Method for industrially and efficiently producing ulinastatin

Abstract

The invention discloses a method for industrially and efficiently generating ulinastatin, which comprises the following steps of: mixing female urine waste liquid from which HCG (Human Chorionic Gonadotropin) is extracted with sodium bentonite to obtain a mixture; carrying out first-stage solid-liquid separation on the mixture, and collecting a liquid part to obtain filtrate; adsorbing the filtrate by using an adsorption column of ion exchange resin, and then eluting the adsorbed resin by using a sodium chloride solution to obtain an eluent; the pH of the eluent is adjusted to be alkaline, diatomite is used as a filter aid, second-stage solid-liquid separation is conducted on the eluent with the pH adjusted, and sediment is collected and is ulinastatin. According to the method, three technical modules of sodium bentonite pretreatment, anion exchange adsorption and high-pH precipitation-diatomite refined filtration are creatively combined, and the HCG production waste liquid is ingeniously used as the raw material, so that many defects in the prior art are successfully overcome, and powerful support is provided for industrial production of ulinastatin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a method for industrialized and efficient production of Ulinastatin. BACKGROUND

[0002] Ulinastatin, also known as Human Urinary Trypsin Inhibitor (H-UTI), is a broad-spectrum serine protease inhibitor isolated and purified from human urine. Its chemical nature is a kind of glycoprotein, and the apparent relative molecular weight is about 66-68 kD. A significant biochemical feature of Ulinastatin is that its isoelectric point is very low, about pI 2-3.

[0003] Due to its structure containing two active functional regions (Kunitz type domain), Ulinastatin has a wide range of enzyme inhibition spectrum, and can efficiently inhibit the activity of multiple hydrolases such as trypsin, phospholipase A2, hyaluronidase, elastase, chymotrypsin, and leukocyte elastase. In addition, Ulinastatin also has many important physiological functions such as stabilizing lysosomal membrane, inhibiting the production of myocardial inhibitory factors, improving the microcirculation state during shock, and inhibiting the release of inflammatory mediators. Based on the above-mentioned mechanism of action, Ulinastatin is widely used as a critical illness treatment drug in clinical practice, especially in the treatment of acute pancreatitis, acute circulatory failure (shock), adjuvant therapy for severe sepsis (Sepsis), and improvement of immune function caused by surgical operation (especially extracorporeal circulation), etc., and shows important clinical value.

[0004] However, the clinical application and production of Ulinastatin are greatly limited by its source. The content of Ulinastatin in human urine is extremely rare, and the efficiency and cost of its separation and purification process become the key bottleneck restricting its large-scale production and application. At present, the existing technologies for purifying Ulinastatin from urine in the field mainly have the following defects: complex process, traditional and mainstream purification methods seriously depend on the combination of multi-step column chromatography technology and the limitation of ultrafiltration / precipitation method, and the source of raw materials is relatively wasteful and has great limitations. According to the search, there are few domestic published patent application documents, such as: Patent publication No. CN103073638A discloses that the use of chitin adsorption, ammonia elution, ammonium sulfate salting-out, adsorption column chromatography and affinity chromatography can improve the total yield of Ulinastatin to more than 70% to prepare high-purity Ulinastatin.

[0005] Patent publication No. CN1824301A uses a combination of anion exchange column, ultrafiltration, metal chelation column, hydrophobic column and gel column. Although these methods can obtain high-purity products, they generally have the following problems: (a) the cost of affinity chromatography medium or hydrophobic chromatography medium is extremely high; (b) the process steps are complicated, involving multiple buffer replacement and equilibration, and the total production cycle is extremely long, for example, the purification step may take more than 12-24 hours; (c) the multi-column chromatography system is difficult to linearly scale up, which is not conducive to large-scale industrial production.

[0006] In order to avoid the complexity of chromatography, patent publication No. CN105753977A develops a method using flocculants (such as chitosan and alginate) mixed with urine, combined with multi-stage ultrafiltration membranes (using different molecular weight cut-offs) and ethanol fractionation precipitation. Although this method avoids chromatography columns, it introduces new process complexity, for example, ultrafiltration membranes are prone to concentration polarization and clogging when processing complex feed such as urine, which requires frequent cleaning and backflushing, increasing the difficulty and time cost of operation. In addition, the large-scale use of ethanol for fractionation precipitation puts extremely high requirements on the explosion-proof level of production equipment and workshop environment, which is difficult to control and poses a safety hazard.

[0007] Most of the existing technologies, such as publication No. CN103073638A or publication No. CN104513308A, specify the use of "fresh urine of healthy adult males" as the starting material. At the same time, there is a common production practice in the biopharmaceutical industry to extract human chorionic gonadotropin (HCG) from female (especially pregnant) urine. After HCG extraction is complete, the remaining urine (HCG waste liquid) still contains a large amount of ulinastatin and other valuable proteins. However, in the existing technology, this part of HCG waste liquid is usually treated as industrial waste and disposed of directly, which not only causes a huge waste of valuable biological resources, but also increases the environmental treatment cost of enterprises.

[0008] The above methods all need to combine multiple steps of chromatography, complex process steps, and are not easy to scale up; in addition, the purification medium used in multi-step chromatography is expensive, which is not conducive to large-scale industrial production. There is an urgent need in the art for a new method for producing ulinastatin, which should overcome the above-mentioned defects of the prior art, achieve a simple process flow, easy to industrialize; use cheap, easily available reagents and equipment, greatly reduce production costs; and be able to recycle existing biological industrial waste (such as urine after HCG extraction). SUMMARY

[0009] The purpose of the present application is to overcome the defects of the prior art, such as complex purification process of ulinastatin, high cost, long cycle, not easy to industrialize and waste of raw materials, and to provide an industrialized and efficient method for producing ulinastatin.

[0010] This invention provides a method for the industrial-scale, efficient production of ulinastatin, comprising the following steps: (1) Mix the female urine waste after HCG extraction with sodium bentonite to obtain a mixture; (2) Perform a first-stage solid-liquid separation on the mixture and collect the liquid portion to obtain the filtrate; (3) The filtrate is adsorbed using an adsorption column of ion exchange resin, and then the resin after adsorption is eluted with sodium chloride solution to obtain the eluent. (4) Adjust the pH of the eluent to alkaline, use diatomaceous earth as a filter aid, and perform a second-stage solid-liquid separation on the pH-adjusted eluent. Collect the precipitate, which is the ulinastatin.

[0011] Preferably, in step (1), the amount of sodium bentonite added is 0.1 to 1% of the mass of female urine waste.

[0012] Preferably, the solid-liquid separation in steps (2) and (4) is achieved by plate and frame filter press.

[0013] Preferably, the ion exchange resin in step (3) is an anion exchange resin; In step (3), the mass ratio of ion exchange resin to filtrate is 0.02~0.05:1.

[0014] Preferably, the adsorption conditions in step (3) are: the flow rate of the filtrate is 1200~1800L / h.

[0015] Preferably, the concentration of the sodium chloride solution in step (3) is 0.5~1.0 mol / L.

[0016] Preferably, in step (3), the amount of sodium chloride solution used is 3 to 4 times the volume of the ion exchange resin; The elution flow rate in step (3) is 0.5 to 1.5 column volumes per hour.

[0017] Preferably, the pH of the eluent in step (4) is adjusted to 10-11; the solution used to adjust the pH is one or more of ammonia, sodium hydroxide solution or sodium bicarbonate solution.

[0018] Preferably, step (4) uses diatomaceous earth as a filter aid and includes the following steps: (4.1) Mix diatomaceous earth and water to obtain a filter aid suspension; (4.2) The filter aid suspension is circulated through the second-stage solid-liquid separation equipment to pre-lay a layer of diatomaceous earth filter membrane on the filter medium of the equipment, i.e., pre-laying layer; (4.3) The pH-adjusted eluent is filtered through the pre-coated layer.

[0019] Preferably, the mass-to-volume ratio of diatomaceous earth to water in step (4.1) is 300~500g:100L.

[0020] Compared with the prior art, the present invention has the following advantages: 1. Turning waste into treasure with low cost: This invention is the first to propose using waste urine from HCG production lines as raw material for the production of ulinastatin, realizing the recycling and maximization of the value of biological resources, greatly reducing the cost of raw material acquisition, and reducing the company's waste liquid treatment costs and environmental pollution.

[0021] 2. Economical reagents and versatile equipment: The key purification steps in this invention (pretreatment and fine filtration) all use extremely inexpensive natural minerals—sodium bentonite and diatomaceous earth—to replace the expensive affinity chromatography media or sophisticated multi-stage ultrafiltration membrane systems used in the prior art. The main equipment used (stirred tanks and plate and frame filter presses) are the most common and mature equipment in the pharmaceutical and chemical industries, which are easy to obtain, maintain, and scale up.

[0022] 3. Synergistic Process and High-Efficiency Protection: This invention innovatively introduces a bentonite pretreatment step, which removes a large amount of lipids and colloidal impurities present in HCG waste liquid at the very beginning of the process. This effectively prevents clogging and contamination of subsequent ion exchange resins, ensuring high adsorption efficiency and long service life of the resins, and guaranteeing the stability of continuous industrial production.

[0023] 4. Simplified process and reliable purity: The innovative combination process of "cation-rich environment + alkaline environment to adjust the isoelectric point of ulinastatin (pH=10) + diatomaceous earth pre-coated membrane filtration" in this invention efficiently removes a large amount of impurities from the ion exchange eluent with an extremely simple and low-cost industrial method. Its effect is comparable to or can replace the complex and expensive ethanol fractionation or multi-step gel chromatography in traditional technologies. While greatly simplifying the process, it significantly improves the purity of the crude product.

[0024] 5. Shorter cycle time, suitable for industrialization: Compared with the time-consuming multi-step chromatography or ultrafiltration operations in the prior art, the core steps of this invention (batch adsorption, plate and frame filtration) are all fast and high-throughput operation units, which significantly shortens the total production cycle and fully meets the requirements of large-scale industrial production for cost, efficiency, stability and safety. Detailed Implementation

[0025] This invention provides a method for the industrial-scale, efficient production of ulinastatin, comprising the following steps: (1) Mix the female urine waste after HCG extraction with sodium bentonite to obtain a mixture; (2) Perform a first-stage solid-liquid separation on the mixture and collect the liquid portion to obtain the filtrate; (3) The filtrate is adsorbed using an adsorption column of ion exchange resin, and then the resin after adsorption is eluted with sodium chloride solution to obtain the eluent. (4) Adjust the pH of the eluent to alkaline, use diatomaceous earth as a filter aid, and perform a second-stage solid-liquid separation on the pH-adjusted eluent. Collect the precipitate, which is the ulinastatin.

[0026] In this invention, the amount of sodium-based bentonite added in step (1) is preferably 0.1 to 1% of the mass of female urine waste, more preferably 0.5 to 1%, and even more preferably 1%.

[0027] In this invention, sodium-based bentonite is rich in sodium ions. The invention uses sodium-based bentonite in the pretreatment stage of the process, resulting in a mixture rich in sodium ions. Under alkaline conditions, the sodium ions and ulinastatin are electrostatically adsorbed to obtain structures with larger molecular weights, which then continuously aggregate. Bentonite is also an inexpensive and readily available natural mineral adsorbent with a large specific surface area and adsorption capacity. HCG urinary wastewater contains a large amount of lipids, pigments, mucopolysaccharides, and other colloidal impurities. These impurities are the main causes of physical blockage and non-specific adsorption (resin contamination) in subsequent ion exchange resins. Simultaneously, bentonite efficiently removes these interfering substances, playing a crucial role in protecting the expensive subsequent ion exchange resins, extending their service life, and ensuring adsorption efficiency.

[0028] In this invention, the mixing time in step (1) is preferably 20-40 min, more preferably 25-35 min, and even more preferably 30 min.

[0029] In this invention, the solid-liquid separation in steps (2) and (4) is achieved by plate and frame filter press. Plate and frame filter press is a mature, stable, high-capacity, and low-cost conventional equipment in the chemical and pharmaceutical fields. Its application ensures that the process of this invention can be easily scaled up to an industrial production scale of tons or more.

[0030] In this invention, the ion exchange resin in step (3) is preferably an anion exchange resin, and more preferably a strong anion exchange (SAX) resin.

[0031] In this invention, the mass ratio of ion exchange resin to filtrate in step (3) is preferably 0.02~0.05:1, more preferably 0.03~0.05:1, and even more preferably 0.05:1.

[0032] In this invention, the adsorption conditions in step (3) are as follows: the flow rate of the filtrate is preferably 1200~1800L / h, more preferably 1300~1700L / h, and even more preferably 1500L / h.

[0033] In this invention, the anion exchange resin can adsorb ulinastatin in the filtrate. Ulinastatin has a very low isoelectric point (about 2 to 3), which causes it to carry a strong net negative charge at a normal pH (e.g., 6 to 8), thereby enabling it to bind efficiently with the anion exchange resin.

[0034] In this invention, the concentration of sodium chloride solution in step (3) is preferably 0.5~1.0 mol / L, more preferably 0.6~0.9 mol / L, and even more preferably 0.8 mol / L.

[0035] In this invention, the amount of sodium chloride solution used in step (3) is preferably 3 to 4 times the volume of the ion exchange resin, more preferably 3 to 3.8 times, and even more preferably 3 to 3.5 times.

[0036] In this invention, the elution flow rate in step (3) is preferably 0.5 to 1.5 column volumes (BV) / h, more preferably 0.7 to 1 BV / h, and even more preferably 0.8 BV / h.

[0037] In this invention, the pH of the eluent in step (4) is adjusted to 10-11, more preferably 10-10.5, and even more preferably 10. During the pH adjustment process, the solution gradually changes from clear to turbid, and ulinastatin precipitates. This process is a key innovation of this invention. In the liquid eluted from the ion exchange column, the pH of the solution is adjusted to 10 (alkaline environment). Ulinastatin, due to its extremely low pI, remains highly soluble and stable (carrying a strong negative charge), thus undergoing a strong electrostatic interaction with the cations in the solution, thereby agglomerating and forming a precipitate. This step utilizes the isoelectric point property of ulinastatin to achieve the extraction of ulinastatin through an extremely simple and inexpensive pH adjustment method.

[0038] In this invention, the solution used to adjust the pH is preferably one or more of ammonia water, sodium hydroxide solution or sodium bicarbonate solution.

[0039] In this invention, step (4) uses diatomaceous earth as a filter aid and includes the following steps: (4.1) Mix diatomaceous earth and water to obtain a filter aid suspension; (4.2) The filter aid suspension is circulated through the second-stage solid-liquid separation equipment to pre-lay a layer of diatomaceous earth filter membrane on the filter medium of the equipment, i.e., pre-laying layer; (4.3) The pH-adjusted eluent is filtered through the pre-coated layer.

[0040] In this invention, the specific method of using diatomaceous earth as a filter aid in step (4) is pre-film filtration. This step is another key innovation that works synergistically with the pH adjustment process. The ulinastatin precipitate produced in step (4) is usually tiny and colloidal. If conventional filter cloth or filter membrane (such as a plate and frame filter press) is used directly for filtration, it will be impossible to retain ulinastatin. In this invention, diatomaceous earth is made into a suspension and circulated through a plate and frame filter press. A diatomaceous earth filter membrane (pre-film layer) with a rigid microporous structure is pre-filmed on the filter cloth, and then the alkaline feed solution is pumped in. This pre-film layer (rather than the filter cloth itself) undertakes the actual filtration function. Its micron-level complex channels can effectively retain all the tiny ulinastatin precipitates, while dissolved impurities can pass through smoothly, thereby obtaining crude ulinastatin.

[0041] In this invention, the mass-to-volume ratio of diatomaceous earth to water in step (4.1) is 300-500g:100L, more preferably 400-500g:100L, and even more preferably 500g:100L. Example 1

[0042] One ton of waste urine from women after HCG (human chorionic gonadotropin) extraction was collected from the HCG production workshop. 10 kg (1% w / w) of sodium bentonite was added to this waste liquid, and the mixture was stirred for 30 minutes to obtain a final mixture. The mixture was then subjected to a first-stage solid-liquid separation using a plate and frame filter press, and the liquid portion, approximately one ton, was collected as filtrate.

[0043] The collected filtrate was pumped into an adsorption column loaded with 50 kg of anion exchange resin (the mass ratio of anion exchange resin to filtrate was 0.05:1). The flow rate was controlled at 1500 L / h to ensure sufficient contact between the filtrate and the resin, allowing ulinastatin to be adsorbed onto the resin. The resin adsorbed with ulinastatin was eluted with 3 column volumes (i.e., 150 L) of 0.8 mol / L sodium chloride solution at a flow rate of 0.8 column volumes / hour (i.e., 40 L / h), and the entire amount was collected as the eluent.

[0044] The collected eluent was slowly stirred while 1 mol / L NaOH solution was added dropwise, and the pH value was monitored in real time until it stabilized at 10.0. During this process, the solution gradually changed from clear to turbid (ulinastatin precipitation). 500g of diatomaceous earth was used as a filter aid, and the diatomaceous earth was mixed with 100L of water to obtain a filter aid suspension. The filter aid suspension was circulated through a second-stage solid-liquid separation device, where a diatomaceous earth filter membrane was pre-laid on the filter media of the device, i.e., a pre-laying layer. The eluent, after pH adjustment, was passed through the pre-laying layer for second-stage solid-liquid separation to obtain the precipitate, which is the ulinastatin.

[0045] Results and testing: The ulinastatin obtained in this embodiment was a gray powder. The total yield of ulinastatin, measured by the activity assay, was 75.1% based on the total activity in the starting material solution. The entire process flowed smoothly without any blockages. Example 2

[0046] One ton of waste urine from women after HCG (human chorionic gonadotropin) extraction was collected from the HCG production workshop. 10 kg (1% w / w) of sodium bentonite was added to this waste liquid, and the mixture was stirred for 30 minutes to obtain a final mixture. The mixture was then subjected to a first-stage solid-liquid separation using a plate and frame filter press, and the liquid portion, approximately one ton, was collected as filtrate.

[0047] The collected filtrate was pumped into an adsorption column loaded with 50 kg of anion exchange resin (the mass ratio of anion exchange resin to filtrate was 0.05:1). The flow rate was controlled at 1500 L / h to ensure sufficient contact between the filtrate and the resin, allowing ulinastatin to be adsorbed onto the resin. The resin adsorbed with ulinastatin was eluted with 3 column volumes (i.e., 150 L) of 0.8 mol / L sodium chloride solution at a flow rate of 0.8 column volumes / hour (i.e., 40 L / h), and the entire amount was collected as the eluent.

[0048] The collected eluent was slowly stirred while 1 mol / L NaOH solution was added dropwise, and the pH value was monitored in real time until it stabilized at 11.0. During this process, the solution gradually changed from clear to turbid (ulinastatin precipitation). 500g of diatomaceous earth was used as a filter aid, and the diatomaceous earth was mixed with 100L of water to obtain a filter aid suspension. The filter aid suspension was circulated through a second-stage solid-liquid separation device, where a diatomaceous earth filter membrane was pre-laid on the filter media of the device, i.e., a pre-laying layer. The eluent, after pH adjustment, was passed through the pre-laying layer for second-stage solid-liquid separation to obtain the precipitate, which is the ulinastatin.

[0049] Results: The ulinastatin obtained in this embodiment was a gray powder. The total yield of ulinastatin, based on the total activity in the starting material solution, was 71.4% according to the activity assay. The entire process flowed smoothly without any blockages. Example 3

[0050] One ton of waste urine from women after HCG (human chorionic gonadotropin) extraction was collected from the HCG production workshop. One kg (0.1% w / w) of sodium bentonite was added to this waste liquid, and the mixture was stirred for 30 minutes to obtain a final mixture. The mixture was then subjected to a plate and frame filter press for the first stage of solid-liquid separation, and the liquid portion, approximately one ton, was collected as filtrate.

[0051] The collected filtrate was pumped into an adsorption column loaded with 50 kg of anion exchange resin (the mass ratio of anion exchange resin to filtrate was 0.05:1). The flow rate was controlled at 1500 L / h to ensure sufficient contact between the filtrate and the resin, allowing ulinastatin to be adsorbed onto the resin. The resin adsorbed with ulinastatin was eluted with 3 column volumes (i.e., 150 L) of 0.8 mol / L sodium chloride solution at a flow rate of 0.8 column volumes / hour (i.e., 40 L / h), and the entire amount was collected as the eluent.

[0052] The collected eluent was slowly stirred while 1 mol / L NaOH solution was added dropwise, and the pH value was monitored in real time until it stabilized at 10.0. During this process, the solution gradually changed from clear to turbid (ulinastatin precipitation). 500g of diatomaceous earth was used as a filter aid, and the diatomaceous earth was mixed with 100L of water to obtain a filter aid suspension. The filter aid suspension was circulated through a second-stage solid-liquid separation device, where a diatomaceous earth filter membrane was pre-laid on the filter media of the device, i.e., a pre-laying layer. The eluent, after pH adjustment, was passed through the pre-laying layer for second-stage solid-liquid separation to obtain the precipitate, which is the ulinastatin.

[0053] Results: The ulinastatin obtained in this embodiment was a gray powder. The total yield of ulinastatin, measured by the activity assay based on the total activity in the starting material solution, was 53.7%. The entire process flowed smoothly without any blockages. The results indicate that the method of this application remains effective under this parameter combination.

[0054] Comparative Example 1

[0055] One ton of female urine waste liquid after HCG (human chorionic gonadotropin) extraction was collected from the HCG production workshop. The female urine waste liquid was subjected to the first stage of solid-liquid separation by plate and frame filter press, and the liquid part was collected as filtrate, with a mass of about 1 ton.

[0056] The collected filtrate was pumped into an adsorption column loaded with 50 kg of anion exchange resin (the mass ratio of anion exchange resin to filtrate was 0.05:1). The flow rate was controlled at 1500 L / h to ensure sufficient contact between the filtrate and the resin, allowing ulinastatin to be adsorbed onto the resin. The resin adsorbed with ulinastatin was eluted with 3 column volumes (i.e., 150 L) of 0.8 mol / L sodium chloride solution at a flow rate of 0.8 column volumes / hour (i.e., 40 L / h), and the entire amount was collected as the eluent.

[0057] The collected eluent was slowly stirred while 1 mol / L NaOH solution was added dropwise, and the pH value was monitored in real time until it stabilized at 10.0. 500g of diatomaceous earth was used as a filter aid. The diatomaceous earth was mixed with 100L of water to obtain a filter aid suspension. The filter aid suspension was circulated through a second-stage solid-liquid separation device. A diatomaceous earth filter membrane was pre-laid on the filter media of the device to form a pre-laying layer. The eluent, after pH adjustment, was passed through the pre-laying layer for second-stage solid-liquid separation, yielding only a small amount of precipitate.

[0058] Results showed that although a large amount of ulinastatin in the eluent was negatively charged in an alkaline environment, it remained dissolved in the eluent and could not be extracted because a cation-rich environment was not provided.

[0059] It can be seen that without the addition of sodium-based bentonite to create a cation-rich environment, negatively charged ulinastatin cannot complete electrostatic adsorption under alkaline conditions and remains in a dissolved state. High-purity ulinastatin cannot be obtained solely through ion exchange and conventional filtration. The sodium-based bentonite pretreatment in this invention is a necessary prerequisite for the smooth implementation of the subsequent ion exchange process and is one of the key steps for achieving the beneficial effects of this invention.

[0060] Comparative Example 2

[0061] One ton of waste urine from women after HCG (human chorionic gonadotropin) extraction was collected from the HCG production workshop. 10 kg (1% w / w) of sodium bentonite was added to this waste liquid, and the mixture was stirred for 30 minutes to obtain a final mixture. The mixture was then subjected to a first-stage solid-liquid separation using a plate and frame filter press, and the liquid portion, approximately one ton, was collected as filtrate.

[0062] The collected filtrate was pumped into an adsorption column loaded with 50 kg of anion exchange resin (the mass ratio of anion exchange resin to filtrate was 0.05:1). The flow rate was controlled at 1500 L / h to ensure sufficient contact between the filtrate and the resin, allowing ulinastatin to be adsorbed onto the resin. The resin adsorbed with ulinastatin was eluted with 3 column volumes (i.e., 150 L) of 0.8 mol / L sodium chloride solution at a flow rate of 0.8 column volumes / hour (i.e., 40 L / h), and the entire amount was collected as the eluent.

[0063] The eluent was directly passed through a plate and frame filter press for a second stage of solid-liquid separation, and a small amount of precipitate was collected.

[0064] Results: Although a large amount of ulinastatin in the eluent was in a cation-rich environment, the solution environment was relatively neutral, so most of the ulinastatin did not exhibit a strong negative charge and remained in a dissolved state in the eluent, thus it could not be extracted.

[0065] The eluent was further filtered through a 0.22 μm microfiltration membrane, but very little substance was filtered out, making extraction impossible.

[0066] Results: Very little solid material was collected, but it contained a large amount of other miscellaneous proteins.

[0067] This demonstrates that without the alkaline conditions regulated by pH for the charge adjustment of ulinastatin and the pre-coated diatomaceous earth membrane for fine filtration, it is impossible to obtain a high-purity and high-yield crude product solely through ion exchange and conventional filtration. The combination of pH adjustment and the pre-coated diatomaceous earth filter aid in this invention is a key synergistic step in achieving the high-purity and high-yield goals of this invention.

[0068] Comparative Example 3

[0069] This comparative example is used to demonstrate the significant advancement of the present invention compared to the prior art (CN105753977A).

[0070] Take 1 ton of clear urine (non-HCG waste liquid), adjust its pH to 6, and filter with silica gel; then adjust the pH to 4.5, add 8 kg of chitosan and 0.4 kg of sodium alginate, stir, let stand, and collect the lower precipitate. Rinse the precipitate with water, add ammonium sulfate buffer to adjust the pH to 5, and perform a single-stage ultrafiltration (molecular weight cutoff 5 × 10⁻⁶). 4 The retentate was collected. Solution A (triethylamine and isopropanol) was added to the retentate, and the pH was adjusted to 7.5. Two-stage ultrafiltration was then performed (molecular weight cutoff 3 × 10⁻⁶). 4 Collect the retentate. Add 1.1 times the volume of 95% ethanol to the retentate for fractionation and precipitate separation, passing the solution through a plate and frame filter; add another 1.9 times the volume of ethanol to the supernatant, pass the solution through a plate and frame filter, and collect the precipitate. Dry the precipitate to obtain pure ulinastatin.

[0071] Results: The yield of this method was 77%.

[0072] Conclusion (compared with Embodiment 1 of the present invention): Process complexity: The method in Comparative Example 3 is extremely cumbersome, involving two pH adjustments, two flocculation and sedimentation processes, two ultrafiltration processes (and the replacement of membranes with different molecular weight cutoffs), and two ethanol fractionation and precipitation processes. This invention (Example 1) involves only one bentonite adsorption process, one resin adsorption process, and one pH adjustment filtration process, greatly simplifying the process flow.

[0073] Cost and Equipment: Comparative Example 3 requires an expensive ultrafiltration membrane system (prone to fouling) and an explosion-proof ethanol precipitation device. This invention (Example 1) uses only inexpensive bentonite, diatomaceous earth, and a conventional plate and frame filter press.

[0074] Raw materials: Comparative Example 3 used conventional urine. This invention (Example 1) utilizes HCG waste liquid to achieve resource regeneration.

[0075] Efficiency: The method in Comparative Example 3 involves time-consuming steps such as ultrafiltration and precipitation (chromatography takes 12-24 hours, and ultrafiltration is also time-consuming). The plate and frame filtration and batch adsorption operations of this invention have a shorter total time.

[0076] Yield and purity: The crude product yield (75.1%) of Example 1 of the present invention reached a high level, which fully met the requirements for subsequent refining as a crude product, and its overall efficiency was better than that of the method of Comparative Example 3.

[0077] In summary, the process of the present invention (bentonite-resin-pH precipitation-diatomite) is far superior to the ultrafiltration method represented by Comparative Example 3 in terms of cost, efficiency, equipment versatility, safety, and environmental friendliness (utilization of waste liquid).

[0078] To more clearly compare the effects of the various embodiments and comparative examples of the present invention, the key results are summarized in Table 1.

[0079] Table 1. Comparison of key results between various embodiments of the present invention and comparative examples. ; Table 1 provides sufficient proof that: Necessity (Comparative Example 1): The bentonite pretreatment used in this invention is essential for treating HCG waste liquid. Without this step, the subsequent extraction process cannot be carried out.

[0080] Synergy (Comparative Example 2): The "high pH precipitation + diatomaceous earth pre-coated membrane fine filtration" adopted in this invention is a synergistic combination of steps, neither of which can be omitted. It is the key to achieving high purity and high yield of crude product.

[0081] Progressiveness (Comparative Example 3): The complete process provided by this invention, compared with the prior art (Comparative Example 3), has outstanding substantive features and significant progress in terms of raw material cost, reagent cost, equipment requirements, process simplicity, safety, environmental protection and production efficiency.

[0082] In summary, the method for industrially and efficiently producing ulinastatin provided by this invention creatively combines three major technical modules: "sodium-based bentonite pretreatment," "anion exchange adsorption," and "high-pH precipitation-diatomaceous earth filtration," and cleverly uses HCG production waste liquid as raw material. This method successfully overcomes many shortcomings of existing technologies and provides strong support for the industrial production of ulinastatin.

[0083] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for the industrialized and efficient production of ulinastatin, characterized in that, Includes the following steps: (1) Mix the female urine waste after HCG extraction with sodium bentonite to obtain a mixture; (2) Perform a first-stage solid-liquid separation on the mixture and collect the liquid portion to obtain the filtrate; (3) The filtrate is adsorbed using an adsorption column of ion exchange resin, and then the resin after adsorption is eluted with sodium chloride solution to obtain the eluent. (4) Adjust the pH of the eluent to alkaline, use diatomaceous earth as a filter aid, and perform a second-stage solid-liquid separation on the pH-adjusted eluent. Collect the precipitate, which is the ulinastatin.

2. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, In step (1), the amount of sodium bentonite added is 0.1-1% of the mass of female urine waste.

3. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, The solid-liquid separation in steps (2) and (4) is achieved by plate and frame filter press.

4. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, The ion exchange resin in step (3) is an anion exchange resin; In step (3), the mass ratio of ion exchange resin to filtrate is 0.02~0.05:

1.

5. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, The adsorption conditions in step (3) are: the flow rate of the filtrate is 1200~1800L / h.

6. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, The concentration of the sodium chloride solution in step (3) is 0.5~1.0 mol / L.

7. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, In step (3), the amount of sodium chloride solution used is 3 to 4 times the volume of the ion exchange resin; The elution flow rate in step (3) is 0.5 to 1.5 column volumes per hour.

8. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, In step (4), the pH of the eluent is adjusted to 10-11; the solution used to adjust the pH is one or more of ammonia, sodium hydroxide solution or sodium bicarbonate solution.

9. The method for industrial-scale, efficient production of ulinastatin according to claim 1, characterized in that, Step (4) uses diatomaceous earth as a filter aid, and includes the following steps: (4.1) Mix diatomaceous earth and water to obtain a filter aid suspension; (4.2) The filter aid suspension is circulated through the second-stage solid-liquid separation equipment to pre-lay a layer of diatomaceous earth filter membrane on the filter medium of the equipment, i.e., pre-laying layer; (4.3) The pH-adjusted eluent is filtered through the pre-coated layer.

10. A method for the industrialized and efficient production of ulinastatin according to claim 9, characterized in that, In step (4.1), the mass-to-volume ratio of diatomaceous earth to water is 300-500g:100L.