Improved method of synthesis and purification of citrulline

Abstract

To provide a synthesis of citrulline, and improved methods for purification of citrulline.SOLUTION: This invention provides a synthesis of citrulline from a transition metal complex of ornithine using cyanate to derivatize the terminal amino group of ornithine. There are also provided improved methods for purification of citrulline produced by reaction of cyanate with ornithine via the steps of reprecipitation of copper complex of citrulline, removal of the complexing metal by sulfide precipitation, activated carbon adsorption and antisolvent crystallization.SELECTED DRAWING: Figure 2A

Description

[Technical Field]

[0001]

[0001] The present invention provides a synthesis of citrulline from a transition metal complex of ornithine using cyanate to derivatize the terminal amino group of ornithine. The present invention also provides an improved method for the purification of citrulline produced by the reaction of cyanate with ornithine through the steps of reprecipitation of a copper complex of citrulline, removal of the complexed metal by sulfide precipitation, activated carbon adsorption, and antisolvent crystallization. [Background technology]

[0002] Ornithine is an α-amino acid with a terminal amino group opposite the α-carbon. Citrulline is an α-amino acid with a terminal carbamide group in the same position as the terminal amino group of ornithine. Dr. A. Kurtz described the synthesis of racemic citrulline from racemic ornithine in 1938 (J. Biol. Chem., Vol. 122: pp. 477-484). Following that disclosure, optically active l-citrulline was synthesized from l-ornithine in 1949 (J. Biol. Chem., Vol. 180: pp. 1253-1267). Optical activity was maintained by complexing the starting material (l-ornithine) in a transition metal complex via the α-amino and carboxyl groups, followed by reaction of the terminal amino group with urea to form the carbamide derivative (see Figure 1). Kurth (1949) describes many other syntheses, all relying on transition metal complexes to maintain the α-amino acid character of the starting compound while derivatizing other parts of the molecule, an example of which is described in Example 1 below. Summary of the Invention

[0003] The present invention provides an improved method for adding a terminal carbamide group to an α-amino acid by using a copper-complexed α-amino acid having a terminal amine and exposing it to excess cyanate in aqueous solution. The cyanate reacts with the terminal amine to form a carbamide derivative of the copper-complexed α-amino acid, which precipitates from the aqueous solution. Preferred α-amino acids having a terminal amine are ornithine or lysine, and the resulting carbamide derivative is citrulline or homocitrulline. In contrast to prior art synthetic schemes that use reflux temperatures to react urea with ornithine, the method of the present invention may be carried out at temperatures between 30°C and 100°C, preferably between 40°C and 80°C, and more preferably between 55°C and 65°C, with the reaction occurring within 0.5 to 5 hours, preferably between 1 and 5 hours, and more preferably between 3 and 4.5 hours. However, at 55°C and 60°C, the reaction preferably proceeds for at least 3.5 hours. When the reaction is complete, the precipitate may be collected by filtration at ambient temperature and the precipitate may be washed with water, preferably until no blue color appears in the filtrate.

[0004]

[0004] The present invention also provides an improved method for reducing ornithine contamination of citrulline by complexing citrulline with copper and washing the precipitated citrulline:copper complex with water, thereby reducing the ornithine:citrulline ratio in the precipitate. In the method of the present invention, the precipitated citrulline:copper complex is suspended in water, and the pH of the suspension is adjusted with an acid until the precipitate redissolves in water. The pH of the citrulline:copper complex solution is adjusted with a base to reprecipitate the citrulline:copper complex. Preferably, the aqueous suspension of the precipitated citrulline:copper complex is acidified with hydrochloric acid, thereby bringing citrulline into solution, and then a base is added to reprecipitate the citrulline-copper complex. The reprecipitated citrulline:copper complex is recovered by filtration, and the recovered precipitate is washed with water until no chloride appears in the filtrate. Heat generated by acidification and neutralization is controlled by active cooling, preferably maintaining a temperature below 45°C.

[0005]

[0005] According to the present invention, citrulline is recovered from the citrulline:copper complex by suspending the citrulline:copper complex in water; introducing hydrogen sulfide to dissolve the complex and produce an aqueous citrulline solution containing precipitated copper salts; and then removing the precipitated copper salts from the solution by filtration. Hydrogen sulfide gas is added to the suspension in a sealed reaction vessel until no further consumption is observed. Consumption is monitored by observing the resulting pressure change. During the reaction, the pH is reduced to less than 4, preferably to about pH 3, and the temperature is maintained below ambient temperature, preferably below 5°C. The temperature is then raised above ambient temperature, preferably above 30°C, to keep the citrulline in solution during filtration and to remove the precipitated copper salts.

[0006]

[0006] The present invention provides further purification of citrulline in the solution recovered from the citrulline:copper complex. The recovered citrulline solution is neutralized, preferably by adjusting the pH to 5.9±0.2, and then treated with activated carbon, preferably by circulating the neutralized citrulline solution through an activated carbon adsorption bed. After activated carbon treatment, the citrulline solution is mixed with a water-miscible antisolvent to precipitate citrulline from the aqueous solution. Suitable antisolvents include 2-propanol, ethanol, methanol, or preferably acetone. The solvent / antisolvent precipitation is carried out at low temperatures, preferably between 0°C and 10°C. All steps following recovery of citrulline from the citrulline:copper complex are preferably carried out in a sealed container to minimize microbial contamination. The citrulline precipitate is dried to remove water and the antisolvent. The resulting product is suitable for use in an injectable therapeutic composition. [Brief explanation of the drawings]

[0007] [Figure 1A]

[0007] Figure 1 shows the chemical structures of reactants and products for the synthesis of citrulline from ornithine by reaction with urea. [Figure 1B]

[0008] 1 shows the chemical structure of the citrulline:copper complex and the citrulline obtained when the complex is treated with inorganic sulfide. [Figure 2A]

[0009] 1 shows the chemical structures of reactants and products for the synthesis of citrulline from ornithine by reaction with cyanate. [Figure 2B]

[0010] 1 shows the chemical structure of the citrulline:copper complex and the citrulline obtained when the complex is treated with hydrogen sulfide gas. [Figure 3]

[0011] A cross-sectional view of a press filter is shown. [Figure 4]

[0012] A photograph of a press filter is shown. DETAILED DESCRIPTION OF THE INVENTION

[0008]

[0013] Developed by the inventors to produce pharmaceutical grade citrulline Details of the various steps in the improved process are discussed below. Synthesis of citrulline from ornithine

[0014] The present inventors have reported that In this study, the α-terminus of the molecule was complexed with a transition metal atom to preserve the stereochemistry around the α-carbon of the α-amino acid during reaction of amino groups elsewhere on the compound. The initial formation of the l-ornithine-copper complex was carried out as described by Kurtz. Kurtz described various transition metals as complexing metals in his 1949 paper, but the preferred metal is copper(II) based on the ease with which stable complexes can be formed and the subsequent ease with which copper(II) can be removed from the product. Copper is usually supplied as cupric sulfate, but complex formation from copper(II) acetate, cupric carbonate, or cupric oxide has also been reported.

[0009]

[0015] The present inventors have used a cyanate reaction rather than the urea reaction reported by Kurth. Kurth discovered an alternative method for derivatizing the terminal amino group of a complexed α-amino acid using urea. An example of this improved synthesis is shown in Figure 2A and described in Example 3 below. It was found that using cyanate as the derivatizing agent produced fewer distinct product compounds, simplifying the purification of the desired citrulline product. Kurth found that the use of excess urea Urea derivatization was carried out by refluxing the copper complex in the presence of urea. Cyanate derivatization may be carried out at lower temperatures (e.g., 55°C to 65°C), which may contribute to a higher yield of citrulline relative to the initial amount of ornithine. Cyanate is preferably provided in excess, and the reaction is driven by precipitation of the citrulline:copper complex. To remove unreacted copper, the precipitated complex is washed with water (e.g., until no blue color is observed in the filtrate). The precipitated copper complex of citrulline may be recovered and dried. Enrichment of citrulline as a copper complex

[0016] The inventors have found that the relative citrulline content of the reaction products is related to the citrulline:copper complex. We have found that the yield can be enhanced by reprecipitation of the complex. Precipitated copper complexes of citrulline (e.g., produced by the reaction of ornithine:copper complex with urea or cyanate in water) may be dried. The citrulline:copper complex may be redissolved by suspending the precipitate in water and acidifying the suspension until the complex dissolves. Acidification can be achieved by adding concentrated acid, preferably hydrogen chloride, to the suspension while stirring. After the copper:citrulline complex solution becomes clear, a base (usually sodium hydroxide) is added to bring the pH to 7-10. During both the acidification and subsequent neutralization steps, active cooling (temperatures below 45°C) is used to protect the citrulline product from hydrolysis or reactions that generate by-products. The precipitate is washed with water (e.g., until the filtrate is chloride-free, as determined by checking the turbidity of the filtrate with silver nitrate), and then the precipitate is dried. Reprecipitation under these conditions is more selective for the citrulline:copper complex than the ornithine:copper complex because the ornithine complex is more soluble in water. If the dried complex contains a higher than desired level of ornithine contamination (e.g., greater than 10 mol % ornithine as measured by NMR), the complex can be redissolved and reprecipitated as necessary to further reduce the relative amount of ornithine. Recovery of citrulline from copper complexes

[0017] Once the ornithine content in the copper:citrulline complex precipitate is sufficiently low (preferred), Ornithine (or less than 10 mol % ornithine), the precipitate is resuspended in water, and citrulline is liberated from the complex by removing the copper as an inorganic precipitate, typically copper sulfide (see Figure 2B). While sulfide can be introduced in various salt forms, the inventors have found it preferable to use hydrogen sulfide gas as the sulfide source. In a preferred method, the aqueous suspension is placed in a stirred pressure vessel. Air is then pumped out of the reactor headspace to create a low pressure. The reactor is then repressurized with hydrogen sulfide gas (preferably at a low temperature, e.g., 0°C to 5°C, to maximize hydrogen sulfide solubility) above the aqueous suspension. Hydrogen sulfide is continuously added to the reactor to maintain atmospheric pressure during gas consumption. The copper salt precipitates, leaving citrulline in solution. The pressure in the vessel decreases as hydrogen sulfide is consumed, and the reaction is complete when the pressure stabilizes. The reaction of hydrogen sulfide with residual copper salts (e.g., chloride or sulfate) results in a decrease in pH. Typically, the pH will be less than 4, preferably about 3. The copper salts typically include copper(II) sulfide, but may also include copper(I) sulfide and copper oxide. The solution temperature is typically raised to about 30°C for filtration to promote citrulline solubility and drive off excess hydrogen sulfide gas, while the precipitated copper salts are removed by filtration. Citrulline Purification

[0018] For pharmaceutical applications, the active compound must be substantially free of contaminants and To produce a pharmaceutical-grade product, further purification steps are required. For the purposes of the present invention, substantially free of contaminants is considered to be 0.8% or less (not more than: NMT) ornithine, 0.15% or less of each specific impurity, 0.1% or less of each unspecified (unknown) impurity, a total of 1.3% or less of related substances, and 10 ppm or less of Cu. With regard to citrulline produced from ornithine using a copper complex to protect α-amino acid function, the inventors have found that the desired purification after citrulline is released from the copper complex can be achieved by activated carbon adsorption of contaminants and solvent / antisolvent crystallization of the active pharmaceutical ingredient.

[0010]

[0019] The aqueous citrulline solution remaining after removal of the precipitated copper salts is resistant to hydrolysis. The solution is neutralized to stabilize citrulline, enhance the adsorption of residual copper to the activated carbon, and promote solvent / antisolvent precipitation of citrulline. The pH is preferably adjusted to 5.9±0.2, the isoelectric point of citrulline. The neutralized citrulline solution may be passed through a nanofilter to remove bacteria and / or bacterial cell wall fragments that may contaminate the solution. The nanofiltered solution may be stored in a semi-sterile reservoir for subsequent purification steps. The neutralized citrulline solution is treated with activated carbon by mixing with carbon dust or by passing the solution through an activated carbon adsorption bed. The aqueous citrulline-containing effluent from the activated carbon is mixed with an antisolvent to cause antisolvent crystallization. Suitable antisolvents include aliphatic alcohols such as 2-propanol, ethanol, or methanol, and acetone, which are miscible with water. A preferred antisolvent for citrulline is acetone when mixed with approximately two volumes of water (e.g., 1.8 volumes of acetone to 1 volume of water). The acetone is preferably pre-cooled so that the resulting suspension is between 0° C. and 10° C. The cooled suspension may be collected in a reservoir or immediately processed by filtration to recover the citrulline precipitate. Microbial control:

[0020] Citrulline synthesis and purification are carried out in aqueous solution, which reduces the risk of microbial contamination in the product. There is an increased risk of contamination and endotoxin accumulation. Washing of the citrulline:copper precipitate and the addition of H2S to the acidic solution minimizes microbial accumulation. From the exposure of the complex to H2S to the treatment with acetone, the aqueous solution of citrulline is preferably kept in a sealed container to limit microbial contamination and growth. Enclosing the purification steps to minimize contact with the environment and using sterile filters to capture potential microbial contamination allows manufacturing to be carried out in an ISO8 cleanroom. Alternatively, the final purification step can be carried out in a sterile GMP environment (e.g., ISO 8001) of the type used for aseptic filling of sterile dosage products. It can be implemented within the framework of the IEEE 802.11a standard (Class 5 / 6).

[0011]

[0021] Testing of the solution prior to antisolvent precipitation determines whether the amount of microorganisms or endotoxin levels is sufficient to support the injectable therapeutic composition. If the product demonstrates an acceptable level (e.g., 50 EU / g API, more preferably 20 EU / g), the product can be subjected to nanofiltration to remove microorganisms and endotoxins before being recovered by anti-solvent precipitation and drying. Citrulline and water molecules pass through the nanofiltration membrane, while larger bacteria and bacterial cell wall fragments are retained by the filter. Press filter

[0022] The reaction mixture may be pumped through a filter press to collect / remove suspended solids. See the overall view in Figure 3 and the accompanying photograph in Figure 4. The press consists of a series of plates 1 which are then hydraulically squeezed together, ensuring a tight seal on the system. The suspension is then pumped through a central tube 2 where it spreads through multiple chambers 3 between the plates. The walls of the plates contain filter sheets which allow the filtrate to pass through and exit via an internal cavity 4.

[0012]

[0023] A general advantage of filter presses is that they provide a high surface area for filtration. This effect significantly accelerates the collection and washing of the complex and API in small amounts. This system may also be used to collect copper salts after exposure to hydrogen sulfide. In the latter case, the suspension is pumped from the reactor to an expeller, and the filtrate is then passed through an in-line 5 μm filter to capture residual copper particles, followed by an in-line 0.2 μm filter for sterilization at the entrance to a semi-sterile holding vessel. The press may be used to collect: Crude citrulline copper complex Complexes after pH-driven reprecipitation Precipitated copper salts (if citrulline remains in solution in the filtrate) Citrulline precipitated from antisolvent precipitation before drying semi-sterile container

[0024] Useful semi-sterile containers are essentially those equipped with a stirrer and ports for adding and removing liquids. The container is a sealed container equipped with a filter, a pH meter, and a pH meter. The container must be sterilized immediately before use (e.g., by treating with an isopropyl alcohol solution and rinsing with water) and must not be opened during use. A sterile air filter attached to the lid allows air to flow into the container when the liquid is being pumped out. pH adjustment may be performed in this container before treatment with activated charcoal. The container is not particularly suitable for long-term storage of the solution. activated carbon adsorption bed

[0025] The solution is pumped from the semi-sterile vessel through an activated carbon bed (a column packed with granular activated carbon) that has been pre-flushed with argon. The liquid is then returned to the semi-sterile vessel through an in-line 5 μm filter and a 0.2 μm sterile filter at the inlet port. When the solution is cyclically pumped with the agitator running for 6 hours or more, the sterile filter acts as a "microbial scrubber" and continuously collects microorganisms in the solution. The activated carbon primarily removes organic impurities, but also removes any remaining dissolved copper ions. The 5 μm filter captures carbon particles that leave the bed. sterile bag

[0026] After treatment with the activated carbon bed, the solution is passed through another sterile filter and then passed through a disposable sterilizer. The solution may be passed through a sterile bag. The solution may be stored in the bag longer than in the semi-sterile container. At this point, testing for the presence of microbial and / or bacterial endotoxins may be performed. If endotoxins are observed, a cut-off (nanofiltration) membrane may be used. If no endotoxins are observed, the citrulline is ready to be recovered from the solution by anti-solvent precipitation. Collecting the solution in a sterile bag allows the citrulline solution to be processed batchwise, with a convenient portion of the citrulline precipitated and recovered in a filter press. Solvent / antisolvent mixtures

[0027] The aqueous citrulline solution is mixed with a pre-cooled anti-solvent to precipitate citrulline from the solution. After mixing with the antisolvent, the threat posed by bacterial growth is no greater than that of other APIs. The addition of an organic solvent makes the resulting solution at least bacteriostatic. This precipitation improves citrulline purity, particularly reducing ornithine levels, and allows for rapid extraction of citrulline from the solution. Final drying

[0028] The precipitate is dried to remove residual acetone and water. This can be done in a cone dryer to first drive off the acetone anti-solvent, then the moisture, and finally the water of crystallization. The cone dryer may also be used to homogenize the product. The final dried product of anti-solvent precipitation can be stored and ultimately dissolved in a sterile aqueous diluent for therapeutic administration.

[0013]

[0029] Once dissolved in a sterile aqueous medium, citrulline prepared as described herein , It may be used to treat pulmonary hypertension (WO / 2000 / 073322), bronchopulmonary dysplasia (WO / 2009 / 099998), sickle cell crisis (WO / 2018 / 157137), cardiac surgery patients (WO / 2005 / 082042), cardiopulmonary bypass patients (WO / 2018 / 125999), and vasospasm as a complication of subarachnoid hemorrhage (WO / 2009 / 099999) by parenteral administration as described in these documents, which are incorporated herein by reference. [Example]

[0014] Example 1 Synthesis of citrulline from ornithine using urea.

[0030] L-citrulline is synthesized from L-ornithine and urea. Reaction flow diagram is shown in Figure 1A.

[0015]

[0031] L-citrulline is synthetically prepared starting from L-ornithine hydrochloride. Ten kilograms of L-ornithine hydrochloride is dissolved in approximately 50 liters of water in a 120 L reactor. This solution is neutralized with potassium hydroxide and then converted to a copper complex by adding 15 kg of copper sulfate (molar equivalent). The copper complex protects the 2-aminocarboxylic acid functional group within the molecule while allowing chemical reactions to occur at the terminal amino group. The L-ornithine copper complex is then exposed to excess urea at reflux to promote the conversion to the L-citrulline copper complex. The resulting L-citrulline copper complex is then precipitated and collected by filtration.

[0016]

[0032] The isolated copper complex of L-citrulline is dried and tested. and conduct in-service performance tests to determine suitability for continued use. Example 2 Purification of citrulline from copper-citrulline complex.

[0017]

[0033] L-Citrulline synthesized from L-ornithine and urea is purified and purified using a resin system. The reaction mixture is purified by recrystallization. The reaction flow diagram is shown in Figure 1B.

[0034] In a 120 L reactor, approximately 13 kilograms of L-cysteine prepared in Example 1 was added. The copper complex is added to a stirred solution of sodium sulfide (NaS) in water (approximately 8 kilograms of NaS in 50 liters of water), causing the precipitation of copper sulfide and the liberation of L-citrulline. The solution is filtered to remove the copper salt. The pH of the resulting aqueous solution, containing the sodium salt of L-citrulline and residual sodium sulfide, is lowered to 4 by the addition of an acidic ion exchange resin (Amberlite™). A constant stream of argon gas is passed through the solution to remove residual sulfide as hydrogen disulfide. The pH of the solution is then raised to 5.9±0.2 using sodium hydroxide, forming isoelectric L-citrulline. Activated carbon is then added to the reaction mixture to remove remaining impurities, particularly residual copper ions. The solids (Amberlite™ and activated carbon) are then removed by filtration, and the filtrate is concentrated (either by evaporation or reverse osmosis) to approximately 50 liters. L-citrulline is then precipitated from the aqueous solution by adding an equal part of acetone and cooling the mixture to near 0° C. The precipitate is collected by filtration and dried in a vacuum oven.

[0018]

[0035] The non-sterile bulk powder is then reconstituted for endotoxin reduction and sterile filtration steps. The sterile bulk powder is then used as the "raw material" for aseptic filling into glass vials to produce a final formulation that can be reconstituted with a sterile diluent prior to use. Example 3 Synthesis of citrulline from ornithine using cyanate

[0036] L-Citrulline was synthetically prepared starting from L-ornithine hydrochloride. L-ornithine hydrochloride (44 kg) was dissolved in a reactor containing sodium hydroxide (11 kg) in ethanol (170 kg). The temperature was maintained below 40°C by active cooling. Next, 0.5 molar equivalents of copper sulfate (33 kg) were added and the mixture was stirred at ambient temperature for at least 15 minutes to convert ornithine to its copper complex. The copper complex protects the 2-aminocarboxylic acid functional group of the molecule while chemical reaction occurs at the terminal amino group. Next, a molar excess of potassium cyanate (32 kg) was added to the L-ornithine copper complex, and the solution was maintained at 55°C–65°C for 4.0–4.5 hours. This promotes the conversion of L-citrulline to its copper complex. The resulting copper complex of L-citrulline precipitates during the reaction and is recovered by filtration. Example 4 Purified therapeutic grade citrulline.

[0019]

[0037] The dried copper:citrulline complex produced in Example 3 was added to a reactor containing water. The mixture is stirred to resuspend the complex. While maintaining the reactor temperature below 45°C with active cooling, concentrated hydrogen chloride solution is added to convert the complex into a solution of copper(II) chloride and citrulline hydrochloride. Once the contents of the reactor are dissolved, sodium hydroxide is added to raise the pH to 7-10 while maintaining the temperature below 40°C. The copper complex of citrulline then precipitates. The precipitate is collected and washed with water until the filtrate no longer exhibits a blue color.

[0020]

[0038] The washed precipitate is tested to determine the relative ornithine content. If the ornithine is greater than 10 mol %, redissolve and resuspend the precipitate as above until the ornithine content drops to 10 mol % or less.

[0021]

[0039] Once the precipitate reaches the desired ornithine content, it is reconstituted in water in a stirred reactor. The suspension is then spun with hydrogen sulfide gas to precipitate copper sulfide and dissolve citrulline. The solution is then heated to 30°C ± 2°C until citrulline is completely solubilized and the precipitated copper salts are removed by filtration. The citrulline-containing filtrate is passed through a micro- and sterile filter and collected in a semi-sterile reactor.

[0022]

[0040] Activated carbon is used to remove residual impurities, especially organic components and residual copper ions. The pH of the resulting aqueous solution containing L-citrulline and residual copper is adjusted to 5.9±0.2 with sodium hydroxide to form an equipotential citrulline solution. The equipotential citrulline solution is treated with activated charcoal granules, preferably by passing the solution through an activated charcoal bed, followed by microfiltration and sterilization.

[0023]

[0041] Next, L-citrulline was precipitated from the aqueous solution by adding acetone as an antisolvent. The mixture is cooled to near 0°C. 1.5 to 2 volume equivalents of acetone are added to produce citrulline dihydrate crystals. The precipitate is collected by filtration. The crystals are dried in a conical oven under vacuum at temperatures up to 45°C to remove acetone and water, yielding an anhydrous crystalline solid. This solid citrulline corresponds to the anhydrous crystals of the orthorhombic δ form reported by Allouchi et al. (2014) (Cryst. Growth Des., Vol. 14: 1279-1286).

[0024]

[0042] Either the dihydrate crystal or the anhydrous crystal can be used therapeutically. The solid or aqueous solution / suspension can be administered enterally, or the solid can be reconstituted for parenteral administration. To produce the final therapeutic agent, the non-sterile bulk powder was reconstituted and subjected to endotoxin reduction and sterile filtration steps, followed by crystallization, drying, and micronization in a sterile environment. The sterile bulk powder was then used as the "raw material" for aseptic filling into glass vials to produce the final formulation, which was reconstituted with a sterile diluent prior to use.

Claims

1. 1. A method for adding a terminal carbamide group to an α-amino acid, comprising: (a) obtaining an α-amino acid having a terminal amine complexed with copper in an aqueous solution; (b) reacting an α-amino acid having a terminal amine with a cyanate; (c) recovering the copper-complexed carbamide derivative of the α-amino acid as a precipitate; A method comprising:

2. 2. The method of claim 1, wherein the α-amino acid having a terminal amine is at least one member selected from the group consisting of ornithine or lysine.

3. 3. The method of claim 1, wherein the carbamide derivative of the α-amino acid is at least one member selected from the group consisting of citrulline or homocitrulline.

4. The method according to any one of claims 1 to 3, wherein the temperature in step (b) is from 30°C to 100°C, preferably from 40°C to 80°C, more preferably from 55°C to 65°C.

5. The process according to any one of claims 1 to 4, wherein step (b) has a reaction time of 0.5 to 5 hours, preferably 1 to 5 hours, more preferably 3 to 4.5 hours.

6. The process according to any one of claims 1 to 5, wherein the reaction time is at least 3.5 hours.

7. The method of any one of claims 1 to 6, wherein step (b) results in a reaction mixture.

8. 8. The reaction mixture of claim 7, wherein the reaction mixture is cooled to at least ambient temperature or below prior to step (c).

9. The method of any one of claims 1 to 8, wherein the precipitate recovered in step (c) is further washed with water.

10. 10. The method of claim 9, wherein the precipitate is washed with water until no blue color appears.

11. 1. A method for reducing ornithine contamination of citrulline, comprising the step of complexing citrulline with copper, wherein the step of complexing citrulline with copper results in a precipitated citrulline-copper complex.

12. 12. The method of claim 11, wherein the precipitated citrulline-copper complex is washed with water.

13. Washing the precipitated citrulline-copper complex with water (a) suspending the precipitated citrulline-copper complex in water; (b) acidifying the suspension of step (a) to dissolve the citrulline; (c) alkalizing the solution of step (b) to reprecipitate the citrulline-copper complex; (d) recovering the citrulline-copper precipitate; The method of claim 12, wherein the method is accomplished by:

14. 14. The method of claim 13, wherein the citrulline-copper precipitate of step (d) is washed with water until no chloride appears.

15. 15. The method of any one of claims 13-14, wherein the citrulline-copper precipitate contains copper in an amount less than or equal to the amount of copper contained in the precipitated citrulline-copper complex of step (a).

16. 16. The method according to any one of claims 13 to 15, wherein the temperature of the redissolved citrulline in steps (b) and (c) is 85°C or less, preferably 55°C or less, more preferably 45°C or less.

17. 17. The method of any one of claims 12 to 16, wherein washing the precipitated citrulline-copper complex results in a decrease in the ratio of ornithine:citrulline in the precipitated citrulline-copper complex.

18. 18. The method of any one of claims 12 to 17, wherein the precipitated citrulline-copper complex is resuspended in water, the pH is adjusted with an acid until the precipitated citrulline-copper complex is redissolved in water, and then the pH is readjusted with a base to reprecipitate the citrulline-copper complex.

19. 19. The method of any one of claims 11 to 18, wherein the citrulline is produced by reacting ornithine with urea.

20. 19. The method of any one of claims 11 to 18, wherein the citrulline is produced by reacting ornithine with cyanate ions.

21. 1. A method for purifying citrulline, comprising: (a) obtaining a citrulline:copper complex, optionally obtained by steps comprising a method according to any one of the preceding claims; (b) suspending the citrulline:copper complex in water; (c) introducing hydrogen sulfide to dissolve the citrulline:copper complex to produce an aqueous citrulline solution containing precipitated copper salts; (d) removing precipitated copper salts from the aqueous citrulline solution by filtration; A method comprising:

22. 22. The method of claim 21 , wherein the citrulline:copper complex of step (a) is produced by forming a carbamide derivative of ornithine complexed with copper, or the citrulline:copper complex is obtained by steps comprising the method of any one of the preceding claims.

23. 23. The method of claim 21 or 22, wherein the citrulline:copper complex of step (c) is exposed to hydrogen sulfide until no further gas consumption is observed.

24. 24. The method of any one of claims 21 to 23, wherein the temperature during step (c) is maintained below ambient temperature, preferably below 5°C, or optionally between 0°C and 5°C.

25. A method according to any one of claims 21 to 24, wherein the temperature in step (d) is increased above ambient temperature, preferably to 30°C or higher.

26. 21 to 22, wherein the copper salts removed in step (d) are removed in a filter press.

6. The method according to any one of claims 5 to 5.

27. (e) adjusting the pH of the aqueous citrulline solution after removing the precipitated copper salt to neutralize the aqueous citrulline solution, preferably adjusting the pH to pH=5.9±0.2; The method of any one of claims 21 to 26, further comprising:

28. (f) treating the neutralized aqueous citrulline solution of step (e) with activated carbon 28. The method of claim 27, further comprising:

29. (f) periodically passing the neutralized aqueous citrulline solution of step (e) through an activated carbon adsorbent bed.

30. The method of claim 28, further comprising:

30. (g) adding a water-miscible anti-solvent to the neutralized aqueous citrulline solution after treatment with activated carbon to precipitate citrulline from the neutralized aqueous citrulline solution; The method of any one of claims 28 to 29, further comprising:

31. 31. The method of claim 30, wherein the water-miscible anti-solvent is a water-miscible organic solvent.

32. 32. The method of claim 31, wherein the water-miscible anti-solvent is at least one member selected from the group consisting of 2-propanol, ethanol, methanol, and acetone, preferably acetone.

33. A process according to any one of claims 30 to 32, wherein the ratio of water-miscible anti-solvent to water is 0.5:3, preferably 1.0:2.5, more preferably 1.5:2.

2.

34. 34. The method of any one of claims 32 to 33, wherein the ratio of acetone to water is about 1.8:

1.

35. 35. The method according to any one of claims 30 to 34, wherein the temperature during step (g) is between -5°C and 20°C, preferably between 0°C and 20°C, more preferably between 0°C and 10°C.

36. 36. The method of any one of claims 27 to 35, wherein steps (e), (f), and / or (g) are carried out in a low microbial contamination environment.

37. 37. The method of claim 36, wherein the low microbial contamination environment is maintained by performing steps (e), (f), and / or (g) in a sealed container.

38. 37. The method of claim 36, wherein the low microbial contamination environment is maintained by performing steps (e), (f), and / or (g) in a clean room environment.

39. A citrulline-containing product suitable for parenteral administration, produced by the method of any one of claims 1 to 38.

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