Process for separating l-arginyl-l-arginine from l-arginine

Abstract

This invention provides a method for efficiently separating L-arginyl-L-arginine and L-arginine, comprising: loading a mixed solution containing L-arginyl-L-arginine and L-arginine onto a weak cation exchange chromatography column; eluting with 0.1-2% ammonia water, collecting the L-arginine solution, concentrating, crystallizing, and drying the resulting solution to obtain L-arginine; eluting with 0.1-1.0% hydrochloric acid aqueous solution, collecting the L-arginyl-L-arginine hydrochloride solution, adjusting the pH of the resulting solution to ≥10 with free base, desalting by nanofiltration, concentrating, and drying to obtain L-arginyl-L-arginine. This separation method is applicable to the industrial production of pharmaceutical intermediates, nutritional supplements, and cosmetic raw materials.

Description

Technical Field

[0001] This invention belongs to the field of separation technology. Specifically, this invention relates to a method for separating L-arginyl-L-arginine and L-arginine. Background Technology

[0002] Arginine (L-arginine, Arg) is a conditionally essential amino acid for the human body and is widely used in wound repair, immune regulation, and sports nutrition. Arginine dipeptide (L-arginyl-L-arginine, Arg-Arg), due to its higher cell membrane permeability and stability, exhibits unique biological activity in promoting collagen synthesis, anti-oxidation, and skin barrier repair. In enzymatic synthesis of Arg-Arg or protein hydrolysis processes, the reaction system often contains unreacted arginine and byproducts. These two substances have highly similar physicochemical properties (both contain a strongly basic guanidine group, pI > 10.5), making separation difficult.

[0003] Existing separation and purification techniques have significant drawbacks: In traditional cation exchange chromatography, both arginine dipeptides and arginine exhibit strong adsorption affinity on the resin, but their adsorption selectivity is poor, making separation difficult (they are eluted simultaneously). Furthermore, arginine dipeptides have extremely high solubility in water (>900 g / mL), making crystal precipitation in water challenging. Adding antisolvents (such as methanol or ethanol) easily forms solvent compounds, leading to oil precipitation and liquid-liquid separation, further hindering the acquisition of arginine dipeptide crystals. Therefore, purification of arginine dipeptides through crystallization is difficult.

[0004] In conclusion, there is an urgent industrial need to develop a highly selective, easily scalable, and green separation process. Summary of the Invention

[0005] This invention aims to solve the above-mentioned technical problems and provides a method for separating L-arginyl-L-arginine and L-arginine that is mild in operation, highly efficient in separation, produces high-purity products, is environmentally friendly, can be industrialized, and has controllable costs.

[0006] At pH 4.5, the net Arg charge ≈ +1.1 (α-carboxyl deprotonation, α-amino and guanidine protonation), and the Arg-Arg net charge ≈ +2.3 (N-terminal amino and biguanidine protonation, C-terminal carboxyl partial deprotonation), with a charge difference of 1.2 units, significantly enhancing the resin's selective adsorption differences. This invention uses a weak cation exchange resin (methyl acrylate type). Arginine has a relatively weak retention capacity on the resin, while arginine dipeptide has a strong retention capacity. A two-stage elution method is employed: first, arginine is eluted with dilute ammonia, and then the arginine dipeptide is eluted with dilute hydrochloric acid, yielding high-purity arginine and arginine dipeptide respectively.

[0007] In one aspect, the present invention provides a method for separating L-arginyl-L-arginine from L-arginine, comprising the following steps: Step a. Load the mixed solution containing L-arginyl-L-arginine and L-arginine onto a weak cation exchange column; Step b. Elute with 0.1-2% ammonia solution and collect the L-arginine solution; Step c. Elute with 0.1-1.0% hydrochloric acid aqueous solution and collect the L-arginyl-L-arginine hydrochloride solution; Step d. Concentrate, crystallize, and dry the L-arginine solution obtained in step b to obtain L-arginine; Step e. Adjust the pH of the L-arginyl-L-arginine hydrochloride solution obtained in step c to ≥10 with free alkali, desalt by nanofiltration, concentrate and dry to obtain L-arginyl-L-arginine; or concentrate and dry the L-arginyl-L-arginine hydrochloride solution obtained in step c to obtain L-arginyl-L-arginine hydrochloride.

[0008] In some implementations, the column packing material is methyl acrylate.

[0009] In some implementations, in step a, the chromatographic column is pre-regenerated with acid or base and rinsed with water until neutral.

[0010] In some embodiments, the mixed solution is obtained by microfiltration of the enzyme catalytic solution through a ceramic membrane, and is first washed with water before step b to remove pigments and inorganic salts from the enzyme catalytic solution.

[0011] In some embodiments, the equilibrium ion on the methyl acrylate weak cation exchange resin is H2O. + Na + NH4 + .

[0012] In some embodiments, the molar ratio of L-arginyl-L-arginine to L-arginine in the mixed solution is 0.1:1 to 5:1. In some embodiments, the pH value is controlled between 2 and 10 before loading.

[0013] In some implementations, step b further includes eluting with water after eluting with ammonia.

[0014] In some implementations, the ammonia concentration in step b is 0.5-2%.

[0015] In some implementations, the concentration of the hydrochloric acid aqueous solution in step c is 0.5-1.0%.

[0016] In some implementations, step e involves adjusting the pH to 10-13.

[0017] In some implementations, the drying in step e is spray drying, with an inlet temperature of 140-180°C and an outlet temperature of 60-90°C.

[0018] In some implementations, the free base is sodium hydroxide.

[0019] In some embodiments, the mixed solution of L-arginyl-L-arginine and L-arginine can be an enzyme catalytic solution for preparing L-arginyl-L-arginine. In some embodiments, the enzyme catalytic solution is pretreated by ceramic membrane microfiltration before loading. In some embodiments, after loading, impurities are washed with water. In some embodiments, impurities are removed from the enzyme catalytic solution by washing with 4-6 BV of pure water.

[0020] In some implementations, separation is achieved during elution by simultaneously monitoring the concentrations of various components (such as L-arginine and L-arginyl-L-arginine) in the eluent. In other implementations, the conductivity, pH, and UV 210 nm signal of the eluent are simultaneously monitored to dynamically and precisely cut the target fraction, achieving efficient separation.

[0021] In some embodiments, step e. adjusts the pH of the L-arginyl-L-arginine hydrochloride solution obtained in step c to 7.0 with a free base, desaltes by nanofiltration, concentrates, and dries to obtain L-arginyl-L-arginine hydrochloride. In some embodiments, the free base is sodium hydroxide. In some embodiments, the drying is spray drying, with an inlet temperature of 170-180°C and an outlet temperature of 50-80°C.

[0022] On the other hand, the present invention provides high-purity L-arginine, which is obtained by the separation method described above. In some embodiments, the purity of the high-purity L-arginine is ≥98.5% or ≥99.0%.

[0023] In another aspect, the present invention provides a high-purity L-arginyl-L-arginine, which is obtained by the separation method described above. In some embodiments, the purity of the high-purity L-arginyl-L-arginine is ≥99.0%.

[0024] In another aspect, the present invention provides a high-purity L-arginyl-L-arginine hydrochloride, which is obtained by the separation method described above. In some embodiments, the purity of the high-purity L-arginyl-L-arginine hydrochloride is >99%.

[0025] In another aspect, the present invention provides the use of high-purity L-arginine, high-purity L-arginyl-L-arginine or high-purity L-arginyl-L-arginine hydrochloride in the preparation of any of the following products: (1) sports nutrition supplements or immune-modulating functional foods; (2) cosmetic active ingredients with skin barrier repair function.

[0026] On the other hand, the present invention provides a continuous separation system, including a weak cation exchange chromatography unit (filled with methyl acrylate resin); and an online multi-parameter monitoring module (for real-time acquisition of conductivity, pH, and UV 210 nm signals). This system is suitable for the industrial-scale implementation of the above-mentioned separation method.

[0027] This invention achieves efficient separation of L-arginyl-L-arginine and L-arginine on an industrial-grade chromatographic column, with a product purity of ≥98.5%. It optimizes the washing and elution conditions, improves the arginine recovery rate (≥95%), and simplifies subsequent processing. The process parameters are adapted to industrial continuous production, reducing the unit processing cost by more than 30%.

[0028] The summary provides a simplified overview of the selected concepts, which will be further described in the detailed description below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. Detailed Implementation

[0029] The present invention will now be further described with reference to preferred embodiments thereof. While the invention will be described in conjunction with preferred embodiments, it should be understood that they are not intended to limit the invention to these embodiments. Rather, the invention is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the invention as defined in the claims.

[0030] As used herein, the term “or” is intended to include both “and” and “or”. In other words, the term “or” can also be replaced with “and / or”.

[0031] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” are intended to include the plural forms as well.

[0032] As used herein, the term “comprising” or “including” or variations thereof means, in its non-restrictive sense, the inclusion of the item following the word, but does not exclude items not specifically mentioned. It also includes the more restrictive verbs 'consistently made up of' and 'comprises from'.

[0033] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein are standard procedures widely used in the relevant fields. Unless otherwise specified, all raw materials and reagents used in this invention are commercially available. All reagents are commercial grade and used according to received standards. Example 1: Laboratory-scale isolation

[0034] Materials: 0.15 mol / L Arg + 0.20 mol / L Arg-Arg aqueous solution (pH 9.0); amino acid ligase, catalyzing the synthesis of arginine dipeptide from arginine. Resin column: column size: 3.5*20 cm, filled with methyl acrylate resin (particle size 120-600 μm). Eluent A: 0.5% ammonia; Eluent B: 0.5% hydrochloric acid.

[0035] The procedure is as follows: Pack the column (3.5 cm × 20 cm, column volume 150 mL). First, pass 500 mL of 4% ammonia solution through the column at a flow rate of 1 mL / h. Then, rinse the resin column with pure water until the pH of the eluent is < 8.0. Step a: Load 300 mL of sample (flow rate 2.5 mL / min), collect the breakthrough solution to verify complete loading. After loading, wash with 4-10 column volumes of pure water to remove impurities (mainly pigments and inorganic salts) that are not retained by the resin. Step b: Elute with 0.5% ammonia solution, mainly collecting the arginine (Arg) fraction. Wash with 4-6 BV of pure water to remove free ammonia from the resin column. Step c: Elute with 0.3-0.5% hydrochloric acid, collecting the arginine dipeptide (Arg-Arg) fraction in the eluent. Step d: The arginine solution obtained from the elution in step b is concentrated, crystallized, filtered, and dried to obtain the finished arginine product with a content >99%. Mass spectrometry verification shows the molecular ion peak is [M+H]⁺ 175.1±0.3. Step e: The arginine dipeptide hydrochloride solution obtained from the elution in step c is adjusted to a pH between 10 and 13 using sodium hydroxide. Then, it is desalted and concentrated using a nanofiltration membrane. The desalted arginine dipeptide solution is then spray-dried at an inlet temperature of 140-180℃ and an outlet temperature of 60-90℃ to obtain the finished arginine dipeptide powder with a dry basis content >99%. Mass spectrometry verification shows the molecular ion peak is [M+H]⁺ 331.4±0.5. Comparative Example 1

[0036] Strong acid cation exchange chromatography (001*7) was used:

[0037] Materials: 0.15 mol / L Arg + 0.20 mol / L Arg-Arg aqueous solution (pH 9.0); amino acid ligase, catalyzing the synthesis of arginine dipeptide from arginine. Resin column: column size: Φ3 cm × 30 cm, column volume: 170 mL (packing weight: 80%), filled with strongly acidic styrene-divinylbenzene resin (001*7). Eluent A: 0.5% ammonia; Eluent B: 0.5% hydrochloric acid.

[0038] The procedure is as follows: Step a, Resin regeneration and column packing: Regenerate the strong acid ion exchange resin with 4-6% hydrochloric acid and pack it into a column (170 mL). Then wash the resin with pure water until neutral (pH > 6.0) and set aside. Step b, Column loading and washing: Separate the mixed solution of arginine and arginine dipeptide onto the column at a flow rate of 2 mL / min. After loading, wash with pure water until the conductivity of the eluent is <0.1 mS / cm. Step c, Washing with 0.5% ammonia water only elutes about 20% of the arginine. Step d, Elution with 0.5% hydrochloric acid is insufficient to elute the arginine dipeptide; the concentration of arginine dipeptide in the eluent is 0.

[0039] Other elution conditions: Because 0.5% hydrochloric acid is insufficient to elute arginine dipeptide from the resin, after elution with 0.5% ammonia, 0.2-0.6% sodium hydroxide solution is used for elution. However, since the eluent contains both arginine dipeptide and arginine, separation between the two is still not possible. Comparative Example 2

[0040] Weakly acidic cation exchange chromatography (methyl acrylate):

[0041] Materials: 0.15 mol / L Arg + 0.20 mol / L Arg-Arg aqueous solution (pH 9.0); amino acid ligase, catalyzing the synthesis of arginine dipeptide from arginine. Resin column: column size: Φ5.4 cm × 20 cm, column volume: 366 mL (packing weight: 80%), filled with a weakly acidic cation exchange resin (methyl acrylate). Eluent A: 0.3~1% sodium chloride.

[0042] The procedure is as follows: Step a, Resin regeneration and column packing: The resin is regenerated with 4% ammonia and then packed into a column (366 mL). The resin is then washed with pure water until neutral (pH > 6.0) and set aside. Step b, Column loading and washing: A mixed solution of arginine and arginine dipeptide is loaded onto the column for separation at a flow rate of 2 mL / min. After loading, the column is washed with pure water until the conductivity of the effluent is <0.1 mS / cm. Step c, Elution with 0.3–1.0% sodium chloride solution: The concentration of arginine dipeptide and arginine in the eluent is only 0.07–0.15 g / L, making it difficult to achieve elution of arginine dipeptide and arginine. When the eluent volume reaches 40 times the column volume, the yield of arginine dipeptide is only 13%, and the elution rate of arginine dipeptide is only 17%. In step d, elution was continued with 0.5% sodium hydroxide. Arginine dipeptide and arginine were eluted simultaneously, but arginine dipeptide is unstable under alkaline conditions, resulting in a yield of only 50%, with a large amount of arginine dipeptide decomposing. This left arginine dipeptide and arginine still mixed together. Example 2: Pilot-scale production and verification of process robustness

[0043] Equipment: Acrylic glass column (Φ20 cm × 200 cm, resin volume 50 L). Sample loading: 150 L of mixture (Arg 3940 g + Arg-Arg 10000 g). Elution: Flow rate 80 L / h.

[0044] Results: Arg component: purity 98.9%, recovery rate 95.1%, conductivity <50 μS / cm (no desalting required); Arg-Arg component: purity 98.5%, recovery rate 98.6%.

[0045] After five consecutive batches of operation, the column loading (the mass of arginine dipeptides processed in each batch) remained essentially unchanged, indicating that the process was stable.

[0046] Economic efficiency: High-purity product (arginine dipeptide) is obtained from the enzyme-catalyzed reaction solution, while high-purity unreacted substrate (arginine) is recovered, improving substrate utilization and reducing costs by more than 30%. Example 3: Separation of arginine dipeptide hydrochloride from catalytic solution

[0047] Step 1: Solid-liquid separation of enzyme catalytic solution. First, the catalytic solution is centrifuged at high speed to remove most of the precipitated solid residue in the solution. The supernatant obtained is then microfiltered using a ceramic membrane to obtain a completely clear and transparent mixed solution.

[0048] Step 2: The catalyst solution is passed through a chromatographic column packed with regenerated methyl acrylate resin. Arginine dipeptides and arginine are retained on the resin, while most impurities (including inorganic salts, pigments, etc.) pass through. Subsequently, the column is washed with pure water to completely remove components not retained by the resin.

[0049] Step 3: Use 0.5-1% ammonia solution to wash the arginine on the resin in the column from Step 2. Stop washing when no arginine is detected in the column eluent by high performance liquid chromatography (HPLC). Then, use 0.5-1% hydrochloric acid to elute the arginine dipeptide on the column to obtain an arginine dipeptide hydrochloride solution with HPLC purity >99%.

[0050] Step 4: Adjust the pH of the purified solution obtained in Step 3 to 7.0 using sodium hydroxide, and then desalinate it using nanofiltration. Stop nanofiltration when the desalination rate is >90%. Concentrate the desalted solution under vacuum.

[0051] Step 5: The arginine dipeptide hydrochloride concentrate obtained in Step 4 is spray-dried with an inlet temperature of 170-180℃ and an outlet temperature of 50-80℃. The resulting spray-dried powder is the finished arginine dipeptide hydrochloride product.

[0052] Alternatively, the pH of the arginine dipeptide hydrochloride solution obtained in step 3 can be adjusted to between 10 and 13 using sodium hydroxide, followed by desalting and concentration using a nanofiltration membrane. The desalted arginine dipeptide solution can then be spray-dried with an inlet temperature of 140-180℃ and an outlet temperature of 60-90℃ to obtain the finished arginine dipeptide powder.

[0053] This invention employs a methyl acrylate-based weak cation exchange resin as the core separation medium, combined with a staged gradient elution strategy: the enzyme catalytic solution is pretreated by ceramic membrane microfiltration before loading, followed by sequential washing with pure water to remove impurities, pigments, and inorganic salts; selective elution of the L-arginine fraction with 0.1–2% ammonia; and directional elution of the L-argininoyl-L-arginine fraction with 0.1–1.0% hydrochloric acid solution. The target fraction is dynamically and precisely separated using online UV and conductivity monitoring. Subsequent processes are specifically optimized: the L-arginine fraction is deammonied by vacuum evaporation, crystallized, and dried to obtain high-purity crystals (HPLC purity ≥98%); the L-argininoyl-L-arginine fraction can be flexibly prepared as hydrochloride (nanofiltration concentration followed by spray drying) or free alkali (alkalization to pH >10 followed by nanofiltration desalting and drying, purity ≥98%). This method avoids prolonged exposure to strong acids and bases throughout the process, effectively protecting peptide bond integrity, achieving a recovery rate >95%, low solvent consumption, no toxic reagent residues, and significantly reduced wastewater salt load. The process parameters are robust and have been adapted to continuous separation systems, making it suitable for the green and large-scale production of high-value-added products such as sports nutrition supplements, immune-modulating functional foods, and skin barrier repair cosmetics.

[0054] The separation and purification method of this invention has high selectivity: it adopts a staged elution method to achieve near-complete separation of arginine and arginine dipeptide; the product is of high quality: under near-neutral conditions throughout the process, no degradation products were detected by mass spectrometry, and it meets USP standards. <1058> Standard; Green process: The buffer system is non-toxic and easily degradable. The arginine eluent is nanofiltration and evaporation to concentrate and recover ammonia water, reducing the salt content in the wastewater by 70%; Industrialization friendly: The parameters have been successfully linearly scaled up, making it suitable for continuous production using a simulated moving bed (SMB).

[0055] While specific embodiments and examples of the invention have been described herein, those skilled in the art will understand that any modifications and variations can be made without departing from the principles of the invention. The above embodiments and descriptions do not limit the scope of the invention. Any combination of embodiments of the invention, as well as any obvious extensions or analogies thereof, are within the scope of the invention. Furthermore, the invention covers any arrangement intended to achieve the same purpose, and all such variations and modifications falling within the scope of the appended claims.

Claims

1. A method for separating L-arginyl-L-arginine and L-arginine, characterized in that, The separation method includes the following steps: Step a. Load the mixed solution containing L-arginyl-L-arginine and L-arginine onto a weak cation exchange column; Step b. Elute with 0.1-2% ammonia solution and collect the L-arginine solution; Step c. Elute with 0.1-1.0% hydrochloric acid aqueous solution and collect the L-arginyl-L-arginine hydrochloride solution; Step d. The L-arginine solution obtained in step b is concentrated, crystallized, and dried to obtain L-arginine; Step e. Adjust the pH of the L-arginyl-L-arginine hydrochloride solution obtained in step c to ≥10 with free alkali, desalt by nanofiltration, concentrate and dry to obtain L-arginyl-L-arginine; or concentrate and dry the L-arginyl-L-arginine hydrochloride solution obtained in step c to obtain L-arginyl-L-arginine hydrochloride.

2. The separation method according to claim 1, characterized in that, The packing material for the chromatographic column is methyl acrylate.

3. The separation method according to claim 1, characterized in that, In step a, the chromatographic column is pre-regenerated with acid or alkali and rinsed with water until neutral.

4. The separation method according to claim 1, characterized in that, The mixed solution is obtained by microfiltration of the enzyme catalytic solution through a ceramic membrane. Before step b, it is first washed with water to remove pigments and inorganic salts from the enzyme catalytic solution.

5. The separation method according to claim 2, characterized in that, The equilibrium ions on the methyl acrylate weak cation exchange resin are H+. + Na + NH4 + .

6. The separation method according to claim 1, characterized in that, The molar ratio of L-arginyl-L-arginine to L-arginine in the mixed solution is from 0.1:1 to 5:

1.

7. The separation method according to claim 1, characterized in that, The ammonia concentration in step b is 0.5-2%.

8. The separation method according to claim 1, characterized in that, The concentration of the hydrochloric acid aqueous solution in step c is 0.5~1.0%.

9. The separation method according to claim 1, characterized in that, In step e, the drying is spray drying, with an inlet temperature of 140-180℃ and an outlet temperature of 60-90℃.

10. The separation method according to claim 1, characterized in that, The free base is sodium hydroxide.