WO2023117673A1

WO2023117673A1 - CRYSTALLIZATION OF LNnT

Info

Publication number: WO2023117673A1
Application number: PCT/EP2022/086108
Authority: WO
Inventors: Getachew S. MOLLA
Original assignee: Dsm Ip Assets B.V.
Priority date: 2021-12-21
Filing date: 2022-12-15
Publication date: 2023-06-29
Also published as: DK202101233A1

Abstract

The invention relates to a method for selective crystallization of LNnT from an aqueous solution comprising LNnT and one or more other fucosylated carbohydrates by adding methanol to the solution.

Description

Crystallization of LNnT

FIELD OF THE INVENTION

The invention relates to a method of crystallizing lacto-N-neotetraose (LNnT) from an aqueous solution, using methanol, particularly from an aqueous solution obtained in a biotechnological production of LNnT.

BACKGROUND OF THE INVENTION

Crystallization or recrystallization is one of the simplest and cheapest methods to isolate a product from a reaction mixture, separate it from contaminations/impurities and obtain pure substance. Isolation or purification that uses crystallization makes the whole technological process more robust and cost-effective and thus advantageous.

LNnT may exist in different crystalline forms, see for example EP-A-1405856, WO 2011/100980 or WO 2014/094783. In this regard, crystallization of LNnT can advantageously be a part of its production, especially in its purification and isolation process from its aqueous culture medium in which it has been produced.

WO 2011/100980 discloses crystallizing LNnT polymorph III from its aqueous methanolic solution obtained after catalytic hydrogenolysis of a suitable precursor compound.

With respect to crystallized LNnT, especially LNnT polymorph III, there is still a need for an improved crystallization method that consumes lower amount of methanol and/or produces crystals with improved properties such as stable crystal morphology, better filterability, lower level of moisture/volatiles, faster powder drying and/or better dry powder physical and rheological properties, while maintaining or even exceeding the yield and/or the purity provided by the prior art process.

SUMMARY OF THE INVENTION

The first aspect of the invention relates to a method for crystallizing LNnT, advantageously polymorph III LNnT, comprising:

1) providing an aqueous solution or syrup comprising LNnT,

2) optionally, adding crystalline LNnT, advantageously polymorph III of LNnT, optionally as a suspension in methanol, to the aqueous solution or syrup comprising LNnT, thereby generating a slurry,

3) adding methanol to the aqueous solution or syrup comprising LNnT according to step 1) or to the slurry according to step 2), to obtain a wet crystalline mass, and 4) filtering the crystalline LNnT, advantageously polymorph III of LNnT, from the wet crystalline mass.

The second aspect of the invention relates to crystalline LNnT, advantageously polymorph III of LNnT, obtained or obtainable according to the first aspect of the invention.

The third aspect of the invention relates to a nutritional or pharmaceutical composition comprising the crystalline LNnT, advantageously polymorph III of LNnT, according to the second aspect of the invention.

The fourth aspect of the invention relates to the use of the crystalline LNnT, advantageously polymorph III of LNnT, according to the second aspect of the invention in the preparation of food compositions or food supplements.

The fifth aspect of the invention relates to the use of the crystalline LNnT, advantageously polymorph III of LNnT, according to the second aspect of the invention as food or food supplement.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows the X-ray powder diffraction pattern of the crystalline LNnT polymorph III.

DETAILED DESCRIPTION OF THE INVENTION

The crystallization protocol of LNnT polymorph III disclosed in WO 2011/100980 can be adapted to aqueous LNnT solutions obtainable in a microbial fermentative production followed by one or more purification steps of the fermentation culture medium (see e.g. WO 2017/221208). However, to achieve a high yield and good purity of the crystallized LNnT, relatively high amount of methanol has been used in the crystallization.

The present inventors have made an effort to reduce the amount of methanol used for the crystallization while at least keeping the acceptable crystallization yield and the purity of LNnT provided by the original method and surprisingly discovered that the crystals so-produced possess beneficial properties and crystal quality.

Accordingly, a method is provided for crystallizing LNnT, advantageously polymorph III LNnT, comprising:

1) providing an aqueous solution or syrup comprising LNnT,

2) optionally, adding crystalline LNnT, advantageously polymorph III of LNnT, optionally as a suspension in methanol, to the aqueous solution or syrup comprising LNnT, thereby generating a slurry, 3) adding methanol to the aqueous solution or syrup comprising LNnT according to step 1) or to the slurry according to step 2), to obtain a wet crystalline mass, and

4) filtering the crystalline LNnT, advantageously polymorph III of LNnT, from the wet crystalline mass.

Preferably, steps 1 ) to 4) in the order of numbering are comprised in the method.

LNnT polymorph III refers to a crystalline modification of LNnT disclosed in WO 2011/100980 that comprises X-ray powder diffraction reflections, based on a measurement using CuKa radiation, at 20.32±0.2020 angle, preferably at 20.32±0.20 and 19.10±0.20 20 angles, more preferably at 20.32±0.20, 19.10±0.20 and 7.98±0.2020 angles, even more preferably at 20.32±0.20, 19.10±0.20, 7.98±0.20 and 21.03±0.20 20 angles, most preferably 20.32±0.20, 19.10±0.20, 7.98±0.20, 21.03±0.20 and 20.95±0.20 20 angles, in particular 20.32±0.20, 19.10±0.20, 7.98±0.20, 21.03±0.20, 20.95±0.20 and 5.66±0.20 20 angles. Figure 1 shows the X-ray powder diffraction pattern of the crystalline LNnT polymorph III, and the list of peaks is given in Table 1 below.

Table 1 .

The claimed method provides crystalline LNnT, preferably LNnT polymorph III, possessing beneficial features compared to a crystalline LNnT known from the prior art. Moreover, the claimed crystallizing method is characterized by lower antisolvent (methanol) consumption (meaning more economical process) while the crystallization yield and the crystal purity (typically measured by HPLC) is not reduced.

The first step of the method is the provision of an aqueous solution or syrup comprising LNnT. The aqueous solution or syrup does not comprise organic solvent(s) and is preferably homogenous. If it is an aqueous solution, it does not need to be a supersaturated solution with

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RECTIFIED SHEET (RULE 91) ISA/EP respect to LNnT. A supersaturated solution with respect to LNnT is a solution that contains more than the maximum amount of LNnT that is capable of being dissolved at a given temperature. “The maximum amount of LNnT that is capable of being dissolved at a given temperature’’ is in fact the solubility of LNnT, that is a thermo-chemical property referring to the ability for LNnT to dissolve in a solvent at that temperature. It is measured in terms of the maximum amount of solute dissolved in a solvent at equilibrium, here in water. The resulting solution is called a saturated solution. Super- or oversaturation occurs when the concentration of LNnT exceeds the concentration specified by the value equilibrium solubility. The concentration of LNnT in the aqueous solution or syrup is at least 20 wt%, preferably at least 30 wt%, more preferably around 35-40 wt% (determined by HPLC).

In one embodiment, the aqueous solution or syrup comprising LNnT is provided at an elevated temperature (higher than room temperature) or heated up to an elevated temperature before conducting the next step. The elevated temperature is between 35-55 °C, preferably at least 45 °C, such as around 45-55 °C or 50±2 °C.

The aqueous solution or syrup containing LNnT disclosed above may comprise one or more other carbohydrates or carbohydrate derivatives, depending on how LNnT has been previously produced. LNnT may be made by chemical total synthesis from simpler carbohydrate precursors which synthesis comprises chemical glycosylations of a suitably protected lactose acceptor; such a method is disclosed e.g. in WO 2011/100980. The carbohydrate derivatives other than LNnT contaminating LNnT are typically intermediary derivatives in the synthetic route towards LNnT disclosed therein, especially the final intermediates before complete deprotection to LNnT. Such final intermediates are fully or partially protected LNnT derivatives wherein the protecting groups may be acyl (mainly acetyl or benzoyl) and/or optionally substituted benzyl. In fermentative production of LNnT (see e.g. WO 2017/221208), typical carbohydrate by-products may comprise lacto-N-triose II (LNTri II, GlcNAcpi-3Gal[31-4Glc) and/or para-lacto-N- neohexaose (pLNnH, Gaipi-4GlcNAcpi-3Gal[31-4GlcNAc|31-3Gal|31-4Glc) and/or lactose and/or glucose and/or sucrose.

The total solid content of the aqueous solution or syrup according to step 1 ) is at least 30 °Bx (degrees Brix) as measured with a refractometer, as disclosed in the experimental part, preferably at least 40 °Bx, more preferably at least 45 °Bx, even more preferably at least 48 °Bx; such as 30-55 °Bx, preferably 40-52 °Bx, more preferably 46-50 °Bx. It refers to any solute dissolved in the aqueous solution, that is it includes LNnT and its accompanying carbohydrate derivates as disclosed above, and e.g. salts. In addition, the amount of LNnT in the total solid content of the aqueous solution or syrup according to step 1 ) is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt% (determined by HPLC).

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RECTIFIED SHEET (RULE 91) ISA/EP In one embodiment, the total solid of the aqueous solution or syrup according to step 1 ) in which the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, contains lacto-N-triose II wherein the LNnT/lacto-N-triose II weight ratio is at least 6, preferably at least 8, more preferably at least 12.

In one embodiment, the total solid of the aqueous solution or syrup according to step 1 ) in which the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, contains lactose wherein the LNnT/lactose weight ratio is at least 5, preferably at least 12.5, more preferably at least 25.

In one embodiment, the total solid of the aqueous solution or syrup according to step 1 ) in which the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, contains pLNnH wherein the LNnT/pLNnH weight ratio is at least 50, preferably at least 12.5, more preferably at least 25.

In one embodiment, the total solid of the aqueous solution or syrup according to step 1 ) in which the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, contains lacto-N-triose II and lactose wherein the LNnT/lacto-N-triose II weight ratio and the LNnT/lactose weight ratios are, respectively, at least 6 and at least 5, preferably at least 8 and at least 12.5, more preferably at least 12 and at least 25.

In one embodiment, the total solid of the aqueous solution or syrup according to step 1 ) in which the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, contains lacto-N-triose II, pLNnH and lactose wherein the LNnT/lacto-N-triose II weight ratio, the LNnT/pLNnH weight ration and the LNnT/lactose weight ratio are, respectively, at least 6, at least 50 and at least 5, preferably at least 8, 50-200 and at least 12.5, more preferably at least 12, 50-200 and at least 25.

In one embodiment of step 1 ), the aqueous solution or syrup containing LNnT has a total solid content of 30-55 °Bx and the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC.

Preferably, the aqueous solution or syrup containing LNnT has a total solid content of 30-55 °Bx, the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC, and is provided at a temperature between 35- 55 °C, preferably between 45-55 °C, more preferably at 50±2 °C.

In a preferred embodiment of step 1), the aqueous solution or syrup containing LNnT has a total solid content of 40-52 °Bx and the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC. Preferably, the aqueous solution or syrup containing LNnT has a total solid content of 40-52 °Bx, the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC, and is provided at a temperature between 35- 55 °C, preferably between 45-55 °C, more preferably at 50±2 °C.

In a more preferred embodiment of step 1 ), the aqueous solution or syrup containing LNnT has a total solid content of 46-50 °Bx and the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC.

Preferably, the aqueous solution or syrup containing LNnT has a total solid content of 46-50 °Bx, the amount of LNnT is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%, of the total solid as estimated by HPLC, and is provided at a temperature between 35- 55 °C, preferably between 45-55 °C, more preferably at 50±2 °C.

In any of the preferred and more preferred embodiments disclosed above, the aqueous solution or syrup containing LNnT contains lacto-N-triose II wherein the LNnT/lacto-N-triose II weight ratio is at least 6, preferably at least 8, more preferably at least 12.

In any of the preferred and more preferred embodiments disclosed above, the aqueous solution or syrup containing LNnT contains pLNnH wherein the LNnT/pLNnH weight ratio is at least 50, for example 50-200.

In any of the preferred and more preferred embodiments disclosed above, the aqueous solution or syrup containing LNnT contains lactose wherein the LNnT/lactose weight ratio is at least 5, preferably at least 12.5, more preferably at least 25.

In any of the preferred and more preferred embodiments disclosed above, the aqueous solution or syrup containing LNnT contains lacto-N-triose II and lactose wherein the LNnT/lacto-N-triose II weight ratio and the LNnT/lactose weight ratios are, respectively, at least 6 and at least 5, preferably at least 8 and at least 12.5, more preferably at least 12 and at least 25.

In any of the preferred and more preferred embodiments disclosed above, the aqueous solution or syrup containing LNnT contains lacto-N-triose II, pLNnH and lactose wherein the LNnT/lacto- N-triose II weight ratio, the LNnT/pLNnH weight ratio and the LNnT/lactose weight ratio are, respectively, at least 6, at least 50 and at least 5, preferably at least 8, 50-200 and at least 12.5, more preferably at least 12, 50-200 and at least 25.

In the second step, to initiate crystallization, crystalline LNnT, preferably LNnT polymorph III, is added to the aqueous solution or syrup comprising LNnT according to step 1 ) at elevated temperature, preferably at the same temperature as that in step 1), and preferably under continuous stirring, thereby generating a slurry. Preferably, the amount of the seeding crystal is at least 0.4 wt% relative to the total solid content of the aqueous solution or syrup provided in step 1), such as 0.4-1 .2 wt%, preferably 0.6-0.8 wt%.

In a preferred realization of step 2), the crystalline LNnT, preferably LNnT polymorph III, is added as a suspension in methanol. The suspension of crystalline LNnT, preferably LNnT polymorph III, in methanol to be added to the aqueous solution or syrup comprising LNnT provided in step 1) is made so that the crystalline LNnT is suspended in small volume of methanol, for example 0.03-0.06 g of crystalline LNnT is suspended in 1 ml of methanol.

Typically, LNnT seed crystal, preferably LNnT polymorph III seed crystal, more preferably as the LNnT/methanol suspension, is added to the aqueous solution or syrup comprising LNnT at once, preferably under agitation. After seeding, the mixture is stirred at the same temperature for 30-60 minutes, to generate a slurry.

During the step of initiation crystallization described above no vacuum or reduced pressure is applied.

In one embodiment of step 2), the amount of seeding LNnT, preferably LNnT polymorph III, is 0.4-1 .2 wt% of the solid content of the aqueous solution or syrup provided in step 1 ) and the LNnT/methanol suspension is of 0.03-0.06 g/ml.

Preferably, the amount of seeding LNnT, preferably LNnT polymorph III, is 0.6-0.8 wt% of the solid content of the aqueous solution or syrup provided in step 1 ) and the LNnT/methanol suspension is of 0.03-0.06 g/ml.

In the next step 3), following step 2), to the slurry obtained above pure methanol is added under continuous stirring. In one embodiment, the temperature at which the pure methanol is added to the slurry is the same as that in the precedent step. In other embodiment, the temperature is lower than that in step 2). In a preferred realization, the temperature is the same as in the precedent step. The amount of methanol added in step 3) is calculated so that the methanol to water weight fraction in the wet crystalline mass at the end of the addition of the methanol in step 3) is 2-4, preferably around 3. In the methanol to water weight fraction, the amount of methanol shall comprise the methanol added in step 3) and the methanol that makes up the LNnT/methanol suspension used for seeding in step 2). The water content of the wet crystalline mass at the end of step 3) is practically the amount of water in the aqueous solution or syrup comprising LNnT provided in step 1 ) and can be calculated as follows: water wt% = 100 - Brix [of the aqueous solution or syrup comprising LNnT provided in step 1].

The pure methanol can be added in step 3) continuously with a given addition rate or in several, preferably equal, portions. The calculated amount of methanol is added relatively slowly, in a couple of hours, but not less than in 10 hours such as 10-27 hours, more preferably not less than in 16 hours such as in 16-27 hours, more preferably in 25±2 hours. At the end of the methanol addition, the slurry becomes a crystalline mass.

During the step of adding methanol described above no vacuum or reduced pressure is applied.

Preferably, the methanol is added in step 3) in two phases. In the first phase the 25-50 % of the overall methanol is added in a slower addition rate than that of the remaining 50-75 % of the overall methanol in the second phase: the 25-50 % of the overall methanol is added in the first 60-80 % of the overall addition time, followed by the 50-75 % of the ethanol in the remaining 20- 40 % of the overall addition time.

More preferably, the first half of the methanol is added during a four times longer period that the second half of the methanol.

Even more preferably, the first half of the methanol is added during a four times longer period that the second half of the methanol, wherein the overall addition time of the methanol is 16-27 hours, more preferably in 25±2 hours.

After addition of the pure methanol and obtaining a crystalline mass of LNnT according to step 3) above, the temperature is decreased to a final temperature in a couple of hours, typically between 0.5-3 hours, which is between 0 °C and room temperature, preferably around 20-25 °C, or the crystalline mass is allowed to cool to around 20-25 °C e.g. in 0.5-3 hours. At this final temperature, optionally, the crystalline mass of LNnT is stirred for a couple of hours, such as at least for 1 -2 hours, for example 5±1 hours. During the step of equilibrating the crystalline mass of LNnT described above no vacuum or reduced pressure is applied.

The crystalline mass of LNnT obtained in step 3) or the optionally equilibrated crystalline mass described above is then filtered in step 4). The crystalline LNnT is separated from the mother liquor conventionally, e.g. dead-end filtration, centrifugation or decantation at a temperature between 0 °C and room temperature, preferably around 20-25 °C. In some embodiments, the separated LNnT crystals, preferably those of LNnT polymorph III, are washed with methanol. The optionally washed crystals are then dried conventionally.

The crystalline LNnT, preferably LNnT polymorph III, obtained or obtainable by the method of the invention, has a purity at least around 85-90 % (determined by HPLC) if the crude LNnT is produced by fermentation and the aqueous solution or syrup containing LNnT before step 1 ) comprises lacto-N-triose II, pLNnH and lactose as accompanying carbohydrates.

The above method provides crystalline LNnT, preferably LNnT polymorph III, possessing at least one of the following beneficial features that the crystalline LNnT polymorph III obtainable by WO 201 1/100980 does not have, as it has been demonstrated by comparative tests disclosed in the examples in further detail:

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RECTIFIED SHEET (RULE 91) ISA/EP - better filterability that leads to shorter filtration period,

- lower volatile (methanol) content (as measured by loss on drying, LoD) of the filtered crystals meaning shorter drying period, preferably, the LoD value is reduced by at least 25 %, more preferably by at least 40 %, even more preferably by at least 50 %,

- better flowability as seen by Carr index and Hausner ratio,

- increased bulk and tapped density, and/or

- higher crystallization yield,

Preferably, at least a better filterability and/or a lower volatile (methanol) content can be achieved.

In addition, the above method consumes significantly less (at least 30 % less, preferably at least 40 % less, such as around 50 % less, and even around 70 % less) methanol compared to the prior art method according to WO 2011/100980 while the crystallization yield is at least maintained.

Having the powder properties considerably improved, downstream operations can be significantly facilitated, for example direct formulation processes such as dry mixing.

The present crystallization method is highly suitable for selectively crystallizing LNnT from an aqueous solution, preferably obtained from fermentation broth, containing LNnT and other carbohydrate-like contaminants, by treating the aqueous solution with methanol as disclosed above. This selective crystallization provides LNnT of acceptable purity in one step, and typically crystallization of batches of at least 100 g of LNnT, such as at least 1 kg, or at least 100 kg, or even at least 1 ton of LNnT, can be achieved despite the wide range of concentrations of contaminating sugar-like compounds in such aqueous solutions. In this regard, LNnT can be crystallized with a yield of at least 85 %, preferably at least 85 %, more preferably at least 90 %.

The aqueous solution or syrup comprising LNnT from which LNnT is crystallized out according to the present invention is preferably a treated/purified fermentation broth, which fermentation provides LNnT by culturing a genetically modified cell. The fermentation is preferably performed as disclosed for example in WO 2017/101958, WO 2017/182965 or WO 2017/221208.

The fermentation broth that comprises the LNnT can be accompanied by other carbohydrate compounds. As disclosed above, typically, another carbohydrate compound is lactose which is used as acceptor in the fermentation process and left unconverted. Although its amount can be substantially reduced in the fermentation broth before subjecting it to the separation/purification steps disclosed below, e.g. as disclosed in WO 2015/036138, it is not necessary to do so. The

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RECTIFIED SHEET (RULE 91) ISA/EP purification method, in one embodiment, is suitable to separate LNnT accompanied by carbohydrate compounds from non-carbohydrate contaminants, while the relative proportion of the carbohydrate compounds does not substantially change in the course of the claimed method. Another embodiment of the claimed method, however, is suitable to purify the LNnT by separating it from carbohydrate and non-carbohydrate contaminations, thereby providing crude LNnT in purer form.

Accordingly, the accompanying carbohydrate in the fermentative production of LNnT is mainly lactose, lacto-N-triose II and pLNnH. Moreover, there can be further non-HMO carbohydrate contaminants in the fermentation broth. These are typically lactulose and its glycosylated derivatives. However, the amount of lactulose and its glycosylated derivatives does not exceed a couple of tenth weight% of the overall dry solid matter of the broth after biomass separation.

From the fermentation broth, non-carbohydrate particles and substances are removed/separated. This process can include a conventional demineralization step during which minerals, salts and other charged molecules are extracted from the fermentation broth containing LNnT and the other carbohydrates. The demineralization can be conducted using conventional ion exchange resins, e.g. passing the fermentation broth through a cation exchange resin in H⁺-form and an anion exchange resin in free base form. The cation exchange resin is preferably a strong exchanger, and the anion exchange resin can be a weak or strong exchanger. The ion exchange resins, besides removing salts and charged molecules from the broth, can physically adsorb proteins, DNA and colorizing/caramel bodies that optionally have been left in the broth after previous purification steps. Alternatively, the demineralization can be conducted by means of a conventional electrodialysis or a conventional membrane filtration/diafiltration system using an appropriate particle size cut-off. The solution obtained in any of the above ways can then be concentrated by either a conventional evaporation step or a conventional nanofiltration step.

In addition, the above process removing/separating non-carbohydrate particles and substances from carbohydrates are can also include a conventional charcoal treatment to remove colour bodies and optionally water soluble biomolecules (e.g. nucleic acids, peptides, proteins, amino acids, exopolysaccharides and lipids) left from previous purification steps. Charcoal has a weaker affinity for carbohydrate compounds in aqueous medium than for some water-soluble lipophilic contaminants (e.g. proteins and amino acids containing lipophilic moieties, lipids and coloured aromatic bodies). Thus, the carbohydrates, free of the lipophilic contaminants on the charcoal, can be easily washed from the charcoal with (distilled) water.

Moreover, the above process can also include a conventional clarification step for removing cells, cells fragments and proteins after fermentation, preferably prior to the charcoal and ion exchanger treatment described above. The clarification can be done in a conventional manner, e.g. by sedimentation in centrifuge producing a clarified or partially clarified supernatant solution. Alternatively, the fermentation broth can be subjected to ultrafiltration in a conventional manner to remove high molecular weight components. The semipermeable membrane used for ultrafiltrating a LNnT fermentation broth can suitably have a cut off of 5-50 kDa, preferably 10- 25 kDa, more preferably around 15 kDa. For biomass separation from the broth see e.g. WO 2021/064629. Depending on the characteristics of the fermentation broth to be clarified combination of higher and lower cut off membranes (in this order) within the above given range can be employed. Optionally, centrifugation or ultrafiltration can be followed by nanofiltration, during which the aqueous solution containing LNnT and accompanying carbohydrates is concentrated in a conventional manner before it is treated with charcoal. In this nanofiltration step, so typically a 200-300 Da cut off membrane can be used. Alternatively, if the UF permeate contains a higher amount of lactose, the membrane suitable for nanofiltration has a MWCO of 600-3500 Da ensuring the retention of LNnT and allowing at least a part of lactose to pass through the membrane, and the active (top) layer of the membrane is composed of polyamide, wherein the MgSC rejection factor on said membrane is around 20-90 %, preferably 50-90 %. In case of a relatively high rejection (about 90 %) of lactose, a subsequent diafiltration with pure water may be necessary to bring all or at least the majority of the lactose in the permeate. The higher the lactose rejection the more diafiltration water is necessary for efficient separation. This nanofiltration membrane shall be tight for LNnT in order that it is efficiently retained. Preferably, the rejection of LNnT is more than 95 %, more preferably 97 %, even more preferably 99 %. Membranes with MWCO of more than 3500 Da are expected to allow more or significant amount of LNnT pass through the membrane thus show a reduced retention of LNnT and therefore are not suitable. It is preferred that the rejection of the lactose is not more than 80-90 %. If the lactose rejection turns to be 90 ± 1 -2 %, the LNnT rejection shall preferably be around 99 % or higher in order to achieve a practically satisfying separation. These requirements are simultaneously fulfilled when the membrane is relatively loose for MgSC , that is its rejection is about 50-90 %. In this regard the above specified membrane is tight for LNnT and loose for monosaccharides and lactose, and as well as for MgSC . Therefore, it is possible to separate lactose, the precursor in making LNnT by fermentation, from LNnT by nanofiltration with a good efficacy, and additionally a substantial part of divalent ions also passes to the permeate. Also preferably, the membrane has a rejection factor for NaCI that is lower than that for MgSC . At a NaCI rejection of around 20-30 %, a substantial reduction of all monovalent salts in the retentate is also achievable. The active or the top layer of the above nanofiltration membrane is preferably made of polyamide, more preferably the polyamide membrane is a polyamide with phenylene diamine or piperazine building blocks as amine, even more preferably piperazine (referred to as piperazine-based polyamide, too). An example of suitable piperazine based polyamide TFC membranes is TriSep® UA60 (see WO 2019/003133).

To obtain one or more benefits mentioned earlier by performing the method disclosed and claimed herein, the amount of the accompanying by-product pLNnH should be maintain relatively low, preferably the pLNnH:LNnT weight ratio is not more than 0.02. If the fermentation produces more pLNnH, an additional chromatographic purification step may be necessary after biomass separation, nanofiltration, ion exchanger treatment and/or active charcoal treatment. Such a chromatographic purification step is advantageously performed on a hydrophobic stationary phase which is a polystyrene cross-linked with divinylbenzene (PS-DVB) and functionalized with bromine on the aromatic ring, as disclosed in WO 2020/128945.

The following numbered aspects of the invention are provided:

Aspect 1. A method for crystallizing LNnT comprising:

1 ) providing an aqueous solution or syrup comprising LNnT,

3) adding methanol to the aqueous solution or syrup comprising LNnT according to step 1 ) or to the slurry according to step 2), to obtain a wet crystalline mass, and

4) filtering the crystalline LNnT from the wet crystalline mass.

Aspect 2. The method of aspect 1 , wherein the crystalline LNnT is polymorph III as disclosed in WO 2011/100980.

Aspect 3. The method of aspect 1 or 2, wherein steps 1 ) to 4) in the order of numbering are comprised.

Aspect 4. The method of any of the preceding aspects, wherein the concentration of LNnT of aqueous solution or syrup comprising LNnT provided in step 1 ) is at least 20 wt%.

Aspect 5. The method of aspect 4, wherein the concentration is at least 30 wt%.

Aspect 6. The method of aspect 5, wherein the concentration is at least 40 wt%.

Aspect 7. The method of any of the preceding aspects, wherein the aqueous solution or syrup comprising LNnT is provided at an elevated temperature or heated up to an elevated temperature.

Aspect 8. The method of aspect 7, wherein the elevated temperature is between 35-55 °C.

Aspect 9. The method of aspect 8, wherein the elevated temperature is between at least 45 °C.

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RECTIFIED SHEET (RULE 91) ISA/EP Aspect 10. The method of aspect 9, wherein the elevated temperature is 45-55 °C.

Aspect 11 . The method of aspect 10, wherein the elevated temperature is 50±2 °C.

Aspect 12. The method of any of the preceding aspects, wherein the aqueous solution or syrup comprising LNnT contains one or more of lacto-N-triose II, pLNnH and lactose.

Aspect 13. The method of any of the preceding aspects, wherein the total solid content of the aqueous solution or syrup comprising LNnT is at least 30 Brix (°Bx) as measured by a calibrated refractometer.

Aspect 14. The method of aspect 13, wherein the total solid content is at least 40 Brix.

Aspect 15. The method of aspect 14, wherein the total solid content is at least 45 Brix.

Aspect 16. The method of aspect 15, wherein the total solid content is at least 48 Brix.

Aspect 17. The method of any of aspects 13 to 16, wherein the total solid content is 30-55 Brix, preferably 40-52 Brix, more preferably 46-50 Brix.

Aspect 18. The method of any of aspects 13 to 17, wherein the amount of LNnT in the total solid content is at least 65 wt%.

Aspect 19. The method of aspect 18, wherein the amount of LNnT in the total solid content is at least 70 wt%.

Aspect 20. The method of aspect 19, wherein the amount of LNnT in the total solid content is at least 75 wt%.

Aspect 21 . The method of any of aspects 18 to 20, wherein the LNnT/lacto-N-triose II weight ratio is at least 6, preferably at least 8, more preferably at least 12.

Aspect 22. The method of any of aspects 18 to 20, wherein the LNnT/lactose weight ratio is at least 5, preferably at least 12.5, more preferably at least 25.

Aspect 23. The method of any of aspects 18 to 20, wherein the LNnT/pLNnH weight ratio is at least 50, for example 50-200.

Aspect 24. The method of aspect 21 or 22, wherein the LNnT/lacto-N-triose II weight ratio and the LNnT/lactose weight ratios are, respectively, at least 6 and at least 5, preferably at least 8 and at least 12.5, more preferably at least 12 and at least 25.

Aspect 25. The method of any of aspects 21 to 24, wherein the LNnT/lacto-N-triose II weight ratio, the LNnT/pLNnH weight ratio and the LNnT/lactose weight ratio are, respectively, at least 6, at least 50 and at least 5. Aspect 26. The method of aspect 25, wherein the LNnT/lacto-N-triose II weight ratio, the LNnT/pLNnH weight ratio and the LNnT/lactose weight ratio are, respectively, at least 8, 50-200 and at least 12.5.

Aspect 27. The method of aspect 26, wherein the LNnT/lacto-N-triose II weight ratio, the LNnT/pLNnH weight ratio and the LNnT/lactose weight ratio are, respectively, at least 12, 50- 200 and at least 25.

Aspect 28. The method of any of the preceding aspects, wherein in step 2) crystalline LNnT, preferably LNnT polymorph III, is added to the aqueous solution or syrup comprising LNnT according to step 1) for seeding.

Aspect 29. The method of aspect 28, wherein the crystalline LNnT is added at the same temperature as that in step 1).

Aspect 30. The method of aspect 28 or 29, wherein the amount of the seeding crystal is at least 0.4 wt% relative to the total solid content of the aqueous solution or syrup provided in step 1).

Aspect 31 . The method of aspect 30, wherein the amount of the seeding crystal is 0.4-1 .2 wt%, preferably 0.6-0.8 wt%.

Aspect 32. The method of any of the aspects 28 to 31 , wherein the crystalline LNnT, preferably LNnT polymorph III, is added as a suspension in methanol.

Aspect 33. The method of aspect 32, wherein the suspension contains 0.03-0.06 g of crystalline LNnT in 1 ml of methanol.

Aspect 34. The method of any of the preceding aspects, wherein in step 3) methanol is added at the same temperature as that in step 2).

Aspect 35. The method of any of the preceding aspects, wherein in step 3) the amount of methanol added in step 3) is calculated so that the methanol to water weight fraction in the wet crystalline mass at the end of the addition of the methanol in step 3) is 2-4, preferably around 3.

Aspect 36. The method of aspect 34 or 35, wherein the methanol is added over at least 10 hours.

Aspect 37. The method of aspect 36, wherein the methanol is added over at least 16 hours.

Aspect 38. The method of aspect 37, wherein the methanol is added over 25±2 hours.

Aspect 39. The method of any of the aspects 34 to 38, wherein the methanol is added in two phases.

Aspect 40. The method of aspect 39, wherein the 25-50 % of the overall methanol is added in a slower addition rate in the first phase than that of the remaining 50-75 % in the second phase. Aspect 41 . The method of aspect 39 or 40, wherein the first phase is 60-80 % of the overall addition time.

Aspect 42. The method of aspect 40 or 41 , wherein 50 % of the overall methanol is added in the first phase.

Aspect 43. The method of any of the aspects 40 to 42, wherein the first phase is four times longer than the second phase.

Aspect 44. The method of any of the preceding aspects, wherein after step 3) the temperature is decreased to a final temperature in a couple of hours, typically between 0.5-3 hours.

Aspect 45. The method of aspect 44, wherein the final temperature is around 20-25 °C.

Aspect 46. The method of aspect 44 or 45, wherein the crystalline mass of LNnT is stirred for a couple of hours, such as at least for 1 -2 hours, for example 5±1 hours, at the final temperature.

EXAMPLES

General:

The term “Brix” refers to degrees Brix, that is the sugar content of an aqueous solution (g of sugar in 100 g of solution). In this regard, Brix of the LNnT solution of this application refers to the overall carbohydrate content of the solution including LNnT and its accompanying carbohydrates and thus practically represents the total dissolved solid (TDS). Brix was measured by a calibrated refractometer at room temperature. The Brix measurement was validated by measuring the remaining water fraction of the solution using Karl-Fischer titration.

HPLC: The concentration of impurities was analysed by HPLC on apHera NH2 polymer (250 mm x 4.6 mm; 5 pm) with 72 v/v % acetonitrile (ACN) at flow rate of 1 .1 ml/min and 25 °C using charged aerosol detector (CAD). The concentration of LNnT was measured by HPLC on TSKgel Amide-80 (150 mm x 4.6 mm, particle size: 3pm) with 64 v/v % acetonitrile at flow rate of 1 .1 ml/min and 25 °C using refractive index detector at 37 °C.

The water content of dried crystal powder was measured by Karl-Fischer titration.

Powder X-ray diffraction investigations were conducted with a Philips PW 1830/PW1050 instrument in transmission geometry, using CuKa radiation made monochromatic by means of a graphite monochromator. D-spacings were calculated from the 20 values, based on a wavelength of 1 .54186 A. As a general rule the 20 values have an error rate of ± 0.2 A. Based on their diffractograms, all crystalline LNnT samples produced according to the examples below proved to be polymorph III as disclosed in WO 2011/100980.

Fermentation and purification: LNnT was made by fermentation using a genetically modified E. coli cell of LacZ- phenotype, wherein said cell comprises a recombinant gene encoding a p-1 ,3-

15

RECTIFIED SHEET (RULE 91) ISA/EP N-acetyl-glucosaminyl transferase which is able to transfer the GIcNAc of UDP-GIcNAc to the internalized lactose, a recombinant gene encoding a p-1 ,4-galactosyl transferase which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl-glucosaminylated lactose, and genes encoding a biosynthetic pathway to UDP-GIcNAc and UDP-Gal. The fermentation was performed by culturing said cell in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNnT which was accompanied by lacto-N-triose II, pLNnH and lactose in the fermentation broth (see WO 2017/101958, WO 2017/182965 or 2017/221208). The broth was subjected to a standard cell removal operation by UF, NF with diafiltration, decolourization with activated charcoal and ion exchange treatment (strong acidic (H⁺) resin and weak basic resin), optionally followed by chromatography on a polystyrene crosslinked with divinylbenzene (PS-DVB) and functionalized with bromine on the aromatic ring, thereby producing LNnT which was accompanied with lacto-N-triose II (=7-13 % to LNnT) and unreacted lactose (=2-20 % to LNnT) as major carbohydrate impurities, and optionally pLNnH (less than 3 % to LNnT).

Pressure filtration: 75 ml suspension taken from the crystalline mass/suspension resulting from the crystallization after homogenization was filtered at 1 bar in a filtration chamber. The time of the filtration was measured until the beginning of the liquid-gas mass transfer from the pressure filtration chamber and the overall filtration rate was calculated (cm/s). The obtained filter cake was ventilated for 1 min after the beginning of liquid-gas mass transfer at 1 bar and this filter cake was dried overnight at 65 °C and 50 mbar generating loss on drying (i.e. LoD). The concentrations of LNnT from the mother liquor and from the dried solid were assayed by HPLC based on which the crystallization yield and the methanol consumption of the crystallization (the weight of methanol per weight of crystallized LNnT) were calculated.

Example 1

This example illustrates a crystallization in accordance with WO 2011/100980.

A crude solid LNnT (50 g, containing 87.5 wt% of LNnT and 1 .4 wt% of lacto-N-triose II among other carbohydrates) was dissolved in 1 10 ml water. 120 ml of MeOH was added into the syrup followed by the addition of 20 ml of MeOH/H₂O (1/1 by volume) mixture. The solution was added into a crystallizer and heated to 50 °C while agitating at 300 rpm. To the above agitated solution, 600 ml of MeOH (prewarmed to 50 °C) was added in three portions over 30 min. The temperature of the crystallizer was allowed to cool down to 40 °C during the addition of the 600 ml of MeOH. After the entire addition of MeOH the slurry was agitated for 3 h while cooling down to room temperature.

After pressure filtration and drying, 47.9 g of a crystalline product was obtained (assay: 90.1 wt% LNnT polymorph III, 1.3 wt% lacto-N-triose II), crystallization yield: 98.7 %.

16

RECTIFIED SHEET (RULE 91) ISA/EP 2

A concentrated LNnT syrup (149.4 g, total dissolved solid (TDS): 48.3 Brix, LNnT: 37.5 wt%, lactose: 1 .3 wt%, lacto-N-triose II: 2.75 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 50 °C while agitating at 300 rpm. To the above agitated solution, a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and pre-warmed to 50 °C was added. The seeded syrup turned to a slurry and was agitated for 1 h at 50 °C. Then methanol was fed into the crystallizer over 25 h in two fractions generating a methanol to water weight fraction of 3 at the end of the crystallization. The first half of methanol was added over 20 h followed by the addition of the second half in 5 h at 50 °C. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium.

After pressure filtration and drying, 62.9 of a crystalline product was obtained (assay: 88.2 wt% LNnT polymorph III, 2.7 wt% lacto-N-triose II, 0.3 wt% lactose), crystallization yield: 98.9 %.

A concentrated LNnT syrup (150.7 g, total dissolved solid (TDS): 48.1 Brix, LNnT: 37.3 wt%, lactose: 0.9 wt%, lacto-N-triose II: 4.65 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 50 °C while agitating at 300 rpm. To the above agitated solution, a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and prewarmed to 50 °C was added. The seeded syrup turned to a slurry and was agitated for 1 h at 50 °C. Then methanol was fed into the crystallizer over 25 h in two fractions generating a methanol to water weight fraction of 3 at the end of the crystallization. The first half of methanol was added over 20 h followed by the addition of the second half in 5 h at 50 °C. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium.

After pressure filtration and drying, 60.6 g of a crystalline product was obtained (assay: 86.3 wt% LNnT polymorph III, 2.8 wt% lacto-N-triose II, 0.1 wt% lactose), crystallization yield: 90.9 %.

A concentrated LNnT syrup (139.9 g, total dissolved solid (TDS): 47.9 Brix, LNnT: 34.15 wt%, lactose: 6.3 wt%, lacto-N-triose II: 2.8 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 50 °C while agitating at 300 rpm. To the above agitated solution, a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and prewarmed to 50 °C was added. The seeded syrup turned to a slurry and was agitated for 1 h at 50 °C. Then methanol was fed into the crystallizer over 25 h in two fractions generating a methanol to water weight fraction of 3 at the end of the crystallization. The first half of methanol was added over 20 h followed by the addition of the second half in 5 h at 50 °C. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium.

After pressure filtration and drying, a crystalline product was obtained (assay: 84.7 wt% LNnT polymorph III, 3.3 wt% lacto-N-triose II, 2.1 wt% lactose), crystallization yield: 97.5 %.

Example 5

A concentrated LNnT syrup (169.4 g, total dissolved solid (TDS): 46.6 Brix, LNnT: 36.2 wt%, lactose: 1 .25 wt%, lacto-N-triose II: 2.65 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 50 °C while agitating at 300 rpm. To the above agitated solution, a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and prewarmed to 50 °C was added. The seeded syrup turned to a slurry and was agitated for 1 h at 50 °C. Then methanol was fed into the crystallizer over 10 h at 50 °C generating a methanol to water weight fraction of 3 at the end of the crystallization. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium.

After pressure filtration and drying, 70.8 g of a crystalline product was obtained (assay: 84.8 wt% LNnT polymorph III, 2.7 wt% lacto-N-triose II, 0.3 wt% lactose), crystallization yield: 97.9 %.

Example 6

A concentrated LNnT syrup (146.6 g, total dissolved solid (TDS): 49.1 Brix, LNnT: 38.1 wt%, lactose: 1 .3 wt%, lacto-N-triose II: 2.8 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 35 °C while agitating at 300 rpm. To the above agitated solution, a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and pre-warmed to 35 °C was added. The seeded syrup turned to a slurry and was agitated for 1 h at 35 °C. Then methanol was fed into the crystallizer over 25 h in two fractions generating a methanol to water weight fraction of 3 at the end of the crystallization. The first half of methanol was added over 20 h followed by the addition of the second half in 5 h at 50 °C. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium.

After pressure filtration and drying, a crystalline product was obtained.

Example 7

A concentrated LNnT syrup (219.2 g, total dissolved solid (TDS): 30.4 Brix, LNnT: 23.6 wt%, lactose: 0.8 wt%, lacto-N-triose II: 1.7 wt%, among other carbohydrates) was added into a crystallizer. The syrup was heated to 50 °C while agitating at 300 rpm. To the above agitated solution, methanol was added to generate a methanol to water weight fraction of 1 , followed by a suspension of LNnT polymorph III seed crystal (0.7 wt% relative to TDS) suspended in methanol (0.045 g/ml) and pre-warmed to 50 °C. The seeded syrup turned to a slurry and was agitated for 0.5 h at 50 °C. Then methanol was fed into the crystallizer over 16 h at 50 °C in two fractions generating a methanol to water weight fraction of 3 at the end of the crystallization: he % parts of methanol was added over 10 h followed by the addition of the % parts in 6 h. At the end of methanol feeding the slurry was cooled down to 25 °C while agitating for 5 h to reach a thermodynamic solid-liquid equilibrium. After pressure filtration and drying, 56.6 g of a crystalline product was obtained (assay: 86.9 wt% LNnT polymorph III, 2.2 wt% lacto-N-triose II, 0.2 wt% lactose), crystallization yield: 95.0 %.

Results of the comparison tests

The table below summarizes the filtration rates determined from the pressure filtration experiment, LoD measured from the filter cake drying and the methanol consumption of the crystallization (the weight of methanol per weight of crystallized LNnT) in each example.

Claims

1. A method for crystallizing LNnT polymorph III comprising:

1) providing an aqueous solution or syrup comprising LNnT,

2) optionally, adding polymorph III of LNnT, optionally as a suspension in methanol, to the aqueous solution or syrup comprising LNnT, thereby generating a slurry,

3) adding methanol to the aqueous solution or syrup comprising LNnT according to step 1) or to the slurry according to step 2), to obtain a wet crystalline mass, and

4) filtering the crystalline LNnT from the wet crystalline mass, wherein:

- the total solid content of the aqueous solution or syrup comprising LNnT provided in step 1) is at least 30 Brix (°Bx) as measured by a calibrated refractometer,

- the amount of LNnT in the total solid content is at least 65 wt%, preferably at least 70 wt%, more preferably at least 75 wt%,

- in step 3), the methanol is added at 35-55 °C over at least 10 hours, and

- the amount of methanol added in step 3) is calculated so that the methanol to water weight fraction in the wet crystalline mass at the end of the addition of the methanol is 2-4, preferably around 3.

2. The method according to claim 1 , wherein the aqueous solution or syrup comprising LNnT contains one or more of lacto-N-triose II, pLNnH and lactose.

3. The method according to claim 2, wherein the LNnT/lacto-N-triose II weight ratio is at least 6, preferably at least 8, more preferably at least 12.

4. The method according to claim 2, wherein the LNnT/lactose weight ratio is at least 5, preferably at least 12.5, more preferably at least 25.

5. The method according to claim 2, wherein the LNnT/pLNnH weight ratio is at least 50, for example 50-200.

6. The method according to any of the preceding claims, wherein in step 2) crystalline LNnT polymorph III is added to the aqueous solution or syrup comprising LNnT according to step 1) for seeding, preferably the amount of polymorph III of LNnT is 0.4-1 .2 wt% relative to the total solid content of the aqueous solution or syrup provided in step 1 ).

7. The method according to claim 6, wherein the crystalline LNnT polymorph III is added as a suspension in methanol.

8. The method according to claim 7, wherein the suspension contains 0.03-0.06 g of crystalline LNnT in 1 ml of methanol.

9. The method according to any of the preceding claims, wherein the methanol is added in step 3) over 25±2 hours.

10. The method according to any of the preceding claims, wherein the methanol is added in two phases.

11 . The method according to claim 10, wherein 25-50 % of the overall methanol is added in a slower addition rate in the first phase than that of the remaining 50-75 % in the second phase.

12. The method according to claim 11 , wherein 50 % of the overall methanol is added in the first phase.

13. The method according to claim 11 or 12, wherein the first phase is four times longer than the second phase.

14. The method according to any of the preceding claims, wherein after step 3) the temperature is decreased to a final temperature of around 20-25 °C and the crystalline mass of LNnT is stirred for at least for 1 -2 hours.

15. The method according to claim 1 , wherein

- the aqueous solution or syrup comprising LNnT provided in step 1) contains lacto-N- triose II and lactose,

- the total solid content of the aqueous solution or syrup comprising LNnT provided in step 1) is 46-50 Brix (°Bx) as measured by a calibrated refractometer,

- in step 3), the methanol is added at 50±2 °C over 25±2 hours in two phases, wherein 50 % of the overall methanol is added in the first phase and the first phase is four times longer than the second phase.