Method for producing glycopeptide-containing composition

Abstract

The present invention addresses the problem of providing a production method capable of fractionating a glycopeptide having a constant sugar content at high yield and good reproducibility in a short production time using a small amount of enzyme as a method for preparing a glycopeptide-containing composition.　Provided is a method for producing a composition containing a glycopeptide, the method being characterized in that the following two steps are performed simultaneously in one reactor. (1) A step for bringing a composition containing a glycoprotein into contact with an endo-type protease and / or an exo-type protease in a solution to obtain a reaction mixture solution. (2) A step for ultrafiltration of the reaction mixture solution obtained in (1).

Description

Method for producing a glycopeptide-containing composition

[0001] The present invention relates to a method for producing a glycopeptide-containing composition, particularly to a method for producing a glycopeptide-containing composition by fractionating a glycopeptide-containing composition from a raw material containing a glycoprotein using an enzyme and an ultrafiltration membrane.

[0002] In recent years, glycopeptides (hereinafter sometimes referred to as glycopeptides) have attracted attention as a new food ingredient with health benefits. Glycopeptides are generally produced by treating glycoproteins (hereinafter sometimes referred to as glycoproteins) with endoproteases and / or exoproteases. However, simply treating glycoproteins with endoproteases and / or exoproteases results in the treatment product containing large amounts of unglycosylated peptides along with glycopeptides. Therefore, this production method only yields compositions with very low contents of the target glycopeptides. Therefore, a new step of separating glycopeptides from peptides is required.

[0003] Methods for separating glycopeptides and peptides using an ultrafiltration membrane have been reported. Patent Document 1 discloses a production method for preparing a glycopeptide (sialylglycopeptide) mixture with a high sialic acid content by treating purified κ-casein with a protease, terminating the enzymatic reaction by heat treatment, and fractionating the resulting solution using an ultrafiltration membrane. Patent Document 2 discloses the following method for preparing a sialylglycopeptide concentrate. First, a composition containing glycomacropeptide (GMP) produced during cheese production is treated with a protease. The enzymatic reaction is then terminated by heat treatment, and the resulting solution is fractionated using an ultrafiltration membrane with a molecular weight cutoff of 500 to 3,000. The production method for compositions containing these glycopeptides (hereinafter sometimes referred to as glycopeptide-containing compositions) involves the following two separate steps: (1) a step of enzymatically hydrolyzing proteins and terminating the enzymatic reaction to obtain an enzymatic hydrolysis solution (in which the enzyme is already inactivated); and (2) a step of passing the enzymatic hydrolysis solution through an ultrafiltration membrane to fractionate glycopeptides. In this method, each step requires approximately 1 to 10 hours, resulting in a long production time, which is the total time required for both steps. Furthermore, to fractionate glycopeptides, glycoproteins must be thoroughly degraded, requiring large amounts of expensive enzymes (see "Comparative Example 1" in the Examples below). Furthermore, because glycopeptides and peptides have very similar molecular weights after the enzymatic reaction, fractionation by ultrafiltration is inefficient, and even when glycopeptides are concentrated to a certain level, the yield remains at around 50% (see Comparative Example 2 in the same Examples). Thus, conventional methods for fractionating glycopeptides have the drawbacks of requiring a long production time, requiring a large amount of enzyme for degradation, and producing low yields of the desired glycopeptides. However, to reproducibly fractionate glycopeptides with a consistent sugar content, it is necessary to prevent the enzymatic reaction from proceeding during membrane treatment, which necessitates the process being carried out in two steps (1) and (2) as described above.

[0004] Patent No. 2980362 WO2019 / 044960 Brochure

[0005] The objective of the present invention is to provide a method for producing a glycopeptide-containing composition that requires a short production time, a small amount of enzyme, and is capable of fractionating glycopeptides with a consistent sugar content in high yield and with good reproducibility.

[0006] The present inventors conducted extensive research to solve the above-mentioned problems and discovered a production method that simultaneously performs two steps that were previously separated. Specifically, they found that the problem can be solved by a method in which an enzymatic reaction step and a step using an ultrafiltration membrane are simultaneously performed in the same reactor. Furthermore, they discovered that glycopeptides can be recovered reproducibly regardless of the treatment volume or treatment speed by performing a concentration treatment using an ultrafiltration membrane and then diafiltration after the concentration treatment, thereby completing the present invention. Specifically, the present invention has the following features: <1> A method for producing a glycopeptide-containing composition, characterized in that the following two steps are simultaneously performed in a single reactor: (1) a step of contacting a glycoprotein-containing composition with an endoprotease and / or an exoprotease in a solution to perform an enzymatic reaction in the mixture; and (2) a step of ultrafiltrating the mixture obtained in (1). <2> The production method according to <1>, wherein the step (2) includes a concentration treatment and / or diafiltration treatment of the mixture. <3> The production method according to <2>, wherein step (2) is a step of performing a dialysis treatment after a concentration treatment. <4> The production method according to <1>, wherein the ultrafiltration membrane used in step (2) has a molecular weight cutoff of 150 to 3,000, and the step separates glycopeptides into a concentrate and peptides into a permeate. <5> The production method according to <1>, wherein the glycopeptide contains a sugar chain bound to a hydroxyl group of a serine and / or threonine residue of the peptide. <6> The production method according to <1>, wherein the glycopeptide contains a sugar chain consisting of sialic acid and galacto-N-biose, the combined amount of which is 4.9% by weight or more. <7> The production method according to <1>, wherein the molecular weight of the glycopeptide is 700 to 4,000. <8> The production method according to <1>, wherein the glycopeptide contains 1 to 16 amino acids constituting the peptide. <9> The production method according to <1>, wherein the glycopeptide contains one or more sugar chain structures selected from the group consisting of (a) to (e) below.(a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 (d) Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (e) Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 <10> The production method according to <9>, wherein, of the sugar chain structures contained in the glycopeptide, (a) accounts for 3.2% by weight or more, (b) accounts for 0.6% by weight or more, and (c) accounts for 0.7% by weight or more. <11> The production method according to <9>, wherein, among the glycan structures contained in the glycopeptide, the proportion of (a) relative to the total weight of (a), (b), and (c) is 70% or more. <12> A production method for a glycopeptide-containing composition, the production method comprising a step of ultrafiltration of a mixture in the course of an enzymatic reaction between a composition containing a glycoprotein and an endoprotease and / or an exoprotease. <13> The production method according to <12>, wherein the ultrafiltration step comprises a concentration treatment and / or diafiltration treatment of the mixture. <14> A glycopeptide-containing composition comprising the following glycan structures (a) to (c), characterized in that the composition contains 3.2% by weight or more of (a), 0.6% by weight or more of (b), and 0.7% by weight or more of (c): (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 <15> A glycopeptide-containing composition comprising the following glycan structures (a) to (c), wherein the weight ratios of (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 satisfy the following relationship: (a) / [(a)+(b)+(c)]≧70% <16> A glycopeptide-containing composition produced by the production method according to any one of <1> to <13>.

[0007] According to the present invention, by simultaneously carrying out an enzymatic reaction in a mixture obtained by contacting a glycoprotein-containing composition with an endoprotease and / or an exoprotease in solution and an ultrafiltration process in the same reactor, it has become possible to fractionate a glycopeptide-containing composition with a constant sugar content in high yield and with good reproducibility in a short time and with a small amount of enzyme. This makes it possible to stably provide glycopeptide-containing compositions with a high sialic acid content at low cost. It has also become possible to provide glycopeptide-containing compositions containing specific sugar chain structures in a predetermined ratio.

[0008] FIG. 1 is a schematic diagram of a bioreactor (reactor) used in the production method of the present invention.

[0009] (Method for Producing Glycopeptides) The method for producing a glycopeptide-containing composition of the present invention is characterized by simultaneously carrying out the following two steps in a single reactor: (1) a step of contacting a composition containing a glycoprotein with an endoprotease and / or an exoprotease in a solution to carry out an enzymatic reaction in the mixed solution; and (2) a step of ultrafiltrating the mixed solution obtained in step (1). The present invention is a method for ultrafiltrating an enzymatic reaction solution in progress while the enzymatic reaction is in progress, rather than the conventional method of ultrafiltrating a solution containing the inactivated enzyme after the enzymatic reaction is terminated and the enzyme is inactivated by heating or the like. That is, the method differs in that a mixed solution of a live enzyme (uninactivated enzyme) and a glycoprotein is ultrafiltrated. In other words, the method for producing a glycopeptide-containing composition of the present invention is a method for ultrafiltrating a solution in the enzymatic reaction of a composition containing a glycoprotein with an endoprotease and / or an exoprotease. Furthermore, the method for producing a glycopeptide-containing composition of the present invention is a method for producing a glycopeptide-containing composition of a specific composition by ultrafiltrating a mixed solution of a glycoprotein and a protease. Figure 1 shows a schematic diagram of a membrane bioreactor as an example of an apparatus for performing this process. The reaction tank is connected to an ultrafiltration membrane (sometimes called a UF membrane), and is piped so that the concentrate side is returned to the reaction tank (also called a reactor). The reaction tank is also piped so that dialysis water is supplied from a water tank, thereby controlling the number of diafiltration cycles. The permeate side of the UF membrane is configured to be discharged. The reaction tank is covered with a jacket, and hot water heated by steam is introduced into the jacket through a water inlet and circulated within the jacket. The circulated hot water is also discharged through a water outlet. This allows the temperature within the tank to be controlled. The temperature can also be controlled by other temperature control mechanisms, such as a heat exchanger.

[0010] A composition containing the raw glycoprotein, protease, and water are supplied to the reaction tank and mixed therein. A protease reaction cleaves glycopeptides from the glycoprotein. The reaction tank is configured so that a concentrated solution is returned to the reaction tank via a UF membrane. The glycoproteins in the returned concentrated solution react with the protease again in the tank, resulting in further glycopeptides. Since the UF membrane has a fixed molecular weight cutoff, when the concentrated protease reaction solution is supplied, peptides with low molecular weights in the reaction solution permeate the UF membrane, while glycopeptides with a molecular weight above a certain level that are unable to permeate are returned to the tank. By repeating this cycle, glycopeptides of the desired molecular weight are concentrated in the concentrated solution (concentration process). The concentration factor (CF factor) of the mixed solution is preferably greater than 1, more preferably greater than 2. A CF factor of 2 or greater improves the reproducibility of the sialic acid content and sialic acid recovery rate regardless of the processing volume and processing speed.

[0011] Furthermore, in the step (2) of the present invention, diafiltration (DF, also referred to as diafiltration) is preferably performed using a UF membrane. In step (2), both the concentration treatment and diafiltration are preferably performed using the same UF membrane. In this case, diafiltration is preferably initiated after the concentration treatment using the UF membrane. "Initiating after the concentration treatment" does not mean that dialysis is initiated after the concentration treatment is completely completed, but also includes the case where dialysis is initiated after concentration has progressed to a certain extent. For example, if the final concentration is three-fold, dialysis may be initiated when concentration has progressed two-fold. The dialysis step is initiated by introducing dialysis water from the water tank into the reaction tank. Dialysis water and concentrate are supplied to the UF membrane from the reaction tank, and repeated circulation between the reaction tank and the UF membrane migrates glycopeptides to the DF retentate (concentrate) side and peptides to the permeate side, allowing glycopeptides with a high sialic acid content to be fractionated into the DF retentate (concentrate) side. The more the number of DF cycles is, the higher the sialic acid content per solid and the smaller the variation, but the lower the recovery rate of sialic acid. Therefore, it is desirable to perform the cycles so that these values ​​fall within the desired range.

[0012] According to conventional methods, an enzymatic reaction is carried out in a first reaction tank, the enzyme is inactivated by heating to terminate the enzymatic reaction, and the enzymatically reacted solution is supplied to a second tank for UF membrane treatment, which requires a total time for the enzymatic reaction and the UF membrane treatment. In contrast, according to the production method of the present invention, the enzymatic reaction is carried out in a single reaction tank, and the reaction solution undergoes UF membrane treatment as it progresses, so that the enzymatic reaction and UF membrane treatment are carried out simultaneously in a single reactor. This saves time during the time-consuming enzymatic reaction treatment. Furthermore, by circulating the enzyme while mixing it with unreacted glycoprotein without deactivating it, it is possible to carry out the enzymatic reaction with a smaller amount of enzyme, which is an additional advantage.

[0013] The UF membrane used in the step (2) may be any membrane capable of separating the glycopeptide of the present invention into the concentrate, and preferably has a molecular weight cutoff of 150 or more and less than 5000. The molecular weight cutoff is more preferably 150 or more and 4000 or less, more preferably 150 or more and 3000 or less, even more preferably 300 or more and 3000 or less, and most preferably 600 or more and 3000 or less.

[0014] (Protease) The type of protease used in the production of the glycopeptide-containing composition of the present invention is not particularly limited as long as it can hydrolyze peptide bonds to yield glycopeptides having the above-mentioned molecular weights and sugar chains. One or more types of endoproteases or exoproteases can be used. Enzymes that can be used in the production of foods and pharmaceuticals are preferred, and examples include Actinase E (Kaken Pharma), Actinase AS (Kaken Pharma), Nucleisin (HBI), Orientase AY (HBI), Sumiteam FP (Shin Nippon Chemical Industry), Sumiteam SPP-G (Shin Nippon Chemical Industry), Protease A (Amano Enzyme), Peptidase R (Amano Enzyme), Alcalase (Novozyme), and Flavorzyme (Novozyme), which can be used alone or in combination. The amount of protease added will vary depending on the type and time required for glycoprotein degradation, other reaction conditions, operating conditions, etc. Specifically, the amount of protease added may be 0.1 to 20% by weight, preferably 1 to 10% by weight, and more preferably 1 to 5% by weight, based on the raw material. When WPI is used as the raw material, the amount of protease added is preferably 1 to 10% by weight, more preferably 1 to 5% by weight, and even more preferably 1 to 3% by weight, based on the weight of WPI. The production method of the present invention has the advantage that a significantly smaller amount of enzyme is required to obtain a glycopeptide-containing composition with the same sialic acid content, compared to conventional methods in which the enzymatic reaction step and the dialysis treatment step using an ultrafiltration membrane are carried out separately.

[0015] (Composition containing glycoprotein) The raw material used in the method for producing a glycopeptide-containing composition of the present invention may be any composition containing glycoprotein, including milk raw materials such as whey materials such as cheese whey, whey protein concentrate, and whey protein isolate, casein materials, and GMP materials. The GMP may be any material obtained from whey derived from cow's milk, as well as from animal milk such as goat's or sheep's milk.

[0016] (Glycopeptide-Containing Composition) The glycopeptide-containing composition of the present invention is a composition containing glycopeptides fractionated by the above-described production method, and may contain components other than glycopeptides (e.g., peptides). The above-described production method of the present invention enables efficient fractionation of glycopeptides with a certain or higher sialic acid content. The sialic acid content is preferably 3.0% by weight or more (based on the solid weight) of the glycopeptide-containing composition, more preferably 10.0% by weight or more, and even more preferably 15.0% by weight or more. More specifically, glycopeptides having one or more of the following characteristics are included: (i) a molecular weight of 700 to 4,000; (ii) a sugar chain is bonded to the hydroxyl group of a threonine and / or serine residue of the peptide; (iii) the sugar chain contained in the glycopeptide is composed of sialic acid and galacto-N-biose, and the combined amount of these is 4.9% by weight or more (based on the solid weight) of the glycopeptide composition; and (iv) a peptide chain consisting of 1 to 16 amino acid residues.

[0017] A more specific embodiment of a glycopeptide having such characteristics is a peptide having a threonine and / or serine residue and a sugar chain bound to the hydroxyl group of the residue, wherein the sugar chain is one or more selected from the group consisting of the following (a) to (e): (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 (d) Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1 (e) Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1 The glycopeptides of the present invention are peptides containing one or more of the above-mentioned sugar chains, and may therefore be peptides containing two or more of them, including peptides containing two or more of the same type of sugar chain and peptides containing two or more different types. For example, the glycopeptide may be a glycopeptide containing two (a)s, or a glycopeptide containing (a) and one or more selected from the group consisting of (b) to (e). The glycopeptide-containing composition of the present invention may be a mixture of peptides containing each of the sugar chains, and is preferably a glycopeptide mixture containing all of the sugar chains.

[0018] The production method of the present invention can also provide a glycopeptide-containing composition containing the above-mentioned sugar chains (a) to (c), wherein the composition contains 3.2 wt % or more of (a), 0.6 wt % or more of (b), and 0.7 wt % or more of (c). The production method of the present invention can also provide a glycopeptide-containing composition containing the following sugar chains (a) to (c), wherein the weight ratio satisfies the following relationship: (a) / [(a) + (b) + (c)] ≧ 70%.

[0019] (Foods, beverages, and pharmaceuticals containing glycopeptide-containing compositions) The glycopeptide-containing composition obtained by the above-described production method of the present invention can be used directly as a material or ingredient for foods and beverages, which can be produced according to standard methods for each food product, except for the addition of the glycopeptide-containing composition. Therefore, the glycopeptide-containing composition of the present invention can be incorporated into any food or beverage, or added to raw materials during the production process of the food or beverage. Examples of foods and beverages include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, and margarine; beverages such as milk drinks, fruit juice drinks, and soft drinks; egg products such as jelly, candy, pudding, and mayonnaise; confectioneries and breads such as butter cake; and various types of powdered milk, as well as infant foods and nutritional compositions.

[0020] The glycopeptide-containing composition obtained by the above-described production method of the present invention can be used directly as a raw material for pharmaceuticals, and can be produced by standard methods such as tablets, capsules, powders, and syrups, except for the addition of the glycopeptide-containing composition. Therefore, when formulating pharmaceuticals containing the glycopeptide-containing composition of the present invention as an active ingredient, the glycopeptide-containing composition can be formulated by appropriately mixing it with excipients, stabilizers, flavoring agents, etc. that are approved for pharmaceutical use. Alternatively, the glycopeptide-containing composition can be dried and used as a powder or sachets. It can also be formulated by mixing it with excipients, binders, disintegrants, lubricants, flavoring agents, suspending agents, coating agents, and other optional drugs. Possible dosage forms include tablets, capsules, granules, powders, syrups, and the like.

[0021] The method for analyzing each sugar in the glycopeptide-containing composition of the present invention is described below. 1. Method for measuring the sialic acid content in the glycopeptide-containing composition: A sialylglycopeptide was dissolved in 50 μL of 50 mM potassium phosphate buffer (pH 5.0) to a concentration of 250 μg / mL and a sialidase (Cosmobio) concentration of 0.2 U / mL. The solution was incubated at 37°C for 24 hours and then diluted 2-10 times with ultrapure water, and the concentration of liberated sialic acid was analyzed by HPLC. <HPLC Analysis Conditions> Apparatus: DIONEX ICS-5000DP System (Thermo Fisher Scientific Inc.) Column: CarboPac PA1 Detector: Electrochemical detector (pulsed amperometric mode) Mobile phase: Ultrapure water (Solution A), 200 mM sodium hydroxide solution (Solution B), 100 mM sodium hydroxide solution containing 600 mM sodium acetate (Solution C) Flow rate: 1 mL / min. Concentration gradient: 45% Solution B and 10% Solution C for the first 7 minutes, then linearly decreasing Solution B to 37.5% by 10 minutes and linearly increasing Solution C to 25%. From 10 to 20 minutes, 37.5% Solution B and 25% Solution C were passed, and from 20.1 to 25 minutes, 45% Solution B and 10% Solution C were passed. The amount of sialic acid in the sample was calculated from a calibration curve prepared using a standard sample of known concentration.

[0022] 2. Measurement of Galacto-N-biose (GNB) Content in Glycopeptide-Containing Compositions Enzymes and substrates were dissolved in 50 μL of 50 mM potassium phosphate buffer (pH 5.0) to achieve a sialylglycopeptide concentration of 250 μg / mL, a sialidase (Cosmobio) concentration of 0.2 U / mL, and an O-glycanase (Prozyme) concentration of 0.125 U / mL. The solution was incubated at 37°C for 24 hours using a thermal cycler (Takara), and the reaction was stopped by cooling on ice. The reaction solution was diluted 2-10 times with ultrapure water, and the concentration of liberated GNB was analyzed by HPLC. <HPLC Analysis Conditions> The apparatus, column, detector, mobile phase, and mobile phase flow rate were the same as those used for sialic acid measurements. Concentration gradient: After elution with 50% solution B for 10 minutes from the start, the proportion of solution C was increased linearly from 0% to 100% by 25 minutes, and elution continued with 100% solution C until 30 minutes. Next, elution continued with 50% solution B at 30.1 minutes, and then with 50% solution B until 45 minutes. The amount of GNB in ​​the sample was calculated from a calibration curve created using standards of known concentrations.

[0023] Comparative Example 1: Preparation of glycopeptide by separate enzymatic reaction and diafiltration using an ultrafiltration membrane 1. Preparation of glycopeptide-containing composition 1-1. Enzymatic reaction 3.0 kg of cheese whey-derived WPI (Provon (registered trademark) 190, Glanbia Co., Ltd.) was dissolved in 27.0 kg of filtered water to a concentration of 10% (w / w). The solution was heated to 55°C in a water bath. To the heated 10% Provon 190 solution, Alcalase (Novozymes) was added as an endoprotease, and Flavorzyme (Novozymes) as an exoprotease. Each enzyme was added at a concentration of 1% (w / w), 3% (w / w), and 5% (w / w) per weight of WPI, respectively. After maintaining the mixture at 55°C for 8 hours, the enzyme activity was inactivated by maintaining it at 85°C for a further 30 minutes, and 30 kg of enzymatic hydrolysis solution with three different amounts of enzyme added was prepared.

[0024] 1-2. Diafiltration using an ultrafiltration membrane. A UF ​​membrane made of organic material with a molecular weight cutoff of 1 kDa (hereinafter referred to as an organic 1 kDa UF membrane) (manufactured by Synder, membrane area 5.39 m) was used. ２Using a membrane treatment system equipped with a dialysis membrane treatment system (1-1), the enzymatic hydrolysis solution prepared in 1-1 above was circulated through the membrane while being heated at 55°C. Diafiltration (DF) was initiated using filtrate heated to 55°C as dialysis water, and the DF retentate was lyophilized to obtain a glycopeptide-containing composition. The DF ratio (DF times) is the value obtained by dividing the volume of the permeate by the volume of the DF retentate (the enzymatic hydrolysis solution retained in the DF membrane).

[0025] 2. Evaluation of glycopeptide-containing compositions The dry weight and sialic acid content of each glycopeptide-containing composition obtained by each level of test were measured. The preparation conditions, sialic acid content per dry weight, and sialic acid recovery rate of each glycopeptide-containing composition are shown in Table 1.

[0026]

[0027] Comparative Example 2 Preparation of glycopeptide by separate enzymatic reaction and diafiltration using an ultrafiltration membrane (confirmation of reproducibility) 1. Enzymatic reaction and diafiltration using an ultrafiltration membrane Glycopeptide was prepared in the same manner as in Comparative Example 1, except that the amount of enzyme added was 5% (w / w) per weight of WPI and 8-fold DF was performed. Three tests were performed under the same preparation conditions.

[0028] 2. Test Results The dry weight and sialic acid content of the glycopeptide-containing composition were measured. Table 2 shows the preparation conditions of the glycopeptide-containing composition, the sialic acid content per dry weight, the sialic acid recovery rate, and the variability (CV).

[0029]

[0030] Example 1: Preparation of glycopeptide by simultaneous enzymatic reaction treatment and ultrafiltration membrane treatment (concentration and diafiltration) 1. Method for preparing a glycopeptide-containing composition (i) Cheese whey-derived WPI (Provon 190, Glanbia) was dissolved in filtered water to a concentration of 10% (w / w). Since the minimum retention capacity of the membrane treatment system was 20 kg, and taking into account the loss of liquid volume due to concentration, the following seven levels of solution volume of 10% (w / w) WPI solution were subjected to membrane treatment: (Level #1) 20 kg (Level #2) 40 kg (Level #3) 60 kg (Level #4) 100 kg (Level #5) 200 kg (Level #6) 400 kg (Level #7) 800 kg (ii) An organic 1 kDa UF membrane (Syndall, membrane area 5.39 m) was used. ２ Using a membrane bioreactor (Figure 1) equipped with a UF membrane filter (Figure 1), the solutions prepared above were circulated through a UF membrane while being heated to 55°C. (iii) When the temperature of the circulated solution reached 55°C, Alcalase (Novozymes) was added as an endoprotease and Flavorzyme (Novozymes) as an exoprotease. Each enzyme was added at 5% (w / w) per WPI weight, and the concentrate was concentrated to 20 kg. (iv) Using filtered water heated to 55°C as the dialysis water for DF, diafiltration (DF) was performed until the sialic acid content per solid exceeded 16%. (v) The resulting DF retentate was lyophilized to obtain a glycopeptide-containing composition. To confirm variability, the experiment was performed three times under the same preparation conditions.

[0031] 2. Evaluation of glycopeptide-containing compositions The dry weight and sialic acid content of the glycopeptide-containing compositions obtained by each level of testing were measured. Table 3 shows the preparation conditions, sialic acid content per dry weight, sialic acid recovery rate, and variability (CV) for each glycopeptide-containing composition. The DF run count is the value obtained by dividing the permeate volume by the volume of the UF membrane concentrate described in 1.(iii) above. These results confirmed that a CF ratio (concentration ratio) of 2 or higher reduced variability (i.e., reproducibility) in the sialic acid content and sialic acid recovery rate, and that a higher CF ratio enabled more stable production of peptide compositions with a high sialic acid content.

[0032]

[0033] Example 2: Effect of the amount of enzyme added on the preparation of glycopeptides by simultaneous enzymatic reaction and ultrafiltration (concentration and diafiltration) 1. Preparation method of glycopeptide-containing composition (i) 40.0 kg of cheese whey-derived WPI (Provon 190, Glanbia) was dissolved in 360.0 kg of filtered water to a concentration of 10% (w / w). (ii) Same as Example 1. (iii) Same as Example 1, except that each enzyme was added to 1% (w / w), 3% (w / w), and 5% (w / w) of the WPI weight, respectively, and the mixture was concentrated 20-fold. (iv) to (v) Same as Example 1.

[0034] 2. Evaluation of Glycopeptide-Containing Compositions The dry weight and sialic acid content of the glycopeptide-containing compositions obtained at each level of test were measured. Table 4 shows the preparation conditions, sialic acid content per dry weight, sialic acid recovery rate, and variability (CV) for each glycopeptide-containing composition. These results demonstrate that even with a low enzyme addition amount of 1% (w / w) relative to the raw material WPI, glycopeptide-containing compositions with a high sialic acid content of 15% (w / w) or more could be reproducibly fractionated. In the conventional method shown in Comparative Example 1, a 1% (w / w) enzyme amount yielded only a glycopeptide-containing composition with a sialic acid content of 5.5% (w / w) even after an 8-hour enzymatic reaction. To achieve a sialic acid content as high as that of Example 1, a 5% (w / w) enzyme amount was required. Therefore, it was found that the method of the present invention, in which diafiltration using an ultrafiltration membrane is simultaneously performed while maintaining the enzymatic reaction, can reduce the amount of expensive enzyme required.

[0035]

[0036] Example 3 Preparation of glycopeptide by simultaneously performing enzymatic reaction treatment and ultrafiltration membrane treatment (concentration and diafiltration) in the same vessel: variability in intermediate steps and determination of content range 1. Method for preparing glycopeptide-containing composition (i) 4.0 kg of cheese whey-derived WPI (Provon 190, Glan-a) was dissolved in 36.0 kg of filtered water to a concentration of 10% (w / w). (ii) Same as Example 1. (iii) Same as Example 1, except that each enzyme was added to a concentration of 1% (w / w) per WPI weight and concentrated twice. (iv) to (v) Same as Example 1.

[0037] 2. Test Results (1) Each glycopeptide-containing composition was collected at each of the DF cycles (0 to 5) using the above preparation method, and the dry weight and sialic acid content were measured. The preparation conditions, sialic acid content per dry weight, sialic acid recovery rate, and variability (CV) for each glycopeptide-containing composition are shown in Table 5. (2) For glycopeptide-containing compositions A (DF cycle 1) and B (DF cycle 5), the galacto-N-biose content, molecular weight, and number of amino acid residues were also measured in addition to sialic acid. The sialic acid and galacto-N-biose contents per dry weight, molecular weight, and number of amino acid residues for glycopeptide-containing compositions A and B are shown in Table 6. These results demonstrate that the higher the DF cycle, the higher the sialic acid content of the resulting glycopeptide-containing composition. Furthermore, by concentrating the composition 2-fold, the sialic acid recovery rate was reproducible even for glycopeptide-containing compositions from intermediate steps.

[0038]

[0039]

[0040] [Example 4] Preparation from cheese whey 1. Method for preparing a glycopeptide-containing composition (i) Camembert cheese whey (0.6% protein) was kept at 50°C for 30 minutes to dissolve lactose crystals. Subsequently, casein fine particles were removed at 6,500 xg using a milk clarifier (AMC-100, Iwai Machinery Co., Ltd.), and then the solution was degreased using a separator. The resulting 800 kg of degreased Camembert cheese whey solution was subjected to membrane treatment. (ii) Same as Example 1. (iii) Same as Example 1, except that each enzyme was added to a concentration of 1% (w / w) per protein weight and the solution was concentrated twice. (iv) to (v) Same as Example 1.

[0041] 2. Evaluation of glycopeptide-containing composition The dry weight, sialic acid content, and galacto-N-biose content of the obtained glycopeptide-containing composition were measured. The preparation conditions, sialic acid content per dry weight, and galacto-N-biose content of the glycopeptide-containing composition are shown in Table 7.

[0042]

[0043] Example 5 Preparation of Glycopeptides by Simultaneous Enzyme Reaction and Ultrafiltration Membrane Treatment (Concentration and Diafiltration): Molecular Weight Cutoff Range of Membrane 1. Method for Preparing a Glycopeptide-Containing Composition (i) 4.0 kg of cheese whey-derived WPI (Provon 190, Glanbia) was dissolved in 36.0 kg of filtered water to a concentration of 10% (w / w). (ii) Same as in Example 1, except that five types of organic UF membranes with different molecular weight cutoffs (Levels #1 to #5) shown in Table 7 were used. (iii) Same as in Example 1, except that each enzyme was added to a concentration of 1% per WPI weight, resulting in a two-fold concentration. (iv) 20 kg of the concentrate was subjected to 5 cycles of DF using filtered water heated to 55°C as the dialysis water for DF. (v) Same as in Example 1.

[0044] 2. Evaluation of Glycopeptide-Containing Compositions The dry weight, sialic acid content, and galacto-N-biose content of the glycopeptide-containing compositions obtained by each test level were measured. Table 8 shows the preparation conditions, sialic acid content, and galacto-N-biose content per dry weight of each glycopeptide-containing composition. According to these results, neither sialic acid nor galacto-N-biose was detected when the molecular weight cutoff was 5,000. However, when the molecular weight cutoff was 150 or higher, the sialic acid content and galacto-N-biose content per solid increased with increasing molecular weight cutoff. Therefore, it was found that the molecular weight cutoff of the ultrafiltration membrane used is preferably 150 or higher but less than 5,000, and more preferably 150 or higher but less than 3,000.

[0045]

[0046] Example 6: Structural Analysis of Glycopeptide-Containing Compositions and Measurement of Glycochain Content (1) Structural Analysis For structural analysis, an Orbitrap mass spectrometer Q-Exactive (Thermo Fisher Scientific) connected to a high-performance liquid chromatograph (HPLC) UltiMate 3000 (Thermo Fisher Scientific) was used (LC-ESI-IT MS), and an InertSustain AQ-C18 (φ2.1 mm × 150 mm, GL Sciences) was installed as the separation column. A 2% acetonitrile solution containing 0.1% formic acid (Solution A) and a 90% acetonitrile solution containing 0.1% formic acid (Solution B) were used as the mobile phase. The glycopeptide-containing composition was dissolved in ultrapure water to a concentration of 100 μg / mL, and 10 μL was introduced into the HPLC. The mobile phase was passed at 200 μL / min. After sample introduction, the proportion of solution B was 0% for 5 minutes, after which the proportion of solution B was linearly increased from 0% to 10% over 25 minutes. The proportion of solution B was then linearly increased from 10% to 100% over 20 minutes. The mass spectrometer performed MS (m / z 200-2000, positive mode) and MS / MS measurements. For MS / MS measurements, the five most intense ions in the MS spectrum were automatically selected as precursor ions, and the m / z values ​​of the resulting product ions were observed after destruction at collision energies of 15, 30, and 45. The acquired MS / MS spectra were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) with the Byonic (Protein Metrics) node. The protein database used was a database consisting only of GMP, and the glycan database used contained 10 types of glycans consisting of one molecule of N-acetylhexosamine (HexNAc), 0 to 1 molecule of hexose (Hex), 0 to 2 molecules of N-acetylneuraminic acid (Neu5Ac), and 0 to 2 O-acetyl groups (O-Ac). Analysis by Byonic revealed that the glycans were classified as (HexNAc), (HexNAc)(Hex), or (HexNAc)(Hex)(Neu5Ac). ２The glycans assigned as "(HexNAc)(Hex)(Neu5Ac)" were assigned to GalNAc, Galβ1-3GalNAc, or Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc, respectively, according to the previously reported glycan structure of GMP. Furthermore, the glycans assigned as "(HexNAc)(Hex)(Neu5Ac)" were manually confirmed by MS / MS spectrum and assigned to Neu5Acα2-3Galβ1-3GalNAc or Galβ1-3(Neu5Acα2-6)GalNAc. Similarly, the MS / MS spectrum of O-acetyl-modified Neu5Ac (O-Ac-Neu5Ac or O,O'-diAc-Neu5Ac) was manually confirmed and assigned to Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAc or Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAc.

[0047] (2) Measurement of Glycan Content. The glycans contained in the sialylglycopeptide-containing composition were measured using the EZGlyco O-Glycan Prep Kit (Sumitomo Bakelite Co., Ltd.). 20 μL of a 10 mg / mL sialylglycopeptide solution was mixed with 15 μL of a glycan-releasing reagent (releasing reagent A:releasing reagent B = 5:2) and incubated at 37°C for 75 minutes. The released glycans were then captured with glycan-capturing beads, which were then washed with acetonitrile. Next, 50 μL of methanol / acetic acid / ultrapure water (9 / 2 / 9) containing 3.9 mg of 2-aminobenzamide (2-AB) and 0.039 mg of reducing reagent was added to the beads, and the glycan solution was recovered by centrifugation at 3,000 × g for 1 minute. This solution was incubated at 50°C for 2.5 hours and then washed with acetonitrile using a clean-up column to remove excess 2-AB. Ultrapure water was added to the cleanup column to recover the 2-AB-labeled glycans, which were then analyzed by LC / MS / FL. The structure of each glycan peak was determined by MS measurement, and the amount of each glycan in the sample was quantified from the area value of the fluorescence measurement.

[0048] <LC / MS analysis conditions> Apparatus: UltiMate 3000 (Thermo Fisher Scientific Inc.) Column: Glycanpac AXH-1 (2.1 mm × 150 mm, particle size 3 μm, Thermo Fisher Scientific Inc.) Detector: Q Exactive (Thermo Fisher Scientific Inc.) and RS Fluorescence Detector (Thermo Fisher Scientific Inc.) Mobile phase: 50 mM ammonium formate (pH 4.4) / acetonitrile (17 / 83) Flow rate: 0.4 mL / min Column temperature: 40°C MS settings: electrospray voltage, 3.5 kV; heat capillary temperature, 250°C; sheath gas, 45; auxiliary gas, 10; scan range, m / z 200-1500; resolution, 70,000.

[0049] 2. Structural Analysis Results Table 9 shows the glycan structures of glycopeptides detected by LC / MS analysis. As a result, all of the detected glycopeptides were peptides bound to glycans containing sialic acid. Many of the detected glycopeptides had a glycan structure of Neu5Acα1-3Galβ1-3(Neu5Acα1-6)GalNAc with two sialic acid molecules bound (Glycan types 3, 4, 5, 6, and 7), but Neu5Acα1-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3(Neu5Acα1-6)GalNAc (Glycan type 2), which have one sialic acid molecule, were also detected. Furthermore, the O-acetyl forms of sialic acid, N,O-diacetylneuraminic acid (O-Ac-Neu5Ac, glycan type 4) and N,O,O-triacetylneuraminic acid (O-diAc-Neu5Ac, glycan type 5), were also detected. Glycopeptides with two glycans attached to a single peptide chain (glycan types 6 and 7) were also detected. All peptide chains detected by MS / MS spectral analysis were those contained in bovine glycomacropeptide. All glycopeptides contained one or more serine or threonine residues, and the peptide chain length ranged from one residue to 16 residues. The molecular weights of the glycopeptides ranged from 745 to 3577.

[0050] 3. Measurement Results of Glycan Content The amounts of glycan types 1, 2, and 3 contained in glycopeptides A and B were measured by LC / MS / FL (Table 10). In all glycopeptides, glycan type 1 was 0.74 wt% or more, glycan type 2 was 0.65 wt% or more, and glycan type 3 was 3.25 wt% or more. Furthermore, the proportion of glycan type 3 in these glycans was 70.0% or more.

[0051]

[0052]

Claims

1. A method for producing a glycopeptide-containing composition, characterized in that the following two steps are carried out simultaneously in one reaction vessel: (1) a step of contacting a composition containing a glycoprotein with an endoprotease and / or an exoprotease in a solution to carry out an enzymatic reaction in the mixture, and (2) a step of ultrafiltering the mixture obtained in (1).

2. The method according to claim 1, wherein the step (2) includes a concentration treatment and / or a diafiltration treatment of the mixture.

3. The method according to claim 2, wherein the step (2) is a step of concentrating the solution and then dialysis.

4. The method according to claim 1, wherein the ultrafiltration membrane used in step (2) has a molecular weight cutoff of 150 or more and 3,000 or less, and the process separates glycopeptides into the concentrate side and peptides into the permeate side.

5. The method of claim 1, wherein the glycopeptide comprises a sugar chain attached to a hydroxyl group of a serine and / or threonine residue of the peptide.

6. The method according to claim 1, wherein the glycopeptide contains a sugar chain consisting of sialic acid and galacto-N-biose, the combined amount of which is 4.9% by weight or more.

7. The method of claim 1, wherein the molecular weight of the glycopeptide is 700 or more and 4,000 or less.

8. The method of claim 1, wherein the number of amino acids constituting the peptide contained in the glycopeptide is 1 to 16.

9. The method according to claim 1, wherein the glycopeptide comprises one or more glycan structures selected from the group consisting of the following (a) to (e): (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1, (b) Galβ1-3(Neu5Acα2-6)GalNAcα1, (c) Neu5Acα2-3Galβ1-3GalNAcα1, (d) Neu5Acα2-3Galβ1-3(O-Ac-Neu5Acα2-6)GalNAcα1, and (e) Neu5Acα2-3Galβ1-3(O-diAc-Neu5Acα2-6)GalNAcα1.

10. The method of claim 9, wherein the glycan structures contained in the glycopeptide are (a) 3.2% by weight or more, (b) 0.6% by weight or more, and (c) 0.7% by weight or more.

11. The method according to claim 9, wherein the proportion of (a) relative to the total weight of (a), (b) and (c) among the glycan structures contained in the glycopeptide is 70% or more.

12. A method for producing a glycopeptide-containing composition, characterized in that the method includes a step of ultrafiltering a mixture during an enzymatic reaction between a composition containing a glycoprotein and an endoprotease and / or an exoprotease.

13. The method according to claim 12, wherein the step of ultrafiltration includes a concentration treatment and / or a diafiltration treatment of the mixture.

14. A glycopeptide-containing composition comprising the following glycan structures (a) to (c), characterized in that the composition contains 3.2% by weight or more of (a), 0.6% by weight or more of (b), and 0.7% by weight or more of (c): (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 15. A glycopeptide-containing composition comprising the following glycan structures (a) to (c), wherein the weight ratio of (a) Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1 (b) Galβ1-3(Neu5Acα2-6)GalNAcα1 (c) Neu5Acα2-3Galβ1-3GalNAcα1 satisfies the following relational formula: (a) / [(a)+(b)+(c)]≧70%.

16. A glycopeptide-containing composition produced by the production method described in any one of claims 1 to 13.

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