FIELD OF THE INVENTION

The present invention relates to the separation and isolation of neutral or sialylated human milk oligosaccharides (HMOs) from a reaction mixture in which they are produced.

BACKGROUND OF THE INVENTION

During the past decades, the interest in the preparation and commercialisation of human milk oligosaccharides (HMOs) has been increasing steadily. The importance of HMOs is directly linked to their unique biological activities. Therefore, HMOs have become important potential products for nutrition and therapeutic uses. As a result, low cost ways of producing industrially HMOs have been sought.

To date, the structures of more than 140 HMOs have been determined, and considerably more are probably present in human milk (Urashima et a\:. Milk oligosaccharides, Nova Biomedical Books, 2011; Chen k/v. Carbohydr. Chem. Biochem. 72, 113 (2015)). The HMOs comprise a lactose (Gaipi-4Glc) moiety at the reducing end and may be elongated with an N-acetylglucosamine, or one or more N-acetyllactosamine moiety /moi eties (Gai i-4GlcNAc) and/or a lacto-N-biose moiety (Gaipi-3GlcNAc). Lactose and the N-acetyllactosaminylated or lacto-N-biosylated lactose derivatives may further be substituted with one or more fucose and/or sialic acid residue(s), or lactose may be substituted with an additional galactose, to produce HMOs known so far.

Direct fermentative production of HMOs, especially of those being a tri saccharide, has recently become practical (Han et al. Biotechnol. Adv. 30, 1268 (2012) and references cited therein). Such fermentation technology has used a recombinant E. coll system wherein one or more types of glycosyl transferases originating from viruses or bacteria have been co-expressed to glycosylate exogenously added lactose, which has been internalized by the LacY permease of the E. coli. However, the use of a recombinant glycosyl transferase, especially series of recombinant glycosyl transferases to produce oligosaccharides of four or more monosaccharide units, has always led to by-product formation hence resulting in a complex mixture of oligosaccharides in the fermentation broth. Further, a fermentation broth inevitably contains a wide range of non-carbohydrate substances such as cells, cell fragments, proteins, protein fragments, DNA, DNA fragments, endotoxins, caramelized by-products, minerals, salts, or other charged molecules. For separating HMOs from carbohydrate by-products and other contaminating components, active carbon treatment combined with gel filtration chromatography has been proposed as a method of choice (WO 01/04341, EP-A-2479263, Dumon et al. Glycoconj. J. 18, 465 (2001), Priem et al. Glycobiology 12, 235 (2002), Drouillard et al. Angew. Chem. Int. Ed. 45, 1778 (2006), Gebus et al. Carbohydr. Res. 361, 83 (2012), Baumgartner et al. ChemBioChem 15, 1896 (2014)). Although gel filtration chromatography is a convenient lab scale method, it cannot be efficiently scaled up for industrial production. Instead, recent methods comprise ion exchange resin treatment for removing charged organic and inorganic substances combined with active charcoal treatment for decolorization (see e.g. WO 2015/106943, WO 2017/182965). However, active carbon treatment generally has the disadvantage that active carbon has the potential to adsorb relatively high amounts of HMOs, which may lead to decreased HMO yields. Moreover, the regeneration of active carbon is complicated, which makes the reuse of active carbon less attractive.

Alternative and/or improved procedures for isolating and purifying neutral or sialylated HMOs from non-carbohydrate components of the fermentation broth in which they have been produced, especially those suitable for industrial scale, are needed to improve the recovery yield of neutral or sialylated HMOs and/or to simplify prior art methods while the purity of the neutral or sialylated HMOs is at least maintained, and preferably, improved. Moreover, such alternative purification procedures preferably lead to purified neutral or sialylated HMOs that are free of proteins and recombinant materials originating from the used recombinant microbial strains, which are thus well suited for use in food, medical food, and feed applications.

SUMMARY OF THE INVENTION

The invention relates to a method for the purification of a neutral or sialylated human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of:

I. separating an HMO-containing stream from biomass;

II. purifying the HMO-containing stream with an acidic cation exchange resin, then

III. treating the resin eluate with an adsorbent resin.

Preferably, the method comprises a. separating the fermentation broth to form an HMO-containing stream and a biomass waste stream; b. purifying the HMO-containing stream by nanofiltration; c. purifying the HMO-containing stream with an acidic cation exchange resin; d. purifying the HMO-containing stream with an adsorbent resin; e. concentrating and drying the purified HMO-containing stream to obtain the neutral or sialylated HMO in solidified form.

Preferably, step b) precedes step c) and/or step d).

Preferably, step c) directly follows step b).

Preferably, step d) directly follows step c).

Even more preferably, the method does not comprise electrodialysis and/or treatment with anion exchange resin.

Even more preferably, electrodialysis step and/or treatment with anion exchange resin are excluded.

Even more preferably, purification/decolourization step with active carbon is excluded.

In another aspect, the invention relates to neutral or sialylated human milk oligosaccharides obtained by the method according to the invention.

Another aspect of the invention relates to neutral or sialylated human milk oligosaccharides obtained by the method according to the invention for use in medicine.

Another aspect of the invention relates to the use of neutral or sialylated human milk oligosaccharides obtained by the method according to the invention for food and/or feed applications.

Another aspect of the invention relates to a food or cosmetic product comprising neutral or sialylated human milk oligosaccharides obtained by the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

1, Terms and definitions

The term "fermentation broth", as used in this specification, refers to a product obtained from fermentation of the microbial organism. Thus, the fermentation product comprises cells (biomass), the fermentation medium, salts, residual substrate material, and any molecules/by -products produced during fermentation, such as the desired neutral or sialylated HMOs. After each step of the purification method, one or more of the components of the fermentation product is removed, resulting in more purified neutral or sialylated HMOs. The term “monosaccharide” means a sugar of 5-9 carbon atoms that is an aldose (e.g. D-glucose, D- galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), a ketose (e.g. D- fructose, D-sorbose, D-tagatose, etc.), a deoxysugar (e.g. L-rhamnose, L-fucose, etc ), a deoxyaminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), a uronic acid, a ketoaldonic acid (e.g. sialic acid) or equivalents.

The term “di saccharide” means a carbohydrate consisting of two monosaccharide units linked to each other by an interglycosidic linkage.

The term “tri- or higher oligosaccharide” means a sugar polymer consisting of at least three, preferably from three to eight, more preferably from three to six, monosaccharide units (vide supra). The oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.

The term "human milk oligosaccharide" or "HMO" means a complex carbohydrate found in human breast milk (Urashima et al. : Milk Oligosaccharides, Nova Medical Books, NY, 2011; Chen Adv. Carbohydr. Chem. Biochem. ’ll, 113 (2015)). The HMDs have a core structure being a lactose unit at the reducing end that is elongated i) by a P-N-acetyl-glucosaminyl group or ii) by one or more P- N-acetyl-lactosaminyl and/or one or more P-lacto-N-biosyl units, and which core structures can be substituted by an a-L-fucopyranosyl and/or an a-N-acetyl-neuraminyl (sialyl) moiety. In this regard, the non-acidic (or neutral) HMDs are devoid of a sialyl residue, and the acidic HMDs have at least one sialyl residue in their structure. The non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNTri, GlcNAc(pi-3)Gal(pi-4)Glc), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N- neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto- N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2’ -FL), lacto- N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3 -FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-in), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N- fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-I), fucosyl-para-lacto-N-neohexaose II (F- pLNnH II) and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3’- sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 3-fucosyl-3’-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

The term “sialyl” or “sialyl moiety” means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac), preferably linked with a-linkage:

The term “fucosyl” means an L-fucopyranosyl group, preferably linked with a-interglycosidic linkage:

“N-acetyl-glucosaminyl” means an N-acetyl-2-amino-2-deoxy-D-glucopyranosyl (GlcNAc) group, preferably linked with P-linkage:

“N-acetyl-lactosaminyl” means the glycosyl residue of N-acetyl-lactosamine (LacNAc, Galppi- 4GlcNAc), preferably linked with P-linkage: Furthermore, the term “lacto-N-biosyl” means the glycosyl residue of lacto-N-biose (LNB, GalpPl-

3GlcNAc), preferably linked with P-linkage: The term “biomass”, in the context of fermentation, refers to the suspended, precipitated, or insoluble materials originating from fermentation cells, like intact cells, disrupted cells, cell fragments, proteins, protein fragments, polysaccharides.

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 human milk oligosaccharide solution of this application refers to the overall carbohydrate content of the solution including the human milk oligosaccharides and its accompanying carbohydrates. Brix is measured by a calibrated refractometer.

“Demineralization” preferably means a process of removing minerals or mineral salts from a liquid. In the context of the present invention, demineralization can occur in the nanofiltration step, especially when it is combined with diafiltration, or by using cation and anion exchange resins (if applicable).

The term “protein-free aqueous medium” preferably means an aqueous medium or broth from a fermentation or enzymatic process, which has been treated to remove substantially all the proteins, as well as peptides, peptide fragments, RNAs and DNAs, as well as endotoxins and glycolipids that could interfere with the eventual purification of the one or more neutral or sialylated HMOs and/or one or more of their components, especially the mixture thereof, from the fermentation or enzymatic process mixture.

The term “HMO-containing stream” means an aqueous medium containing neutral or sialylated HMOs obtained from a fermentation process, which has been treated to remove suspended particulates and contaminants from the process, particularly cells, cell components, insoluble metabolites and debris that could interfere with the eventual purification of the one or more hydrophilic oligosaccharides, especially one or more neutral or sialylated HMOs and/or one or more HMO components, especially mixtures thereof.

The term “biomass waste stream” preferably means suspended particulates and contaminants from the fermentation process, particularly cells, cell components, insoluble metabolites, and debris.

Rejection factor of a salt (in percent) is calculated as (1-K _p /K _r )- 100, wherein K _P is the conductivity of the salt in the permeate and K _r is the conductivity of the salt in the retentate. Rejection factor of a carbohydrate (in percent) is calculated as (1-C _p /C _r )- 100, wherein C _p is the concentration of the carbohydrate in the permeate and C _r is the concentration of the carbohydrate in the retentate.

The term “diafiltration” refers to solvent addition (water) during the membrane filtration process. If diafiltration is applied during ultrafiltration, it improves the yield of the desired HMO in the permeate. If diafiltration is applied during nanofiltration, it improves the separation of small size impurities and salts to the permeate. The solute yield and therefore the product enrichment could be calculated based on the formulas known to the skilled person based on rejection factors and relative amount of water added.

The term “concentrating” refers to the removal of liquid, mostly water, thus resulting in a higher concentration of the neutral or sialylated HMOs in the purified HMO-containing product stream.

The term “decolorization” refers to the process of removing colour bodies from a solution to the extent required by product specifications. The decolorization of carbohydrate-containing solutions is mainly based on Van-der-Waals type interactions of the colour bodies with the adsorbent. In the context of the present invention, the colour of the solution is quantified by absorption of visible light at 400 nm (Abs_400) and normalized by the concentration and the path length. Thereby, the colour index CI_400 is defined as 1000 x Abs_400/Brix with the path length = 1 cm. Normally, if the CI_400 < 5, then a solid product isolated from its solution appears as colourless (white) solid. A crude supernatant solution containing HMO product after fermentation usually has a colour index CI_400 in the range from 100 to 400.

2, Method for the purification of a neutral or sialylated HMO from a fermentation broth:

The invention relates to a method for the purification of a neutral or sialylated human milk oligosaccharide (HMO) from a fermentation broth, comprising the steps of:

I. separating an HMO-containing stream from biomass;

II. purifying the HMO-containing stream with an acidic cation exchange resin, then

III. treating the resin eluate with an adsorbent resin.

Preferably, the method comprises a. separating the fermentation broth to form an HMO-containing stream and a biomass waste stream; b. purifying the HMO-containing stream by nanofiltration; c. purifying the HMO-containing stream with an acidic cation exchange resin; d. purifying the HMO-containing stream with an adsorbent resin; e. concentrating and drying the purified HMO-containing stream to obtain the neutral or sialylated HMO in solidified form.

In one embodiment, the method according to the invention consists of steps a)-e).

In a preferred embodiment, the method does not comprise an anion exchanger treatment step.

In a preferred embodiment, an anion exchanger treatment step is excluded from the method according to the invention.

In a preferred embodiment, method steps a)-e) are performed in the consecutive order a)-e) as given above.

The fermentation broth:

In an embodiment, the neutral or sialylated HMO being present in the fermentation broth has been obtained by culturing a genetically modified microorganism capable of producing said neutral or sialylated human milk oligosaccharide from an internalized carbohydrate precursor. Preferably, the microbial organism is a genetically modified bacterium or yeast such as a Saccharomyces strain, a Candida strain, a Hansenula strain, a Kluyveromyces strain, a Pichia strain, a Schizosaccharomyces stain, a Schwanniomyces strain, a Torulaspora strain, a Yarrowia strain, or a Zygosaccharomyces strain. More preferably, the yeast is Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii, or Zygosaccharomyces bailii; and the Bacillus is Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis.

In an embodiment, at least one neutral or sialylated human milk oligosaccharide being present in the fermentation broth has not been obtained by microbial fermentation, but has been e.g. added to the fermentation broth after it has been produced by a non-microbial method, e.g. chemical and/or enzymatic synthesis.

In an embodiment, the purity of the neutral or sialylated HMO in the fermentation broth is <70%, preferably <60%, more preferably <50%, most preferably <40%. Preferably, the HMO is a neutral HMO. In an embodiment, the neutral HMO is preferably selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N- triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V (alternative name: lacto-N-fucopentaose VI), lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N- difucohexaose III, 6'-galactosyllactose, 3 '-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, and any mixture thereof. More preferably, the HMO is 2'-fucosyllactose, 3-fucosyllactose, 2',3- difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose or a lacto-N-fucopentaose, more preferably 2'-fucosyllactose, LNT, LNnT or a lacto-N-fucopentaose.

In an embodiment, the sialylated HMO is selected from the group consisting of 3’-sialyllactose (3’- SL) and 6’-sialyllactose (6’-SL).

In an embodiment, the HMO in the fermentation broth is a single neutral or sialylated HMO.

In an embodiment, the HMO in the fermentation broth is a mixture of various individual neutral or sialylated HMOs.

In an embodiment, the HMO is a mixture of two individual neutral or sialylated HMOs. In another embodiment, the HMO is a mixture of three individual neutral or sialylated HMOs. In another embodiment, the HMO is a mixture of four individual neutral or sialylated HMOs. In another embodiment, the HMO is a mixture of five individual neutral or sialylated HMOs.

In an embodiment, the HMO in the fermentation broth is a mixture of a neutral or sialylated HMO obtained by microbial fermentation and a HMO that has not been obtained by microbial fermentation, but e g. by chemical and/or enzymatic synthesis.

Separating the fermentation broth to form an HMO-containing stream and a biomass waste stream in step i) or step a) of the method according to the invention

In step a) of the method according to the invention, the HMO-containing stream is separated from the biomass waste stream.

The fermentation broth typically contains, besides the desired neutral or sialylated HMO, the biomass of the cells of the used microorganism together with proteins, protein fragments, peptides, DNAs, RNAs, endotoxins, biogenic amines, amino acids, organic acids, inorganic salts, unreacted carbohydrate acceptors such as lactose, sugar-like by-products, monosaccharides, colorizing bodies, etc. In step a) of the method according to the invention, the biomass is separated from the neutral or sialylated HMO.

In a preferred embodiment, the biomass is separated from the neutral or sialylated HMO in step a) by ultrafiltration. The ultrafiltration step is to separate the biomass and, preferably, also high molecular weight components and suspended solids from the lower molecular weight soluble components of the broth, which pass through the ultrafiltration membrane in the permeate. This ultrafiltration permeate is an aqueous solution containing the neutral or sialylated human milk oligosaccharide also referred to as the HMO-containing stream, whereas the ultrafiltration retentate comprises the biomass waste stream.

Any conventional ultrafiltration membrane can be used having a molecular weight cut-off (MWCO) range between about 1 and about 500 kDa, such as 10-250, 50-100, 200-500, 100-250, 1-100, 1-50, 10-25, 1-5 kDa, or any other suitable sub-range. The membrane material can be a ceramic or made of a synthetic or natural polymer, e.g. polysulfone, polyvinylidene fluoride, polyacrylonitrile, polypropylene, cellulose, cellulose acetate or polylactic acid. The ultrafiltration step can be applied in dead-end or cross-flow mode. Step a) of the method according to the invention may comprise more than one ultrafiltration step using membranes with different MWCO as defined above, e.g. applying two ultrafiltration separations, wherein the first membrane has a higher MWCO than that of the second membrane. This arrangement may provide a better separation efficacy of the higher molecular weight components of the broth. After this separation step, the permeate contains materials that have a molecular weight lower than the MWCO of the second membrane, including the neutral or sialylated human milk oligosaccharides of interest.

In one embodiment, the fermentation broth is ultrafiltered using a membrane having a MWCO of 5 to 30 kDa, such as 10-25, 15 or 20 kDa.

In a preferred embodiment, the yield of the desired neutral or sialylated HMO in the permeate after the ultrafiltration step performed in step a) is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

In another embodiment, the broth obtained from fermentation is subjected to centrifugation to separate the biomass from the neutral or sialylated HMO (HMO-containing stream) in step a) of the method according to the invention. In said embodiment, the supernatant represents the HMO- containing stream, while the remaining material, i.e. the “biomass waste stream” can be separated out. By centrifugation, a clear supernatant comprising the neutral or sialylated HMO can be obtained, which represents the HMO-containing stream.

The centrifuging can be lab scale or, advantageously over previous centrifuging methods, commercial scale (e.g. industrial scale, full production scale).

In some embodiments, a multi-step centrifugation can be used. For example, a series of 2, 3, 4, 5, 6, 7, 8, 9, or 10 centrifugation steps can be performed. In other embodiments, the centrifugation may be a single step. Centrifugation provides for a quick biomass-removal.

In certain embodiments, Sedicanter® centrifuge designed and manufactured by Flottweg can be used.

The particular type of centrifuge is not limiting, and many types of centrifuges can be used. The centrifuging can be a continuous process. In some embodiments, the centrifuging can have feed addition. For example, the centrifuging can have a continuous feed addition. In certain embodiments, the centrifuging can include a solid removal, such as a wet solid removal. The wet solid removal can be continuous in some implementations, and periodic in other implementations.

For example, a conical plate centrifuge (e.g. disk bowl centrifuge or disc stack separator) can be used. The conical plate centrifuge can be used to remove solids (usually impurities) from liquids, or to separate two liquid phases from each other by means of a high centrifugal force. The denser solids or liquids which are subjected to these forces move outwards towards the rotating bowl wall while the less dense fluids move towards the centre. The special plates (known as disc stacks) increase the surface settling area which speeds up the separation process. Different stack designs, arrangements and shapes are used for different processes depending on the type of feed present. The concentrated denser solid or liquid can then be removed continuously, manually, or intermittently, depending on the design of the conical plate centrifuge. This centrifuge is very suitable for clarifying liquids that have small proportion of suspended solids.

The centrifuge works by using the inclined plate setter principle. A set of parallel plates with a tilt angle 9 with respect to horizontal plane is installed to reduce the distance of the particle settling. The reason for the tilted angle is to allow the settled solids on the plates to slide down by centrifugal force so they do not accumulate and clog the channel formed between adjacent plates. This type of centrifuge can come in different designs, such as nozzle-type, manual-cleaning, selfcleaning, and hermetic. The particular centrifuge is not limiting.

Factors affecting the centrifuge include disk angle, effect of g-force, disk spacing, feed solids, cone angle for discharge, discharge frequency, and liquid discharge.

Alternatively, a solid bowl centrifuge (e.g. a decanter centrifuge) can be used. This is a type of centrifuge that uses the principle of sedimentation. A centrifuge is used to separate a mixture that consists of two substances with different densities by using the centrifugal force resulting from continuous rotation. It is normally used to separate solid-liquid, liquid-liquid, and solid-solid mixtures. One advantage of solid bowl centrifuges for industrial uses is the simplicity of installation compared to other types of centrifuge. There are three design types of solid bowl centrifuge, which are conical, cylindrical, and conical-cylindrical.

Solid bowl centrifuges can have a number of different designs, any of which can be used for the disclosed method. For example, conical solid bowl centrifuges, cylindrical solid bowl centrifuges, and conical-cylindrical bowl centrifuges can be used.

The centrifuging can be performed at a number of speeds and residence times. For example, the centrifuging can be performed with a relative centrifugal force (RCF) of 20000g, 15000g, 10000g, or 5000g. In some embodiments, the centrifuging can be performed with a relative centrifugal force (RCF) of less than 20000g, 15000g, 10000g or 5000g. In some embodiments, the centrifuging can be performed with a relative centrifugal force (RCF) of greater than 20000g, 15000g, 10000g or 5000g.

In some embodiments, the centrifuging can be characterized by working volume. In some embodiments, the working volume can be 1, 5, 10, 15, 20, 50, 100, 300, or 5001. In some embodiments, the working volume can be less than 1, 5, 10, 15, 20, 50, 100, 300, or 500 1. In some embodiments, the working volume can be greater than 1, 5, 10, 15, 20, 50, 100, 300, or 5001.

In some embodiments, the centrifuging can be characterized by feed flow rate. In some embodiments, the feed flow rate can be 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 1/hr. In some embodiments, the feed flow rate can be greater than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 100000 1/hr. In some embodiments, the feed flow rate can be less than 100, 500, 1000, 1500, 2000, 5000, 10000, 20000, 40000, or 1000001/hr. The amount of time spent centrifuging (e.g. residence time) can vary as well. For example, the residence time can be 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the residence time can be greater than 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the residence time can be less than 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes.

Any of the above supernatant properties can be produced through a single instance of centrifuging. Alternatively, it can be produced through multiple instances of centrifuging.

In view of the above, step a) of the method according to the invention can be performed via ultrafiltration as defined above or centrifugation, or via a combination of ultrafiltration and centrifugation. Preferably, method step a) is carried out by ultrafiltration as defined above to obtain the HMO-containing stream separate from the biomass waste stream.

Before the ultrafiltration and/or centrifugation step, the fermentation broth can be subjected to a pre-treatment step. Pre-treatment of the fermentation broth can include pH adjustment, and/or dilution, and/or heat treatment. In certain implementations, all three of pH adjustment, dilution, and heat treatment can be performed. In alternative embodiments, pH adjustment and dilution can be performed. In alternative embodiments, pH adjustment and heat treating can be performed. In alternative embodiments, heat treating and dilution can be performed. Advantageously, a combination of a plurality of pre-treatment methods can provide an improved synergistic effect not found in individual pre-treatments.

In certain embodiments, one or more of the aforementioned pre-treatment steps can occur during the biomass removal in step a) by centrifuging and/or ultrafiltration as defined above. For example, between steps in a multi-step centrifuging, or the centrifuging vessel may be able to heat the fermentation broth during centrifuging.

Advantageously, the pre-treatment can increase the settling velocity of the solid particles (biomass) in the fermentation broth by a factor of 100 to 20000, making the biomass separation by centrifugation much more efficient and thus applicable in industrial scale. In addition to settling velocity, at least three other parameters are substantially improved due to pre-treatment that are, improved neutral or sialylated HMO yield in the HMO-containing stream, reduced protein and DNA content in the supernatant, and further residual suspended solid content can be substantially reduced. Purifying the HMO-containing stream in step b ) by nanofiltration:

In step b) of the method according to the invention, the neutral or sialylated HMO-containing stream is purified by nanofiltration.

Nanofiltration (NF) can be used to remove low molecular weight molecules smaller than the desired neutral or sialylated HMOs, such as mono- and disaccharides, short peptides, small organic acids, water, and salts.

The product stream, i.e. the neutral or sialylated HMO-containing steam, is the NF retentate. The nanofiltration membrane thus has a MWCO or a pore size that ensures the retention of the neutral or sialylated human milk oligosaccharide of interest, i.e. the MWCO of the nanofiltration membrane is adjusted accordingly.

Typically, the pore size of the nanofiltration membrane is from 0.5 nm to 2 nm and/or from 150 dalton (Da) molecular weight cut-off (MWCO) to 3500 Da MWCO.

In an embodiment, the membranes are in the range of 150-300 Da MWCO, which are defined as “tight” NF membranes.

In a preferred embodiment, the membranes are above 300 Da MWCO, and preferably not higher than 3500 Da MWCO. In said embodiment, the membranes are considered “loose” NF membranes.

In another preferred embodiment, the “loose” nanofiltration membrane has a molecular weight cutoff (MWCO) of 500-3500 Da and the active (top) layer of the membrane is preferably composed of polyamide, more preferably piperazine-based polyamide. Thereby, the retention of tri- or higher oligosaccharides is ensured and at least part of the disaccharides is allowed to pass the membrane. In this embodiment, the applied nanofiltration membrane shall be tight for tri- and higher oligosaccharides for them to be efficiently retained. At the same time, the membrane shall be relatively loose for MgSC , that is its rejection is about 50-90 %, in order that disaccharides can pass the membrane. This way, it is possible to separate e.g. lactose, which is a precursor in making human milk oligosaccharides e.g. by fermentation, from the neutral or sialylated human milk oligosaccharides product with a good efficacy, and additionally a substantial part of divalent ions also passes to the permeate. In some embodiments, the MgSC rejection factor is 60-90 %, 70-90 %, 50-80 %, 50-70 %, 60-70 % or 70-80 %. Preferably, the MgSCU rejection factor on said membrane is 80-90 %. Preferably, the membrane has a rejection factor for NaCl that is lower than that for MgSCU. In one embodiment, the rejection factor for NaCl is not more than 50 %. In another embodiment, the rejection factor for NaCl is not more than 40 %. In another embodiment, the rejection factor for NaCl is not more than 30 %. In this latter embodiment, a substantial reduction of all monovalent salts in the retentate is also achievable. In said embodiment, the membrane is a thin- film composite (TFC) membrane. An example of a suitable piperazine-based polyamide TFC membrane is TriSep® UA60. Other examples of suitable NF membranes include Synder NFG (600- 800 Da), Synder NDX (500-700 Da), and TriSep® XN-45 (500 Da).

Preferably, the yield of the desired neutral or sialylated HMO in the retentate after a nanofiltration step is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

In a preferred embodiment, step b) comprises a diafiltration step, that is the nanofiltration step conducted in diafiltration mode. Preferably, the diafiltration step follows the aforementioned (conventionally conducted) nanofiltration step.

Diafiltration is a process that involves the addition of purified water to a solution during membrane filtration process in order to remove membrane permeable components more efficiently. Thus, diafiltration can be used to separate components on the basis of their properties, in particular molecular size, charge or polarity by using appropriate membranes, wherein one or more species are efficiently retained and other species are membrane permeable.

In a preferred embodiment, diafiltration and nanofiltration is combined within one step (referred to as nanofiltration/diafiltration or NF/DF) in which diafiltration is executed while using a nanofiltration membrane that is effective for the separation of low molecular weight compounds and/or salts from the neutral or sialylated HMDs. Diafiltration with “loose” NF membrane as defined above, is particularly efficient for both mono- and divalent salts removal and disaccharides removal from neutral or sialylated HMDs.

In a preferred embodiment, the DF step or the NF/DF step is performed so that the pH is set below 5.0, preferably, below 4.5, advantageously below 4.0, but preferably not less than 3.0. Under this condition, salts comprised of monovalent cations such as sodium salts (that is sodium ion together with the co-anion(s)) are effectively removed, giving rise to a low-salt or a practically salt-free purified solution containing a neutral or sialylated HMO in the retentate.

In an embodiment, a second nanofiltration step is carried out in the method according to the invention so that it is comprised in step b). In said second nanofiltration step, the nanofiltration membrane is either a “loose” NF membrane or a “tight” NF membrane. The second optional nanofiltration step is performed after the first nanofiltration step (step b), but is preferably performed before step c) of the method according to the invention.

Likewise, a second diafiltration can be performed in the method according to the invention. This second optional diafiltration step can also be combined with the second nanofiltration step. This second NF/DF step, when “loose” NF membrane is applied as disclosed above, is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0.

In an embodiment, the purified solution obtained after step b) of the method according to the invention, contains the neutral or sialylated HMO at a purity of >80%, preferably >85%, more preferably >90%.

In an embodiment, the purified solution obtained after step b) of the method according to the invention is free proteins and/or recombinant genetic material.

Step c): Purifying the HMO-containing stream with an acidic cation exchange resin:

The method according to the invention comprises the purification of the HMO-containing stream with an acidic cation exchange resin (step c)).

In the cation exchanger treatment step, positively charged materials can be efficiently removed from the HMO-containing stream, either before or after nanofiltration, as they bind to the resin, while the HMOs will not be retained by the acidic cation exchange resin. Thereby, also the amounts of salts and/or colorizing agents and/or proteins can be further reduced.

In a preferred embodiment, the stationary phase (resin) comprises sulfonate groups that are negatively charged in aqueous solution and that tightly bind cationic compounds.

In a preferred embodiment, the acidic cation exchange resin is a strongly acidic cation exchange resin, preferably a polystyrene-divinylbenzene cation exchange resin with sulfonic acid functional groups.

In a further preferred embodiment, the acidic cation exchange resin is in H ⁺ -form.

The binding capacity of an acidic cation exchange resin is generally from 1.2 to 2.2 eq/1.

When using a cationic ion exchange resin, its degree of crosslinking can be chosen depending on the operating conditions of the ion exchange column. A highly crosslinked resin offers the advantage of durability and a high degree of mechanical integrity, however, suffers from a decreased porosity and a drop off in mass-transfer. A low-crosslinked resin is more fragile and tends to swell by absorption of mobile phase. The particle size of the ion exchange resin is selected to allow an efficient flow of the eluent, while the charged materials are still effectively removed. A suitable flow rate may also be obtained by applying a negative pressure to the eluting end of the column or a positive pressure to the loading end of the column, and collecting the eluent. A combination of both positive and negative pressure may also be used. The cationic ion exchange treatment can be carried out in a conventional manner, e.g. batch-wise or continuously.

Non-limiting examples of a suitable acidic cation exchange resin can be e g. Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet 1200.

Preferably, the cation exchange resin treatment step is performed after the nanofiltration step.

In a preferred embodiment, step c) results in a purified solution containing the neutral or sialylated HMOs at a purity of > 80%, preferably > 85%, more preferably > 90%.

In a preferred embodiment, step c) results in a purified solution that is free of proteins and/or recombinant genetic material.

In a particularly preferred embodiment, the NF or NF/DF purification of the HMO-containing stream obtained in step a) is followed by a strong cation exchange resin treatment (H ⁺ -form) of the retentate from the NF or NF/DF step b).

Purifying the HMO-containing stream in step d) with an adsorbent resin:

“Adsorbent” is an insoluble solid porous material with high specific surface area, i.e. >400 m ² /g (as determined using the nitrogen sorption BET technique) capable of removing certain compounds from solution by physical adsorption and/or chemical sorption, whereas adsorption occurs on the pore surface of the adsorbent. Physical adsorption involves non-stoichiometric process with non- covalent interaction comprising of e.g. van der Waals, polar or ionic interactions without ion exchange. Chemical sorption or chemisorption proceeds due to a chemical reaction, i.e. involving electron exchange with chemical bond formation on the surface of the adsorbent. Examples of adsorbents are activated carbon (activated charcoal or AC) and adsorbent resins.

“Adsorbent resin” or “synthetic adsorbent” is specially designed macroporous polymer usually provided in spherical bed form with high specific surface area, e.g. > 400 m ² /g (as determined using the nitrogen sorption BET technique) and defined pore structure for selective removal of specific substances from an aqueous solution. In the context of the present application, the adsorbent resin suitable in step d) is partially functionalized with hydrophilic groups for easier regeneration. The adsorbent resin is therefore an acid adsorbent derived from cross-linked polystyrene or polyacrylic polymers and partially functionalized with primary, secondary or tertiary amines as weak base. In a preferred embodiment, the adsorbent resin used in step d) is a polystyrene-divinylbenzene copolymer with tertiary amine functional groups.

In the context of the present invention adsorbent resins are distinguished from ion exchangers or ion-exchange resins by a non-stoichiometric physical adsorption or by chemical adsorption such as acid adsorption by amines on polymeric support, where in both cases the molecules are removed from the feed solution as whole entities without releasing of ions or other substances from the adsorbent. In addition, an adsorbent resin functionalized with basic amino groups in free-base form does not contain any exchangeable ions and therefore could not be an ion exchanger if used in the free-base form. Also these resins usually have low basic capacity such as 0.1-0.3 eq/1 (or around 0.6-1.0 eq/kg on dry weight), which is substantially below the typical capacity of an ion exchange resin, i .e. 1-2 eq/1. A resin capacity is a measurement of total capacity, as determined by a test performed in the lab by a titration methodology. A measured quantity of a basic (anion) resin, including a polystyrene-divinylbenzene copolymer adsorbent resin with tertiary amine functional groups used in step d), is fully converted to the basic form with an excess of strong base, e.g. NaOH- solution, and then well rinsed. A measured quantity of a strong inorganic acid, e.g. HC1 or sulphuric acid solution, is then passed through the resin so as to totally exhaust the resin. The effluent is captured. The acid that passed through the column represents the amount that was not captured by the resin. This solution is then titrated with base to neutralize it and the amount of base required is expressed in equivalents. The difference between the total equivalents of acid passed through the column and the acid exiting the column represents the total equivalents of acid captured by the resin. The capacity of the resin is then determined.

In a preferred embodiment, the adsorbent resin has a surface area of > 400 m ² /g. In an embodiment, the adsorbent resin has a surface of from 400 to 1200 m ² /g. More preferably, the adsorbent resin has a basic capacity of 0.1-0.3 eq/1 (or around 0.6-1.0 eq/kg on dry weight). Further preferably, the adsorbent resin used in step d) is a polyacrylic, more preferably a methacrylic polymer adsorbent resin. Advantageously, the polymer is functionalized polystyrene (PS), more preferably cross-linked PS with divinylbenzene (DVB). Suitable adsorbent resins are Dowex Optipore SD-2, Purolite MN100 or MN102.

In the context of the present invention, the substances to be removed are numerous coloured compounds, hydrophobic impurities, large biomolecules such as proteins, DNAs, RNAs, polysaccharides and their fragments.

In a preferred embodiment, purification with the adsorbent resin leads to an HMO-containing stream having a CI_400 <5, preferably CI_400 <4.

In a preferred embodiment, step d) results in a purified solution containing the HMO at a purity of >80%, preferably >85%, more preferably >90%.

The above defined adsorbent resin, in the method according to the invention, can be used as a replacement of activated carbon that is frequently used in known methods for purifying HMO streams. The adsorbent resin is as efficient as activated charcoal to remove colour, large biomolecules (proteins, DNAs etc.) and hydrophobic compounds, and has the advantages over the use of active carbon, that it does not adsorb HMOs like charcoal. Charcoal may adsorb HMOs up to 30-35 wt% in relation of the weight of the charcoal used, which reduces the HMO recovery yield; it could be easily regenerated and thus re-used many times unlike the active charcoal, where regeneration of charcoal requires harsh conditions or the use of organic solvents; due to regeneration and lack of product adsorption, the amount of the adsorbent resin, unlike that of charcoal, is not limited, i.e. it could be used in any amount as long as it is sufficient to remove the impurities; it lacks trace toxic elements such as nickel, lead and arsenic that can be present in the active carbon and that could be released into the product stream; and due to the presence of the weakly basic sites, it is used as an insoluble base with acid adsorbent property, and thus replaces the active carbon together with the basic anion exchange resin treatment step that characterize the prior art methods.

Concentrating and drying the purified HMO-containing stream to obtain a neutral or sialylated HMO in solidified form in step e):

Preferably after all separation/purification steps have been applied, in particular after method steps a)-d), the neutral or sialylated HMO of interest is provided in its solid form via a concentration and drying step (step e)). A concentration step is used to economically remove significant quantities of liquid, mostly water, from the neutral or sialylated HMO-containing stream using e.g. evaporation, nanofiltration, or reverse-osmosis filtration. Evaporation processes can include, e.g. falling film evaporation, climbing film evaporation and rotary evaporation. The evaporation can also be conducted under vacuum. The incoming solids concentration to the process is preferably approximately 5 to 30 wt.%. The exit solids concentration from such a process is typically greater than 30 wt.%., preferably greater than 50 wt.%. More preferably, the solids concentration exiting the dewatering operation is 60 to 80 wt.%. The solids portion of the recovered material is preferably greater than 80 wt.% of neutral or sialylated HMO.

In an embodiment, the purified neutral or sialylated HMO-containing stream is concentrated to a concentration of > 100 g/1 of neutral or sialylated HMO, preferably of > 200 g/1, more preferably of > 300 g/1.

When the purified neutral or sialylated HMO-containing stream is concentrated by evaporation, the evaporation is preferably carried out at a temperature of from about 20 to about 80 °C. In some embodiments, the evaporation is carried out at a temperature of from 25 to 75 °C. In some embodiments, the evaporation is carried out at a temperature of from 30 to 70 °C. In some embodiments, the evaporation is carried out at a temperature of from 30 to 65 °C. Preferably, the evaporation is carried out under vacuum.

When the purified neutral or sialylated HMO-containing stream is concentrated by membrane filtration, any membrane, typically nanofiltration membrane, is suitable that sufficiently rejects the neutral or sialylated HMO. Concentration by membrane filtration usually provides an HMO- solution of around 30-35 wt%. This concentration may be suitable for conducting the subsequent drying-solidification step, e.g. freeze-drying. However, other drying methods may require more concentrated solutions, e.g. spray-drying or crystallization. In this case, concentration by evaporation, preferably under vacuum, is the preferred embodiment. Alternatively, the neutral or sialylated HMO-containing stream obtained in the previous step is concentrated to around 30-35 wt% using a nanofiltration membrane, and the solution is further concentrated by evaporation.

In one embodiment of the concentration by membrane filtration, the membrane of choice is a “tight” NF with 150-300 Da MWCO.

In other embodiment of the concentration by membrane filtration, the membrane of choice is a nanofiltration membrane that has a molecular weight cut-off (MWCO) of 500-3500 Da and an active (top) layer of polyamide (“loose” NF membrane); and the concentration step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0. In this latter embodiment, a substantial reduction of all monovalent salts in the retentate is also achievable. In said embodiment, the membrane is preferably a thin-film composite (TFC) membrane which is a piperazine-based polyamide membrane, more preferably its MgSC rejection is about 50-90 %, even more preferably its NaCl rejection is not more than 50 %. An example of such a membrane is TriSep® UA60. Under this condition, remaining salts are also effectively removed, giving rise to a low-salt or a practically salt-free purified neutral or sialylated HMO- concentrate. In this embodiment, after completing the concentration step, the pH of the neutral or sialylated HMO-concentrate is advantageously set between 4-6 before performing the next step (e.g. evaporation, drying-solidification, sterile filtration).

The concentration step may be optional when the drying step is freeze-drying.

In an embodiment, the method according to the invention further comprises a step, wherein the HMO-containing solution, preferably after a concentration step is sterile filtered and/or subjected to endotoxin removal, preferably by filtration of the purified solution through a 3 kDa filter. Said optional step is preferably conducted before the drying step. The sterile filtration step does not affect the purity of the HMO-containing solution.

In a preferred embodiment, the drying step comprises spray-drying of the neutral or sialylated HMO-containing stream, preferably consists of spray-drying of the neutral or sialylated HMO- containing stream.

Preferably, spray-drying leads to solidified neutral or sialylated HMO having an amorphous structure, i.e. an amorphous powder is obtained.

In an embodiment, spray-drying is performed at a concentration of the neutral or sialylated HMO of 20-60 % (w/v), preferably 30-50 % (w/v), more preferably 35-45 % (w/v), and an inlet temperature of 110-150 °C, preferably 120-140 °C, more preferably 125-135 °C and/or an outlet temperature of 60-80 °C, preferably 65-70 °C.

In some embodiment, the neutral or sialylated HMO-containing stream fed into the spray-dryer has a Brix value of from about 8 to about 75% Brix. In some embodiments, the Brix value is from about 30 to about 65% Brix. In some embodiments, the Brix value is from about 50 to about 60% Brix. In some embodiments, the feed into the spray-dryer is at a temperature of from about 2 to about 70 °C immediately before being dispersed into droplets in the spray-dryer. In some embodiments, the feed into the spray-dryer is at a temperature of from about 30 to about 60 °C immediately before being dispersed into droplets in the spray-dryer. In some embodiments, the feed into the spray-dryer is at a temperature of from about 2 to about 30 °C immediately before being dispersed into droplets in the spray-dryer. In some embodiments, the spray-drying uses air having an air inlet temperature of from 120 to 280 °C. In some embodiments, the air inlet temperature is from 120 to 210 °C. In some embodiments, the air inlet temperature is from about 130 to about 190 °C. In some embodiments, the air inlet temperature is from about 135 to about 160 °C. In some embodiments, the spray-drying uses air having an air outlet temperature of from about 80 to about 110 °C. In some embodiments, the air outlet temperature is from about 100 to about 110 °C. In some embodiments, the spraydrying is carried out at a temperature of from about 20 to about 90 °C. In some embodiments, the spray-dryer is a co-current spray-dryer. In some embodiments, the spray-dryer is attached to an external fluid bed. In some embodiments, the spray-dryer comprises a rotary disk, a high-pressure nozzle, or a two-fluid nozzle. In some embodiments, the spray-dryer comprises an atomizer wheel. In some embodiments, spray-drying is the final purification step for the desired neutral or sialylated HMO.

Alternatively, the drying-solidification step comprises an indirect drying method. For the purposes of this specification, indirect dryers include those devices that do not utilize direct contact of the material to be dried with a heated process gas for drying, but instead rely on heat transfer either through walls of the dryer, e.g. through the shell walls in the case of a drum dryer, or alternately through the walls of hollow paddles of a paddle dryer, as they rotate through the solids while the heat transfer medium circulates in the hollow interior of the paddles. Other examples of indirect dryers include contact dryers and vacuum drum dryers.

Alternatively, the drying-solidification step comprises freeze-drying.

Alternatively, the drying-solidification step comprises crystallization (provided that the HMO is obtainable in crystalline form).

Further optional method steps:

In an embodiment, the HMO-containing stream is neutralized to a pH value of approximately 7 after the acidic cation exchange resin treatment (step c)) by the addition of sufficient quantities of sodium hydroxide. In this case, the concentration step is advantageously a membrane filtration, the membrane of choice is a nanofiltration membrane that has a molecular weight cut-off (MWCO) of 500-3500 Da and an active (top) layer of polyamide (“loose” NF membrane); and the concentration step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but not less than 3.0. Under this condition a removal of all monovalent (e.g. sodium) salts in the retentate is achievable.

In another embodiment, the method according to the invention including its preferred and more preferred realizations further comprises a step, wherein the purified neutral or sialylated HMO- containing solution, preferably after concentration and before the drying step comprised in step e), is sterile filtered and/or subjected to endotoxin removal, preferably by filtration of the purified solution through a 3 kDa filter or using a membrane having less than 0.5 pm, less than 0.4 pm, less than 0.3 pm, or less than 0.2 pm pore size. It is noted though that the sterile filtration step does not contribute to the solution of the technical problem, namely to purify HMOs from fermentation broth in which they have been produced and separate them from biomass, proteins, protein fragments, fragments of genetic material originated from genetically modified microorganism, salts, fermentation additives, metabolic by-products, especially non-carbohydrate by-products, colour bodies, etc., and thus to make them suitable for human administration. When the method disclosed above is carried out under suitably sterile conditions, sterile filtration is not necessary. Nevertheless, according to one embodiment, the sterile filtration step, disclosed above, may be part of the method of the invention.

Particular embodiments of the invention:

In a preferred embodiment, the method according to the present invention does not include an anion exchange resin treatment step.

In a preferred embodiment, the method according to the invention does not comprise a purification/decolourization step with active carbon.

In a further preferred embodiment, the method according to the invention comprises or consists of the following steps (preferably in consecutive order): i. separating the fermentation broth to form an HMO-containing stream and a biomass waste stream; ii. purifying the HMO-containing stream by nanofiltration, wherein the nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3500 Da; iii. purifying the nanofiltration retentate with a strongly acidic cation exchange resin in H ⁺ - form; iv. purifying the cation exchange resin eluate with an adsorbent resin; v. concentrating the adsorbent resin eluate and drying the purified HMO-concentrate to obtain the neutral or sialylated HMO in solidified form.

In a further preferred embodiment, the method according to the invention comprises or consists of the following steps (preferably in consecutive order): i. separating the fermentation broth to form an HMO-containing stream and a biomass waste stream; ii. purifying the HMO-containing stream by nanofiltration, wherein the nanofiltration membrane has a molecular weight cut-off (MWCO) of 500-3500 Da; iii. purifying the nanofiltration retentate with a strongly acidic cation exchange resin in H ⁺ - form; iv. purifying the cation exchange resin eluate with an adsorbent resin; v. concentrating the adsorbent resin eluate and drying the purified HMO-concentrate to obtain the neutral or sialylated HMO in solidified form, wherein the cation exchange resin eluate is neutralized to a pH value of approximately 7 before conducting step iv).

More preferably, the nanofiltration membrane has an active (top) layer composed of polyamide, more preferably piperazine-based polyamide, a MgSO4 rejection factor of about 50-90 % and preferably a NaCl rejection factor of not more than 50 %. More preferably, the nanofiltration step is performed so that the pH is set below 5.0, preferably below 4.5, advantageously below 4.0, but preferably not less than 3.0, ensuring the retention of the neutral or sialylated HMO to be purified and allowing the mono-and divalent salts to pass and accumulate in the permeate, and also allowing at least a part of lactose to pass and accumulate in the permeate.

Also preferably, including the preferred and more preferred embodiments disclosed above, the adsorbent resin is an acid adsorbent derived from cross-linked polystyrene or polyacrylic polymers and partially functionalized with tertiary amine functional groups. More preferably, the adsorbent resin has a surface area of > 400 m ² /g, such as 400 to 1200 m ² /g. Even more preferably, the adsorbent resin has a capacity of 0.1-0.3 eq/1. 3, Neutral or sialylated human milk oligosaccharide produced by the method according to the invention

In another aspect, the invention relates to a neutral or sialylated human milk oligosaccharide obtained by the method according to the invention.

The neutral or sialylated HMO recovered and purified according to the method described in this specification can be amorphous or crystalline, preferably amorphous.

In a preferred embodiment, the purity of the neutral or sialylated HMO on a dry basis is greater than 80 wt.% for a single neutral or sialylated HMO based on dry matter; or for mixtures of HMOs, greater than 70% purity based on dry matter, for the combination. More preferably, the purity of a single neutral or sialylated HMO is greater than 90 wt.%.

In a preferred embodiment, the neutral or sialylated HMO has at least one of the following characteristics (by weight): < 2% lactulose, < 3% fucose, < 1% galactose, or < 3% glucose.

In an embodiment, the neutral or sialylated HMO has a fines fraction (less than or equal to 10 pm), of less than 10%, preferably less than 5%, more preferably less than 1%, most preferably less than 0.1%. The neutral or sialylated HMO also preferably has an average particle size (d50), of greater than 100 pm, more preferably greater than 150 pm, even more preferably greater than 200 pm.

The neutral or sialylated HMO produced by the method according to the invention, demonstrates good flowability. Preferably, the HMO has a Carr index of less than 30, where the Carr index (C) is determined by the formula C = 100(1 - pB/pT), where pB is the freely settled bulk density of the powder, and pT is the tapped bulk density of the powder after “tapping down”. For free-flowing solids, the values bulk and tapped density would be similar, so the value is small. For poorer flowing solids, the differences between these values would be larger, so that the Carr index would be larger.

In a preferred embodiment, the neutral or sialylated HMO has a water content of less than 15 wt.%, less than 10 wt.%, less than 9 wt.%, less than 8 wt.%, less than 7 wt.%, or less than 6 wt.%. In order to optimize product recovery, preferably, the neutral or sialylated HMO has a pH greater than 3.0 in at least 5% solution. Typically, this is achieved by adjusting the pH of the HMO-containing stream to greater than 3.0 prior to the drying step. Preferably, the neutral or sialylated HMO has a pH of from 4 to 7, more preferably from 4.5 to 5.5. Preferably, the HMO is a neutral HMO. In an embodiment, the neutral HMO is preferably selected from the group consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N- triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V (alternative name: lacto-N-fucopentaose VI), lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N- difucohexaose III, 6'-galactosyllactose, 3 '-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, and any mixture thereof More preferably, the HMO is 2'-fucosyllactose, 3-fucosyllactose, 2',3- difucosyllactose, lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose or a lacto-N-fucopentaose, more preferably 2'-fucosyllactose, LNT, LNnT or a lacto-N-fucopentaose.

In an embodiment, the sialylated HMO is selected from the group consisting of 3’-sialyllactose (3’- SL) and 6’-sialyllactose (6’-SL).

In an embodiment, the neutral or sialylated HMO obtained by the method according to the invention, is incorporated into a food product (e g. human or pet food), dietary supplement or medicine product.

In some embodiments, the neutral or sialylated HMO is incorporated into a human baby food (e g. infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to roughly mimic human breast milk. In some embodiments, a neutral or sialylated HMO purified by a method in this specification is included in infant formula to provide nutritional benefits similar to those provided by one or more neutral or sialylated HMOs in human breast milk. In some embodiments, the neutral or sialylated HMO is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include skimmed milk, carbohydrate sources (e.g. lactose), protein sources (e.g. whey protein concentrate and casein), fat sources (e.g. vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, B, B2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate).

Hence, another aspect of the invention relates to a neutral or sialylated human milk oligosaccharide obtained by the method according to the invention for use in medicine. Hence, another aspect of the invention relates to the use of a neutral or sialylated human milk oligosaccharide obtained by the method according to the invention for food and/or feed applications.

Hence, another aspect of the invention relates to a food or cosmetic product comprising the neutral or sialylated human milk oligosaccharide obtained by the method according to the invention.

EXAMPLES

Example 1 : Production and purification of lacto-N-fucopentaose I (LNFP-I) and 2'-fucosyllactose (2’-FL)

Fermentation: LNFP-I -containing broth was generated on 20 L scale by fermentation using a genetically modified E. coli strain ofLacZ', LacY ⁺ phenotype, wherein said strain comprises a recombinant gene encoding p- l ,3-N-acetyl-glucosaminyl transferase which is able to transfer the GlcNAc of UDP-GlcNAc to the internalized lactose, a recombinant gene encoding a P-1, 3- galactosyl transferase which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl- glucosaminylated lactose (lacto-N-triose II or LNT-2) forming LNT (lacto-N-tetraose), alpha-1, 2- fucosyltransferase enzyme which is able to transfer fucose of GDP -fucose to LNT and genes encoding a biosynthetic pathway to UDP-GlcNAc, UDP-Gal, and GDP -fucose. The fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNFP-I which was accompanied with 2’ -FL, traces of LNT and unreacted lactose as major carbohydrate impurities in the fermentation broth.

Purification by ultrafiltration, nanofiltration, and strong acidic cation exchange (UF/NF/SAC(H ⁺ )): The obtained fermentation broth was acidified with H2SO4 to pH=4.0. A part of the broth (12 kg in 4x3 kg sublots) was processed by ultrafiltration with a Kerasep 15 kDa membrane (BE-8 channels, size 1178x25mm, area 0.21 m ² ) in a batch mode at a temperature of 60 °C, trans membrane pressure (TMP) of 2-2.5 bar, and cross-flow of 600 1/h in two steps: first the volume was reduced by removing permeate to a concentration factor CF= 2 followed by continuous diafiltration with 6 kg deionized water generating ultrafiltration permeate of 7.00 kg each time (4x7 kg sublots, total 28 kg).

The combined permeate (27.7 kg) was processed by cross-flow loose nanofiltration with Trisep UA60 membrane (spiral-wound size 1812, area 0.23 m ² , pore size/MWCO of 1000-3500 Da) at 30- 40 °C, and a TMP of 38 bar: first 23 kg permeate was collected to give concentrated retentate (Brix 19.4, conductivity 17.3 mS/cm), followed by continuous diafiltration with 25 kg of de-ionized water to give additional 27 kg diafiltration permeate. The obtained retentate was collected from the system (2.680 kg, Brix 22.0) with additional washes (de-ionized water, 500 ml) to give final nanofiltration/diafiltration retentate with substantially reduced conductivity (3.17 kg, Brix 18.8, conductivity 1.30 mS/cm, pH 4.11).

Part of the obtained nanofiltration retentate (2.67 kg) was passed through the strong acidic cation exchange resin Dowex-88 (H ⁺ -form, bed volume (BV)= 800 ml), followed by water (600 ml) to give 3.21 kg of a yellow-brown solution (Brix 15.2, conductivity 4.22 mS/cm, pH 1.85).

Purification by an adsorbent resin (AR): A small sample after the Dowex-88 treatment (119 g, pH 1.85) was passed through a column packed with Dowex Optipore SD-2 adsorbent resin (15.6 g, water content 58 %, surface area: around 800 m ² /g, dry weight capacity: 0.8 eq/kg, BV=24 ml) at 250 ml/h flow rate and eluted with water to give 167 g of a solution (Brix 10.0, conductivity 0.110 mS/cm, pH 3.64), the column was regenerated by washing with 4% HC1, 4% NaOH, and 4% NaCl with water washes after each step and the obtained solution was re-processed again through the regenerated resin at 75 ml/h (3BV/h) to give 215 g of completely colourless solution (Brix 7.5, conductivity 0.024 mS/cm, pH 5.0, Abs_400=0.0000, proteins < 0.5 mg/1) and freeze-dried.

Analytical data showed that the AR step not only removed the colour completely, but also reduced the protein concentration by an order of magnitude.

Example 2: Production and purification of 2'-fucosyllactose (2’-FL) and difucosyllactose (DFL)

Fermentation: 2’-FL-containing broth was generated by fermentation using a genetically modified E. col strain of LacZ", LacY ⁺ phenotype, wherein said strain comprises a recombinant gene encoding an a-l,2-fucosyltransferase enzyme which is able to transfer fucose of GDP -fucose to the internalized lactose and genes encoding a biosynthetic pathway to GDP -fucose. The fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing 2’-FL which was accompanied with DFL and unreacted lactose as major carbohydrate impurities in the fermentation broth.

Purification by ultrafiltration, nanofiltration, and strong acidic cation exchange (UF/NF): the obtained fermentation broth was processed with regard to UF/NF as described in Example 1, i.e. by pH-adjustment, cross-flow UF and NF with diafiltration to give a NF retentate with the following parameters: Brix 23.3, conductivity 3.80 mS/cm, pH 3.65, Abs_400 2.899, colour index CI_400 124. Purification by strong acidic cation exchange and adsorbent resin (SAC(H ⁺ )/AR): a portion of the NF retentate (1.11 kg, 1020 ml, 6 BV) was passed through Dowex-88 (H ⁺ -form, BV= 170 ml) packed in a XK 16/100 column (ID = 16mm), immediately followed by Dowex Optipore SD-2 adsorbent resin (120 g of wet resin, water content 58 %, BV= 172 ml) packed in another XK16/100 column at approximately 500 ml/h flow rate (3 BV/h), followed by water (2 BV, 340 ml). 35 ml (0.2 BV) fractions were collected and analysed for Brix, conductivity, pH, Abs_400 and protein content.

Fractions #6-23 were combined (630 ml, 3.66 BV, Brix 18.3, CI_400 1.78), pH-adjusted with 4 % NaOH-solution to 4 8 and freeze-dried (protein 17 mg/kg). The remaining fractions #24-40 were also combined and freeze-dried.

Example 3: Production and purification of lacto-N-tetraose (LNT). lacto-N-triose II (LNTri II) and para-lacto-N-hexaose II (pLNH II)

Fermentation: LNT-containing broth was generated on 201 scale by fermentation using a genetically modified A. coli strain ofLacZ', LacY ⁺ phenotype, wherein said strain comprises a recombinant gene encoding |3-l,3-N-acetyl-glucosaminyl transferase which is able to transfer the GlcNAc of UDP-GlcNAc to the internalized lactose, a recombinant gene encoding a (3-1,3- galactosyl transferase which is able to transfer the galactosyl residue of UDP-Gal to the N-acetyl- glucosaminylated lactose (lacto-N-triose II) forming LNT (lacto-N-tetraose) and genes encoding a biosynthetic pathway to UDP-GlcNAc, UDP-Gal. The fermentation was performed by culturing the strain in the presence of exogenously added lactose and a suitable carbon source, thereby producing LNT which was accompanied with lacto-N-triose II, pLNH II and unreacted lactose as major carbohydrate impurities in the fermentation broth.

Ultrafiltration (UF): The obtained broth was acidified with H2SO4 to pH=3.7. A part of the broth (12.86 kg) was processed by UF with Tami 15 kDa membrane (39 channels, size 1178x25mm, area 0.5 m ² ) in a batch mode at a temperature of 60 °C, a trans membrane pressure of 6-7 bar, and crossflow of 600 1/h in two steps: 1) 9190 g of permeate was collected followed by 2) continuous diafiltration with 14.55 kg of water at ca 5-6 1/h feeding rate. Total 24.52 kg of permeate was collected and the retentate (ca 2.85 kg) was discarded.

Nanofiltration (NF): The obtained UF permeate (24.43 kg, Brix 9.9, conductivity 9.10 mS/cm, pH 4.58, Ab s 400 0.9465) was processed by cross-flow loose nanofiltration with Trisep UA60 membrane (spiral-wound size 1812, area 0.23 m ² , MWCO of 1000-3500 Da) at 30-48 °C, TMP = 30-31 bar to collect 14.85 kg of permeate and a concentrated retentate (Brix 23.0, conductivity 10.10 mS/cm, pH 4.30, Abs_4002.0552).; The pH of the retentate was adjusted with 50 % NaOH- solution to 5.6 followed by diafiltration with 301 of water which was added continuously at 5.1 1/h flow rate to give 46 kg combined permeate and intermediate retentate (ca. 8.38 kg, Brix 21.1, conductivity 3.05 mS/cm, pH 5.45, Ab s 400 1.6358). Then, the pH of the intermediate retentate was adjusted with 25 % H2SO4-solution to 3.71, followed by additional diafiltration with 10 1 of water. The obtained retentate (Brix 20.4, conductivity 1.77 mS/cm, pH 3.83, Abs_400 1.3853) was further concentrated at TMP= 39 bar to obtain the final retentate (5.48 kg, Brix 30.4, conductivity 1.35 mS/cm, pH 4.06, Abs_400 2.0307).

Strong acidic cation exchange (SAC(H ⁺ )): The obtained NF retentate (5.41 kg) was passed through Dowex-88 (H ⁺ -form, BV= 800 ml) and eluted with water to give a yellow-brown solution (6.1 kg, Brix 26.6, conductivity 5.21 mS/cm, pH 1.62, Abs_400 0.8269)

Adsorbent resin (AR) treatment: A small sample after SAC(H ⁺ ) treatment (260 g) was passed through a column packed with Dowex Optipore SD-2 adsorbent resin (170 ml) at 510 ml/h flow rate and eluted with water to give 415 g of a solution (Brix 15.5, conductivity 0.023 mS/cm, pH 8.4, Abs_400 0.0450), it was pH-adjusted to 4.4 with 25 % H2SO4-solution and freeze-dried to give 60.9 g of a white solid.

Example 4: Determination of a substance rejection factor on a membrane

The NaCl and MgSCU rejection on a membrane is determined as follows: in a membrane filtration system, a NaCl (0.1 %) or a MgSCU (0.2 %) solution is circulated across the selected membrane sheet (for Tami: tubular module) while the permeate stream is circulated back into the feed tank. The system is equilibrated at 10 bars and 25 °C for 10 minutes before taking samples from the permeate and retentate. The rejection factor is calculated from the measured conductivity of the samples: (1-K _p /K _r )- 100, wherein K _P is the conductivity of NaCl or MgSCU in the permeate and K _r is the conductivity of NaCl or MgSCE in the retentate. A carbohydrate rejection factor is determined in a similar way with the difference that the rejection factor is calculated from the concentration of the samples (determined by HPLC): (1-C _p /C _r ) - 100, wherein C _P is the concentration of the carbohydrate in the permeate and C _r is the concentration of the carbohydrate in the retentate. Example 5: Comparison of MgSCE and rejection

2.01 of 0.2 % MgSCU solution were loaded into an MMS SW18 system equipped with 1812-size spiral wound Trisep UA60 element (piperazine-amide, MWCO 1000-3500 Da, membrane area 0.23 m ² ). The system was run at 400 1/h cross-flow with permeate circulating back to the feed tank. It was equilibrated for at least 5 min or until constant conductivity in the permeate under each condition. The pH was adjusted by adding a small amount of 25 % H2SO4 solution. The conductivity of the permeate and the retentate are disclosed in the table below.

The same experiment was performed with 0.2 % ISfeSCU solution. It was demonstrated that the sodium salt rejection with divalent counter-ion such as sulphate is strongly pH dependent in case of NF with polyamide membrane. Therefore, inorganic anions with sodium cation can be effectively removed in a NF/DF step when the DF is conducted at a pH of less than 4.5, advantageously less than 4.0, resulting in a practically salt-free solution (as assessed from conductivity). In this regard, no basic anionic resins are necessary to use to obtain a salt-free solution.