Method for regulating crystal form in crystallization process of L-isoleucine

Abstract

The invention discloses a method for regulating and controlling a crystal form in an L-isoleucine crystallization process, and belongs to the technical field of biochemical engineering. According to the method, the thermodynamically stable B crystal form or A + B crystal form can be directionally prepared without depending on any additive by using the 20-50% ethanol aqueous solution as the solvent and regulating the conditions in the crystallization process of the L-isoleucine, the prepared L-isoleucine crystal is regular in crystal form, the angle of repose is as low as 39 degrees or below, the fluidity is remarkably improved, and the method is simple in process, clean, environmentally friendly and suitable for industrial production. The method is suitable for industrial crystallization and purification production of L-isoleucine.

Description

Technical Field

[0001] This application belongs to the field of biochemical technology, and in particular relates to a method for regulating the crystal form of L-isoleucine during crystallization. Background Technology

[0002] L-Isoleucine is one of the eight essential amino acids for the human body. It was first isolated and refined from beet molasses in 1904. Due to its unique structure and function, it plays a vital role in human metabolism and is widely used in medicine, cosmetics, and food. For example, it can be used to fortify foods, balance amino acids, and improve the nutritional value of food. In particular, L-isoleucine can promote the release of growth hormone and insulin, making it suitable for therapeutic and nutritional amino acid infusions; therefore, the requirements for its purity are increasingly stringent.

[0003] L-Isoleucine, along with leucine and valine, is collectively known as branched-chain amino acids, which are mostly commercially synthesized through fermentation. The fermentation process often produces small amounts of other amino acid impurities, which must be separated and purified from the fermentation broth through crystallization. The crystallization process significantly affects the drug's crystal form; different solvents and process conditions can result in different crystal structures.

[0004] Traditional crystallization methods often yield L-isoleucine as poorly crystallized flaky crystals or crystalline powders, exhibiting poor flowability and unsatisfactory bulk density. One of the most prominent problems in the crystallization of amino acid raw materials is polymorphism. Drug polymorphism directly affects the safety, efficacy, and quality controllability of drugs. Different polymorphs of the same drug molecule may possess different physicochemical properties (such as solubility, stability, and bioavailability), thus impacting formulation processes and clinical efficacy. Therefore, the 2020 edition of the Chinese Pharmacopoeia, Volume II, explicitly requires the control of the effective polymorphism of polymorphic drugs. L-isoleucine exists in two amorphous forms, A and B, but a technical method for targeted control of its polymorphism is lacking.

[0005] The prior art discloses a variable-temperature and isothermal crystallization technique. Under the assistance of potentials of 5V, 9V and 20V, and a cooling rate of 0.1℃ / min ~ 0.7℃ / min, the possibility of generating L-isoleucine crystals under potential assistance and its influence on the nucleation kinetics of L-isoleucine were studied. Isothermal crystallization yielded crystal form B, while multi-temperature crystallization yielded a mixture of crystal forms A and B. However, the path is complex, the crystal form control is weak, and the performance of L-isoleucine is not optimized.

[0006] Therefore, developing a simple, safe, easily industrialized method for regulating the L-isoleucine crystal form that improves product flowability is of significant research importance and application value. Summary of the Invention

[0007] To address the technical problems existing in the prior art, the primary objective of this invention is to provide a method for regulating the crystal form of L-isoleucine during crystallization.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention protects a method for regulating the crystal form during the crystallization process of L-isoleucine, comprising the following steps: S1. Add L-isoleucine raw material to a solvent to obtain an L-isoleucine solution; S2. Add inorganic acid to adjust the pH of the solution to 5.5~6.5; Cooling and crystallization were carried out at a cooling rate of 3.01~1.0℃ / min, and L-isoleucine crystals were obtained by maintaining the temperature and growing crystals. In step S1, the solvent is an ethanol solution with a volume fraction of 20-50%.

[0009] The method for regulating the crystal form of L-isoleucine provided by this invention uses a 20-50% ethanol solution as a crystallization medium. It has the ability to regulate the crystal form in both directions in synergy with other crystallization conditions. Different crystal forms, including A+B crystal form and B crystal form, can be obtained by adjusting the reaction parameters. Other crystallization conditions include cooling rate and pH conditions.

[0010] In step S2 of this invention, crystallization is carried out near the isoelectric point of L-isoleucine. Taking advantage of its lowest solubility in this pH range, it provides the maximum and consistent thermodynamic driving force for crystal nucleation and growth.

[0011] Preferably, after adding the solvent in step S1, the method further includes the following steps: heating to 70~80℃ and stirring to completely dissolve the solvent, and continuing to stir for 30~40 min to obtain an L-isoleucine solution.

[0012] Preferably, the stirring rate is 300~500 r / min.

[0013] Preferably, the concentration of the L-isoleucine solution in step S1 is 20~40 g / L.

[0014] Preferably, the solvent in step S1 is an ethanol solution with a volume fraction of 50%.

[0015] Preferably, the cooling rate is 0.1 ℃ / min, 0.5 ℃ / min, or 1.0 ℃ / min.

[0016] Preferably, the inorganic acid is selected from hydrochloric acid and has a concentration of 1~2 mol / L.

[0017] Preferably, the crystal growth time in step S3 is 30 min to 120 min.

[0018] Preferably, the L-isoleucine crystals in step S3 are obtained by filtering the crystal slurry through a Buchner funnel, washing with 5 to 10 times the amount of deionized water, and then drying.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The method of this invention uses a 20%~50% ethanol aqueous solution as a solvent to control the conditions during the crystallization process of L-isoleucine. Without relying on any additives, thermodynamically stable B crystal form or A+B crystal form can be prepared in a directional manner. The prepared L-isoleucine crystals have regular crystal forms, a repose angle as low as below 39°, and significantly improved fluidity. This method is simple, clean and environmentally friendly, and suitable for the industrial crystallization and purification production of L-isoleucine. Attached Figure Description

[0020] Figure 1 (A) Schematic diagram of L-isoleucine crystal shape and measured interior angle; (B) Type A crystal and (C) Type B crystal; Figure 2 (A) L-Isoleucine type A crystals; (B) L-Isoleucine type B crystals; Figure 3 (A) XRD patterns of two crystal forms of L-isoleucine (from literature); (B) X-ray powder diffraction patterns of type A and type B; Figure 4 Differential scanning calorimetry (DSC) curves of L-isoleucine A and B crystal forms. Detailed Implementation

[0021] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from the conventional market. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0022] I. Reagent Instructions L-Isoleucine Raw Material - Jiahe Crude Product: Shantou Jiahe Biotechnology Co., Ltd. 004-240401A.

[0023] II. Experimental Methods Example 1: S1. At room temperature, add 20g of L-isoleucine solid raw material and 1000mL of 20% ethanol solution to the crystallizer to obtain an L-isoleucine solution with a concentration of 20g / L. Control the crystallizer temperature at 80℃ using a super constant temperature water bath, mix evenly using magnetic stirring, set the stirring speed to 300 r / min, and continue stirring the solution for 30 min after the solid is completely dissolved. S2. Under magnetic stirring, slowly add 2 mol / L hydrochloric acid to adjust the pH of the solution to 6.0, which is near the isoelectric point of L-isoleucine. Continue stirring the solution for 60 min. S3. Cool the system at a cooling rate of 1.0 ℃ / min. Stop cooling when crystals appear and the solution becomes turbid. Keep the system at this temperature for 30 min to allow crystals to grow. The crystal slurry was filtered using a Buchner funnel, and the filter cake was washed with five times the volume of deionized water. The resulting solid was dried at 60°C and atmospheric pressure for 24 hours.

[0024] The crystallization conditions of L-isoleucine in Examples 2-12 are shown in Table 1 below.

[0025] Comparative Example 1 The experimental conditions were the same as in Example 1, except that the solvent in step S1 was 1000 mL of deionized water.

[0026] Comparative Example 2 The experimental conditions were the same as in Example 1, except that the solvent in step S1 was a 15% (v / v) ethanol solution.

[0027] Table 1. Parameters and conditions for examples / comparative examples

[0028] III. Crystal form product testing: (1) Optical microscope and scanning electron microscope This invention employs both optical microscopy and scanning electron microscopy to observe the morphology of L-isoleucine crystals. The L-isoleucine polycrystals have a flattened hexagonal shape, with each corner approximately 120°. Figure 1 A). These two polymorphs are morphologically difficult to distinguish because they exhibit twice the crystal symmetry. However, to facilitate differentiation, two lateral angles were named φ and δ, and the vertex angle was named ω. As shown in Figures 1B and 1C, the results indicate that the vertex angle ω of polymorph A is significantly larger than that of polymorph B (121.845°), at 128.182°. Figure 1 (B and C); while the other two angles φ and δ are almost identical for both crystal forms, this difference may be due to experimental error. Additionally, the scanning electron microscope (SEM) images are as follows: Figure 2The display clearly shows the ω-vertex angle characteristics of crystal forms A and B. Figure 1 Consistent.

[0029] (2) X-ray diffraction (XRD) and differential scanning calorimetry (DSC) spectra The crystal was characterized using XRD and DSC in this invention. The XRD detection conditions were: 2θ angle range of 5–90°; scan step size of 0.02°; scan rate of 2° / min. For DSC measurement, the temperature range was 40–350°C, the heating rate was 10°C / min, and nitrogen protection was used throughout the process.

[0030] XRD characterization of crystals can provide supporting evidence for the existence of crystal forms. Currently, more detailed XRD data include... Figure 3 As shown in B, XRD analysis was performed on samples from Examples 2 and 10 of this invention. The characteristic peaks of the B-type solid were 2θ = 18.5° and 25.56°, and four peaks (2θ) of 18.35°, 20.09°, 23.51°, and 25.28°, which were missing compared to the A-type. The 2θ peaks of 6.37° and 12.68° corresponding to (001) and (002) respectively in the A-type showed no significant change, indicating that the c-axis lattice sizes of these two forms are quite similar. The XRD results of this invention correspond to the characteristic peak positions in the references. Figure 3 Since the initial concentration and cooling rate are basically consistent, it can be determined that changing the initial concentration and cooling rate will affect the results of L-isoleucine polymorphism. This invention prepared L-isoleucine in both A and B crystal forms. Figure 4 The DSC curve results showed that crystal form A had two endothermic peaks. The first peak was at T 267.42 ℃, corresponding to the solid-solid phase transformation from crystal form A to crystal form B, i.e., the optical isomerization transformation from type A to type B. The second peak (T 287.76 ℃) was the melting peak of crystal form B, which was the same as the melting point of pure crystal form B. Furthermore, crystal form B showed only one endothermic peak (T 287.76 ℃), i.e., its melting point, indicating that crystal form B did not undergo a crystal form transformation during heating and melted directly, representing a typical characteristic of a stable crystal form. The differential scanning calorimetry (DSC) results of the sample in Example 2 also confirmed the X-ray powder diffraction (XRD) data.

[0031] (3) Determination of crystal properties Bulk density: determined according to the Chinese Pharmacopoeia 2020 edition, Part 4, 0993, method for determination of bulk density and tapped density; Angle of repose: Determined in accordance with GB / T 11986-1989 "Measurement of Angle of Repose of Surfactant Powders and Particles" or "Guidelines for Determination of Powder Flowability"; Amino acid dissolution time: As a condition-dependent kinetic parameter, the dissolution time of amino acids is of great significance for crystallization process development, production scale-up, and product quality control. The dissolution time was determined by setting a fixed concentration, temperature, and stirring speed. The results showed that at 30℃, the dissolution time for L-isoleucine with a mass concentration of 0.5%, 1%, and 2% was 62 s, 65 s, and 163 s, respectively, when stirred at 500 r / min. At 50℃, the dissolution time for 2% L-isoleucine was 52 s, 59 s, and 141 s, respectively, when stirred at 500 r / min.

[0032] IV. Experimental Results Table 2. Results of Experiments in Examples / Comparative Examples

[0033] According to Table 2, Comparative Example 1 uses pure water, but water is too polar. The solubility and crystallization behavior of L-isoleucine differ in water, easily forming flaky, irregular crystals with a loose packing structure, low bulk density, high angle of repose, and poor flowability. The resulting product is also flaky and easily absorbs moisture. Comparative Example 2 uses 15% ethanol, which has a concentration below 20%, resulting in poor flowability, flaky crystals, and easy moisture absorption. Using ethanol with a concentration above 50% poses a safety hazard as it can easily cause explosions in industrial applications. Jiahe crude product is a commercially available ordinary L-isoleucine product that has not undergone the optimization process of this invention. It has a high bulk density, high angle of repose, very poor flowability, and is prone to clumping.

[0034] Table 3. Dissolution time experimental results of the examples / comparative examples

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for controlling the crystalline form of L-isoleucine in a crystallization process, characterized by, Includes the following steps: S1. Add L-isoleucine raw material to a solvent to obtain an L-isoleucine solution; S2. Add inorganic acid to adjust the pH of the solution to 5.5~6.5; S3. Cooling and crystallizing at a cooling rate of 0.1~1.0℃ / min, and maintaining the temperature to grow crystals to obtain L-isoleucine crystals; In step S1, the solvent is an ethanol solution with a volume fraction of 20-50%.

2. The method of claim 1, wherein, The concentration of the L-isoleucine solution in step S1 is 20~40 g / L.

3. The method of claim 1, wherein, The solvent mentioned in step S1 is an ethanol solution with a volume fraction of 50%.

4. The method of claim 1, wherein, After adding the solvent in step S1, the following steps are also included: heating to 70~80℃ and stirring to completely dissolve, continuing to stir for 30~40 min to obtain an L-isoleucine solution.

5. The method of claim 4, wherein, The stirring rate is 300~500 r / min.

6. The method of claim 1, wherein, After adjusting the pH of the solution as described in step S2, maintain the solution for 0.5 to 2.0 hours.

7. The method of claim 1 wherein, The inorganic acid mentioned in step S2 is hydrochloric acid with a concentration of 1~2 mol / L.

8. The method of claim 1, wherein, The cooling rate mentioned in step S3 is one of 0.1 ℃ / min, 0.5 ℃ / min or 1.0 ℃ / min.

9. The method of claim 1 wherein, The crystal growth time mentioned in step S3 is 30 min to 120 min.

10. The method of claim 1, wherein, The L-isoleucine crystals described in step S3 are obtained by filtering the crystal slurry through a Buchner funnel, washing with 5 to 10 times the amount of deionized water, and then drying.

Images