A method for extracting crude product of ulinastatin based on macroporous resin

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a method for extracting crude urokinase based on macroporous resin. The method significantly improves the specific activity and shelf life of the crude urokinase through a dynamic pH segmentation protection technology. In the process of lectin affinity chromatography, the high flow rate condition of the first stage pH 8.5-9.5 ensures the efficient and specific binding of urokinase and lectin, and the rapid removal of impure proteins; the dynamic adjustment of the second stage pH from 8.5-9.5 linearly to 7.5-8.5 in combination with low flow rate operation provides a mild microenvironment for urokinase molecules, effectively protecting the integrity of the sugar chain structure of the molecules. The added polyol and disaccharide complex protective agent can stabilize the protein conformation, prevent sugar chain degradation and modification, and reduce the impact of pH mutation on the protein structure. The addition of the complex protective agent further enhances the stability of the sugar chain structure, preventing enzymatic and chemical degradation during the chromatography process.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a method for extracting crude ulinastatin based on macroporous resin. Background Technology

[0002] Ulinastatin is an acidic glycoprotein isolated and purified from fresh urine of healthy individuals. It consists of 143 amino acids with a relative molecular mass of approximately 37,000-43,000. It is primarily synthesized in the liver and metabolized by the kidneys, then excreted in the urine. Ulinastatin is a protease inhibitor, inhibiting serine proteases such as trypsin and α-chymotrypsin, as well as various enzymes including granulocyte elastase, hyaluronidase, thiolase, and plasmin. It also stabilizes lysosomal membranes, inhibits the release of lysosomal enzymes, inhibits the production of myocardial depressant factors, scavenge oxygen free radicals, and inhibits the release of inflammatory mediators.

[0003] Ulinastatin has a wide range of applications in clinical medicine, including acute pancreatitis, acute exacerbations of chronic relapsing pancreatitis, acute circulatory failure, and adjuvant treatment in thoracic surgery, digestive system surgery, tumor surgery, and organ transplantation. Japan first successfully developed injectable ulinastatin in 1985, and China began research and industrial production of high-purity ulinastatin in the 1990s.

[0004] In recent years, there has been limited research on the technological optimization of crude ulinastatin. Although many companies produce crude ulinastatin and other urinary protein products, they generally suffer from problems such as suboptimal extraction technology, low activity, high content of impurities and proteins, and relatively short shelf life. The integrity of the sugar chain structure in crude ulinastatin is a key factor affecting its specific activity and shelf life, but existing processes are significantly inadequate in protecting the sugar chain, leading to easy degradation and rapid decline in activity during storage. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a method for extracting ulinastatin using macroporous resin.

[0006] This application provides a method for extracting crude ulinastatin from macroporous resin, comprising the following steps:

[0007] a. Adsorb urine from healthy adults using macroporous adsorption resin, then desorb using a salt solution to obtain the eluent;

[0008] b. The eluent is concentrated by ultrafiltration to prepare a first concentrate;

[0009] c. The concentrated solution is purified by fractional binding and elution using a lectin affinity chromatography column, and the liquid phase is collected.

[0010] The segmented binding purification includes a first stage and a second stage;

[0011] The first stage involves rapid binding at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min.

[0012] The second stage involves glycan protection under conditions of linearly decreasing pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. In the second stage buffer, 0.04-0.06 mol / L of polyol and 0.008-0.012 mol / L of disaccharide are added as a complex protectant. The duration of the second stage is 50-70 min.

[0013] d. The obtained liquid phase is concentrated by ultrafiltration to prepare a second concentrate;

[0014] e. Salt out the second concentrate and filter to obtain the crude ulinastatin product.

[0015] In some embodiments, in step a, the macroporous resin is DIAION HP20 and / or ZGA455-H16.

[0016] In some embodiments, in step a, the salt solution is a 0.5-0.8 mol / L NaCl aqueous solution.

[0017] In some embodiments, in steps b and d, the ultrafiltration concentration is performed using an ultrafiltration membrane with a density of 2000-4000 Da.

[0018] In some embodiments, step c specifically includes the following steps:

[0019] c1. The first concentrated solution was subjected to the first stage of binding using a lectin affinity chromatography column at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min for rapid binding.

[0020] c2. Perform the second stage of glycan protection under the conditions of linearly decreasing the pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. Add 0.04-0.06 mol / L polyol and 0.008-0.012 mol / L disaccharide to the second stage buffer as a complex protectant. The duration of the second stage is 50-70 min.

[0021] c3. Elute the chromatography column with eluent and collect the liquid phase.

[0022] In some embodiments, before step c1, the pH of the first concentrate is adjusted to 8.5-9.5, and the conductivity is controlled within the range of 50-80 ms / cm.

[0023] In some embodiments, the lectin affinity chromatography column is Capto™ lentil lectin affinity chromatography resin and / or Pierce™ jackfruit lectin agarose.

[0024] In some embodiments, in step c3, the eluent is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl, and 0.05 mol / L mannitol at pH 7.0-7.4.

[0025] In some embodiments, the parameters for dynamic pH adjustment in step c2 are: initial pH 9.0, final pH 8.0, adjustment time 60 min, and pH decrease rate 0.0167 pH / min.

[0026] In some embodiments, in step c2, the polyol is mannitol and the disaccharide is trehalose.

[0027] This application provides a method for preparing crude ulinastatin, comprising the following steps: a. adsorbing urine from healthy adults using macroporous adsorption resin, followed by desorption using a salt solution to obtain an eluent; b. concentrating the obtained eluent by ultrafiltration to obtain a first concentrate; c. purifying the concentrate obtained in step b using a lectin affinity chromatography column for fractional binding and elution, and collecting the liquid phase; wherein the fractional binding purification includes a first stage and a second stage, the first stage being rapid binding at pH 8.5-9.5 and a flow rate of 3-5 mL / min, and the second stage being glycan protection at a linear decrease in pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min, wherein 0.04-0.06 mol / L polyol and 0.008-0.012 mol / L disaccharide are added as a complex protectant to the buffer solution in the second stage, and the duration of the second stage is 50-70 minutes; d. concentrating the obtained liquid phase by ultrafiltration to obtain a second concentrate; e. The second concentrate was subjected to salting out and filtered to obtain the crude ulinastatin product.

[0028] This method significantly improves the specific activity and shelf life of crude ulinastatin through dynamic pH segmentation protection technology. During lectin affinity chromatography, the high flow rate conditions (pH 8.5-9.5) in the first stage ensured efficient and specific binding of ulinastatin to lectin, rapidly removing contaminating proteins. The dynamic adjustment of pH in the second stage, linearly decreasing from 8.5-9.5 to 7.5-8.5, combined with low flow rate operation, provided a mild microenvironment for ulinastatin molecules, effectively protecting the integrity of its glycan structure. The added polyol and disaccharide complex protectant stabilized the protein conformation, preventing glycan degradation and modification. This segmented binding strategy cleverly resolves the contradiction between binding efficiency and glycan protection in traditional processes. The high pH environment in the first stage facilitates the specific binding of lectin to ulinastatin, while the gradual pH decrease in the second stage simulates natural environmental changes in proteins, reducing the impact of pH abrupt changes on protein structure. The addition of the complex protectant further enhances the stability of the glycan structure, preventing enzymatic and chemical degradation during chromatography. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] the term

[0032] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0033] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0034] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0036] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0037] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0038] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0039] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0040] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0041] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0042] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0043] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0044] This application provides a method for extracting crude ulinastatin from macroporous resin, comprising the following steps:

[0045] a. Adsorb urine from healthy adults using macroporous adsorption resin, then desorb using a salt solution to obtain the eluent;

[0046] b. The eluent is concentrated by ultrafiltration to prepare a first concentrate;

[0047] c. The concentrated solution is purified by fractional binding and elution using a lectin affinity chromatography column, and the liquid phase is collected.

[0048] The segmented binding purification includes a first stage and a second stage;

[0049] The first stage involves rapid binding at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min.

[0050] The second stage involves glycan protection under conditions of linearly decreasing pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. In the second stage buffer, 0.04-0.06 mol / L of polyol and 0.008-0.012 mol / L of disaccharide are added as a complex protectant. The duration of the second stage is 50-70 min.

[0051] d. The obtained liquid phase is concentrated by ultrafiltration to prepare a second concentrate;

[0052] e. Salt out the second concentrate and filter to obtain the crude ulinastatin product.

[0053] In some embodiments, in step a, the macroporous resin is DIAION HP20 and / or ZGA455-H16. This macroporous resin has a large specific surface area and suitable pore size distribution, enabling it to effectively adsorb ulinastatin in urine, thus improving adsorption efficiency and recovery rate.

[0054] In some embodiments, in step a, the salt solution is a 0.5-0.8 mol / L NaCl aqueous solution. This concentration range of NaCl solution effectively desorbs ulinastatin while avoiding the adverse effects of excessively high salt concentrations on protein structure.

[0055] In some embodiments, in steps b and d, the ultrafiltration concentration is performed using an ultrafiltration membrane with a molecular weight cutoff of 2000-4000 Da. This ultrafiltration membrane with a molecular weight cutoff effectively retains ulinastatin molecules while removing small molecule impurities, thus improving product purity.

[0056] In some embodiments, step c specifically includes the following steps:

[0057] c1. The first concentrated solution was subjected to the first stage of binding using a lectin affinity chromatography column at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min for rapid binding.

[0058] c2. Perform the second stage of glycan protection under the conditions of linearly decreasing the pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. Add 0.04-0.06 mol / L polyol and 0.008-0.012 mol / L disaccharide to the second stage buffer as a complex protectant. The duration of the second stage is 50-70 min.

[0059] c3. Elute the chromatography column with eluent and collect the liquid phase. This dynamic pH segmentation protection method, through precise control of pH changes and flow rate, combined with the use of a composite protectant, effectively protects the sugar chain structure of ulinastatin, thereby improving product activity and stability.

[0060] In some embodiments, prior to step c1, the pH of the first concentrate is adjusted to 8.5-9.5, and the conductivity is controlled within the range of 50-80 mS / cm. Suitable pH and conductivity conditions facilitate the specific binding of ulinastatin to lectins, thereby improving binding efficiency.

[0061] In some embodiments, the lectin affinity chromatography column is Capto™ lentil lectin affinity chromatography resin and / or Pierce™ jackfruit lectin agarose. These lectin affinity chromatography media have high affinity and specificity for ulinastatin, enabling efficient separation and purification of ulinastatin.

[0062] In some embodiments, in step c3, the eluent is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl, and 0.05 mol / L mannitol at pH 7.0-7.4. This eluent composition enables efficient elution of ulinastatin under mild conditions while maintaining its activity.

[0063] In some embodiments, the parameters for dynamic pH adjustment in step c2 are: initial pH 9.0, final pH 8.0, adjustment time 60 min, and pH decrease rate 0.0167 pH / min. Precise pH control can maximally protect the glycan structure of ulinastatin and reduce protein damage caused by pH mutations.

[0064] In some embodiments, in step c2, the polyol is mannitol and the disaccharide is trehalose. The combined use of mannitol and trehalose can synergistically protect the sugar chain structure of ulinastatin, preventing its degradation during purification and improving product stability.

[0065] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0066] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0067] It should be understood that, in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0068] Example 1: Preparation of crude ulinastatin by dynamic pH segmentation protection method

[0069] This embodiment provides a crude ulinastatin product and its preparation method.

[0070] The raw materials for preparing crude ulinastatin include: 2.5 tons of urine from healthy adults, 25 kg of macroporous adsorption resin DIAIONHP20, an ultrafiltration membrane (molecular weight cutoff 3000 Da), a lectin affinity chromatography column (10 L Pierce™ jackfruit lectin agarose), pH adjuster (0.1 mol / L NaOH solution, 0.1 mol / L HCl solution), buffer components (Tris, NaCl, PBS, mannitol, trehalose), and salting-out reagent (ammonium sulfate).

[0071] The preparation method of crude ulinastatin includes the following steps:

[0072] Step a: Adsorption and desorption of macroporous adsorption resin

[0073] 2.5 tons of urine from healthy adults were collected and filtered through a 100-mesh sieve to remove solid impurities, resulting in clear urine. The urine was transferred to a 10-ton stainless steel reaction vessel, and 25 kg of DIAION HP20 macroporous adsorption resin was added. Adsorption was carried out at 200 rpm for 4 hours at room temperature. After adsorption, the mixture was allowed to stand for 30 minutes, the supernatant was discarded, and the resin containing adsorbed urinary proteins was collected. The resin was then desorbed using a 0.7 mol / L NaCl aqueous solution. Specifically, 500 L of 0.7 mol / L NaCl solution was added, and the mixture was stirred at room temperature for 2 hours. After standing, the supernatant was collected. This process was repeated three times, and the eluent was combined to obtain 210 L.

[0074] Step b: First ultrafiltration concentration

[0075] 210 L of eluent was transferred to an ultrafiltration system and concentrated using an ultrafiltration membrane (10 m²) with a molecular weight cutoff of 3000 Da. The operating pressure was controlled at 2.5 bar, and the temperature at 4-8 °C, concentrating to approximately 4 L to obtain the first concentrate. The concentrate was measured to have a pH of 7.2, a conductivity of 65 mS / cm, and a protein concentration of 15.2 mg / mL.

[0076] Step c: Dynamic pH-protected lectin affinity chromatography

[0077] c1: Preparation and Equilibration of Chromatography Column

[0078] 10 L of Pierce™ jackfruit lectin agarose was packed into a chromatography column (15 cm diameter, 57 cm height) and connected to an ÄKTA avant 25 chromatography system. The column was equilibrated with 5 column volumes (50 L) of equilibration buffer (pH 9.0, 0.05 mol / L Tris + 0.1 mol / L NaCl) at a flow rate of 5 mL / min until the UV280 baseline, pH, and conductivity stabilized.

[0079] c2: Sample pretreatment

[0080] Take 4 L of the first concentrate and adjust the pH to 9.0 with 0.1 mol / L NaOH solution. Simultaneously detect the conductivity within the range of 50-80 ms / cm (actual measurement was 68 ms / cm). Filter the sample through a 0.22 μm filter membrane to remove particulate impurities.

[0081] c3: First-stage rapid integration

[0082] The pretreated sample was loaded onto the chromatography column at a flow rate of 4 mL / min, maintaining a constant pH of 9.0. UV280 absorbance was monitored in real time; loading was complete when the absorbance reached a plateau, which took approximately 25 minutes. During this phase, lectin binds efficiently and specifically to ulinastatin, rapidly removing most of the contaminating proteins.

[0083] c4: Phase II dynamic pH glycan protection

[0084] After sample loading, a dynamic pH adjustment program was immediately initiated, linearly decreasing the buffer pH from 9.0 to a target endpoint of 8.0. Simultaneously, the flow rate was reduced to 1 mL / min, and a combined protective agent (0.05 mol / L mannitol + 0.01 mol / L trehalose) was added to the buffer. Specific parameters for dynamic pH adjustment were: initial pH: 9.0, endpoint pH: 8.0, adjustment time: 60 minutes, pH decrease rate: 0.0167 pH / min, flow rate: 1 mL / min, buffer composition: 0.05 mol / L Tris + 0.1 mol / L NaCl + 0.05 mol / L mannitol + 0.01 mol / L trehalose. Throughout the second stage, pH changes were monitored in real-time using an online pH monitor to ensure a linear decreasing trend. UV280 absorption was also monitored to observe protein elution.

[0085] c5: Washout

[0086] After the second stage, the column was eluted with elution buffer (pH 7.0, 0.05 mol / L PBS + 0.5 mol / L NaCl + 0.05 mol / L mannitol) at a flow rate of 2 mL / min. The eluent corresponding to the UV280 absorption peak was collected, including the sample ramp-up buffer and equilibration buffer, totaling approximately 60 L.

[0087] Step d: Second ultrafiltration concentration

[0088] The collected 60 L of liquid phase was concentrated by ultrafiltration using an ultrafiltration membrane (membrane area 2.5 m²) with a molecular weight cutoff of 3000 Da. The operating pressure was controlled at 2.0 bar and the temperature at 4-8 °C, and the concentrate was concentrated to approximately 4 L to obtain a second concentrate. The protein concentration of the concentrate was determined to be 18.5 mg / mL, and the pH was 7.1.

[0089] Step e: Salting out

[0090] At 4°C, ammonium sulfate powder was slowly added to the second concentrate while stirring until the final concentration reached 60 wt%. After the addition was complete, stirring was continued at 2-8°C for 2 hours to allow ulinastatin to precipitate completely. After standing for 1 hour, the precipitate was filtered using a plate and frame filter press (40 cm × 40 cm) and collected. The precipitate was washed twice with pre-cooled deionized water to remove residual ammonium sulfate. The precipitate was then dried in a fume hood to obtain crude ulinastatin powder.

[0091] Example 2: Preparation of crude ulinastatin using a dynamic pH-fractional protection method with Capto™ lentil lectin affinity chromatography resin.

[0092] The difference between this embodiment and Embodiment 1 is that in step c, 10L Capto™ lentil lectin affinity chromatography resin (Cytiva) is used instead of Pierce™ jackfruit lectin agarose; the other steps are the same as in Embodiment 1.

[0093] Example 3: Preparation of crude ulinastatin using a dynamic pH segmentation protection method with different pH ranges

[0094] The difference between this embodiment and Embodiment 1 is that the parameters for dynamic pH adjustment in step c4 are: initial pH: 8.5, final pH: 7.5, adjustment time: 50 minutes, pH decrease rate: 0.02 pH / minute. The other steps are the same as in Embodiment 1.

[0095] Example 4: Preparation of crude ulinastatin using a dynamic pH-segmented protection method with different concentrations of protective agents.

[0096] The difference between this embodiment and Embodiment 1 is that the composite protective agent added to the buffer solution in step c4 is: 0.06 mol / L mannitol + 0.012 mol / L trehalose. The other steps are the same as in Embodiment 1.

[0097] Example 5: Preparation of crude ulinastatin using a dynamic pH-segmented protection method with different flow rates

[0098] The difference between this embodiment and Embodiment 1 is that the flow rate in the first stage of step c3 is 5 mL / min, and the flow rate in the second stage of step c4 is 1.5 mL / min. The other steps are the same as in Embodiment 1.

[0099] Example 6: Preparation of crude ulinastatin using a dynamic pH-segmented protection method with different molecular weight cutoffs of ultrafiltration membranes.

[0100] The difference between this embodiment and Embodiment 1 is that an ultrafiltration membrane with a molecular weight cutoff of 2000 Da is used for ultrafiltration concentration in steps b and d, while the other steps are the same as in Embodiment 1.

[0101] Example 7: Preparation of crude ulinastatin using a dynamic pH-segmented protection method with different molecular weight cutoffs of ultrafiltration membranes.

[0102] The difference between this embodiment and Embodiment 1 is that an ultrafiltration membrane with a molecular weight cutoff of 4000 Da is used for ultrafiltration concentration in steps b and d, while the other steps are the same as in Embodiment 1.

[0103] Example 8: Preparation of crude ulinastatin using a dynamic pH-segmented protection method with different salting-out conditions

[0104] The difference between this embodiment and Embodiment 1 is that the salting-out conditions in step e are: adding 55wt% ammonium sulfate, stirring at 2°C for 3 hours, and then allowing to stand and filter. The other steps are the same as in Embodiment 1.

[0105] Comparative Example 1: Preparation of crude ulinastatin by conventional single-pH lectin affinity chromatography

[0106] This comparative example provides a conventional single-pH lectin affinity chromatography method for preparing crude ulinastatin.

[0107] The raw materials used to prepare crude ulinastatin are the same as those in Example 1.

[0108] The preparation method of crude ulinastatin includes the following steps:

[0109] Steps a and b are the same as in Example 1.

[0110] Step c: Conventional single-pH lectin affinity chromatography

[0111] c1: Preparation and Equilibration of Chromatography Column

[0112] 10 L of Pierce™ jackfruit lectin agarose was packed into a chromatography column (15 cm diameter, 57 cm height) and connected to an ÄKTA avant 25 chromatography system. The column was equilibrated with 5 column volumes (50 L) of equilibration buffer (pH 8.0, 0.05 mol / L Tris + 0.1 mol / L NaCl) at a flow rate of 2 mL / min until the UV280 baseline, pH, and conductivity were stable.

[0113] c2: Sample pretreatment

[0114] Take 4 L of the first concentrate and adjust the pH to 8.0 with 0.1 mol / L NaOH solution. Simultaneously detect the conductivity within the range of 50-80 ms / cm (actual measurement was 68 ms / cm). Filter the sample through a 0.22 μm filter membrane to remove particulate impurities.

[0115] c3: Sample loading and combination

[0116] The pretreated sample was loaded onto the chromatography column at a flow rate of 2 mL / min, maintaining a constant pH of 8.0. UV280 absorbance was monitored in real time; loading was complete when the absorbance reached a plateau, which took approximately 50 minutes.

[0117] c4: Washout

[0118] After sample loading, the column was eluted with elution buffer (pH 7.0, 0.05 mol / L PBS + 0.5 mol / L NaCl) at a flow rate of 2 mL / min. The eluent corresponding to the UV280 absorption peak was collected, including the sample ramp-up buffer and equilibration buffer, totaling approximately 60 L.

[0119] Steps d and e are the same as in Example 1.

[0120] Comparative Example 2: Preparation of crude ulinastatin by dynamic pH segmentation protection method without protective agent

[0121] The difference between this comparative example and Example 1 is that mannitol and trehalose protectants are not added to the buffer solution in step c4, while the other steps are the same as in Example 1.

[0122] Comparative Example 3: Preparation of crude ulinastatin by constant pH lectin affinity chromatography

[0123] The difference between this comparative example and Example 1 is that dynamic pH adjustment is not performed in step c, but the pH is kept constant at 9.0 throughout the process, and the flow rate is 2 mL / min. The other steps are the same as in Example 1.

[0124] Data aggregation and data analysis

[0125] Performance testing methods

[0126] Specific activity assay: The potency of ulinastatin was determined using the method in patent CN 102353640A, and the protein content was determined using the Bradford method. Specific activity = potency / protein content.

[0127] Glycan integrity analysis: High performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) was used to analyze the glycan composition.

[0128] Analysis of miscellaneous protein content: The contents of galactolectin, pepsin and kininogen were analyzed by SDS-PAGE and HPLC.

[0129] Shelf life test: Store the sample at 25°C and test the specific activity periodically. When the specific activity drops to 80% of the initial value, it is determined to be the end of the shelf life.

[0130] Data Analysis

[0131] The test results show that the crude ulinastatin prepared using the dynamic pH segmented protection method (Examples 1-8) is significantly superior to the traditional process (Comparative Examples 1-3) in terms of specific activity, glycan integrity, shelf life, and removal rate of contaminants. Example 1 achieved a specific activity of 759.4 U / mg protein, an increase of 8.9% compared to the traditional process (Comparative Example 1); glycan integrity was improved by 8%; shelf life was extended to 10 months, 4 months longer than the traditional process; and the removal of contaminants was also significantly improved, with a 92% reduction in galactolectin content, a 95% reduction in pepsin content, and a 75% reduction in kininogen content.

[0132] Comparative Example 1, using the conventional single-pH lectin affinity chromatography method, showed lower specific activity, glycan integrity, and shelf life compared to Examples 1-8, indicating that the dynamic pH segmented protection method has significant advantages over the conventional single-pH method. Comparative Example 2, without adding a protective agent during the dynamic pH segmented protection process, showed lower performance in all aspects compared to Example 1, demonstrating the crucial role of the mannitol and trehalose composite protective agent in protecting the glycan structure of ulinastatin. Comparative Example 3, using the constant-pH lectin affinity chromatography method, also showed significantly lower performance than Example 1, indicating that dynamic pH adjustment is a key factor in improving the quality of ulinastatin.

[0133] Of Examples 1-8, Examples 4 (using a higher concentration of protective agent) and 6 (using a 2000 Da ultrafiltration membrane) exhibited the best overall performance, with specific activities of 761.2 U / mg protein and 763.5 U / mg protein, respectively, and shelf lives of 10.5 months and 11 months, respectively. This indicates that appropriately increasing the concentration of the protective agent and using an ultrafiltration membrane with a smaller molecular weight cutoff is beneficial for improving the quality of ulinastatin. Example 7 (using a 4000 Da ultrafiltration membrane) showed relatively lower performance, possibly because the larger pore size of the ultrafiltration membrane could not effectively retain small molecule impurities, leading to a decrease in product purity.

[0134] Example 2 (using Capto™ lentil lectin affinity chromatography resin) showed similar performance to Example 1 (using Pierce™ jackfruit lectin agarose), indicating that both lectin affinity chromatography media are suitable for the dynamic pH segmentation protection method of this application. Example 3 (using different pH ranges) and Example 5 (using different flow rates) showed slightly lower performance than Example 1, suggesting that a dynamic adjustment range of pH 9.0-8.0 and a flow rate combination of 4 mL / min / 1 mL / min are optimal process parameters.

[0135] The performance of Example 8 (using different salting-out conditions) was similar to that of Example 1, indicating that within a certain range, the adjustment of salting-out conditions has a relatively small impact on the quality of ulinastatin.

[0136] In summary, the dynamic pH segmentation protection method of this application effectively protects the sugar chain structure of ulinastatin by precisely controlling pH changes, flow rate, and adding composite protective agents, thereby improving the specific activity and shelf life of the product, while significantly reducing the content of impurity proteins. This provides an innovative technical path for the high-quality preparation of crude ulinastatin.

[0137] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. 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 scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for extracting crude ulinastatin from macroporous resin, characterized in that, Includes the following steps: a. Adsorb urine from healthy adults using macroporous adsorption resin, then desorb using a salt solution to obtain the eluent; b. The eluent is concentrated by ultrafiltration to prepare a first concentrate; c. The concentrated solution is purified by fractional binding and elution using a lectin affinity chromatography column, and the liquid phase is collected. The segmented binding purification includes a first stage and a second stage; The first stage involves rapid binding at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min. The second stage involves glycan protection under conditions of linearly decreasing pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. In the second stage buffer, 0.04-0.06 mol / L of polyol and 0.008-0.012 mol / L of disaccharide are added as a complex protectant. The duration of the second stage is 50-70 min. d. The obtained liquid phase is concentrated by ultrafiltration to prepare a second concentrate; e. Salt out the second concentrate and filter it to obtain the crude ulinastatin product; The polyol is mannitol, and the disaccharide is trehalose.

2. The method for extracting crude ulinastatin based on macroporous resin according to claim 1, characterized in that, In step a, the macroporous resin is DIAION HP20 and / or ZGA455-H16.

3. The method for extracting crude ulinastatin based on macroporous resin according to claim 1, characterized in that, In step a, the salt solution is a 0.5-0.8 mol / L NaCl aqueous solution.

4. The method for extracting crude ulinastatin based on macroporous resin according to claim 1, characterized in that, In steps b and d, the ultrafiltration concentration is performed using an ultrafiltration membrane with a capacity of 2000-4000 Da.

5. The method for extracting crude ulinastatin based on macroporous resin according to any one of claims 1-4, characterized in that, Step c specifically includes the following steps: c1. The first concentrated solution was subjected to the first stage of binding using a lectin affinity chromatography column at a pH of 8.5-9.5 and a flow rate of 3-5 mL / min for rapid binding. c2. Perform the second stage of glycan protection under the conditions of linearly decreasing the pH from 8.5-9.5 to 7.5-8.5 and a flow rate of 0.5-1.5 mL / min. Add 0.04-0.06 mol / L polyol and 0.008-0.012 mol / L disaccharide to the second stage buffer as a complex protectant. The duration of the second stage is 50-70 min. c3. Elute the chromatography column with eluent and collect the liquid phase; The polyol is mannitol, and the disaccharide is trehalose.

6. The method for extracting crude ulinastatin based on macroporous resin according to claim 5, characterized in that, Before step c1, the pH of the first concentrate is adjusted to 8.5-9.5, and the conductivity is controlled within the range of 50-80 ms / cm.

7. The method for extracting crude ulinastatin based on macroporous resin according to claim 5, characterized in that, The lectin affinity chromatography column is Capto™ lentil lectin affinity chromatography resin and / or Pierce™ jackfruit lectin agarose.

8. The method for extracting crude ulinastatin based on macroporous resin according to claim 5, characterized in that, In step c3, the eluent is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl and 0.05 mol / L mannitol at pH 7.0-7.

4.

9. The method for extracting crude ulinastatin based on macroporous resin according to claim 5, characterized in that, In step c2, the parameters for dynamic pH adjustment are: initial pH 9.0, final pH 8.0, adjustment time 60 min, and pH decrease rate 0.0167 pH / min.