A method for extracting monophasic menotropin from human urine

Abstract

This invention relates to the field of biopharmaceutical technology, and particularly to a method for extracting unimodal human chorionic gonadotropin (hCG) from human urine. The method includes: loading urine from a healthy pregnant woman onto a YKW214 resin chromatography column, treating it with a washing buffer and eluent containing sodium chloride, and collecting the eluent; ultrafiltration and desalting to obtain a first ultrafiltrate; adjusting the pH and loading it onto a monoclonal antibody chromatography column with a ligand specifically binding to an HCG β subunit antibody, washing and eluting, and collecting the eluent; ultrafiltration and desalting again to obtain a second ultrafiltrate; performing virus-free filtration; adjusting the pH and loading it onto an RPC15 chromatography column, selectively removing HCG containing only the β subunit by utilizing the difference in the number of hydrophobic amino acids between intact HCG molecules and free β subunits, collecting the eluent; adding ethanol for precipitation, centrifuging, and vacuum drying to obtain unimodal hCG. This method effectively removes impurities such as free β subunits, resulting in a product with high purity, high yield, good safety, simple operation, and suitability for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a method for extracting unimodal human chorionic gonadotropin from human urine. Background Technology

[0002] Human chorionic gonadotropin (HCG) is a gonadotropin extracted from the urine of healthy pregnant women and is widely used clinically for inducing ovulation and treating luteal insufficiency. Due to the complex composition of human urine, containing large amounts of impurities such as proteins, endotoxins, and other hormones, extracting high-purity, high-activity HCG from urine has always been a challenge in purification processes. Currently, reported purification methods mostly employ multi-step chromatographic combinations, such as ion exchange, hydrophobic chromatography, and affinity chromatography. However, existing processes generally suffer from cumbersome procedures, long production cycles, and low yields, making it difficult to simultaneously achieve high purity and high yield.

[0003] Furthermore, products obtained using existing technologies often contain structurally similar impurities, such as free β subunits or other degradation products. These impurities not only affect the product's potency but may also lead to abnormal toxic reactions or allergic risks during clinical use. Simultaneously, bacterial endotoxins in the product are often difficult to control at ideal levels, impacting medication safety. Therefore, how to effectively remove these key impurities while simplifying the process and improving yield to obtain chorionic gonadotropin products with stable quality and higher safety remains a pressing technical problem to be solved in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting unimodal human chorionic gonadotropin from human urine, comprising the following steps: S1. Load the urine of a healthy pregnant woman onto a YKW214 resin chromatography column, wash with a washing solution containing sodium chloride, elute with an elution solution containing sodium chloride, and collect the eluent. S2. The eluent collected in step S1 is subjected to ultrafiltration and desalting to obtain the first ultrafiltrate; S3. After adjusting the pH of the first ultrafiltrate, load it onto a monoclonal antibody chromatography column, wash and elute sequentially, and collect the eluent; wherein, the ligand of the monoclonal antibody chromatography column is an antibody that specifically binds to the β subunit of human chorionic gonadotropin. S4. The eluent collected in step S3 is subjected to ultrafiltration and desalting to obtain the second ultrafiltrate; S5. Filter the second ultrafiltrate to remove viruses; S6. After adjusting the pH of the solution after virus removal filtration, load the sample onto an RPC15 chromatography column, wash and elute sequentially, and collect the eluent; wherein, the RPC15 chromatography is based on the difference in the number of hydrophobic amino acids between the intact human chorionic gonadotropin molecule and its free β subunit, selectively removing human chorionic gonadotropin containing only the β subunit. S7. Add ethanol to the eluent collected in step S6 to precipitate, and collect the precipitate by centrifugation. S8. The precipitate from step S7 is vacuum dried to obtain unimodal human chorionic gonadotropin.

[0005] Furthermore, in step S1, the washing solution is tap water containing 4-8 g / L sodium chloride; the eluent is tap water containing 51-65 g / L sodium chloride; and the loading flow rate is 640-800 L / h.

[0006] Furthermore, in step S2, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa, and the conductivity at the desalination endpoint is controlled to be 8-12 ms / cm.

[0007] Further, in step S3, the pH is adjusted to 5.5-6.5; the washing solution used for washing is (0.015±0.0015) mol / L phosphate buffer containing (0.1±0.01) mol / L sodium chloride, with a pH of 5.5-6.5; the elution solution used for elution is (0.015±0.0015) mol / L phosphate buffer containing (0.5±0.05) mol / L sodium chloride, with a pH of 3.0-4.0.

[0008] Furthermore, in step S4, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa, and the conductivity at the desalination endpoint is controlled to be 1-3 ms / cm.

[0009] Furthermore, in step S5, the virus removal filtration uses a Pall™ Pegasus™ Prime membrane with a filtration pressure not exceeding 2.1 bar.

[0010] Further, in step S6, the pH is adjusted to 5.0-6.0; the washing solution used for washing is (0.08±0.008) mol / L sodium acetate buffer, containing (7±0.7)% (v / v) alcohol, with a pH of 5.0-6.0; the elution solution used for elution is (0.08±0.008) mol / L sodium acetate buffer, containing (25±2.5)% (v / v) alcohol, with a pH of 5.0-6.0.

[0011] Furthermore, in step S7, the ethanol is cold anhydrous ethanol, and its added volume is 2-3 times the volume of the eluent collected in step S6; the relative centrifugal force of the centrifugation is 4000-5000 g, and the centrifugation time is 5-15 minutes.

[0012] Furthermore, in step S8, the vacuum degree of the vacuum drying is lower than -0.8 bar, the drying temperature is room temperature, and the drying time is 36-48 hours.

[0013] Furthermore, in step S1, the urine treated by the YKW214 resin chromatography is returned to the wastewater treatment system.

[0014] Compared with existing technologies, this invention provides a method for extracting unimodal human chorionic gonadotropin (HCG) from human urine. This method employs YKW214 resin chromatography for initial capture, followed by highly selective purification using monoclonal antibody affinity chromatography specifically targeting the HCG β subunit. This is then tandemly coupled with RPC15 reverse-phase chromatography, based on the difference in the number of hydrophobic amino acids between the intact HCG molecule and its free β subunit, to precisely remove free β subunit impurities. Furthermore, it incorporates ultrafiltration desalting, virus removal filtration, ethanol precipitation, and vacuum drying steps, forming a complete purification process. This process can directly obtain high-purity unimodal HCG from human urine, effectively removing free β subunits, endotoxins, and abnormal toxicity-related impurities, significantly improving product purity and safety. Simultaneously, the efficient integration of each step reduces target protein loss, achieving high yield and batch-to-batch quality stability. Moreover, the process is simple to operate, has a short production cycle, and low environmental impact, making it suitable for large-scale production. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 HPLC chromatogram of the chorionic gonadotropin product prepared in Example 1; Figure 2 HPLC chromatogram of the chorionic gonadotropin product prepared in Comparative Example 1; Figure 3 HPLC chromatogram of the chorionic gonadotropin product prepared in Comparative Example 2; Figure 4 HPLC chromatogram of the chorionic gonadotropin product prepared in Comparative Example 3; Figure 5 HPLC chromatogram of the chorionic gonadotropin product prepared in Comparative Example 4; Figure 6 HPLC chromatogram of the chorionic gonadotropin product prepared in Comparative Example 5. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: A method for extracting unimodal human chorionic gonadotropin from human urine (1) Raw materials: 10 tons of urine from healthy pregnant women were collected and the total potency was determined to be 49.0 MIU.

[0019] (2) YKW214 resin chromatography: The urine sample was loaded into a YKW214 resin chromatography column with a packing volume of 80 L and a loading flow rate of 640-800 L / h. After loading, the sample was washed with 800 L of tap water containing 6 g / L sodium chloride at a flow rate of 80 L / h. Then, the sample was eluted with 400 L of tap water containing 58 g / L sodium chloride at a flow rate of 80 L / h. The eluent was collected. The chromatographically treated urine was returned to the wastewater treatment system.

[0020] (3) Ultrafiltration I (Desalination): The above eluent was subjected to ultrafiltration desalting using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa and purified water as the ultrafiltration buffer. The conductivity of the feed solution was controlled at the desalting endpoint to be 10 ms / cm (range 8-12 ms / cm) to obtain the first ultrafiltrate.

[0021] (4) Monoclonal antibody affinity chromatography: The pH of the first ultrafiltrate was adjusted to 6.0 (range 5.5-6.5) with dilute acid, and the conductivity was 10 ms / cm (range 8-12 ms / cm). The adjusted feed solution was loaded onto a monoclonal antibody chromatography column. The ligand of the column was an antibody that specifically binds to the β subunit of the HCG molecule. The packing volume was 2 L and the loading flow rate was 10 L / h. After sample loading, wash thoroughly with 10 L of washing buffer at a flow rate of 2 L / h; then elute with 10 L of elution buffer at a flow rate of 2 L / h. The washing solution was a (0.015±0.0015) mol / L phosphate buffer containing (0.1±0.01) mol / L sodium chloride, with a pH of 6.0 (range 5.5-6.5). The eluent was (0.015±0.0015) mol / L phosphate buffer containing (0.5±0.05) mol / L sodium chloride, with a pH of 3.5 (range 3.0-4.0). The eluent was collected.

[0022] (5) Ultrafiltration II (Desalination): The above eluent was subjected to ultrafiltration desalting using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa and purified water as the ultrafiltration buffer. The conductivity of the feed solution was controlled at the desalting endpoint to be 2 ms / cm (range 1-3 ms / cm) to obtain the second ultrafiltrate.

[0023] (6) Virus filtration: The second ultrafiltrate was filtered for virus removal using a Pall™ Pegasus™ Prime (1 m²) virus removal membrane, with the filtration pressure controlled to not exceed 2.1 bar during filtration.

[0024] (7) RPC15 reverse phase chromatography: Adjust the pH of the virus-free filtered solution to 5.5 (range 5.0-6.0) with dilute acid or dilute alkali, and the conductivity to 2 ms / cm (range 1-3 ms / cm). Load the adjusted solution onto a 2 L RPC15 chromatography column at a flow rate of 10 L / h. After loading, wash thoroughly with 20 L of washing buffer at a flow rate of 2 L / h. Then elute with 10 L of elution buffer at a flow rate of 2 L / h. The washing buffer is (0.08±0.008) mol / L NaAc + (7±0.7)% ethanol, pH=5.0-6.0; the elution buffer is (0.08±0.008) mol / L NaAc + (25±2.5)% ethanol, pH=5.0-6.0. Collect the elution buffer.

[0025] This step is based on the difference in the number of hydrophobic amino acids between the complete HCG molecule (α+β subunit) and HCG containing only the β subunit (the complete molecule has 16 more hydrophobic amino acids), and selectively removes HCG containing only the β subunit.

[0026] (8) Sedimentation and centrifugation: Add at least 2 times the volume (preferably 2-3 times) of cold anhydrous ethanol to the above eluent, stir well, centrifuge the precipitate suspension at a relative centrifugal force of 4000-5000 g for 10 minutes, discard the supernatant, and repeat the process several times until all the product is collected in the centrifuge cup to obtain the precipitate.

[0027] (9) Vacuum drying: The precipitate after centrifugation is placed in a vacuum drying oven, and the vacuum degree is kept below -0.8 bar. It is then vacuum dried at room temperature for 36-48 hours to obtain the unimodal human chorionic gonadotropin product.

[0028] Finished product test results: Related substances (purity): A Shodex PROTEIN KW-803 column was used for detection. The mobile phase was 0.1 mol / L phosphate + 0.2 mol / L sodium chloride, pH=7.0, flow rate 0.5 ml / min, column temperature 25℃, and wavelength 280 nm. The results showed a purity of 100%, meaning the impurity content was below the detection limit of the liquid chromatography column (4.7 μg / ml). The chromatogram is shown below. Figure 1 .

[0029] Bacterial endotoxin: <0.001 EU / IU, which is 10 times lower than the bacterial endotoxin limit (0.01 EU / IU) in the 2025 edition of the Chinese Pharmacopoeia.

[0030] Unit potency: 12096 IU / mg.

[0031] Total potency: 47.5 MIU (total potency of raw urine was 49.0 MIU), yield: 96.9%.

[0032] Abnormal toxicity: 10,000 IU / ml of human chorionic gonadotropin still meets the requirements, and the detection concentration is 5 times the drug dose in the 2025 edition of the Chinese Pharmacopoeia.

[0033] Comparative Example 1: YKW214 resin chromatography was replaced with DEAE Sepharose Fast Flow chromatography. Replace the YKW214 resin chromatography in step (2) of Example 1 with DEAE Sepharose Fast Flow chromatography: Take 10 tons of urine from healthy pregnant women (total titer 50.0 MIU) and inject it into a DEAE Sepharose Fast Flow chromatography column with a packing volume of 80 L and a flow rate of 640-800 L / h. Then, wash thoroughly with 800 L of washing buffer at a flow rate of 80 L / h. Next, elute with 400 L of eluent at a flow rate of 80 L / h. The washing buffer is tap water containing 2-4 g sodium chloride / L, and the eluent is tap water containing 81-95 g sodium chloride / L. The remaining steps are the same as in Example 1.

[0034] Finished product test results: Related substances (purity): Purity is 100%, meaning impurities are below the detection limit (4.7 μg / ml). See chromatogram below. Figure 2 .

[0035] Bacterial endotoxin: 0.005 EU / IU < bacterial endotoxin < 0.01 EU / IU, which meets the bacterial endotoxin limit of the Chinese Pharmacopoeia 2025 edition, but is not less than twice the limit.

[0036] Unit potency: 11895 IU / mg.

[0037] Total potency: 31.0 MIU (total potency of raw urine was 50.0 MIU), yield: 62.0%.

[0038] Abnormal toxicity: 2000 IU / ml of human chorionic gonadotropin meets the requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0039] Comparative Example 2: Ultrafiltration I and Ultrafiltration II were replaced with dialysis desalination. Replace ultrafiltration desalination in steps (3) and (5) of Example 1 with dialysis desalination: The YKW214 resin elution solution was dialyzed and desalted using an MD40 (6000-8000) dialysis bag. Simultaneously, the monoclonal antibody elution solution was also dialyzed and desalted using an MD40 (6000-8000) dialysis bag. Cold purified water needed to be changed frequently during dialysis. The remaining steps were the same as in Example 1.

[0040] Finished product test results: Related substances (purity): Purity is 100%, meaning impurities are below the detection limit (4.7 μg / ml). See chromatogram below. Figure 3 .

[0041] Bacterial endotoxin: <0.001 EU / IU, which is 10 times lower than the bacterial endotoxin limit in the 2025 edition of the Chinese Pharmacopoeia.

[0042] Unit potency: 12005 IU / mg.

[0043] Total potency: 43.3 MIU (total potency of raw urine was 48.0 MIU), yield: 90.2%.

[0044] Abnormal toxicity: 10,000 IU / ml of human chorionic gonadotropin still meets the requirements, and the detection concentration is 5 times the drug dose in the 2025 edition of the Chinese Pharmacopoeia.

[0045] Process comparison: The first step of dialysis desalination time is about 105 hours longer than that of ultrafiltration I, and the second step of dialysis desalination time is about 48 hours longer than that of ultrafiltration II, resulting in a significant extension of the production cycle; the dialysis bags are smaller, which consumes a lot of manpower; a large number of dialysis bags are used, making the operation cumbersome and leading to a decrease in yield.

[0046] Comparative Example 3: Monoclonal antibody chromatography was replaced with Blue Sepharose 6 Fast Flow chromatography. Replace the monoclonal antibody chromatography in step (4) of Example 1 with Blue Sepharose 6 Fast Flow chromatography: Adjust the pH of the ultrafiltration inner solution from ultrafiltration I to 7.0-8.0 and the conductivity to 3.0-5.0 ms / cm. Load the sample into a 5 L BlueSepharose 6 Fast Flow column at a flow rate of 25 L / h. Then wash the column with 25 L of washing buffer (0.02 mol / L PB, pH=7.0-8.0) at a flow rate of 5 L / h. Gradient elution is then used: eluent A is 0.02 mol / L PB (phosphate buffer, hereinafter the same) (pH=7.0-8.0), and eluent B is 0.02 mol / L PB + 2.5 mol / L NaCl (pH=7.0-8.0). Collect the active ingredient. The remaining steps are the same as in Example 1.

[0047] Finished product test results: Related substances (purity): Purity is 95.52%. See chromatogram. Figure 4 .

[0048] Bacterial endotoxin: <0.001 EU / IU, which is 10 times lower than the bacterial endotoxin limit in the 2025 edition of the Chinese Pharmacopoeia.

[0049] Unit potency: 10924 IU / mg.

[0050] Total potency: 46.3 MIU (total potency of raw urine was 49.0 MIU), yield: 94.5%.

[0051] Abnormal toxicity: 2000 IU / ml of human chorionic gonadotropin does not meet the requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0052] Phenomenon observation: Blue Sepharose 6 Fast Flow is a dye affinity chromatography. The presence of a large amount of blue in the effluent indicates a high risk of ligand detachment, which could lead to severe abnormal toxicity.

[0053] Comparative Example 4: Replacing RPC15 chromatography with Phenyl Sepharose 6 Fast Flow chromatography Replace the RPC15 chromatography in step (7) of Example 1 with Phenyl Sepharose 6 Fast Flow chromatography: The virus-free filtered solution was added to ammonium sulfate and stirred until the conductivity reached 150 ± 2 ms / cm. The pH was adjusted to 7.0-8.0, and the solution was loaded onto a 4 L Phenyl Sepharose 6 Fast Flow chromatography column at a flow rate of 20 L / h. The column was then thoroughly washed with 20 L of washing buffer at a flow rate of 4 L / h. Elution was then performed with 20 L of eluent at a flow rate of 4 L / h. The washing buffer consisted of 0.01 mol / L PB + 1.2 mol / L AS (ammonium sulfate), with a pH of 6.0-7.0. The eluent consisted of 0.01 mol / L PB + 0.5 mol / L AS, with a pH of 6.0-7.0. The remaining steps were the same as in Example 1.

[0054] Finished product test results: Related substances (purity): Purity is 82.51%. See chromatogram. Figure 5 .

[0055] Bacterial endotoxin: <0.001 EU / IU, which is 10 times lower than the bacterial endotoxin limit in the 2025 edition of the Chinese Pharmacopoeia.

[0056] Unit potency: 9552 IU / mg.

[0057] Total potency: 40.7 MIU (total potency of raw urine was 48.0 MIU), yield: 84.8%.

[0058] Abnormal toxicity: 2000 IU / ml of human chorionic gonadotropin meets the requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0059] Analysis: Phenyl Sepharose 6 Fast Flow has a weak binding ability with the product, resulting in some product flow through and a reduced yield; during washing, only a very weak washing intensity can be used, which is insufficient to remove HCG containing only the β subunit, thus reducing purity.

[0060] Comparative Example 5: Replacing precipitation and drying with ultrafiltration and lyophilization. Replace the ethanol precipitation + vacuum drying in steps (8) and (9) of Example 1 with ultrafiltration + lyophilization: The RPC15 eluent was subjected to 10 KD or 30 KD ultrafiltration ten times using purified water as the ultrafiltration buffer to remove as much alcohol as possible. The ultrafiltrate after alcohol removal was then lyophilized, and the lyophilization curve is shown below:

[0061] The remaining steps are the same as in the embodiment.

[0062] Finished product test results: Related substances (purity): Purity is 99.94%. See chromatogram. Figure 6 .

[0063] Bacterial endotoxin: <0.001 EU / IU, which is 10 times lower than the bacterial endotoxin limit in the 2025 edition of the Chinese Pharmacopoeia.

[0064] Unit potency: 12007 IU / mg.

[0065] Total potency: 46.1 MIU (total potency of raw urine was 48.0 MIU), yield: 96.0%.

[0066] Abnormal toxicity: 10,000 IU / ml of human chorionic gonadotropin still meets the requirements, and the detection concentration is 5 times the drug dose in the 2025 edition of the Chinese Pharmacopoeia.

[0067] Process Comparison: Using ultrafiltration freeze drying to replace alcohol precipitation vacuum drying to remove alcohol and water requires the addition of expensive freeze dryer equipment, and the time consumption and energy consumption are significantly higher than those of the alcohol precipitation vacuum drying method.

[0068] Test Method Description The detection method of this invention is based on the relevant provisions and standard methods for human chorionic gonadotropin in the 2025 edition of the Chinese Pharmacopoeia. The detection results are shown in Table 1: Related substances (purity) determination: High performance liquid chromatography (HPLC) was used. The chromatographic column was Shodex PROTEIN KW-803; the mobile phase was 0.1 mol / L phosphate + 0.2 mol / L sodium chloride, pH=7.0; the flow rate was 0.5 ml / min; the column temperature was 25℃; and the wavelength was 280 nm. Peak purity was required to be ≥99.8%, and 100% purity meant that the impurity content was below the HPLC column detection limit (4.7 μg / ml).

[0069] Potency testing: The assay was performed according to the 2025 edition of the Chinese Pharmacopoeia for human chorionic gonadotropin (hCG).

[0070] Abnormal toxicity testing: conducted according to the abnormal toxicity testing method for human chorionic gonadotropin in the 2025 edition of the Chinese Pharmacopoeia.

[0071] Bacterial endotoxin detection: performed according to the 2025 edition of the Chinese Pharmacopoeia for the test of bacterial endotoxins of human chorionic gonadotropin (gel limit method).

[0072] It should be noted that the human chorionic gonadotropin (HCG) involved in this invention is composed of an α subunit and a β subunit. The amino acid sequence of the α subunit is shown in SEQ ID NO:1, and the amino acid sequence of the β subunit is shown in SEQ ID NO:2.

[0073] SEQ ID NO:1 (HCG α subunit): Ala-Pro-Asp-Val-Gln-Asp-Cys-Pro-Glu-Cys-Thr-Leu-Gln-Glu-Asp-Pro-Phe-Phe-Ser-Gln-Pro-Gly-Ala-Pro-Ile-Leu-Gln-Cys-Met-Gly-Cys-Cys-Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Leu-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val-Gln-Lys-Asn-Val-Thr-Ser-Glu-Ser-Thr-Cys-Cys-Val-Ala-Lys-Ser-Tyr-Asn-Arg-Val-Thr-Val-Met-Gly-Gly-Phe-Lys-Val-Glu-Asn-His-Thr-Ala-Cys-His-Cys-Ser-Thr-Cys-Tyr-Tyr-His-Lys-Ser SEQ ID NO:2 (HCG β subunit): Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile-Asn-Ala-Thr-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn-Thr-Thr-Ile-Cys-Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr-Arg-Val-Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Tyr-Arg-Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-Cys-Pro-Arg-Gly-Val-Asn-Pro-Val-Val-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln-Cys-Ala-Leu-Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln Table 1 Summary of Results from Examples and Comparative Examples

[0074] in conclusion A comparison of the results of Example 1 and Comparative Examples 1-5 shows that the technical solution of the present invention, through the synergistic effect of YKW214 resin chromatography, monoclonal antibody affinity chromatography, and RPC15 reverse-phase chromatography, combined with ultrafiltration, virus removal filtration, and ethanol precipitation-vacuum drying, can achieve a total yield of up to 96.9% while maintaining ultra-high purity (100%), extremely low endotoxin levels (10 times lower than the pharmacopoeia limit), and high abnormal toxicity safety (passing at 5 times the pharmacopoeia dose). Substituting any key step in Comparative Examples 1-5 did not simultaneously achieve the aforementioned superior effects, demonstrating that the overall technical solution of the present invention has significant progress and inventiveness.

[0075] Although this document frequently uses terms such as YKW214 resin chromatography, monoclonal antibody affinity chromatography, and RPC15 reversed-phase chromatography, the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of this invention; interpreting them as any additional limitation would be contrary to the spirit of this invention.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting unimodal human chorionic gonadotropin from human urine, characterized in that, Includes the following steps: S1. Load the urine of a healthy pregnant woman onto a YKW214 resin chromatography column, wash with a washing solution containing sodium chloride, elute with an elution solution containing sodium chloride, and collect the eluent. S2. The eluent collected in step S1 is subjected to ultrafiltration and desalting to obtain the first ultrafiltrate; S3. After adjusting the pH of the first ultrafiltrate, load it onto a monoclonal antibody chromatography column, wash and elute sequentially, and collect the eluent; wherein, the ligand of the monoclonal antibody chromatography column is an antibody that specifically binds to the β subunit of human chorionic gonadotropin. S4. The eluent collected in step S3 is subjected to ultrafiltration and desalting to obtain the second ultrafiltrate; S5. Filter the second ultrafiltrate to remove viruses; S6. After adjusting the pH of the solution after virus removal filtration, load the sample onto an RPC15 chromatography column, wash and elute sequentially, and collect the eluent; wherein, the RPC15 chromatography is based on the difference in the number of hydrophobic amino acids between the intact human chorionic gonadotropin molecule and its free β subunit, selectively removing human chorionic gonadotropin containing only the β subunit. S7. Add ethanol to the eluent collected in step S6 to precipitate, and collect the precipitate by centrifugation. S8. The precipitate from step S7 is vacuum dried to obtain unimodal human chorionic gonadotropin.

2. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S1, the washing solution is tap water containing 4-8 g / L sodium chloride; the eluent is tap water containing 51-65 g / L sodium chloride; and the loading flow rate is 640-800 L / h.

3. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S2, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa, and the conductivity at the desalination endpoint is controlled to be 8-12 ms / cm.

4. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S3, the pH is adjusted to 5.5-6.5; the washing solution used for washing is (0.015±0.0015) mol / L phosphate buffer containing (0.1±0.01) mol / L sodium chloride, with a pH of 5.5-6.5; the elution solution used for elution is (0.015±0.0015) mol / L phosphate buffer containing (0.5±0.05) mol / L sodium chloride, with a pH of 3.0-4.

0.

5. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S4, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa or 30 kDa, and the conductivity at the desalination endpoint is controlled to be 1-3 ms / cm.

6. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S5, the virus removal filtration uses a Pall™ Pegasus™ Prime membrane with a filtration pressure not exceeding 2.1 bar.

7. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S6, the pH is adjusted to 5.0-6.0; the washing solution used for washing is (0.08±0.008) mol / L sodium acetate buffer, containing (7±0.7)% (v / v) alcohol, with a pH of 5.0-6.0; the elution solution used for elution is (0.08±0.008) mol / L sodium acetate buffer, containing (25±2.5)% (v / v) alcohol, with a pH of 5.0-6.

0.

8. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S7, the ethanol is cold anhydrous ethanol, and its added volume is 2-3 times the volume of the eluent collected in step S6; the relative centrifugal force of the centrifugation is 4000-5000 g, and the centrifugation time is 5-15 minutes.

9. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S8, the vacuum degree of the vacuum drying is lower than -0.8 bar, the drying temperature is room temperature, and the drying time is 36-48 hours.

10. The method for extracting unimodal human chorionic gonadotropin from human urine according to claim 1, characterized in that: In step S1, the urine treated by YKW214 resin chromatography is returned to the wastewater treatment system.

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