Low-inorganic salt citicoline sodium feed liquid and preparation method thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to low-inorganic-salt citicoline sodium feed liquid and a preparation method thereof. The content of inorganic salt in the low-inorganic-salt citicoline sodium feed liquid provided by the invention is less than 3%, calcium chloride and calcium hydroxide are accurately added into the fermentation liquid through a'calcium salt precipitation-nanofiltration membrane separation 'combined strategy, and a nanofiltration membrane is combined, so that the problem that phosphate and sulfate are intercepted by the nanofiltration membrane in the traditional process is solved, and the yield of citicoline sodium is improved. Accurate separation of salt and products is realized; calcium hydroxide is fed step by step, the single feeding amount and interval time are strictly controlled, OH <-> is released step by step, H < + > is neutralized step by step, it is ensured that precipitation is completely subjected to the synergistic effect of concentration and alkaline conditions, double removal of residual salt and alkaline protein is achieved, and the content of inorganic salt is further reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a low-inorganic-salt sodium cytidine diphosphate solution and a preparation method thereof. Background Art

[0002] Sodium cytidine diphosphate is an important pharmaceutical ingredient, which is generally prepared by fermentation. During the production of sodium cytidine diphosphate by fermentation, inorganic salts such as sulfates and phosphates need to be added to provide mineral elements for the growth and metabolism of microorganisms and buffer the pH, etc. However, after fermentation, most of the inorganic salts still remain in the fermentation broth, resulting in a high content of inorganic salts in the fermentation broth, seriously affecting the purity and quality of the product.

[0003] At present, the main methods for treating residual inorganic salts in the fermentation broth are as follows: 1. Discard the fermentation broth and only retain the intracellular part. However, this operation will result in a yield loss of 30%-50%; 2. Use membrane filtration technology to remove inorganic salts, but its removal effect on phosphates and sulfates is poor and cannot really reduce the content of inorganic salts; 3. Use electrodialysis technology to remove inorganic salts, but its removal rate of phosphates and sulfates is low and it is difficult to meet the requirements of industrial production.

[0004] Therefore, how to further reduce the content of inorganic salts in the fermentation broth on the basis of retaining the fermentation broth to improve the purity and quality of sodium cytidine diphosphate has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a low-inorganic-salt sodium cytidine diphosphate solution and a preparation method thereof. By combining "calcium salt precipitation - nanofiltration membrane separation", the effective removal of inorganic salts in the sodium cytidine diphosphate fermentation broth is achieved, and the content of inorganic salts in the treated solution is kept within 3%.

[0006] The technical solution of the present invention is as follows: On the one hand, the present invention provides a low-inorganic-salt sodium cytidine diphosphate solution, and the low inorganic salt is: m 无机盐 / m 胞磷胆碱钠 ×100% < 3%.

[0007] Preferably, the m 无机盐 refers to the weight of the inorganic salt anion calculated as its sodium salt.

[0008] Preferably, the inorganic salt anion is selected from at least one of phosphate, sulfate, chloride, and oxalate.

[0009] On the other hand, the present invention provides a preparation method of the above low-inorganic-salt sodium cytidine diphosphate solution, comprising the following steps: (1)Take the cytidine choline sodium fermentation broth, add diatomaceous earth, heat it, filter it, and obtain the filtrate for standby; (2)Transfer the filtrate into a reaction kettle. After the feed liquid cools down to room temperature, add calcium chloride to the feed liquid and stir; then add calcium hydroxide in batches, stir, filter to remove the solid precipitate, and obtain the filtrate; add oxalic acid to adjust the pH of the filtrate to neutral, and then filter to obtain the filtrate; (3)Perform membrane filtration and rotary evaporation concentration on the collected filtrate to obtain the concentrated filtrate; (4)Adjust the pH of the concentrated filtrate to 9.5 - 11.5 with solid calcium hydroxide, stir, filter, collect the filtrate, and obtain the low-inorganic-salt cytidine choline sodium feed liquid.

[0010] Preferably, in the cytidine choline sodium fermentation broth described in (1), dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and ammonium sulfate are added during the preparation of the culture medium; the input amounts of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and ammonium sulfate are 30 g / L, 15 g / L, and 12 g / L respectively, the molar concentration of potassium dihydrogen phosphate is 0.11 mol / L, and the sum of the molar concentrations of dipotassium hydrogen phosphate and ammonium sulfate is 0.26 mol / L.

[0011] Preferably, the content of cytidine choline sodium in the fermentation broth described in (1) is 15.2 g / L.

[0012] Preferably, in (1), the diatomaceous earth added is 100 mesh, the dosage of diatomaceous earth is 5% of the weight of the fermentation broth, heat it up to 60 - 90 °C, treat it for 30 min, perform plate and frame filtration, and then wash it with water with a volume 0.2 times that of the feed liquid, and combine all the filtrates to obtain the cytidine choline sodium filtrate.

[0013] Preferably, in (2), the stirring time after adding calcium chloride is 30 min, and the molar dosage of calcium chloride is 1 - 1.2 times the molar amounts of ammonium sulfate and potassium hydrogen phosphate in the fermentation broth.

[0014] Preferably, in (2), the stirring time after adding calcium hydroxide is 30 min, and the molar dosage of calcium hydroxide is 1 - 1.2 times the molar amount of potassium dihydrogen phosphate added to the fermentation broth; the amount of calcium hydroxide added each time is 1.0 - 3.0 g / L (calculated based on the volume of the filtrate), and the interval between each feeding is 10 - 30 min.

[0015] Preferably, in (3), the membrane filtration is a 300 Da nanofiltration membrane.

[0016] Preferably, in (3), the concentrated filtrate is 1 / 8 - 1 / 12 of the volume of the original fermentation broth.

[0017] Preferably, in (4), the stirring time is 30 min.

[0018] The beneficial effects of the present invention are as follows: (1) By using the combined strategy of "calcium salt precipitation - nanofiltration membrane separation", calcium chloride and calcium hydroxide are accurately added to the original fermentation broth, and combined with the nanofiltration membrane to solve the problem that phosphates and sulfates are intercepted by the nanofiltration membrane in the traditional process, and achieve the accurate separation of salts and products; (2) Calcium hydroxide is fed in steps, strictly controlling the single - feed amount and the interval time, and releasing OH - step by step, gradually neutralizing H + , ensuring complete precipitation; (3) Through the synergistic action of concentration and alkaline conditions, the dual removal of residual salts and alkaline proteins is achieved, further reducing the content of inorganic salts. Brief Description of the Drawings

[0019] Figure 1 is the HPLC chromatogram of five salts in the low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid obtained in Example 1; Figure 2 is the HPLC chromatogram of five salts in the low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid obtained in Comparative Example 1; Figure 3 is the HPLC chromatogram of five salts in the low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid obtained in Comparative Example 2; Figure 4 is the HPLC chromatogram of five salts in the low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid obtained in Comparative Example 3; Figure 5 is the HPLC chromatogram of five salts in the low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid obtained in Comparative Example 5. Detailed Description of the Embodiments

[0020] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in conjunction with specific embodiments.

[0021] Example 1 A low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid, m 无机盐 / m 胞磷胆碱钠 × 100% = 2.94%.

[0022] The preparation method of the above - mentioned low - inorganic - salt cytidine - 5'-diphosphate choline sodium feed liquid includes the following steps: (1) Take the cytidine - 5'-diphosphate choline sodium fermentation broth (when preparing the culture medium, add 30 g / L of dipotassium hydrogen phosphate, 15 g / L of potassium dihydrogen phosphate, 12 g / L of ammonium sulfate, the molar amount of potassium dihydrogen phosphate is 0.11 mol / L, the molar amount of dipotassium hydrogen phosphate + ammonium sulfate is 0.26 mol / L, and the content of cytidine - 5'-diphosphate choline sodium in the fermentation broth is 15.2 g / L), add 5% of 100 - mesh diatomite, heat up to 80 °C, maintain for 30 min, perform plate - and - frame filtration, and then wash with an appropriate amount of water to obtain a filtrate, and the volume of the filtrate is the same as the volume of the fermentation broth; (2) Transfer 5 L of the filtrate in (1) into a reaction kettle. After the feed liquid cools down to room temperature, add 146.1 g of calcium chloride (1.0 times the molar amount of ammonium sulfate and monopotassium phosphate) to the feed liquid and stir for 30 min. Take 40.8 g of calcium hydroxide (1.0 times the molar amount of potassium dihydrogen phosphate) and add it in portions of 5.0 g at intervals of 10 min; after the feeding is completed, stir for 30 min, filter to remove the solid precipitate to obtain a filtrate; use oxalic acid to adjust the pH of the filtrate to neutral, and then filter to obtain a filtrate; (3) Collect the filtrate obtained in (2), perform 300 Da membrane filtration and rotary evaporation concentration to obtain a concentrated filtrate that is 1 / 10 of the volume of the original fermentation broth; (4) Use solid calcium hydroxide to adjust the pH of the concentrated filtrate to 10.5, stir for 30 min, filter, and collect the filtrate to obtain a low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid.

[0023] Detect the contents of cytidine-5'-diphosphate choline sodium, phosphate, sulfate, chloride, and oxalate in the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, calculate the weight based on its sodium salt, and calculate the inorganic salt content according to the formula: inorganic salt ratio = total weight of inorganic salts / weight of cytidine-5'-diphosphate choline sodium × 100%.

[0024] For the inorganic salts in the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, use high-performance liquid chromatography. The conditions for high-performance liquid chromatography detection are as follows: Chromatographic column: Intertsil ODS 3 / 3V (Shimadzu) 250 × 4.6 mm, 5 μm; Injection volume: 20 μL; column temperature: room temperature; flow rate: 1.0 mL / min; wavelength: 260 nm Mobile phase: 0.2 M cobalt formate - 0.1% sodium octanesulfonate, isocratic elution; The detected chloride ion residue, m 无机盐 / m 胞磷胆碱钠 × 100% is 2.94%, meeting the standard of the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid; the recovery rate of cytidine-5'-diphosphate choline sodium is 79.28%.

[0025] Example 2 The difference from Example 1 is that in (2), add 175.4 g of calcium chloride (1.2 times the molar amount of ammonium sulfate and monopotassium phosphate) to the feed liquid, take 49.0 g of calcium hydroxide (1.2 times the molar amount of potassium dihydrogen phosphate), and the other steps are the same as in Example 1.

[0026] In the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, the detected chloride ion residue, m 无机盐 / m 胞磷胆碱钠 × 100% is 2.81%, meeting the standard of the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid; the recovery rate of cytidine-5'-diphosphate choline sodium is 79.21%.

[0027] Comparative Example 1 The difference from Example 1 is that in (2), 116.9 g of calcium chloride (0.8 times the molar amount of ammonium sulfate and monopotassium phosphate) is added to the feed liquid, and 32.6 g of calcium hydroxide (0.8 times the molar amount of potassium dihydrogen phosphate) is taken. Other steps are the same as in Example 1.

[0028] In the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, residues of phosphate and chloride ions are detected, m 无机盐 / m 胞磷胆碱钠 ×100% is 11.66%, m 无机盐 / m 胞磷胆碱钠 ×100% value is higher than 3%, not meeting the standard of the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid; the yield of cytidine-5'-diphosphate choline sodium is 77.96%.

[0029] Comparative Example 2 The difference from Example 1 is that in step (2), specifically: 5 L of the fermentation filtrate in Example 1 is transferred into a reaction kettle. After the feed liquid cools to room temperature, 207.4 g of calcium chloride (1.0 times the molar amount of ammonium sulfate, monopotassium phosphate, and potassium dihydrogen phosphate) is added to the feed liquid, and it is stirred for 30 min; the solid precipitate is filtered off to obtain a filtrate; oxalic acid is used to adjust the pH of the filtrate to neutral, and then it is filtered again to obtain a filtrate; other steps are the same as in Example 1.

[0030] In the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, residues of phosphate, chloride, and oxalate ions are detected, m 无机盐 / m 胞磷胆碱钠 ×100% is 23.28%, m 无机盐 / m 胞磷胆碱钠 ×100% value is higher than 3%, not meeting the standard of the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid; the yield of cytidine-5'-diphosphate choline sodium is 76.91%.

[0031] Comparative Example 3 The difference from Example 1 is that in step (2), specifically: 5 L of the fermentation filtrate in Example 1 is transferred into a reaction kettle. After the feed liquid cools to room temperature, 138.2 g of calcium hydroxide (1.0 times the molar amount of ammonium sulfate, monopotassium phosphate, and potassium dihydrogen phosphate) is taken and fed in at 7.5 g / time with an interval of 10 min; after the feeding is completed, it is stirred for 30 min, the solid precipitate is filtered off to obtain a filtrate; oxalic acid is used to adjust the pH of the filtrate to neutral, and then it is filtered again to obtain a filtrate; other steps are the same as in Example 1.

[0032] In the low-inorganic-salt cytidine-5'-diphosphate choline sodium feed liquid, residues of sulfate, phosphate, and chloride ions are detected, m 无机盐 / m 胞磷胆碱钠 ×100% is 106.71%, m 无机盐 / m 胞磷胆碱钠The value of ×100% is higher than 3%, and basically no sulfate radical is removed, which does not meet the standard of the low-inorganic-salt sodium cytidine diphosphate solution; the yield of sodium cytidine diphosphate is 75.92%.

[0033] Comparative Example 4 The difference from Example 1 is that in step (2), calcium hydroxide is fed in at 15 g / time, and other steps are the same as those in Example 1.

[0034] In the low-inorganic-salt sodium cytidine diphosphate solution, phosphate radical and chloride ion residues are detected, m 无机盐 / m 胞磷胆碱钠 ×100% is 12.53%, m 无机盐 / m 胞磷胆碱钠 The value of ×100% is higher than 3%, which does not meet the standard of the low-inorganic-salt sodium cytidine diphosphate solution; the yield of sodium cytidine diphosphate is 77.24%.

[0035] Comparative Example 5 The difference from Example 1 is that in step (3), the collected filtrate is subjected to 300 Da membrane filtration and rotary evaporation concentration to obtain a concentrated filtrate with a volume of 1 / 5 of the original fermentation broth volume, and other steps are the same as those in Example 1.

[0036] In the low-inorganic-salt sodium cytidine diphosphate solution, phosphate radical and chloride ion residues are detected, m 无机盐 / m 胞磷胆碱钠 ×100% is 8.63%, m 无机盐 / m 胞磷胆碱钠 The value of ×100% is higher than 3%, which does not meet the standard of the low-inorganic-salt sodium cytidine diphosphate solution; the yield of sodium cytidine diphosphate is 76.12%; after adding calcium hydroxide in step (3), the production of white flocculent precipitate is much less than that in Example 1.

[0037] Comparative Example 6 (1) Take 5 liters of the fermentation filtrate in Example 1, transfer it to a reaction kettle, and after the feed liquid drops to room temperature, perform 300 Da membrane filtration and rotary evaporation concentration to obtain a concentrated filtrate with a volume of 1 / 15 of the original fermentation broth volume.

[0038] (2) Adjust the pH of the concentrated filtrate to 10.5 with solid sodium hydroxide, stir for 30 min, filter, and collect the filtrate.

[0039] (3) Detect the content of sodium cytidine diphosphate and the contents of phosphate radical, sulfate radical, chloride ion, and oxalate radical in the filtrate obtained in (2), and calculate the value of m 无机盐 / m 胞磷胆碱钠 ×100%.

[0040] In the filtrate, phosphate and sulfate residues were detected, and the inorganic salt / citicoline sodium was 404.69%. The purpose of removing sulfate and phosphate could not be achieved only by membrane treatment. During membrane filtration, the passing rate was very slow.

[0041] The total amount of remaining inorganic salts in each example and comparative example is shown in Table 1.

[0042] Table 1 Total amount of remaining inorganic salt anions in each example and comparative example Sulfate radical (g) Phosphate radical (g) Chloride ion (g) Oxalate radical (g) Total amount of citicoline sodium (g) Yield Proportion of inorganic salts Example 1 - - 1.08 - 60.25 79.28% 2.94% Example 2 - - 1.03 - 60.20 79.21% 2.81% Comparative example 1 - 2.63 1.28 - 59.25 77.96% 11.66% Comparative example 2 - 5.12 1.12 1.75 58.45 76.91% 23.28% Comparative example 3 33.7 5.30 1.23 - 57.70 75.92% 106.71% Comparative example 4 - 2.83 1.33 - 58.70 77.24% 12.53% Comparative example 5 - 1.40 1.48 - 57.85 76.12% 8.63% Comparative example 6 41.5 132.20 - - 75.03 98.68% 404.69%

[0043] - indicates not detected.

[0044] According to the data results in the table, the present invention adopts the combined strategy of "calcium salt precipitation - nanofiltration membrane separation" to solve the problem of removing polyvalent salts. In Examples 1 and 2, the inorganic salt / citicoline sodium was reduced to less than 3%, and the yield also reached 79%, meeting the requirements of industrial production.

[0045] Membrane filtration and electrodialysis, as traditional purification means, are difficult to be used for removing polyvalent salts. It can be seen from Comparative Examples 2 and 3 that when calcium chloride and calcium hydroxide are used alone, effective removal effects cannot be achieved. It can be seen from Comparative Example 4 that by slowly adding calcium hydroxide to ensure the stepwise release of OH - , gradually neutralizing the H of dihydrogen phosphate + , is beneficial to the removal of dihydrogen phosphate. It can be seen from Comparative Example 5 that a low concentration of concentration is also not conducive to the removal of residual inorganic salts. It can be seen from Comparative Example 6 that phosphates and sulfates simply cannot pass through a 300 Da membrane. The present invention adopts a desalting process of calcium chloride + calcium hydroxide + membrane filtration + concentration + alkali precipitation to convert polyvalent salts into monovalent salts, and remove basic proteins through concentration and alkali precipitation, obtaining a feed solution of inorganic salt citicoline sodium, which is beneficial to subsequent resin adsorption and crystallization.

Claims

1. A low-inorganic-salt citicoline sodium feed liquid, characterized in that The low inorganic salt is: m 无机盐 / m 胞磷胆碱钠 ×100% < 3%.

2. The liquid material according to claim 1, wherein, Said m 无机盐 refers to the weight of the inorganic salt anion calculated as its sodium salt.

3. The liquid material according to claim 2, wherein The inorganic salt anions are selected from at least one of phosphate, sulfate, chloride, and oxalate.

4. The preparation method of the liquid material according to claim 1, characterized in that It includes the following steps: (1) Take the cytidine monophosphate sodium fermentation broth, add diatomaceous earth, heat, filter, and obtain the filtrate for standby; (2) Transfer the filtrate to a reaction kettle. After the feed liquid cools to room temperature, add calcium chloride to the feed liquid and stir; Then add calcium hydroxide in batches, stir, filter to remove the solid precipitate, and obtain the filtrate; add oxalic acid to adjust the pH of the filtrate to neutral, and then filter to obtain the filtrate; (3) Perform membrane filtration and rotary evaporation concentration on the collected filtrate to obtain the concentrated filtrate; (4) Adjust the pH of the concentrated filtrate to 9.5 - 11.5 with solid calcium hydroxide, stir, filter, collect the filtrate, and obtain the low-inorganic-salt cytidine monophosphate sodium feed liquid.

5. The preparation method according to claim 4, characterized in that, In the cytidine monophosphate sodium fermentation broth described in (1), dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and ammonium sulfate are added during the preparation of the culture medium; the input amounts of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and ammonium sulfate are 30 g / L, 15 g / L, and 12 g / L respectively, the molar concentration of potassium dihydrogen phosphate is 0.11 mol / L, and the sum of the molar concentrations of dipotassium hydrogen phosphate and ammonium sulfate is 0.26 mol / L.

6. The preparation method according to claim 4, characterized in that, The content of cytidine monophosphate sodium in the fermentation broth described in (1) is 15.2 g / L.

7. The preparation method according to claim 4, wherein, In (1), the added diatomaceous earth is 100 mesh, the dosage of diatomaceous earth is 5% of the weight of the fermentation broth, heat up to 60 - 90 °C, treat for 30 min, perform plate and frame filtration, and then wash with water with a volume 0.2 times that of the feed liquid, and combine all the filtrates to obtain the cytidine monophosphate sodium filtrate.

8. The preparation method according to claim 4, characterized in that, In (2), the stirring time after adding calcium chloride is 30 min, and the molar dosage of calcium chloride is 1 - 1.2 times the molar amounts of ammonium sulfate and potassium hydrogen phosphate in the fermentation broth; The stirring time after adding calcium hydroxide is 30 min, and the molar dosage of calcium hydroxide is 1 - 1.2 times the molar amount of potassium dihydrogen phosphate added to the fermentation broth; calculated based on the volume of the filtrate, the amount of calcium hydroxide added each time is 1.0 - 3.0 g / L, and the interval between each feeding is 10 - 30 min.

9. The preparation method according to claim 4, characterized in that, In (3), the membrane filtration is a nanofiltration membrane with a molecular weight cut-off of 300 Da; the concentrated filtrate is 1 / 8 - 1 / 12 of the volume of the original fermentation broth.

10. The preparation method according to claim 4, characterized in that, In (4), the stirring time is 30 min.

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