Crystallization of lacto-n-triose ii

Abstract

Disclosed is a method for the production of crystalline LNT-II, a novel polymorph of a crystalline LNT-II, and its uses.

Description

[0001] Crystallization of lacto-N-triose II

[0002] The present disclosure concerns a method for providing crystalline lacto-N-triose II (LNT-II), a novel polymorph of crystalline lacto-N-triose II (LNT-II), and uses thereof.

[0003] Background

[0004] Mother’s milk is regarded as the best food for the development of her infant. Human milk comprises fats, proteins, saccharides, vitamins, minerals, and trace elements. Besides the disaccharide lactose, human milk contains a family of structurally distinct oligosaccharides which are also known as human milk oligosaccharides (HMOs) (Urashima T. et al., (2011) Milk Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1). To date more than 150 structurally distinct oligosaccharides were identified in human milk. With very few exceptions, HMOs are characterized by a lactose disaccharide moiety at their reducing end. Many HMOs further contain one or more fucose moieties, at least one / V-acetylglucosamine moiety and / or one or more / V-acetylneuraminic acid moieties. Furthermore, there are linear as well as branched representative molecules within the family of HMOs. Generally, the monosaccharide residues of HMOs are selected from the group consisting of D-glucose, D-galactose, / V-acetylglucosamine, L-fucose and / V-acetylneuraminic acid.

[0005] The importance of HMOs for infant nutrition, health and development is directly linked to their biological activities which include protecting the neonate from pathogens, supporting development of the infant’s immune system and cognitive abilities. In addition, HMOs serve as a substrate for beneficial bacteria in the infant’s evolving microbiome such as Bifidobacteria and Lactobacilli.

[0006] The health benefits associated with HMOs render them a valued ingredient for infant formula. However, this requires an industrial scale production for at least some HMOs. Due to challenges and drawbacks that are associated with the chemical synthesis of oligosaccharides, industrial scale production of HMOs currently focuses on biochemical and especially fermentative approaches utilizing metabolically engineered microbial cells for intracellular biosynthesis of an HMO of interest. The industrial scale production of certain HMOs for infant formula requires the recovery of the HMO of interest from a complex composition, i.e. a cell lysate and / or a fermentation broth. Typically, the HMO of interest is dried at the end of the recovery process, either by means of spray-drying or by means of crystallization.

[0007] A member of the HMO family is the trisaccharide lacto- N-triose II (LNT-II; 2- Acetamido-2-deoxy-b-D-glucopyranosyl-(1->3)-b-D-galactopyranosyl-(1->4)-D- glucose; GlcNAc(pi,3)Gal(pi,4)Glc, CAS No. 75645-27-1). Lacto- N-triose II is able to modulate the growth of Bifidobacterium longum subsp. infantis, may increase intestinal immunity, may increase adhesion of the commensal bacterium L plantarum WCFS1 to intestinal epithelial cells, and appears to support the crosstalk between commensal bacteria and epithelial cells.

[0008] Early isolation and characterization of LNT-II took place in the 1950s by Richard Kuhn and co-workers. Those days, HMOs were isolated from the carbohydrate fraction of mother’s milk via chromatographic purification on activated charcoal I celite columns. Among several other HMOs, Kuhn and co-workers crystallized lacto- N-triose I & II (Kuhn et al., Chem. Ber. 1956, 89(4), 1027-1033). LNT-II was crystallized in that amorphous LNT-II powder had been dissolved in hot methanol, the thus obtained solution had been treated with animal charcoal and filtered. Then ethanol had been added dropwise to the filtrate until cloud formation. Slow evaporation of the solvent had led to a white microcrystalline powder (herein referred to as “LNT-II polymorph A”).

[0009] International Publication WO 2002 / 100875 discloses also a process for producing crystals of an oligosaccharide such as LNT-II, wherein an aqueous solution containing the oligosaccharide is added to a water-miscible organic solvent. LNT-II crystals were obtained in that 100 ml of an aqueous LNT-II solution (cone. 200 g / L) was gradually added to 500 ml methanol heated to 60°C in about 30 minutes, and the resulting mixture was refluxed at 60°C for about 3 hours, then cooled to 20°C and stirred for 1 hour. LNT-II crystals were obtained and washed with methanol, then dried by airflow. A Powder X-ray diffraction spectrum was taken and the data presented in table 3 of the document describes a crystal form of LNT-II (herein referred to as “LNT-II polymorph B”).

[0010] Due to the very similar conditions for crystallization from a boiled methanol solution, there is a high probability that LNT-II polymorph A of Kuhn’s disclosure and LNT-II polymorph B of WO 2002 / 100875 are the same crystalline polymorph form of LNT-II.

[0011] Crystallization or recrystallization is one of the simplest and cheapest methods to separate a product from contaminants and obtain a pure substance. In addition, providing one or more crystalline modifications (polymorphs) of a solid compound is an important factor in product development, because the different crystalline forms affect the compound's properties — for example, solubility, dissolution rate, thermodynamic stability, density, hygroscopicity, electrical properties (such as dielectric constant, conductivity), mechanical properties (such as friability, hardness, breaking strength, elasticity), optical properties (such as colour, transparency, refraction), etc. — diversely. Polymorphs enlarge the repertoire of materials that are available for improving the product's characteristics.

[0012] For this reason, ways have been sought for obtaining other polymorphs of LNT-II which may be easier or less expensive to produce in crystalline form.

[0013] In another aspect, there is a need for a crystallizing method for obtaining a crystalline LNT-II, optionally easily included in the general process of recovering LNT-II from an aqueous solution containing LNT-II and one or more contaminants, educts and / or by-products of its synthesis, without the use of a toxic and volatile solvent.

[0014] The present invention provides a method for producing a crystalline form of LNT-II. The problem is further solved by providing a novel crystalline LNT-II polymorph as described herein below. Surprisingly, the method described herein below resulted in crystalline LNT-II possessing a crystal structure that differs from the structures of LNT-II crystals that were obtained by crystallization utilizing an organic solvent, i.e. utilizing a crystallization from a methanol solution.

[0015] Summary

[0016] In a first aspect, provided is a method for producing crystalline LNT-II.

[0017] In a second aspect, provided is a novel polymorph C of LNT-II obtainable by a process as characterized below.

[0018] In a third aspect, provided is a crystalline form of LNT-II as novel polymorph.

[0019] In a fourth aspect, provided is the use of the polymorph C of LNT-II for manufacturing pharmaceutical and / or nutritional compositions.

[0020] In a further aspect, provided are nutritional and / or pharmaceutical compositions containing polymorph C LNT-II.

[0021] Brief description of the drawings

[0022] FIG. 1 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 1.

[0023] FIG. 2 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 2.

[0024] FIG. 3 displays the X-ray powder diffraction pattern of the crystalline LNT-II polymorph C obtained in example 3.

[0025] FIG. 4 is a schematic representation which illustrates the method for producing crystalline LNT-II pursuant to the present invention. Detailed description

[0026] The present invention will be described with respect to particular embodiments and with reference to drawings, but the invention is not limited thereto but only by the claims. Furthermore, the terms “first”, “second” and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0027] It is to be noticed that the term “comprising”, used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

[0028] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0029] Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

[0030] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0031] Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention. In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

[0032] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the true spirit or technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0033] 1. Method for producing crystalline LNT-II

[0034] According to the first aspect, provided is a method for producing a crystalline LNT-II.

[0035] The method comprises the following steps: i) crude lacto-N-triose II (LNT-II) is dissolved in water, or an aqueous solution of lacto-N-triose II (LNT-II) is provided, ii) the aqueous solution of lacto-N-triose II (LNT-II) is adjusted to a concentration of at least about 40 wt.-%, iii) the adjusted aqueous solution is cooled to a temperature of about 2 °C to about 15 °C, iv) the cooled aqueous solution is kept at a temperature in the range of from about 2°C to about 15°C for at least 2 days to allow formation of crystals, v) optionally washing the crystals with a water / ethanol mixture; and vi) drying the crystals at ambient pressure.

[0036] In a first step an aqueous solution of lacto-N-triose II (LNT-II) is provided. The aqueous solution can be a crude aqueous solution from a work-up process of a fermentation method to produce LNT-II. The aqueous solution can as well be a solution of an amorphous or crystalline crude form of LNT-II in water. The water used to prepare the solution is preferably a kind of purified, for instance HPLC grade, water. The concentration of the aqueous LNT-II solution is adjusted to at least 40 wt.-%, in particular to about 45 wt.-%, 50 wt.-%, 55 wt.-%, 60 wt.-%, or 65wt.-%.

[0037] In a preferred embodiment of the inventive method the solution from which lacto-N- triose II is crystallized does not contain methanol, and preferably does not contain an organic solvent.

[0038] In a preferred embodiment of the method the concentration of the aqueous solution of lacto-N-triose II (LNT-II) is adjusted to a concentration of more than 50 wt.-%, preferably more than 60 wt.-%, most preferably to a concentration of about 65 wt.- %.

[0039] As an example, solid LNT-II can be dissolved in an appropriate amount of water by rotating and moderate heating to a temperature of about 30°C to about 40°C, preferably to about 35°C, until completely dissolved.

[0040] In another step the aqueous solution of LNT-II is cooled to a temperature of about 2 °C to about 15 °C, and the cooled aqueous solution is kept at a temperature in the range of from about 2°C to about 15°C for at least 2 days to allow formation of crystals. In a preferred embodiment of the inventive method the aqueous solution of lacto-N- triose II (LNT-II) is cooled to a temperature of between 3°C and 10°C, preferably to about 4°C to 6°C.

[0041] This temperature range is advantageous in terms of optimizing the speed of crystal growth, overall yield and purity of the resulting crystal structure, which can often be considered to be interdepentend features of the crystallization process.

[0042] As an example, the crystallization time is from about 5 hours to about 50 days, or from about 1 day to about 40 days, preferably from about 2 days to about 30 days, or from about 5 days to about 25 days.

[0043] In a final step the resulting crystal mass is dried at ambient pressure. Preferably, the drying is carried out at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.

[0044] Generally, the drying of the crystals is continued until a constant weight of the crystal mass is observed.

[0045] The preferred drying temperature range is advantageous in terms of speed of drying, overall yield of crystals with constant weight, and preserving the crystal structure without risking a part of the material to become amorphous.

[0046] The inventive method can be distinguished from the previous known methods of crystallizing LNT-II especially in that the crystallization is carried out from an aqueous crystallization solution rather than from a methanol solution. In general, water is not considered to be a good choice as a crystallization medium for oligosaccharides because the crystallization tends to be slow and sluggish, often ending in more honey-like outcomes or hydrogels. Further drying of such outcomes often requires harsher conditions, and in the end then leads to amorphous solid forms. Therefore, all previously known crystallization methods of LNT-II started from a solution having methanol as solvent. However, with the present invention, it was surprisingly found that LNT-II can be nicely crystallized from an aqueous solution resulting in obtaining crystals in a good yield.

[0047] The inventive method for producing crystalline LNT-II is thus advantageous in that the use of an organic solvent and / or an antisolvent for crystallizing the LNT-II is not necessary. Neither methanol as toxic and harmful solvent nor ethanol or another organic solvent is required as medium from which the crystallization is carried out. This reduces costs in manufacturing crystalline LNT-II, makes the production process safer and more environment friendly, and also makes the product itself safer because no traces of any solvent other than water can be left, for instance as part of the crystal structure.

[0048] These advantages also apply with the optional step of washing the crystals with an ethanol / water mixture because the washing solvent is generally not included in the crystal structure once the crystals are grown, and surface adherent traces of the ethanol used as washing liquid can be easily removed, free of any residue .

[0049] Moreover, it was surprising that the obtained crystals were formed in a novel polymorph form (polymorph C) of LNT-II. As outlined below in more detail, the novel crystal structure of the crystalline LNT-II obtained by the method of the present invention was determined by X-Ray diffraction analysis and compared to the details of the X-Ray diffraction pattern of the LNT-II polymorph B as described in WO 2002 / 100875, and thus found to be novel.

[0050] Different polymorphs of a crystalline material are known often to possess different physical behavior, for instance in respect to solubility, dissolution rate, thermodynamic stability, density, hygroscopicity, electrical properties (such as dielectric constant, conductivity), mechanical properties (such as friability, hardness, breaking strength, elasticity), optical properties (such as colour, transparency, refraction), etc. Like that, the direct product of the novel method of the invention is able to improve the workablity and / or the features of various special uses thereof, in particluar in respect to a nutritional or pharmaceutical use.

[0051] 2. Crystalline LNT-II Polymorph C

[0052] In a second aspect, provided is crystalline LNT-II polymorph C. The LNT-II polymorph C is a polymorph that exhibits an X-ray powder diffraction reflections, based on a measurement using Cu Ka (1 ,54056 A) radiation, at 8,19°, 19,29°, and 19,60° ±0.2 20 angle, preferably at 8,19°, 19,29°, 19,60°, and 19,86° ±0.2 20 angles, more preferably at 8,19°, 19,29°, 19,60°, 19,86°, and 9,92° ±0.2 20 angles, yet more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, and 21 ,37° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, and 21 ,56° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, and 20,14° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, and 24,98° ±0.2 20 angles, most preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, and 30,18° ±0.2 20 angles, particularly at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, 30,18°, and 14,98° ±0.2 20 angles.

[0053] In one embodiment, the LNT-II polymorph C is a polymorph that exhibits an X-ray powder diffraction reflections, based on a measurement using Cu Ka (1 ,54056 A) radiation, at at 8,19°, 19,29°, and 19,60° ±0.1 20 angle, preferably at 8,19°, 19,29°, 19,60°, and 19,86° ±0.1 20 angles, more preferably at 8,19°, 19,29°, 19,60°, 19,86°, and 9,92° ±0.1 20 angles, yet more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, and 21 ,37° ±0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, and 23,05° ±0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, and 21 ,56° ±0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, and 20,14° ±0.1 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, and 24,98° ±0.1 20 angles, most preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, and 30,18° ±0.1 20 angles, particularly at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, 30,18°, and 14,98° ±0.1 20 angles.

[0054] Preferably, the measurement has a range of measurements from 2° to 40° 20 with a step size of 0.013° 20.

[0055] In other embodiment, the LNT-II polymorph C has an X-ray powder diffraction diagram as shown in FIG. 1 or FIG. 2 or FIG. 3.

[0056] The X-ray powder diffraction (XRPD) reflections of LNT-II polymorph C as shown in Fig. 1 are set forth in Table 1.

[0057] Table 1

[0058] The powder X-ray diffraction pattern of the crystalline LNT-II polymorph C of this invention is different from the known X-ray diffraction pattern of LNT-II polymorph B as there are notable differences which show in their respective diffractogram. In comparison, the prior art LNT-II polymorph B has peak positions of the three peaks with the highest relative intensity at 4.78°, 4.49°, and 9.25° 20 angles, which are all not present in the X-ray pattern of the polymorph C of the present invention. Moreover, the most characteristic peaks at 8,19°, 19,29°, and 19,60° 20 angles of the LNT-II polymorph C in the present invention are all absent in the X-ray pattern of the prior art polymorph B.

[0059] The crystalline LNT-II polymorph C can be considered as an anomeric mixture of a- and p-anomers or even a pure form of one of the anomers. Preferably, the crystalline LNT-II polymorph C of this invention is substantially pure. The expression “substantially pure” preferably means that the crystalline LNT-II polymorph contains less than 10 w / w % of impurity, preferably less than 5 w / w % of impurity, more preferably less than 2 w / w % or less than 1 w / w% of impurity, most preferably less than 0.5 w / w % of impurity, wherein “impurity” refers to any physical entity different from the crystalline LNT-II polymorph, such as an amorphous LNT-II, different LNT-II polymorph(s) or form(s), unreacted intermediate(s) remained from the synthesis of LNT-II, by-product(s), degradation product(s), inorganic salt(s) and / or other contaminants different from water.

[0060] Preferably, the crystalline LNT-II polymorph C of this invention is free from an organic solvent. In particular, the crystalline LNT-II polymorph C of this invention is free from methanol.

[0061] Advantageous, the novel LNT-II polymorph C does not contain any traces of an organic solvent, and especially no traces of methanol. Thus, no further washing steps are needed and a high product safety can be quite easily achieved. Especially in view of the field of its main use as part of a nutritional formula for infants or other vulnerable humans in need of a special nutrition this is very beneficial as even very small traces of methanol are regarded as being harmful.

[0062] 3. Crystalline LNT-II polymorph C obtainable by a process for its production

[0063] According to a third aspect, provided is polymorph C as crystalline LNT-II obtainable by a process for its production comprising the following consecutive steps: a) Providing an aqueous solution of lacto-N-triose II (LNT-II), b) Increasing the concentration of the aqueous solution of step a) to a concentration of more than 40 wt.-%, preferably more than 50 wt.-%, most preferably more than 60 wt.-%, in particular to a concentration of about 65 wt.-%, thereby obtaining a concentrated solution or slurry, c) Setting the resulting concentrated solution or slurry to crystallization at a temperature between 2°C and 15°C, preferably between 3°C and 10°C, more preferably at about 4°C to 6°C, for a period of time, thereby obtaining a white crystal mass, and d) Drying the resulting crystal mass at ambient pressure at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.

[0064] In step a) an aqueous solution of lacto-N-triose II (LNT-II) is provided. The aqueous solution can be a crude aqueous solution from a work-up process of a fermentation method to produce LNT-II. The aqueous solution can as well be a solution of an amorphous or crystalline form of LNT-II in water. The water used to prepare the solution is preferably a kind of purified, for instance HPLC grade, water. The concentration of the aqueous LNT-II solution in step a) is not critical and can range from about 0.5 wt.-% to about 50.0 wt.-%, preferably from about 1.0 wt.-% to about 45.0 wt.-%, and most preferred from about 30 wt.-% to about 45 wt.-%, in particular about 40 wt.-%. As an example, solid LNT-II can be dissolved in an appropriate amount of water by rotating and moderate heating to a temperature of about 30°C to about 40°C, preferably to about 35°C, until completely dissolved.

[0065] In step b) the concentration of the aqueous solution of LNT-II is increased to a concentration of more than 40 wt.-%, preferably more than 50 wt.-%, most preferably more than 60 wt.-%, in particular to a concentration of about 65 wt.-%. At such a concentration, it is observed that this is a point where a slurry is obtained.

[0066] The increase in concentration can be achieved by any means known to the skilled person. Preferably, the increase in concentration is achieved by applying reduced pressure while moderately heating and rotating. In step c) the resulting concentrated solution or slurry is set to crystallization at a temperature between 2°C and 15°C, preferably between 3°C and 10°C, more preferably at about 4°C to 6°C, for a period of time, thereby obtaining a white crystal mass. As an example, the crystallization time is from about 5 hours to about 50 days, or from about 1 day to about 40 days, preferably from about 2 days to about 30 days, or from about 5 days to about 25 days.

[0067] In step d) the resulting crystal mass is dried at ambient pressure at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.

[0068] In general, water is not considered to be a good choice as a crystallization medium for oligosaccharides because the crystallization tends to be slow and sluggish, often ending in more honey-like outcomes or hydrogels. Therefore, all previously known crystallization methods of LNT-II started from a solution having methanol as solvent. However, with the present invention, it was surprisingly found that LNT-II can be nicely crystallized from an aqueous solution resulting in obtaining crystals in a good yield. Moreover, it was surprising that the obtained crystals were formed in a novel polymorph form (polymorph C) of LNT-II.

[0069] Like that, the method is not only clearly easier and safer than the methods for producing crystal LNT-II because no harmful organic solvent is used, and especially no methanol, but also a novel polymorph C can be produced which shows a combination of different physical behaviors that can be beneficial in regard to its use in nutritional and pharmaceutical applications.

[0070] In another embodiment of the present invention, after step c), the crystal mass is washed with a water / ethanol mixture before the drying of step d). Like that, any possibly remaining traces of the production process of the LNT-II, especially by fermentation process and its work-up, can be removed. Washing with a mixture of ethanol and water, preferably in a relation of about 60 % (v / v) to about 75 % (v / v) ethanol, is much less harmful in the production process than methanol.

[0071] 4. Use of crystalline LNT-II polymorph C for manufacturing compositions

[0072] In a fourth aspect, provided is the use of crystalline LNT-II polymorph C as described herein before and / or as obtained by a method as described herein before for the manufacturing of a nutritional and / or pharmaceutical composition.

[0073] 4. 1 for manufacturing nutritional compositions

[0074] The crystalline LNT-II polymorph C according to this invention is suitable for nutritional use. In this regard, LNT-II, alone or in combination with other N- acetyllactosamine and / or fucose and / or sialic acid containing human milk oligosaccharides, is particularly effective in the education and / or maturation of the immune system of neonatal infants, and have preventive effect against secondary infections following viral infections such as influenza. The use of LNT-II as prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and / or asthma in infants, in preventing and treating pathogenic infections in such as diarrhoea in infants.

[0075] In a further embodiment, the crystalline LNT-II polymorph C of the invention is used for the preparation of nutritional formulations, such as foods, drinks and feeds, preferably infant formulas, food supplements and digestive health functional food. The nutritional formulations can be prepared in a conventional manner. For example, it can be prepared by admixing micronutrient components in appropriate proportions, and then adding vitamins and minerals while avoiding thermal degradation or decomposition of heat sensitive vitamins by adding them after homogenization. Lipophilic vitamins can be dissolved in a fat source before mixing. A liquid mixture is formed using water, whose temperature is preferably about 50- 80° C. to help dissolution or dispersal of the ingredients. The crystalline LNT-II polymorph C can be suitably added at this stage. The resulting mixture is then homogenized by flash heating to about 80-150° C. by means of steam injection, heat exchanger or autoclave. This thermal treatment also reduces significantly the bacterial load. The hot mixture is then cooled rapidly to about 60-80° C. If needed, further homogenization can be carried out at this temperature under high pressure of about 2-30 MPa. After cooling, heat sensitive constituents can be added, and the pH and the solids content can be conveniently adjusted. The resulting mixture is then dried in a conventional manner, such as by spray drying or freeze drying to powder. Probiotics can be added at this point by dry-mixing.

[0076] 4.2 for manufacturing pharmaceutical compositions

[0077] The crystalline LNT-II polymorph C according to this invention is suitable for pharmaceutical use. In this regard, LNT-II, alone or in combination with other N- acetyllactosamine and / or fucose and / or sialic acid containing human milk oligosaccharides, is particularly effective in the education and / or maturation of the immune system of neonatal infants, and have preventive effect against secondary infections following viral infections such as influenza. The use of LNT-II as prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and / or asthma in infants, in preventing and treating pathogenic infections in such as diarrhoea in infants.

[0078] Also in accordance with this invention, the crystalline LNT-II polymorph C of the invention, can be used for the preparation of pharmaceutical compositions. Pharmaceutical compositions can be manufactured in a conventional manner, e.g. as described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field. 5. Compositions containing the crystalline LNT-II

[0079] 5. 1 Nutritional compositions

[0080] Further in accordance with this invention, a nutritional formulation (such as food, drink or feed) is provided comprising the crystalline LNT-II polymorph C of this invention. The nutritional formulation can also contain edible micronutrients, vitamins and minerals as well. The amounts of such ingredient can vary depending on whether the formulation is intended for use with normal, healthy infants, children, adults or subjects having specialized needs (e.g. suffering from metabolic disorders). Micronutrients include for example edible oils, fats or fatty acids (such as coconut oil, soy-bean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid, linolenic acid etc.), carbohydrates (such as glucose, fructose, sucrose, maltodextrin, starch, hydrolysed cornstarch, etc.) and proteins from casein, soy-bean, whey or skim milk, or hydrolysates of these proteins, but protein from other source (either intact or hydrolysed) may be used as well. Vitamins may be chosen from the group consisting of vitamin A, B1 , B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid. The nutritional formulation can also contain the following minerals and trace elements: Ca, P, K, Na, Cl, Mg, Mn, Fe, Cu, Zn, Se, Cr or I.

[0081] In a preferred embodiment, the nutritional formulation is an infant formula, i.e. a foodstuff intended for use by infants during the first 4-6 months of life and satisfying, by itself, the nutritional requirements of infants. It can contain one or more probiotic Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk, carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof, lipids (e.g. palm olein, sunflower oil, safflower oil) and vitamins and minerals essential in a daily diet. The infant formula preferably contains the crystalline LNT-II polymorph C of the invention in a concentration mimicking the natural concentration in breast milk. The concentration of LNT-II in human breast milk is generally around 2 - 3 mg / L in the colostrum and sinks to around 0.05 mg / L during lactation. Like that, the infant formula preferably contains the novel crystalline LNT-II polymorph C in an amount which results in a concentration of between 2 - 3 mg / L and 0.05 mg / L in the final liquid foodstuff. In another preferred embodiment, the nutritional formulation can be a food supplement containing the crystalline LNT-II polymorph C. The food supplement can also contain one or more probiotics in an amount sufficient to achieve the desired effect in an individual, preferably in children and adults. The food supplement can also contain vitamins, minerals, trace elements and other micronutrients as well. The food supplement may be for example in the form of tablets, capsules, pastilles or a liquid. The supplement can also contain conventional additives, such as binders, coatings, emulsifiers, solubilising agents, encapsulating agents, film forming agents, adsorbents, carriers, fillers, dispersing agents, wetting agents, jellifying agents, gel forming agents, etc. The daily dose of LNT-II typically ranges from 0.01 to 3.0 g.

[0082] According to a more preferred embodiment, the food supplement is a digestive health functional food as the administration of LNT-II provides a beneficial effect on digestive health. Digestive health functional food is a processed food used with intention enhance and preserve digestive health by crystalline LNT according to the present invention as physiologically functional ingredient or component in forms of tablet, capsule, powder, etc. Different terms such as dietary supplement, nutraceutical, designed food, health product may also be used to refer to functional food.

[0083] 5.2 Pharmaceutical compositions

[0084] Also in accordance with this invention, a pharmaceutical composition is provided comprising the crystalline LNT-II polymorph C of the invention as an active ingredient and one or more pharmaceutically acceptable carriers such as additives, adjuvants, excipients and diluents (water, gelatine, talc, sugars, starch, gum arabic, vegetable gums, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, lubricants, colorants, fillers, wetting agents, etc.). Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field. The dosage form for administration includes, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions, capsules, syrups, injections, liquids, elixirs, extracts and tincture. EXAMPLES

[0085] XRPD investigation of the collected crystals was conducted with the following specifics:

[0086] Instrument: PANalytical X'Pert Pro, QS-Nr. 02204, geometry: transmission, detector: Pixcel (3D), radiation: Cu Ka (1 ,54056 A), tube settings: 40 kV, 40 mA, range: 2-40° 2Theta, step width: 0,013° 2Theta, measuring time per step: 25 s, preparation: sample carrier with acetate foils, software: PANalytical HighScorePlus v.4, reference databank: ICDD PDF-4 (2021) / external standards, evaluation according to: DIN EN 13925:2003.

[0087] Example 1 Crystallization of lacto-N-triose II from a crude aqueous solution

[0088] An aqueous solution having a dry solid content of 31.09 % (w / v) and containing LNT-II at a purity of 74 % based on the peak areas of the HPLC chromatogram was concentrated by using a rotary evaporator at 60 °C and a reduced pressure of 42 mbar. The dry solid content of the concentrate was adjusted to a solids content of 61.48 weight-% and the resulting solution was cooled to about 4 to 6 °C. A crystal mass was obtained over time and washed with two volumes of 80 vol.% ethanolic solution (80 vol.% EtOH 120 vol.% H2O) at a temperature in the range of between 4 and 6 °C. Residual washing solution was removed from the crystals by using a funnel frit filter under reduced pressure. The process step of washing the crystals was repeated twice resulting in a drained and purified lacto-N-triose II crystal mass which was then dried in an oven at 35°C and ambient pressure. The thus obtained lacto-N-triose II (LNT-II) had a purity - based on HPLC area % - of 94.5%. The XRPD diffractogram of the crystals is shown in Fig. 1. Example 2 Recrystallization of lacto-N-triose II from an aqueous solution

[0089] Almost pure LNT-II already obtained from previous crystallization was recrystallized at a sugar concentration of 65% from pure deionized water. Therefore, 200 g of LNT-II were dissolved in 500 mL of water and dissolved by rotating at a temperature of 35 °C. Then, by applying reduced pressure while still heating and rotating the LNT-II concentration was set to 65% dry mass. The highly viscous slurry was then removed from the heating source and some seed crystals were given to it and quickly mixed. The slurry was then set to crystallization at 4 °C for three days which resulted in a homogenous white crystal mass.

[0090] From the obtained crystal mass aliquots of roughly 10 to 12 g were taken and spread on previously weighted sample carrier and exposed to two different drying methods for 20 to 30 days while weighting after certain time intervals. The two methods were firstly drying at standard air exposure (20 °C, 40 - 60% humidity), and secondly drying in a ventilated oven at 37 °C.

[0091] Constant weight was observed after 3 days in both of the two drying methods and the yield was 73 %. All of the thus obtained crystals were analyzed by XRPD and it was shown that in all probes the same crystal structure of polymorph C LNT-II was achieved. One of the thus measured XRPD diffractograms is shown in Fig. 2.

[0092] Example 3 Recrystallization of lacto-N-triose II from an aqueous solution with washing of crystals

[0093] Almost pure LNT-II already obtained from previous crystallization was recrystallized at a sugar concentration of 65% (w / v) from pure deionized water. Therefore, 200 g of LNT-II were dissolved in 500 mL of water and dissolved by rotating at a temperature of 35 °C. Then, by applying reduced pressure while still heating and rotating the LNT-II concentration was set to 65% dry mass (w / v). The highly viscous slurry was then removed from the heating source and some seed crystals were given to it and quickly mixed. The slurry was then set to crystallization at 4 °C for three days which resulted in a homogenous white crystal mass. From the obtained crystal mass aliquots of roughly 10 to 12 g were taken and spread on previously weighted sample carrier and before being exposed to two different drying methods for 20 to 30 days while weighting after certain time intervals, the crystal mass was washed with 60% EtOH in water. The two methods were firstly drying at standard air exposure (20 °C, 40 - 60% humidity), and secondly drying in a ventilated oven at 37 °C.

[0094] Constant weight was observed after 13 days in both of the two drying methods and the yield was 65 %. All of the thus obtained crystals were analyzed by XRPD and it was shown that in all probes the same crystal structure of polymorph C LNT-II was achieved. One of the thus measured XRPD diffractograms is shown in Fig. 3.

Claims

CLAIMS1. A method for producing crystalline lacto-N-triose II (LNT-II), wherein i) crude lacto-N-triose II (LNT-II) is dissolved in water, or an aqueous solution of lacto-N-triose II (LNT-II) is provided, ii) the aqueous solution of lacto-N-triose II (LNT-II) is adjusted to a concentration of at least about 40 wt.-%, iii) the adjusted aqueous solution is cooled to a temperature of about 2 °C to about 15 °C, iv) the cooled aqueous solution is kept at a temperature in the range of from about 2°C to about 15°C for at least 2 days to allow formation of crystals, v) optionally washing the crystals with a water / ethanol mixture; and vi) drying the crystals at ambient pressure.

2. The method of claim 1, wherein the solution from which lacto-N-triose II is crystallized does not contain methanol, and preferably does not contain an organic solvent.

3. The method of claim 1 or 2, wherein the aqueous solution of lacto-N-triose II (LNT-II) is adjusted to a concentration of more than 50 wt.-%, preferably more than 60 wt.-%, most preferably to a concentration of about 65 wt.-%.

4. The method of claim 1 to 3, wherein the aqueous solution of lacto-N-triose II (LNT-II) is cooled to a temperature of between 3°C and 10°C, preferably to about 4°C to 6°C.

5. The method of claim 1 to 4, wherein the crystals are dried at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.

6. A crystalline form of lacto-N-triose II (polymorph C) obtainable by a crystallization process comprising the following consecutive steps: a) Providing an aqueous solution of lacto-N-triose II (LNT-II), b) Increasing the concentration of the aqueous solution of step a) to a concentration of more than 40 wt.-%, preferably more than 50 wt.-%, most preferably more than 60 wt.-%, in particular to a concentration of about 65 wt.-%, thereby obtaining a slurry, c) Setting the resulting slurry to crystallization at a temperature between 2°C and 15°C, preferably between 3°C and 10°C, more preferably at about 4°C to 6°C, for a period of time, thereby obtaining a white crystal mass, and d) Drying the resulting crystal mass at ambient pressure at a temperature below 50°C, preferably below 40°C, more preferably at about 35°C - 37°C.

7. The crystalline form of lacto-N-triose II (polymorph C) of claim 6, wherein no methanol is used, and preferably wherein no organic solvent is used.

8. The crystalline form of lacto-N-triose II (polymorph C) of claim 6 or 7, wherein after step c) the crystal mass is washed with a water / ethanol mixture before the drying of step d).

9. Crystalline lacto-N-triose II (LNT-II) polymorph C, wherein the crystalline lacto- N-triose II polymorph C displays X-ray powder diffraction reflections, based on a measurement using CuKa radiation, at 8,19°, 19,29°, and 19,60° ±0.2 20 angle, preferably at 8,19°, 19,29°, 19,60°, and 19,86° ±0.2 20 angles, more preferably at 8,19°, 19,29°, 19,60°, 19,86°, and 9,92° ±0.2 20 angles, yet more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, and 21 ,37° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21,37°, and 23,05° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21,37°, 23,05°, and 21,56° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, and 20,14° ±0.2 20 angles, even more preferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, and 24,98° ±0.2 20 angles, mostpreferably at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, and 30,18° ±0.2 20 angles, particularly at 8,19°, 19,29°, 19,60°, 19,86°, 9,92°, 21 ,37°, 23,05°, 21 ,56°, 20,14°, 24,98°, 30,18°, and 14,98° ±0.2 20 angles, as measured by x-ray powder diffraction using an x- ray wavelength of 1.5406 A.

10. The crystalline lacto-N-triose II according to claim 9, wherein the lacto-N-triose II polymorph C is substantially pure.

11. The crystalline lacto-N-triose II polymorph C according to any one of claims 9 to 10, wherein the lacto-N-triose II polymorph C is substantially free from an organic solvent, and especially free from methanol.

12. Use of crystalline lacto-N-triose II polymorph C as obtained by a method of any one of the claims 1 to 8, or as defined in any one of claims 9 to 11 as an ingredient in a nutritional composition, preferably an infant formula.

13. A nutritional composition containing crystalline LNT-II polymorph C as obtained by a method of any one of the claims 1 to 8, or as defined in any one of claims 9 to 11.

14. The nutritional composition according to claim 13, wherein the nutritional composition is an infant formula.

15. A pharmaceutical composition containing crystalline LNT-II polymorph C as obtained by a method of any one of the claims 1 to 8, or as defined in any one of claims 9 to 11.

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