried out using a polypropylene filter cloth with a pore size of about 3 μm and a filtration area of 13 cm 2 at ΔP = 1 bar, and the time until the start of liquid - gas mass transfer was measured. The filterability of the slurry was compared by calculating the total filtration rate according to the following equation: filtrate volume [cm 3 / (time [seconds] · filtration area [cm 2 )

[0153] Filtration was then continued for 60 seconds. The solid wet filter cake thus obtained was dried overnight (14 hours) at 65 °C and 50 mbar, and the drying loss was determined (see the data in the table below).

[0154]

[0155] Data indicate that the new crystallization method according to the present invention produces crystals that are easier to filter and dry. The crystals producible by the new method have much lower amounts of volatile residues, especially acetic acid, after drying.

[0156] Further variation: Under the crystallization conditions disclosed above, substantially the same improved powder properties can be achieved from a concentrated syrup of 71 - 74 °Bx containing 64 - 68 wt% of 2'-FL (usually when DFL is less than 2%, relative to 2'-FL).

Claims

1. A method for crystallizing 2’-FL, the method comprising: 1) providing an aqueous solution or syrup containing 2’-FL at a temperature of 35 - 60 °C, 2) at the same temperature, adding a suspension of 10 - 30 wt% crystalline 2’-FL in acetic acid to the aqueous solution or syrup containing 2’-FL, thereby producing a slurry such that after adding the suspension of crystalline 2’-FL in acetic acid, the amount of acetic acid in the slurry is 1.5 - 4 wt%, 3) adding acetic acid to the slurry at a temperature of 35 - 60 °C to obtain a crystalline substance, wherein the added amount of acetic acid is calculated such that the weight fraction of acetic acid to water in the crystalline suspension at the end of adding acetic acid is 4 - 6, and 4) filtering crystalline 2’-FL from the crystalline substance, wherein the crystalline 2’-FL is polymorph II, and polymorph II refers to a crystalline variant of 2’-FL which, based on measurements using CuKα radiation, contains X-ray powder diffraction reflections at 2θ angles of 16.98 ± 0.20, 13.65 ± 0.20, and 18.32 ± 0.

20.

2. The method according to claim 1, wherein the acetic acid is glacial acetic acid.

3. The method according to claim 1 or 2, wherein measured with a calibrated refractometer, the total solids content of the aqueous solution or syrup according to step 1) is 57 - 80 °Bx (Brix), and the concentration of 2’-FL in the aqueous solution or syrup according to step 1) is 40 - 70 wt%.

4. The method according to claim 3, wherein the aqueous solution or syrup containing 2’-FL further contains DFL and optionally lactose.

5. The method according to claim 4, wherein in the aqueous solution or syrup containing 2’-FL, the 2’-FL:DFL weight ratio is greater than 2, and / or the 2’-FL:lactose weight ratio is 10 - 110, and the DFL:lactose weight ratio is 1 - 10.

6. The method according to claim 5, wherein the 2’-FL:DFL weight ratio is greater than 4.

7. The method according to claim 5, wherein the 2’-FL:DFL weight ratio is greater than 6.

8. The method according to claim 5, wherein the 2’-FL:DFL weight ratio is between 8 and 13.

9. The method according to claim 3, wherein the total solids content of the aqueous solution or syrup according to step 1) is 67 - 77 °Bx, and the concentration of 2’-FL in the aqueous solution or syrup according to step 1) is 52 - 62 wt%.

10. The method according to claim 9, wherein the total solids content of the aqueous solution or syrup according to step 1) is 70 - 75 °Bx, and the concentration of 2’-FL in the aqueous solution or syrup according to step 1) is 56 - 62 wt%.

11. The method according to claim 10, wherein the aqueous solution or syrup is provided at at least 45 °C.

12. The method according to claim 11, wherein the aqueous solution or syrup is provided at 50 ± 2 °C.

13. The method according to claim 9, wherein the total solids content of the aqueous solution or syrup according to step 1) is 67 - 75 °Bx, the concentration of 2'-FL in the aqueous solution or syrup according to step 1) is 52 - 62 wt%, and the aqueous solution or syrup is provided at at least 45 °C.

14. The method according to claim 13, wherein the aqueous solution or syrup is provided at 50 ± 2 °C.

15. The method according to claim 3, wherein the aqueous solution or syrup containing 2'-FL further contains DFL and optionally lactose.

16. The method according to claim 15, wherein in the aqueous solution or syrup containing 2'-FL, the 2'-FL:DFL weight ratio is greater than 13, and the DFL:lactose weight ratio is less than 1.

17. The method according to claim 16, wherein the 2'-FL:DFL weight ratio is greater than 25.

18. The method according to claim 16, wherein the 2'-FL:DFL weight ratio is greater than 50.

19. The method according to claim 3, wherein the total solids content of the aqueous solution or syrup according to step 1) is 67 - 77 °Bx, and the concentration of 2'-FL in the aqueous solution or syrup according to step 1) is 62 - 70 wt%.

20. The method according to claim 19, wherein the concentration of 2'-FL in the aqueous solution or syrup is 64 - 68 wt%.

21. The method according to claim 19, wherein the aqueous solution or syrup is provided at at least 45 °C.

22. The method according to claim 21, wherein the aqueous solution or syrup is provided at 50 ± 2 °C.

23. The method according to claim 1 or 2, wherein, relative to the total solids content of the aqueous solution or syrup provided in step 1), the amount of the crystalline 2'-FL in the suspension of crystalline 2'-FL in acetic acid added in step 2) is 0.5 - 2 wt%.

24. The method according to claim 23, wherein after adding the suspension, the amount of acetic acid in the slurry is 2 - 3 wt%.

25. The method according to claim 24, wherein the 2'-FL / acetic acid suspension added in step 2) is 24 ± 2 wt%.

26. The method according to claim 1 or 2, wherein the addition of acetic acid in step 3) is carried out at a temperature of 30 - 40 °C.

27. The method according to claim 26, wherein the acetic acid is added continuously or in portions over 12 ± 2 hours.

28. The method according to claim 26, wherein at the end of the addition of acetic acid, the weight fraction of acetic acid to water in the crystalline suspension is 4.6 - 6.

29. The method according to claim 28, wherein at the end of the addition of acetic acid, the weight fraction of acetic acid to water in the crystalline suspension is 4.6 - 5.

5.

30. The method according to claim 1 or 2, wherein the crystalline material obtained in step 3) is cooled to 0 - 25 °C and stirred for 5 ± 1 hour.

31. The method according to claim 30, wherein the crystalline substance obtained in step 3) is cooled to 20-25 °C and stirred for 5±1 hours.

32. The method according to claim 1 or 2, wherein the aqueous solution or syrup provided in step 1) is a purified fermentation broth.

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