SIMPLE METHOD FOR THE PURIFICATION OF A SIALYLLACTOSE

Abstract

Provided is a method for the purification of a sialyllactose from other carbohydrates, characterized in that the method comprises the steps of subjecting an aqueous solution containing the sialyllactose to two membrane filtration steps using different membranes, a membrane having a molecular weight cut-off of between about 300 Dalton to about 500 Dalton and a membrane having a molecular weight cut-off of between about 600 Dalton to about 800 Dalton.

Claims

1. A method for purification of a sialyllactose from one or more other carbohydrates, wherein the method comprises subjecting an aqueous solution comprising the sialyllactose and other carbohydrates to two membrane filtrations using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.

2. The method according to claim 1, wherein the aqueous solution is obtained from a fermentation or enzymatic process for production of the sialyllactose.

3. The method according to claim 1, wherein the aqueous solution is obtained from a fermentation by separating the biomass from the fermentation broth.

4. The method according to claim 3, wherein separating the biomass from the fermentation broth comprises at least one ultrafiltration, optionally two ultrafiltrations, optionally a first ultrafiltration using a membrane having a molecular weight cut-off of about 500 kDa and a second ultrafiltration using a membrane having a molecular weight cut-off of about 150 kDa.

5. The method according to claim 1, wherein the aqueous solution comprising the sialyllactose is treated with a cation exchange resin, optionally in H.sup.+ form, and an anion exchange resin, optionally in Cl.sup. form, optionally before subjecting the aqueous solution to the membrane filtration.

6. The method according to claim 1, wherein the method further comprises dialysis, optionally electrodialysis.

7. The method according to claim 1, wherein the purity of sialyllactose in the aqueous solution is 70%, 60%, 50%, 40%, 30%, 20%; 10% or 5% prior to the purification and/or the aqueous solution comprises the sialyllactose at a purity of 80%, optionally of 85% or optionally 90% after the purification.

8. The method according to claim 1, wherein the sialyllactose is 3-sialyllactose of 6-sialyllactose.

Description

DETAILED DESCRIPTION

[0015] Provided is a method for the purification of a sialyllactose from other carbohydrates, wherein the method comprises the steps of subjecting an aqueous solution containing a sialyllactose and said other carbohydrates to two membrane filtration steps using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and wherein the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.

[0016] The membrane having a molecular weight cut-off of between about 300 to about 500 Dalton allows removal of the bulk of carbohydrates having a molecular weight that is smaller than that of a sialyllactose. Upon filtration, SL and carbohydrates having a molecular weight larger than that of SL are retained in the retentate.

[0017] In an additional and/or alternative embodiment, the membrane having a molecular weight cut-off of between about 300 to about 500 Dalton has a pore size of 1 to 2 nm.

[0018] Suitable membranes having a molecular weight cut-off that is smaller than the molecular weight of SL arefor exampleTriSep XN-45 (TriSep Corporation, USA), Dairy DK (Suez Water Technologies, formerly GE) and Filmtech NF270 (Dow).

[0019] The membrane having a molecular weight cut-off of between about 600 to about 800 Dalton possesses permeability for sialyllactose and carbohydrates having a molecular weight smaller than that of SL. Upon filtration, SL is present in the filtrate whereas carbohydrates having a molecular weight larger than that of SL remain in the retentate.

[0020] In an additional and/or alternative embodiment, the membrane having a molecular weight cut-off of between about 600 to about 800 Dalton has a pore size of 2.5 to 3 nm.

[0021] Suitable membranes for possessing permeability for 6-SL and retaining carbohydrates having a molecular weight larger than that of 6-SL in the retentate arefor exampleTangenX SIUS TFF 0.65 kDa membrane (Repligen Corporation), Zirkonia modules 3 nm (Pervatech BV) and S-CUT YSNF-YS600 (CUT/Burkert).

[0022] The method circumvents the use of expensive discontinuous chromatographic steps and also renders precipitation or crystallisation steps using organic solvents unnecessary. Thus, in an additional and/or alternative embodiment, the method does not comprise one or more discontinuous chromatography steps and/or one or more steps of precipitating and/or crystallizing 6-SL by using an organic solvent.

[0023] In an additional and/or alternative embodiment, the aqueous solution is obtained from a fermentation or enzymatic process for the production of a sialyllactose.

[0024] The method described herein is suitable for the purification of the human milk oligosaccharides 3-sialyllactose or 6-sialyllactose from a microbial fermentation or biocatalysis reaction in multi-ton amounts, because it is economically feasible and scalable.

[0025] In an additional and/or alternative embodiment, the aqueous solution is obtained from a fermentation, i.e. cultivating microbial cells that are able to produce a sialyllactose in a culture medium (fermentation broth) and under conditions that are permissive for the microbial cells to produce the sialyllactose, by separating the biomass from the fermentation broth. Preferably, separating the biomass from the fermentation broth comprises at least one step of ultrafiltration, preferably two steps of ultrafiltration, more preferably a first ultrafiltration using a membrane having a molecular weight cut-off of about 500 kDa and a second ultrafiltration using a membrane having a molecular weight cut-off of about 150 kDa.

[0026] In an additional and/or alternative embodiment, the aqueous solution is treated with a cation exchange resin, preferably in H.sup.+ form, and with an anion exchange resin, preferably in Cl.sup. form. Preferably, the aqueous solution containing the sialyllactose and other carbohydrates is treated with ion exchange resins before being subjected to the membrane filtration steps for removal of other carbohydrates.

[0027] In an additional and/or alternative embodiment, the method further comprises a step of dialysis, preferably a step of electrodialysis, for removal of ions. Preferably, the aqueous solution containing the sialyllactose is subjected to dialysis and/or electrodialysis after said other carbohydrates have been removed from the aqueous solution.

[0028] The product can be most conveniently supplied as a sterile concentrate or as a spray-dried product.

[0029] The method allows purifying 3-sialyllactose or 6-sialyllactose, i.e. separating 3-SL or 6-SL from other carbohydrates, wherein the purity of the sialyllactose in the aqueous solution is 70%, 60%, 50%, 40%, 30%, 20%; 10% or 5% prior to the purification and/or the aqueous solution contains the sialyllactose at a purity of 80%, preferably of 85% or more preferably 90% after the purification.

[0030] In an additional and/or alternative embodiment, the purification comprises the following steps: [0031] i.) separating the biomass from the fermentation broth; [0032] ii.) subjecting the cell-free fermentation broth to at least one ion-exchange resin treatment for the removal of charged material, preferably to an anion-exchanges resin treatment and a cation-exchange resin treatment; [0033] iii.) subjecting the aqueous solution obtained in step ii. to a membrane filtration step using a membrane having a molecular weight cut-off of about 300 to about 500 Dalton to remove carbohydrates having a molecular weight smaller than that of a sialyllactose; [0034] iv.) subjecting the retentate obtained in step iii. to a membrane filtration step using a membrane having a molecular weight cut-off of about 600 to about 800 Dalton to remove carbohydrates having a molecular weight larger than that of a sialyllactose; andoptionally [0035] v.) increasing the concentration of the sialyllactose present in the filtrate of step iv.

[0036] Whereas other procedures used for the purification of sialyllactose are rather complex and therefore expensive the method described herein before relies mainly on the use of two membrane filtration steps.

[0037] Certain embodiments comprise one or more further steps, such as dialysis steps (for the removal of salts), electrodialysis (for the removal of charged molecules), activated charcoal treatment (for the decolorization of the product solution) and/or other filtration processes (like endotoxin removal and/or sterile filtration). Although not necessary, the process may comprise treatment of the aqueous solution containing the sialyllactose with an organic solvent (such as short chain alcohols like methanol) for the precipitation of contaminating oligosaccharides or for elution after adsorption to activated charcoal with short chain alcohols and water mixtures, and/or for crystallisation of the sialyllactose.

[0038] The aqueous solution resulting from the method contains the sialyllactose, but no nucleic acids and polypeptides of the microbial cells. In addition, said aqueous solution hardlyif at allcontains monosaccharides and/or disialyllactose.

[0039] The present invention will be described with respect to particular embodiments, 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

EXAMPLES

Example 1

Fermentative Production of 6-Sialyllactose

[0047] A 6-sialyllactose feed-batch fermentation employing a recombinant 6-sialyllactose synthesizing E. coli strain (E. coli BL21(DE3) lacZ) expressing the -2,6-sialyltransferase gene plsT6 from Photobacterium leiognathi JT-SHIZ-119 was performed. To enhance the biosynthesis of CMP-sialic acid, genes encoding the glucosamine-6-phosphate synthase GlmS from E. coli, the N-acetylglucosamine 2-epimerase Slr1975 from Synechocystis sp., the glucosamine 6-phosphat N-acetyltransferase Gna1 from Saccharomyces cerevisiae, the phosphoenolpyruvate synthase PpsA from E. coli, the N-acetylneuraminate synthase NeuB, and the CMP-sialic acid synthetase NeuA, the latter both from Campylobacter jejuni, were chromosomally integrated into the E. coli BL21(DE3) host. Furthermore, the gene encoding the lactose permease LacY from E. coli, and the genes cscB (sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase), and cscR (transcriptional regulator) from E. coli W were integrated into the BL21 genome such that transcription of the integrated genes is initiated from constitutive promotors, either from the tetracycline promotor P.sub.tet or the PT5 promotor. A functional gal-operon consisting of and expression the genes galE (UDP-glucose-4-epimerase), galT (galactose-1-phosphate uridylyltransferase), galK (galactokinase), and galM (galactose-1-epimerase) was transferred from E. coli K12 to the genome of the BL21 strain. To prevent degradation of N-acetylglucosamine 6-phosphate, genes coding for N-acetylglucosamine-6-phosphate deacetylase (NagA), glucosamine-6-phosphate deaminase (NagB), and the N-acetylglucosamine specific PTS protein IIABC (NagE) were deleted from the bacterial chromosome. Additionally, the operon manXYZ, encoding a sugar transporter of the E. coli PTS system for mannose, glucose, glucosamine and N-acetylglucosamine, was deleted, as well as the genes nanA, nanK, nanE, and nanT, encoding the N-acetylneuraminate lyase, the N-acetylmannosamine kinase, the N-acetylmannosamine-6-phosphate epimerase, and the sialic acid transporter, respectively. The gene encoding the N-acetylgalactosamine-6-phosphate deacetylase (AgaA) was also deleted.

[0048] The 6-sialyllactose producing E. coli strain was grown in a defined mineral salts medium, comprising 7 g.Math.l.sup.1 NH.sub.4H.sub.2PO.sub.4, 7 g.Math.l.sup.1 K.sub.2HPO.sub.4, 2 g.Math.l.sup.1 KOH, 0.3 g.Math.l.sup.1 citric acid, 5 g.Math.l.sup.1 NH.sub.4Cl, 1 ml.Math.l.sup.1 antifoam (Struktol J673, Schill+Seilacher), 0.1 mM CaCl.sub.2, 8 mM MgSO.sub.4, trace-elements and 2% sucrose as carbon source. Trace elements consisted of 0.101 g.Math.l.sup.1 nitrilotriacetic acid, pH 6.5, 0.056 g.Math.l.sup.1 ammonium ferric citrate, 0.01 g.Math.l.sup.1 MnCl.sub.24 H.sub.2O, 0.002 g.Math.l.sup.1 CoCl.sub.26H.sub.2O, 0.001 g.Math.l.sup.1 CuCl.sub.22H.sub.2O, 0.002 g.Math.l.sup.1 boric acid, 0.009 g.Math.l.sup.1 ZnSO.sub.47 H.sub.2O, 0.001 g.Math.l.sup.1 Na.sub.2MoO.sub.42H.sub.2O, 0.002 g.Math.l.sup.1 Na.sub.2SeO.sub.3, 0.002 g.Math.l.sup.1 NiSO.sub.46H.sub.2O.

[0049] The sucrose feed (500 g.Math.l.sup.1) was supplemented with 8 mM MgSO.sub.4, 0.1 mM CaCl.sub.2, trace elements, and 5 g.Math.l.sup.1 NH.sub.4Cl. For 6-sialyllactose formation, a lactose feed of 216 g.Math.l.sup.1 was employed. The pH of the culture medium was controlled by using ammonia solution (25% v/v). Fed batch fermentation was conducted at 30 C. under constant aeration and agitation for 72 hours by applying a sucrose feeding rate of 5.5-7 ml.Math.l.sup.1.Math.h.sup.1, referring to the starting volume. At 72 hours after the start of the fermentation, most of the added lactose was converted into 6-sialyllactose. In order to remove residual lactose in the fermentation supernatant, -galactosidase was added to the fermentation vessel.

Example 2

Preparation of a Cell Free Fermentation Broth

[0050] The cell-mass (about 10% of the fermentation broth) was separated from the medium by ultrafiltration (0.05 m cut-off) (CUT membrane technology, Erkrath, Germany) followed by a cross-flow filtrations using a filter having a MWCO of 150 kDa (Microdyn-Nadir, Wiesbaden, Germany).

Example 3

Ion Exchange Resin Treatment of a 6-Sialyllactose-Containing Cell Free Fermentation Broth

[0051] The cell-free fermentation broth as obtained from example 2 contained 6-sialyllactose (at a purity of 9% by dry weight) in a volume of 1000 liters and was passed over a strong cationic ion exchange resin (Lewatit S2568 obtained from the company Lanxess, Germany) in H.sup.+ form in order to remove positive charged contaminants (size of the ion exchanger bed volume was 200 L). The elution of 6-SL from the ion exchange resin was continued with deionized water. The obtained solution was then set to pH 7 by the addition of sodium hydroxide solution. The solution was then (without delay) passed over an anionic ion exchanger column (bed volume of the ion exchanger was 200 L). The used strong anionic ion exchanger Lewatit S6368 A (Lanxess, Germany) was in the chloride (Cl.sup.) form. The elution of 6-SL was continued with deionized water. The obtained solution was again neutralized to pH 7.

Example 4

First Filtration for the Removal of Smaller Molecules/Carbohydrates

[0052] The solution obtained by ion exchange resin treatment set forth in example 3 was diafiltrated using a TriSep XN-45 (TriSep Corporation, USA) nanofiltration membrane and 500 L of deionized water. The resulting solution was further concentrated using the nanofiltration membrane (RE 8040-BE, CSM-Saehan) wherein a 6-sialyllactose solution with a purity of 31% (by dry weight) and a conductivity of 8 mS/cm was obtained.

Example 5

Second Filtration for the Removal of Larger Molecules/Carbohydrates

[0053] To separate the 6-sialyllactose from fermentative produced by-product disialyllactose a second membrane treatment was employed. A TangenX SIUS TFF membrane with a nominal cut-off 0.65 kDa (Repligen Corporation) was used for filtration. The solution containing 94.4% 6-sialyllactose and 6.6% disialyllactose (ratio 15:1) was cross-flow filtered with the described system and the concentration of 6-sialyllactose and disialyllactose was determinate inside permeate and retentate. The results show a significant reduction of disialyllactose amount inside the permeate from 6.6% to 0.2% (ratio 6-sialyllactose to disialylactose was 55:1) whereas the ratio of 6-sialyllactose to disialyllactose inside the retentate was 14:1. This result shows that the 6-sialyllactose could be separated from the disialyllactose by using an additional filtration step. This observation could be also shown by using a second kind of membrane with a similar cut-off from a different supplier. By using a S-CUT YSNF-YS600-2540-M46D-ATD membrane (CUT Membrane Technology GmbH) with a nominal cut-off of 600-800 Da the 6-sialyllactose was also separated from disialyllactose by cross-flow filtration.

Example 6

Electrodialysis Treatment

[0054] The permeate obtained in example 5 was electrodialysed to 2 mS/cm using a PC-Cell 15 electrodialysis apparatus (PC-Cell, Heusweiler, Germany) equipped with an PC-Cell ED 1000H membrane stack. This membrane stack was equipped with the following membranes: cation exchange membrane CEM: PK SK and the anion exchange membrane AEM:PcAcid60 with an size exclusion limit of 60 Da. A 0.025 M sulfamic acid (amidosulfonic acid) solution was used as an electrolyte in the electrodialysis process. After electrodialysis a solution containing 6-sialyllactose at a purity of 83% (by dry weight) was obtained. Alternatively to the electrodialysis treatment, a diafiltration process could be employed.

Example 7

Concentration and Activated Carbon Treatment

[0055] Some water was removed from the aqueous solution using a reversed osmosis filter and rotary evaporation at 45 C. to obtain a 25% (m/v) 6-sialyllactose solution. The resulting aqueous solution was then treated with activated carbon (Norit SA2). 125 g activated carbon was employed for 1 L 25% (m/v) 6-sialyllactose.

Example 8

Sterile Filtration and Endotoxin Removal

[0056] Subsequently, the solution obtained in example 7 was subjected to sterile filtration by passing the solution through a 6 kDa filter (Pall Microza ultrafiltration hollow fiber module SEP-2013, Pall Corporation, Dreieich, Germany). The sterile solution was then stored at RT until spray-drying.

Example 9

Spray-Drying of 6-Sialyllactose

[0057] The so obtained (example 8) sterile 6-sialyllactose were then spray-dried using a NUBILOSA LTC-GMP spray dryer (NUBILOSA, Konstanz, Germany). For the spray-drying of the 6-sialyllactose the solution was passed under pressure with 3.5 bar through the spray dryer nozzles set to 130 C. and flow was adjusted to maintaining an exhaust temperature between 67 C. to 69 C. Using these settings a spray-dried powder with less than 9% moisture could be obtained. The moisture contents were determined by Karl-Fischer titration.

Example 10

LC-MS/MS Analysis of 6-Sialyllactose

[0058] For quantification mass spectrometry analysis was performed by MRM (multiple reaction monitoring) using a LC Triple-Quadrupole MS detection system. Precursor ions are selected and analyzed in quadrupole 1, fragmentation takes place in the collision cell using argon as CID gas, selection of fragment ions is performed in quadrupole 3. Chromatographic separation of saccharides was performed on a XBridge Amide HPLC column (3.5 m, 2.150 mm (Waters, USA) with a XBridge Amide guard cartridge (3.5 m, 2.110 mm) (Waters, USA). Column oven temperature of the HPLC system was 50 C. The mobile phase was composed of acetonitrile/H.sub.2O with 10 mM ammonium acetate. A 1 l sample was injected into the instrument; the run was performed for 3.60 min with a flow rate of 400 l/min. 6-sialyllactose was analyzed by MRM in ESI positive ionization mode. The mass spectrometer was operated at unit resolution. Sialyllactose forms an ion of m/z 656.2 [M+Na]. The precursor ion of sialyllactose was further fragmented in the collision cell into the fragment ions m/z 612.15, m/z 365.15 and m/z 314.15. Collision energy, Q1 and Q3 Pre Bias were optimized for each analyte individually. Quantification methods were established using commercially available standards (Carbosynth, Compton, UK).