Method for extracting and purifying blood gonadotropin

Abstract

The present application relates to the field of biological products and veterinary medicine, and discloses a method for extracting and purifying blood gonadotropin, which comprises the following steps: 1) adjusting the pH of pregnant mare serum to 4.5-5.0 by using metaphosphoric acid, and performing solid-liquid separation to collect supernatant; 2) precipitating the supernatant by using a first precipitant, and collecting filtrate; 3) precipitating the filtrate by using a second precipitant, collecting precipitate, and drying the precipitate to obtain crude blood gonadotropin; 4) dissolving the crude blood gonadotropin, and sequentially performing first ultrafiltration, affinity chromatography, second ultrafiltration, and cation exchange chromatography to obtain crude blood gonadotropin filtrate; precipitating the crude blood gonadotropin filtrate by using a third precipitant, collecting precipitate, and drying the precipitate to obtain pure blood gonadotropin. The purity of the blood gonadotropin prepared by the extraction and purification method can reach more than 99.9%, the total yield can reach 88.9%, and the titer can reach 11000 IU / mg.

Description

Technical Field

[0001] This invention relates to the fields of biological products and veterinary drugs, specifically to a method for extracting and purifying gonadotropins. Background Technology

[0002] Serum gonadotropins (PMSG) are serum gonadotropins extracted from the serum or plasma of pregnant mares. They are purified products derived from the serum (plasma) of pregnant mares during days 40-120 of gestation and possess the dual properties of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in reproductive applications. In females, PMSG promotes follicle development and corpus luteum formation in the ovaries; in males, it promotes sperm production in the seminiferous tubules and secretion by interstitial cells in the testes. Experiments have demonstrated that PMSG is highly effective in estrus synchronization, superovulation, embryo transfer, and the treatment of anestrus, weak estrus, and estrus-related infertility. In 1992, the Ministry of Agriculture of my country first included veterinary serum gonadotropins in the "Chinese Veterinary Drug Standards" (1992 edition), and in 2011, it was also included in the Chinese Pharmacopoeia.

[0003] With the expanding application of gonadotropins and the widespread adoption of large-scale farming, the market demand for gonadotropins has increased significantly. Currently, the main methods for extracting and purifying gonadotropins from pregnant mare serum include metaphosphate separation-salting-ethanol precipitation-dialysis, metaphosphate separation-ion exchange, and ammonium sulfate salting-dialysis. However, existing gonadotropin extraction and purification methods are cumbersome, labor-intensive, costly, and result in low gonadotropin yields. Furthermore, the prepared gonadotropins have low purity, low potency, and poor efficacy. Therefore, there is an urgent need to provide a more efficient purification and extraction method for gonadotropins. Summary of the Invention

[0004] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a method for extracting and purifying gonadotropins.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0006] A method for extracting and purifying serum gonadotropins includes the following steps:

[0007] (1) The pH of pregnant mare serum was adjusted to 4.5-5.0 using metaphosphate, and then solid-liquid separation was performed to collect the supernatant;

[0008] (2) The supernatant is precipitated using a first precipitant to separate the solid and liquid, and the filtrate is collected.

[0009] (3) The filtrate is precipitated using a second precipitant to separate the solid and liquid, and the precipitate is collected. The precipitate is dried to obtain crude gonadotropin.

[0010] (4) Dissolve crude gonadotropin to obtain crude gonadotropin solution; subject the crude gonadotropin solution to first ultrafiltration, affinity chromatography, second ultrafiltration and cation exchange chromatography in sequence to obtain crude gonadotropin filtrate; use a third precipitant to precipitate the crude gonadotropin filtrate, collect the precipitate, and dry the precipitate to obtain pure gonadotropin.

[0011] Preferably, the chromatographic matrix of the affinity chromatography column in the affinity chromatography is Blue Sepharose 6Fast Flow.

[0012] Preferably, in the affinity chromatography process, after sample loading, the affinity chromatography column is first rinsed with washing buffer, and then eluted with elution buffer. The washing buffer used in the affinity chromatography is a mixed aqueous solution of sodium chloride and sodium dihydrogen phosphate, with a conductivity of 10–15 mS / cm, a sodium dihydrogen phosphate concentration of 0.05 mol / L, and a sodium chloride concentration of 0.07 mol / L. The elution buffer used in the affinity chromatography is a sodium chloride solution with a conductivity of 40–50 mS / cm and a sodium chloride concentration of 0.45 mol / L. More preferably, the pH of the washing buffer used in the affinity chromatography is 6.0–7.0; the pH of the elution buffer used in the affinity chromatography is 6.0–7.0.

[0013] Preferably, the chromatographic matrix of the cation exchange chromatography column in the cation exchange chromatography is SP-SepharoseFast Flow.

[0014] Preferably, after sample loading during cation exchange chromatography, the column is first rinsed with a washing buffer, followed by elution with an elution buffer. The washing buffer used in the cation exchange chromatography is a mixed aqueous solution of sodium chloride and sodium acetate, with a conductivity of 10–15 mS / cm, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.13 mol / L. The elution buffer used in the cation exchange chromatography is a mixed aqueous solution of sodium chloride and sodium acetate, with a conductivity of 30–40 mS / cm, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.35 mol / L. More preferably, the pH of the washing buffer used in the cation exchange chromatography is 4.0–5.0; and the pH of the elution buffer used in the cation exchange chromatography is 7.0–8.0.

[0015] Preferably, the filter membrane used in the first ultrafiltration has a molecular weight cutoff of 10 kDa, and the conductivity of the crude gonadotropin solution after the first ultrafiltration treatment is 10-15 mS / cm.

[0016] Preferably, the filter membrane used in the second ultrafiltration has a molecular weight cutoff of 10 kDa, and the conductivity of the crude gonadotropin solution after the second ultrafiltration treatment is 7–10 mS / cm.

[0017] Preferably, before affinity chromatography, the pH of the crude gonadotropin solution after the first ultrafiltration treatment is adjusted to 6.0-7.0; before cation exchange chromatography, the pH of the crude gonadotropin solution after the second ultrafiltration treatment is adjusted to 4.0-5.0.

[0018] Preferably, the first precipitant is ethanol and diatomaceous earth; the operation of precipitating the supernatant with the first precipitant is as follows: ethanol is added to the supernatant until the volume fraction of ethanol in the supernatant is 55% to 60%, and then diatomaceous earth is added while stirring. More preferably, the ethanol in the first precipitant is anhydrous ethanol; the amount of diatomaceous earth used is 0.3% to 0.5% of the total weight of pregnant mare serum.

[0019] Preferably, both the second and third precipitants are ethanol. More preferably, both the second and third precipitants are anhydrous ethanol.

[0020] Preferably, the operation of using a second precipitant to precipitate the filtrate is as follows: anhydrous ethanol is added to the filtrate until the volume fraction of ethanol in the filtrate is 70% to 80%, while stirring.

[0021] Preferably, the operation of precipitating the crude gonadotropin filtrate with a third precipitant is as follows: anhydrous ethanol is added to the crude gonadotropin filtrate until the volume fraction of ethanol in the crude gonadotropin filtrate is 80% to 90%, while stirring.

[0022] Preferably, in step (4), the drying is carried out in a vacuum drying oven at a temperature of 25°C.

[0023] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows:

[0024] (1) In the method for extracting and purifying gonadotropins of the present invention, when using metaphosphoric acid to separate pregnant mare serum, the pH of the pregnant mare serum is set to 4.5-5.0. This pH range can reduce the decomposition and inactivation of gonadotropin glycans during the metaphosphoric acid separation process, resulting in a high yield and high potency of extracted gonadotropins. This effectively avoids the technical problem of low yield and low potency of gonadotropins caused by the decomposition and inactivation of gonadotropin glycans due to excessively low pH (pH 3.0-3.5) during the metaphosphoric acid separation process.

[0025] (2) In this invention, the crude gonadotropin solution is subjected to a first ultrafiltration treatment before affinity chromatography and a second ultrafiltration treatment before cation exchange chromatography. Ultrafiltration can remove small molecule protein impurities in the solution and reduce the conductivity of the crude gonadotropin solution. The conductivity of the crude gonadotropin solution can be reduced to 10-15 mS / cm through the first ultrafiltration treatment. This conductivity can ensure that gonadotropin can be adsorbed on the chromatography column and eluted with elution buffer during the subsequent affinity chromatography process. This effectively solves the technical problem that the high concentration of ethanol in the second precipitation process causes some metaphosphate to precipitate, resulting in a high concentration of crude gonadotropin salt, which prevents gonadotropin from being adsorbed on the chromatography column and flowing directly through the chromatography column during affinity chromatography. The second ultrafiltration process can reduce the conductivity of the crude gonadotropin solution to 7–10 mS / cm. This conductivity ensures that gonadotropin can be adsorbed onto the cation exchange chromatography column and eluted with elution buffer during subsequent cation exchange chromatography. This effectively solves the technical problem that the gonadotropin-containing elution buffer obtained by affinity chromatography has a high salt concentration, and gonadotropin cannot be adsorbed onto the cation exchange chromatography column and flow directly through the column during cation exchange chromatography.

[0026] (3) The present invention uses a method for extracting and purifying gonadotropins by sequentially performing metaphosphoric acid separation, first precipitation treatment, second precipitation treatment, first ultrafiltration, affinity chromatography, second ultrafiltration, cation exchange chromatography, and third precipitation treatment on pregnant mare serum. The resulting gonadotropins have high purity (purity can reach over 99.9%), high yield (yield can reach 88.9%), and high potency (potency can reach 11000 IU / mg). At the same time, the operation is simple, with little environmental pollution and low cost. It solves the technical problems of cumbersome extraction process, low yield, low purity, and low potency of existing preparation processes for gonadotropins.

[0027] (4) For the extracted crude gonadotropin, the present invention first performs affinity chromatography and then cation exchange chromatography. Affinity chromatography can effectively remove impurities in crude gonadotropin by specifically adsorbing gonadotropin and then eluting it, thereby improving the purity of gonadotropin. Cation exchange chromatography can further remove impurities in gonadotropin that cannot be removed by affinity chromatography.

[0028] (5) In this invention, Blue Sepharose 6Fast Flow is used as the chromatography matrix of the affinity chromatography column. Blue Sepharose 6Fast Flow can specifically affinity adsorb gonadotropins. After purification of gonadotropins, the gonadotropins obtained have high purity and high yield.

[0029] (6) The present invention uses SP-Sepharose Fast Flow as the chromatography matrix for ion exchange chromatography column, which has the advantages of high resolution and large loading capacity. Detailed Implementation

[0030] The following detailed description is exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form as well. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof. Experimental methods in the following embodiments that do not specify specific conditions employ conventional techniques in this art or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: pH screening experiment during the separation and treatment of pregnant mare serum metaphosphate

[0034] To investigate the effect of pH on the extraction and purification of gonadotropins during the separation and treatment of pregnant mare serum metaphosphate, this invention conducted experimental studies in Examples 1-1 to 1-6. The specific experimental contents of Examples 1-1 to 1-6 are as follows:

[0035] Example 1-1:

[0036] A method for extracting and purifying serum gonadotropins includes the following steps: adding metaphosphate to pregnant mare serum while stirring, adjusting the pH of the pregnant mare serum to 3.0 using metaphosphate, then performing solid-liquid separation and collecting the supernatant.

[0037] The contents of Examples 1-2 to 1-6 are basically the same as those of Example 1-1, except that the pH of the pregnant mare serum is adjusted by metaphosphate in step (1) in different ways; the pH of the pregnant mare serum adjusted by metaphosphate in Examples 1-2 to 1-6 are 3.5, 4.0, 4.5, 5, and 5.5, respectively.

[0038] The purity and potency of gonadotropins were tested in the supernatants collected in Examples 1-1 to 1-6, and the yield of gonadotropins was calculated. The results are shown in Table 1.

[0039] Table 1. Screening results for pH regulation of pregnant mare serum using metaphosphate.

[0040]

[0041] Table 1 shows that when the pH is below 3.5, the purity of gonadotropins in the supernatant is high, but the yield is low; as the pH increases, the yield of gonadotropins gradually increases. Considering both the purity and yield of gonadotropins, the optimal pH for adjusting the pH of pregnant mare serum using metaphosphate is 4.5–5.0, with 5.0 being even more preferred.

[0042] Example 2: Screening experiment for affinity chromatography elution buffer

[0043] To investigate the effect of affinity chromatography elution buffer components on the extraction and purification of gonadotropins, this invention conducted experimental studies in Examples 2-1 to 2-6. The specific details of Examples 2-1 to 2-6 are as follows:

[0044] Example 2-1:

[0045] A method for extracting and purifying serum gonadotropins includes the following steps:

[0046] (1) Add metaphosphate to pregnant mare serum while stirring. Adjust the pH of the pregnant mare serum to 5.0 with metaphosphate, then perform solid-liquid separation and collect the supernatant.

[0047] (2) Add anhydrous ethanol to the supernatant collected in step (1) until the volume fraction of ethanol in the supernatant is 58%, and add diatomaceous earth at the same time. The amount of diatomaceous earth is 0.35% of the total weight of pregnant mare serum. Stir while adding; then let stand for 10 hours. After standing, perform solid-liquid separation and collect the filtrate.

[0048] (3) Add anhydrous ethanol to the filtrate obtained in step (2) until the volume fraction of ethanol in the filtrate is 78%, while stirring. Then let it stand for 10 hours. After standing, perform solid-liquid separation, collect the precipitate, and dry the precipitate to obtain crude gonadotropin.

[0049] (4) Dissolve the crude gonadotropin obtained in step (3) in purified water to obtain a crude gonadotropin solution. Perform a first ultrafiltration on the crude gonadotropin solution. The molecular weight cutoff of the filter membrane used for the first ultrafiltration is 10 kDa. After the first ultrafiltration, the conductivity of the crude gonadotropin solution is 10-15 mS / cm.

[0050] (5) Adjust the pH of the crude gonadotropin solution after the first ultrafiltration to 6.5, and then start affinity chromatography. The specific operation of affinity chromatography is as follows: load the crude gonadotropin solution after the first ultrafiltration onto the affinity chromatography column. After loading, wash the affinity chromatography column with washing buffer to remove impurities. After washing, elute the gonadotropin adsorbed on the affinity chromatography column with elution buffer. Detection is performed during elution. When the A280 absorbance shows a continuous upward trend, start collecting the elution fraction. After collection, obtain the collected solution. The affinity chromatography used a mixed aqueous solution of sodium chloride and sodium dihydrogen phosphate as the washing buffer. The washing buffer had a conductivity of 13 mS / cm, a pH of 6.5, a sodium dihydrogen phosphate concentration of 0.05 mol / L, and a sodium chloride concentration of 0.07 mol / L. The affinity chromatography used a sodium chloride solution as the elution buffer. The sodium chloride solution had a conductivity of 45 mS / cm, a sodium chloride concentration of 0.45 mol / L, and a pH of 6.5.

[0051] Example 2-2:

[0052] The content of Example 2-2 is basically the same as that of Example 2-1, except that in step (5), the elution buffer used for affinity chromatography is sodium dihydrogen phosphate solution with a conductivity of 45 ms / cm, a concentration of 0.35 mol / L, and a pH of 6.5.

[0053] Examples 2-3:

[0054] The contents of Examples 2-3 are basically the same as those of Example 2-1, except that in step (5), the elution buffer used for affinity chromatography is potassium dihydrogen phosphate solution, the conductivity of potassium dihydrogen phosphate solution is 45 ms / cm, the concentration of potassium dihydrogen phosphate solution is 0.35 mol / L, and the pH of potassium dihydrogen phosphate solution is 6.5.

[0055] Examples 2-4:

[0056] The contents of Examples 2-4 are basically the same as those of Example 2-1, except that: in step (5), the elution buffer used for affinity chromatography is a mixed solution of potassium dihydrogen phosphate and sodium chloride; the concentration of potassium dihydrogen phosphate in the elution buffer is 0.15 mol / L, the concentration of sodium chloride is 0.25 mol / L; the conductivity of the elution buffer is 45 ms / cm, and the pH of the elution buffer is 6.5.

[0057] Examples 2-5:

[0058] The contents of Examples 2-5 are basically the same as those of Example 2-1, except that in step (5), the elution buffer used for affinity chromatography is sodium acetate solution with a concentration of 0.48 mol / L, a conductivity of 45 ms / cm, and a pH of 6.5.

[0059] Examples 2-6:

[0060] The contents of Examples 2-6 are basically the same as those of Example 2-1, except that: in step (5), the elution buffer used for affinity chromatography is a mixed solution of sodium acetate and sodium chloride; the concentration of sodium acetate in the elution buffer is 0.15 mol / L, the concentration of sodium chloride is 0.32 mol / L; the conductivity of the elution buffer is 45 ms / cm, and the pH of the elution buffer is 6.5.

[0061] The purity and potency of gonadotropin-containing collection solutions extracted in Examples 2-1 to 2-6 were tested, and the yield of gonadotropin was calculated. The results are shown in Table 2.

[0062] Table 2. Screening results of affinity chromatography elution buffers

[0063]

[0064] As shown in Table 2, when affinity chromatography uses a 0.45 mol / L sodium chloride solution as the elution buffer, the purity and yield of gonadotropins in the collected eluent fraction are the highest. Therefore, the preferred eluent for affinity chromatography is a 0.45 mol / L sodium chloride solution.

[0065] Example 3: pH Screening Experiment for Affinity Chromatography Elution Buffer

[0066] To investigate the effect of pH of affinity chromatography elution buffer on the extraction and purification of gonadotropins, this invention conducted experimental studies in Examples 3-1 to 3-6.

[0067] The experimental contents of Examples 3-1 to 3-6 are basically the same as those of Example 2-1. The difference is that in step (5), the pH of the elution buffer solution used for affinity chromatography—sodium chloride solution—is 5.0, 5.5, 6.0, 7.0, 7.5, and 8.0, respectively.

[0068] The purity and potency of the gonadotropins extracted in Examples 3-1 to 3-6 were tested, and the yield of gonadotropins was calculated. The results are shown in Table 3.

[0069] Table 3. pH screening results for affinity chromatography elution buffer

[0070]

[0071] As shown in Table 3, the purity of gonadotropins in the elution fraction containing gonadotropins gradually decreases with increasing pH of the elution buffer. Considering all factors, the optimal pH of the elution buffer—sodium chloride solution—used in affinity chromatography is 6.0-7.0, and more preferably 6.5.

[0072] Example 4: Screening Experiment of Cation Exchange Chromatography Elution Buffer

[0073] To investigate the effect of the cation exchange chromatography elution buffer composition on the extraction and purification of gonadotropins, the present invention conducted experimental studies in Examples 4-1 to 4-4. The specific details of Examples 4-1 to 4-4 are as follows:

[0074] Example 4-1:

[0075] A method for extracting and purifying serum gonadotropins includes the following steps:

[0076] (1) Add metaphosphate to pregnant mare serum while stirring. Adjust the pH of the pregnant mare serum to 5.0 with metaphosphate, then perform solid-liquid separation and collect the supernatant.

[0077] (2) Add anhydrous ethanol to the supernatant collected in step (1) until the volume fraction of ethanol in the supernatant is 58%, and add diatomaceous earth at the same time. The amount of diatomaceous earth is 0.35% of the total weight of pregnant mare serum. Stir while adding; then let stand for 10 hours. After standing, perform solid-liquid separation and collect the filtrate.

[0078] (3) Add anhydrous ethanol to the filtrate obtained in step (2) until the volume fraction of ethanol in the filtrate is 78%, while stirring. Then let it stand for 10 hours. After standing, perform solid-liquid separation, collect the precipitate, and dry the precipitate to obtain crude gonadotropin.

[0079] (4) Dissolve the crude gonadotropin obtained in step (3) in purified water to obtain a crude gonadotropin solution. Perform a first ultrafiltration on the crude gonadotropin solution. The molecular weight cutoff of the filter membrane used for the first ultrafiltration is 10 kDa. After the first ultrafiltration, the conductivity of the crude gonadotropin solution is 10-15 mS / cm.

[0080] (5) Adjust the pH of the crude gonadotropin solution after the first ultrafiltration to 6.5, and then start affinity chromatography. The specific operation of affinity chromatography is as follows: load the crude gonadotropin solution after the first ultrafiltration onto the affinity chromatography column. After loading, wash the affinity chromatography column with washing buffer to remove impurities. After washing, elute the gonadotropin adsorbed on the affinity chromatography column with elution buffer. Detection is performed during elution. When the A280 absorbance shows a continuous upward trend, start collecting the elution fraction. After collection, obtain the collected solution. The affinity chromatography used a mixed aqueous solution of sodium chloride and sodium dihydrogen phosphate as the washing buffer. The washing buffer had a conductivity of 13 mS / cm, a pH of 6.5, a sodium dihydrogen phosphate concentration of 0.05 mol / L, and a sodium chloride concentration of 0.07 mol / L. The affinity chromatography used a sodium chloride solution as the elution buffer. The sodium chloride solution had a conductivity of 45 mS / cm, a sodium chloride concentration of 0.45 mol / L, and a pH of 6.5.

[0081] (6) The collected solution collected by affinity chromatography was subjected to a second ultrafiltration treatment. The filter membrane used for the second ultrafiltration had a molecular weight cutoff of 10 kDa, and the conductivity of the collected solution after the second ultrafiltration treatment was 7 to 10 mS / cm.

[0082] (7) Adjust the pH of the collected solution after the second ultrafiltration to 4.0-5.0, and then start cation exchange chromatography. The specific operation of cation exchange chromatography is as follows: load the collected solution after the second ultrafiltration onto the cation exchange chromatography column. After loading, wash the cation exchange chromatography column with washing buffer to remove impurities and proteins. After washing, elute with elution buffer. Detect the elution at the same time. When the A280 absorbance shows a continuous upward trend, start collecting the elution fraction to obtain the crude gonadotropin filtrate. The washing buffer used in cation exchange chromatography was a mixed aqueous solution of sodium chloride and sodium acetate with a conductivity of 14 mS / cm, a pH of 4.5, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.13 mol / L. The elution buffer used in cation exchange chromatography was a mixed aqueous solution of sodium chloride and sodium acetate with a conductivity of 35 mS / cm, a pH of 7.5, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.35 mol / L.

[0083] Example 4-2:

[0084] The content of Example 4-2 is basically the same as that of Example 4-1, except that in step (7), the elution buffer used for cation exchange chromatography is sodium acetate solution with a concentration of 0.36 mol / L, a conductivity of 35 ms / cm, and a pH of 7.5.

[0085] Example 4-3:

[0086] The content of Example 4-3 is basically the same as that of Example 4-1, except that in step (7), the elution buffer used for cation exchange chromatography is sodium chloride solution with a concentration of 0.35 mol / L, a conductivity of 35 ms / cm, and a pH of 7.5.

[0087] Example 4-4:

[0088] The content of Example 4-4 is basically the same as that of Example 4-1, except that in step (7), the elution buffer used for cation exchange chromatography is ammonium acetate solution with a concentration of 0.42 mol / L, a conductivity of 35 ms / cm, and a pH of 7.5.

[0089] The purity and potency of the crude gonadotropin filtrates obtained in Examples 4-1 to 4-4 were tested, and the yield of gonadotropin was calculated. The results are shown in Table 4.

[0090] Table 4. Screening results of cation exchange chromatography elution buffer.

[0091]

[0092] Table 4 shows that the highest purity and yield of gonadotropin were obtained from the crude gonadotropin filtrate when the elution buffer used in cation exchange chromatography was a mixed solution of sodium chloride and sodium acetate. Therefore, the preferred elution buffer for cation exchange chromatography is a mixed solution of sodium chloride and sodium acetate, with a sodium acetate concentration of 0.01 mol / L and a sodium chloride concentration of 0.35 mol / L.

[0093] Example 5: pH Screening Experiment for Cation Exchange Chromatography Elution Buffer

[0094] To investigate the effect of cation exchange chromatography elution buffer pH on the extraction and purification of gonadotropins, the present invention conducted experimental studies in Examples 5-1 to 5-6. The specific details of Examples 5-1 to 5-6 are as follows:

[0095] The experimental contents of Examples 5-1 to 5-7 are basically the same as those of Example 4-1, except that in step (7), the pH of the cation exchange chromatography elution buffer is 6.0, 6.5, 7.0, 8.0, 8.5 and 9.0 respectively.

[0096] The purity and potency of the crude gonadotropin filtrates obtained in Examples 5-1 to 5-6 were tested, and the yield of gonadotropin was calculated. The results are shown in Table 5.

[0097] Table 5. pH screening results for cation exchange chromatography elution buffer.

[0098]

[0099] As shown in Table 5, when the pH of the elution buffer used in cation exchange chromatography is 7.0–8.0, the purity and yield of gonadotropin in the crude filtrate are both high. Therefore, the preferred pH of the elution buffer for cation exchange chromatography is 7.0–8.0, and more preferably 7.5.

[0100] Example 6: Discussion on Ultrafiltration Treatment

[0101] To investigate the effect of ultrafiltration on the extraction and purification of gonadotropins, this invention conducted experimental studies in Examples 6-1 to 6-4. The specific details of Examples 6-1 to 6-4 are as follows:

[0102] Example 6-1:

[0103] A method for extracting and purifying serum gonadotropins includes the following steps:

[0104] (1) Add metaphosphate to pregnant mare serum while stirring. Adjust the pH of the pregnant mare serum to 5.0 with metaphosphate, then perform solid-liquid separation and collect the supernatant.

[0105] (2) Add anhydrous ethanol to the supernatant collected in step (1) until the volume fraction of ethanol in the supernatant is 58%, and add diatomaceous earth at the same time. The amount of diatomaceous earth is 0.35% of the total weight of pregnant mare serum. Stir while adding; then let stand for 10 hours. After standing, perform solid-liquid separation and collect the filtrate.

[0106] (3) Add anhydrous ethanol to the filtrate obtained in step (2) until the volume fraction of ethanol in the filtrate is 78%, while stirring. Then let it stand for 10 hours. After standing, perform solid-liquid separation, collect the precipitate, and dry the precipitate to obtain crude gonadotropin.

[0107] (4) Dissolve the crude gonadotropin obtained in step (3) in purified water to obtain a crude gonadotropin solution. Perform a first ultrafiltration on the crude gonadotropin solution. The molecular weight cutoff of the filter membrane used for the first ultrafiltration is 10 kDa. After the first ultrafiltration, the conductivity of the crude gonadotropin solution is 10-15 mS / cm.

[0108] (5) Adjust the pH of the crude gonadotropin solution after the first ultrafiltration to 6.5, and then start affinity chromatography. The specific operation of affinity chromatography is as follows: load the crude gonadotropin solution after the first ultrafiltration onto the affinity chromatography column. After loading, wash the affinity chromatography column with washing buffer to remove impurities. After washing, elute the gonadotropin adsorbed on the affinity chromatography column with elution buffer. Detection is performed during elution. When the A280 absorbance shows a continuous upward trend, start collecting the elution fraction. After collection, the elution fraction containing gonadotropin is obtained. The affinity chromatography used a mixed aqueous solution of sodium chloride and sodium dihydrogen phosphate as the washing buffer. The washing buffer had a conductivity of 13 mS / cm, a pH of 6.5, a sodium dihydrogen phosphate concentration of 0.05 mol / L, and a sodium chloride concentration of 0.07 mol / L. The affinity chromatography used a sodium chloride solution as the elution buffer. The sodium chloride solution had a conductivity of 45 mS / cm, a sodium chloride concentration of 0.45 mol / L, and a pH of 6.5.

[0109] (6) The collected solution collected by affinity chromatography was subjected to a second ultrafiltration treatment. The filter membrane used for the second ultrafiltration had a molecular weight cutoff of 10 kDa, and the conductivity of the collected solution after the second ultrafiltration treatment was 7 to 10 mS / cm.

[0110] (7) Adjust the pH of the collected solution after the second ultrafiltration to 4.0-5.0, and then start cation exchange chromatography. The specific operation of cation exchange chromatography is as follows: load the collected solution after the second ultrafiltration onto the cation exchange chromatography column. After loading, wash the cation exchange chromatography column with washing buffer to remove impurities and proteins. After washing, elute with elution buffer. Detect the elution at the same time. When the A280 absorbance shows a continuous upward trend, start collecting the elution fraction to obtain the crude gonadotropin filtrate. The washing buffer used in cation exchange chromatography was a mixed aqueous solution of sodium chloride and sodium acetate with a conductivity of 14 mS / cm, a pH of 4.5, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.13 mol / L. The elution buffer used in cation exchange chromatography was a mixed aqueous solution of sodium chloride and sodium acetate with a conductivity of 35 mS / cm, a pH of 7.5, a sodium acetate concentration of 0.01 mol / L, and a sodium chloride concentration of 0.35 mol / L.

[0111] (8) Add anhydrous ethanol to the crude gonadotropin filtrate obtained in step (7) until the ethanol volume fraction in the filtrate is 86%, while stirring. Then let it stand for 10 hours to separate the solid and liquid, collect the precipitate, and put the precipitate into a vacuum drying oven for drying to obtain pure gonadotropin.

[0112] Example 6-2:

[0113] The content of Example 6-2 is basically the same as that of Example 6-1, except that: in step (4), the crude gonadotropin solution is not subjected to the first ultrafiltration treatment; in step (5), after obtaining the collection liquid by affinity chromatography, no second ultrafiltration treatment is performed, and the pH of the collection liquid obtained by affinity chromatography is directly adjusted to 4.0-5.0 before starting cation exchange chromatography.

[0114] Example 6-3:

[0115] The content of Example 6-3 is basically the same as that of Example 6-1, except that: after obtaining the collection liquid by affinity chromatography in step (5), a second ultrafiltration process is not performed. Instead, the pH of the collection liquid obtained by affinity chromatography is adjusted to 4.0-5.0 and then cation exchange chromatography is started.

[0116] Example 6-4:

[0117] The content of Example 6-4 is basically the same as that of Example 6-1, except that the crude gonadotropin solution is not subjected to the first ultrafiltration treatment in step (4).

[0118] The purity and potency of the gonadotropins extracted in Examples 6-1 to 6-4 were tested, and the yield of gonadotropins was calculated. The results are shown in Table 6.

[0119] Table 6. Effects of ultrafiltration on the extraction and purification of gonadotropins.

[0120]

[0121] Table 6 shows that the purity of the gonadotropin product is low if ultrafiltration is not performed or only a second ultrafiltration is performed during the extraction process. This is because the high concentration of ethanol during the second precipitation process causes some metaphosphate to precipitate, resulting in a high concentration of crude gonadotropin salt. During affinity chromatography, gonadotropin cannot be adsorbed onto the chromatography column and flows directly through the column, failing to effectively remove impurities from the gonadotropin. Although the purity of the gonadotropin product is significantly improved by performing only the first ultrafiltration, the highest purity can only reach 85.3%. When two ultrafiltrations are combined, the purity of the prepared gonadotropin product is the highest (reaching 99.9%), and the yield can reach 88.9%. Therefore, the preferred method for gonadotropin extraction is to perform two ultrafiltrations, with the first ultrafiltration performed before affinity chromatography and the second ultrafiltration performed before cation exchange chromatography.

[0122] Example 7: Exploration of affinity chromatography and cation exchange chromatography treatment

[0123] To investigate the effects of affinity chromatography and cation exchange chromatography on the extraction and purification of gonadotropins, the present invention conducted experimental studies in Examples 7-1 to 7-3. The specific details of Examples 7-1 to 7-3 are as follows:

[0124] Example 7-1:

[0125] The content of Example 7-1 is basically the same as that of Example 6-1, except that: after the first ultrafiltration treatment of the crude gonadotropin solution in step (4), the crude gonadotropin solution is directly subjected to the second ultrafiltration treatment in step (6), and after the second ultrafiltration treatment, it is directly processed in step (8); that is, the crude gonadotropin solution is not subjected to affinity chromatography or cation exchange chromatography.

[0126] Example 7-2:

[0127] The content of Example 7-2 is basically the same as that of Example 6-1, except that: after the second ultrafiltration treatment in step (6), no cation exchange chromatography treatment is performed, and the filtrate after the second ultrafiltration treatment is directly treated according to step (8); that is, the crude gonadotropin solution is not subjected to cation exchange chromatography treatment.

[0128] Example 7-3:

[0129] The content of Example 7-3 is basically the same as that of Example 6-1, except that: after the first ultrafiltration in step (4), affinity chromatography is not performed, and the crude gonadotropin solution after the first ultrafiltration is directly subjected to a second ultrafiltration; that is, the crude gonadotropin solution is not subjected to affinity chromatography.

[0130] The purity and potency of the gonadotropins extracted in Examples 7-1 to 7-4 were tested, and the yield of gonadotropins was calculated. The results are shown in Table 7.

[0131] Table 7. Effects of affinity chromatography and cation exchange chromatography on the extraction and purification of gonadotropins.

[0132]

[0133] As shown in Table 7, the gonadotropin product with the highest purity and yield was obtained by performing affinity chromatography followed by ion exchange chromatography during the extraction of gonadotropins.

[0134] In summary, the extraction and purification method for gonadotropins of this invention can greatly improve the purity and yield of the prepared gonadotropins.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with similar variations. However, any simple modifications, equivalent variations, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. A method for extracting and purifying gonadotropins, characterized in that, Includes the following steps: (1) The pH of pregnant mare serum was adjusted to 4.5-5.0 using metaphosphate, and then solid-liquid separation was performed to collect the supernatant; (2) The supernatant is precipitated using a first precipitant to separate the solid and liquid, and the filtrate is collected; the first precipitant is ethanol and diatomaceous earth; the operation of precipitating the supernatant using the first precipitant is as follows: ethanol is added to the supernatant until the volume fraction of ethanol in the supernatant is 55% to 60%, and then diatomaceous earth is added while stirring. The amount of diatomaceous earth used is 0.3% to 0.5% of the total weight of pregnant mare serum. (3) The filtrate is precipitated using a second precipitant to separate the solid and liquid, and the precipitate is collected. The precipitate is dried to obtain crude gonadotropin. (4) Dissolve the crude gonadotropin to obtain a crude gonadotropin solution; The crude gonadotropin solution was subjected to a series of ultrafiltration processes: first ultrafiltration, affinity chromatography, second ultrafiltration, and cation exchange chromatography, to obtain a crude gonadotropin filtrate. The filtrate was then precipitated using a third precipitant, and the precipitate was collected and dried to obtain pure gonadotropin. The conductivity of the crude gonadotropin solution after the first ultrafiltration was 10–15 mS / cm, and the conductivity after the second ultrafiltration was 7–10 mS / cm. The affinity chromatography used BlueSepharose 6 Fast Flow as the chromatographic matrix. During affinity chromatography, the column was first washed with washing buffer after sample loading, followed by elution with sodium chloride solution, which has a conductivity of 40–50 mS / cm. The cation exchange chromatography used SP-Sepharose Fast Flow as the chromatographic matrix. In the cation exchange chromatography process, after sample loading, the cation exchange chromatography column is first rinsed with washing buffer, and then eluted with elution buffer. The elution buffer used in the cation exchange chromatography is a mixed aqueous solution of sodium chloride and sodium acetate, and the conductivity of the elution buffer is 30-40 mS / cm. The second and third precipitants are both ethanol. The operation of precipitating the filtrate with the second precipitant is as follows: ethanol is added to the filtrate until the ethanol volume fraction in the filtrate is 70%-80%, while stirring. The operation of precipitating the crude gonadotropin filtrate with the third precipitant is as follows: ethanol is added to the crude gonadotropin filtrate until the ethanol volume fraction in the crude gonadotropin filtrate is 80%-90%, while stirring.

2. The method for extracting and purifying gonadotropins according to claim 1, characterized in that, The affinity chromatography uses a mixed aqueous solution of sodium chloride and sodium dihydrogen phosphate. The conductivity of the washing solution is 10–15 mS / cm, and the concentration of sodium dihydrogen phosphate in the washing solution is 0.05 mol / L, while the concentration of sodium chloride is 0.07 mol / L.

3. The method for extracting and purifying gonadotropins according to claim 1 or 2, characterized in that, The washing solution used in the cation exchange chromatography is a mixed aqueous solution of sodium chloride and sodium acetate with a conductivity of 10–15 mS / cm. The concentration of sodium acetate in the washing solution is 0.01 mol / L and the concentration of sodium chloride is 0.13 mol / L. The concentration of sodium acetate in the elution buffer is 0.01 mol / L and the concentration of sodium chloride is 0.35 mol / L.

4. The method for extracting and purifying gonadotropins according to claim 3, characterized in that, The first ultrafiltration membrane has a molecular weight cutoff of 10 kDa.

5. The method for extracting and purifying gonadotropins according to claim 4, characterized in that, The second ultrafiltration uses a filter membrane with a molecular weight cutoff of 10 kDa.

6. The method for extracting and purifying gonadotropins according to claim 5, characterized in that, The washing buffer used in the affinity chromatography has a pH of 6.0–7.0; the elution buffer used in the affinity chromatography has a pH of 6.0–7.0; the washing buffer used in the cation exchange chromatography has a pH of 4.0–5.0; and the elution buffer used in the cation exchange chromatography has a pH of 7.0–8.0.

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