Method for endotoxin reduction of lysate, low endotoxin lysate and use thereof

Abstract

The present application relates to a kind of lysate endotoxin reduction processing method, low endotoxin lysate and its application;Lysate endotoxin reduction processing method includes the following steps: protein hydrolytic enzyme is dissolved in buffer solution, and form lysate stock solution;Wherein, the buffer solution contains phosphate ion;Lysate stock solution is carried out tangential flow circulation ultrafiltration using ultrafiltration membrane, and phosphate buffer solution is dynamically diluted to reduce its concentration in the process of ultrafiltration, remove endotoxin using the difference of material molecular weight separation, and collect refined liquid containing protease, obtain lysate.So, endotoxin removal rate is higher, can be stock solution 300EU / mg High endotoxin background is stably reduced to finished product lysate 5EU / mg Below, simultaneously relying on the synergistic effect of phosphate buffer system and ultrafiltration screening process, protease enzyme activity retention rate after processing is stably maintained at more than 75%.

Description

Technical Field

[0001] This invention relates to the field of lysis buffer technology, and in particular to a method for treating lysis buffer with reduced endotoxin, a low-endotoxin lysis buffer, and its applications. Background Technology

[0002] Gelatin microcarriers are currently the core solid-phase carrier material for high-density cell culture in suspension bioreactors, industrial production of inactivated vaccines, and mass expansion of adult stem cells / mesenchymal stem cells. After the culture cycle is completed, the gelatin polymer backbone usually needs to be degraded by lysis buffer to completely dissociate the carrier and release the live cells attached to the carrier at the same time, so as to achieve complete peeling and collection of the adhered live cells.

[0003] The core functional component of the lysis buffer is proteolytic enzymes, including six major categories: collagenase, caseinase, neutral protease, papain, trypsin, and metalloproteinases. The protease raw materials have extremely high natural endotoxin content, and the proteases are highly heat-sensitive; the endotoxin level in industrial raw material solutions can typically reach over 300 EU / mg. Conventional lysis buffer formulations are not suitable for endotoxin reduction and purification processes, and the finished product cannot be directly used in compliant biological cell culture processes. Therefore, the endotoxin content of the lysis buffer must be reduced beforehand to meet subsequent application requirements. Proteolytic enzymes are typical heat-sensitive globular proteins with extremely poor thermal stability of their spatial enzyme activity structure. Industry standard 121℃ saturated steam sterilization and long-acting pasteurization processes directly destroy the active sites of the proteases, resulting in complete loss of enzyme activity. Therefore, the entire production process of the gelatin microcarrier lysis buffer can only use non-heated, room-temperature processes to remove endotoxins, and the lysis buffer formulation must be compatible with room-temperature membrane separation and purification conditions.

[0004] Existing room-temperature, non-heated endotoxin removal processes mainly fall into four categories: first, solid-phase adsorption, which uses activated carbon, chelating resins, and hydrophobic resins to adsorb free endotoxins; second, chromatographic purification, which uses ion exchange, hydrophobic chromatography, and gel size exclusion chromatography to separate endotoxin aggregates; third, universal ultrafiltration, which uses membrane filtration with fixed pore size and fixed solvent system to remove impurities; and fourth, additive modification, which adds ionic / nonionic surfactants to disperse endotoxin micelles. However, existing lysis buffer formulations are poorly compatible with the above-mentioned room-temperature endotoxin removal processes, exhibiting multiple drawbacks. Commercial lysis buffer formulations are crudely designed, mostly using physiological saline, pure water, or calcium-magnesium Tris buffer as solvents. The ionic strength and osmotic pressure cannot match the ultrafiltration purification conditions, resulting in significant protease inactivation and a substantial decrease in lysis performance after treatment. Solid-phase adsorption has poor selectivity; activated carbon and resins indiscriminately adsorb proteases, altering the proportion of effective enzyme components in the lysis buffer and reducing the lysis capacity of the finished product. Chromatographic purification equipment is expensive, has low throughput per batch, and a long commissioning cycle, making it only suitable for small-scale laboratory preparation and difficult to achieve industrial-scale production. Traditional general-purpose ultrafiltration does not perform customized parameter optimization for lysis buffer solvents and protease molecular weights. Under the combined action of ultrafiltration shear force and unsuitable solvents, the spatial conformation of proteases is easily destabilized, and only 30%~40% of the enzyme activity remains after purification. The lysis time of microcarriers is extended several times, and the efficiency of live cell recovery is significantly reduced. Surfactant modification methods pose biosafety risks, as surfactant residues are difficult to completely remove. They can inhibit protease catalytic activity and damage cell membrane structure, inducing apoptosis of stem cells and somatic cells. Cell compatibility does not meet medical standards. Furthermore, existing technologies lack an integrated synergistic design scheme for formulation and endotoxin removal processes, making it impossible to simultaneously achieve multiple industrial-grade hard indicators such as cell friendliness, room-temperature ultrafiltration compatibility, no high-temperature treatment throughout the process, and endotoxin control of the finished product ≤5 EU / mg.

[0005] CN108066784A discloses a method for removing endotoxins from biopharmaceutical preparations. The method involves ultrafiltration of the pretreated biopharmaceutical preparation using an ultrafiltration membrane. The ultrafiltration membrane is an external pressure hollow fiber ultrafiltration membrane made of polysulfone. The hollow fiber outer diameter of the membrane is 380-420 micrometers, and the fiber wall thickness is 90-100 micrometers. This method not only removes endotoxins but also removes bacteria from the biopharmaceutical preparation, and is safe, reliable, effective, and cost-effective. However, when using traditional ultrafiltration to remove endotoxins from lysis buffers, the activity of proteolytic enzymes remains unstable, resulting in a low enzyme activity retention rate and consequently, an excessively long lysis time. Summary of the Invention

[0006] Therefore, it is necessary to provide a lysate-reducing endotoxin treatment method, lysate, and its application that can significantly reduce endotoxin content, has a relatively mild treatment process, a high enzyme activity retention rate after treatment, and a significantly shortened lysis time.

[0007] A method for treating lysate to reduce endotoxin levels includes the following steps: The proteolytic enzyme is dissolved in a buffer solution to form a lysis stock solution; wherein the buffer solution contains phosphate ions; The lysate was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane. During the ultrafiltration process, the buffer was dynamically diluted to reduce its concentration. Endotoxins were removed by utilizing the molecular weight difference of the materials, and the purified feed containing protease was collected to obtain the lysate.

[0008] In one embodiment, the proteolytic enzyme is selected from one or more of collagenase, caseinase, neutral protease, papain, trypsin, and metalloproteinase.

[0009] In one embodiment, the buffer solution is any one of phosphate buffer, Hanks balanced salt solution, and serum-free cell basal culture medium; In one embodiment, when the proteolytic enzyme is a non-metal-dependent protease, the buffer solution is free of calcium and magnesium ions; the buffer solution free of calcium and magnesium ions is suitable for papain, trypsin, and neutral protease; the buffer solution containing calcium and / or magnesium ions is suitable for collagenase and metalloproteinase.

[0010] In one embodiment, the concentration of proteolytic enzyme in the lysis stock solution is 1-320 g / L.

[0011] In one embodiment, the ultrafiltration membrane has a molecular weight cutoff of 10-1000 kDa, preferably 20-500 kDa. In one embodiment, the ultrafiltration membrane is a hydrophilic modified polyethersulfone membrane or a polyvinylidene fluoride membrane; In one embodiment, the tangential flow circulation ultrafiltration is performed at room temperature, which is 4-35°C, preferably 15-30°C. In one embodiment, the tangential flow ultrafiltration operating pressure is controlled at 0.5–1.5 bar, preferably 0.5–1 bar; In one embodiment, the dynamic dilution method is any one of one-time feed dilution, continuous closed-loop percolation, or segmented gradient dilution; the diluent used for dilution is sterile water for injection; preferably, the concentration of the stock solution buffer is ≤1×, and the concentration of the finished buffer solution after dynamic dilution is ≤1×. Preferably, the concentration of the stock solution buffer is 0.1× to 1×, and the concentration of the finished buffer solution after dynamic dilution is 0.1× to 1×.

[0012] In one embodiment, the process of separating and removing endotoxins and collecting the purified liquid containing protease by utilizing the molecular weight difference of the materials is as follows: When the apparent molecular weight of the proteolytic enzyme is lower than the first retention threshold of 10-100 kDa, an ultrafiltration membrane with a molecular weight cutoff not higher than the first retention threshold is selected, and the membrane module retention concentrate is collected as the purification feed solution; wherein, 10 kDa ≤ first retention threshold < 100 kDa.

[0013] When the apparent molecular weight of the proteolytic enzyme is higher than the second retention threshold of 100–500 kDa, an ultrafiltration membrane with a molecular weight cutoff not lower than the second retention threshold is selected, and the permeate from the membrane module is collected as the purification feed. Wherein, 100 kDa ≤ second retention threshold ≤ 500 kDa.

[0014] In one embodiment, before ultrafiltration of the lysate, the treatment method further includes the step of pre-filtering the lysate through a 0.2-0.45 μm filter. In one embodiment, after collecting the refined feed liquid, the processing method further includes a finished product preparation step: the collected refined feed liquid is aseptically filtered through a 0.22 μm terminal filter and then dispensed to prepare a liquid lysis buffer; or the refined feed liquid is freeze-dried at low temperature under vacuum to prepare a freeze-dried lysis buffer product. Preferably, after collecting the refined feed liquid, the processing method further includes desalting the liquid lysis buffer.

[0015] In one embodiment, in the ultrafiltration operation, the ultrafiltration device is an inner-pressure type hydrophilic hollow fiber membrane column with an ultrafiltration membrane built therein; preferably, before the ultrafiltration operation is performed on the crude lysate, the treatment method further comprises the following step of pre-treating the ultrafiltration device: the pre-treatment step sequentially includes the processes of alkali washing, water washing, membrane column integrity detection, and buffer solution equilibration; the alkali washing step: the hollow fiber membrane column is circularly washed for 1 h with 0.2 to 0.5 M sterile sodium hydroxide solution, the alkali washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and after the alkali washing is completed, the residual alkali liquor in the hollow inner cavity and the permeate end of the membrane column is drained; the water washing step: the membrane column is continuously rinsed for 1.5 to 4 h with sterile water for injection, the water washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and after the water washing is completed, the residual water for injection in the hollow inner cavity and the permeate end of the membrane column is drained; the membrane column integrity detection step: the liquid outlet pipeline of the membrane column is closed, sterile gas is introduced from the liquid inlet pipeline, the pressure is increased to no more than 2 bar, the pressure drop is observed under the maintained pressure, and if the pressure drop per minute is less than 0.1 to 0.2 bar, the membrane column is judged to be qualified in integrity; if it is unqualified, the steps of alkali washing, water washing and integrity detection are repeated, and if the re-detection is still unqualified, the membrane column is replaced; the membrane column integrity detection can be carried out before alkali washing and water washing, but the integrity detection must be carried out both before the ultrafiltration feeding and after the ultrafiltration is completed; the buffer solution equilibration process: the retentate end and the permeate end of the membrane column are jointly communicated with a buffer solution storage tank, the membrane column is circularly rinsed with the buffer solution for 5 to 10 min, the pH value and the conductivity of the filtrate are detected in real time until the pH value and the conductivity of the filtrate are consistent with those of the buffer solution, and the pre-equilibration of the membrane column is completed; In one embodiment, after the ultrafiltration is finished, the treatment method further comprises the step of recovering and preserving the membrane column for the ultrafiltration device; the step of recovering and preserving the membrane column comprises: after the ultrafiltration is finished, the membrane column is first washed with sterile water for injection for 0.5 to 1 h, then replaced with brand-new sterile water for injection and washed again for 0.5 to 1 h, the washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and after the washing is completed, the residual water for injection in the hollow inner cavity and the permeate end of the membrane column is drained; subsequently, the membrane column is circularly alkali-washed or soaked for 0.5 to 1 h with 0.5 to 1 M sodium hydroxide solution, and the alkali washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar; finally, the membrane column is immersed and preserved by replacing the solution with 0.1 to 0.2 M sodium hydroxide solution.

[0016] A low-endotoxin gelatin microcarrier lysate is prepared by the method for reducing endotoxin in a lysate according to any one of the above embodiments; the finished product is in a sterile liquid form or a vacuum freeze-dried solid form; the endotoxin content of the lysate is ≤5 EU / mg, and the retention rate of proteolytic enzyme activity is 75% to 90% or more.

[0017] Application of the low endotoxin gelatin microcarrier lysis buffer in the preparation of cell harvesting reagents after cell culture, as described in any of the above embodiments.

[0018] A cell-scale culture kit comprising a low-endotoxin gelatin microcarrier lysis buffer as described in any of the above embodiments, for use in the lysis of gelatin microcarriers and the non-destructive release and recovery of live cells.

[0019] The above-mentioned method for reducing endotoxins in lysate involves dissolving proteolytic enzymes in a phosphate-containing buffer solution to prepare the lysate stock solution. This is combined with tangential flow circulating ultrafiltration, dynamically diluting the buffer solution during ultrafiltration to lower the system concentration. Endotoxins are removed by sieving based on the molecular weight differences of the materials. Phosphate ions synergistically stabilize the protease hydration membrane and spatial conformation with the equilibrium salt ions in the buffer system, reducing the damage to the heat-sensitive protease structure caused by fluid shear forces during ultrafiltration. Simultaneously, the phosphate buffer system optimizes the osmotic pressure of the feed solution, preventing protease denaturation due to sudden changes in osmotic pressure. Combined with a room-temperature ultrafiltration physical separation mode relying solely on particle size sieving, the entire process operates under a mild 4-35℃ environment with no high temperatures, no resin adsorption, and no added exogenous surfactants. This avoids significant activity loss due to non-specific binding of the adsorption medium to the protease, while precisely distinguishing protease molecules from high-molecular-weight endotoxin aggregates through the ultrafiltration membrane. The endotoxin removal rate is high, stabilizing the endotoxin background of 300 EU / mg in the stock solution to 5 EU / mg in the finished lysate. The following method utilizes the synergistic effect of the phosphate buffer system and ultrafiltration sieving process to maintain a stable protease activity retention rate of over 75% after treatment. The dynamic dilution step simultaneously adjusts the buffer concentration and removes endotoxins. The process is streamlined and can be scaled up to GMP standards. The resulting lysis buffer has no harmful additive residues, excellent cell biocompatibility, and can effectively lyse gelatin microcarriers and release and recover live cells without damage. This synergistic approach achieves the core technical effects of significantly reducing endotoxins, providing mild treatment conditions, maintaining high enzyme activity, and greatly shortening lysis time.

[0020] The lysis stock solution prepared according to the proprietary enzyme and balanced salt ion formula of this invention has a native endotoxin level as high as 300 EU / mg. After purification using a proprietary endotoxin removal process, the measured endotoxin level of the finished lysis solution is 0.125-2 EU / mg, with a batch-wide batch stability of ≤5 EU / mg, meeting the pharmacopoeia limits for biological agents. The endotoxin content of the finished lysis solution is ≤5 EU / mg; the enzyme activity retention rate after purification is 75-90%; there are no significant differences in the enzyme components, functional salt ions, and lysis kinetic parameters between the formulated and unrefined stock solution; the effective shelf life of the liquid product is ≥6 months when stored frozen, protected from light, and sealed; the freeze-dried product, when stored at low temperature and protected from light, shows no decrease in formulation performance, enzyme activity, or lysis performance after reconstitution. It is suitable for large-scale cell culture in GMP workshop bioreactors, industrial mass production of human vaccines, mesenchymal stem cell / immune cell expansion and preparation, and degradation and dissociation of medical-grade gelatin solid carriers, enabling controlled lysis of gelatin microcarriers at the end of the culture period and non-destructive release and recovery of live cells. This application utilizes a proprietary balanced salt ion composition to stabilize the protease's spatial conformation and withstand ultrafiltration shear forces. Unlike commercially available salt-free, single-solvent, crude lysis buffers, this formulation offers greater originality. The proprietary lysis buffer formulation is compatible with a room-temperature ultrafiltration endotoxin reduction process. This process removes endotoxins without altering the enzyme composition or beneficial salt ion ratio, requiring no external modifying reagents or subsequent recombining of the lysis buffer. The entire process is physically purified at room temperature (4-35℃), with zero high-temperature treatment, zero heat treatment, and zero high-pressure sterilization, preventing protease denaturation and inactivation from the source. It can reliably refine the stock solution from a high background concentration of 300 EU / mg to a finished lysis buffer of ≤5%. With an EU / mg concentration and an endotoxin removal rate ≥98%, meeting the GMP excipient access standards for cell therapy, the formulation utilizes three optimal solvent formulations: PBS, Hanks' solution, and MSC culture medium, combined with suitable salt ions. After ultrafiltration, enzyme activity is stably retained at 75-90%, far superior to the 30%-40% enzyme activity retention rate of physiological saline and pure water systems. Based on molecular weight sieving logic, it can adapt to small molecule, large molecule, and compound protease lysis buffer formulations, offering strong process versatility. Ultrafiltration simultaneously completes endotoxin removal and lysis buffer solvent concentration adjustment, retaining functional salt ions to directly obtain a usable finished product formulation, simplifying production processes, reducing membrane material costs, and facilitating GMP industrial scale-up. High biosafety: the enzyme and balanced salt ion composition of the lysis buffer formulation is controllable, with no exogenous adjuvants or toxic additives. The purification process does not introduce impurities, resulting in excellent cell compatibility of the finished product and no damage to lysed ex vivo cells. Versatile product storage and transportation: the formulation can be prepared in liquid and lyophilized forms, suitable for on-site use, remote storage and transportation, and long-term stockpiling scenarios, with stable shelf life. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of an ultrafiltration device used in a lysate endotoxin reduction treatment method according to an embodiment of the present invention. Detailed Implementation

[0022] To facilitate understanding of the present invention and to make the above-mentioned objects, features, and advantages of the present invention more apparent, a detailed description of specific embodiments of the present invention is provided below in conjunction with the accompanying drawings. Numerous specific details are set forth in the following description to provide a thorough understanding of the present invention, and preferred embodiments are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. The present invention can be implemented in many other ways than those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention; therefore, the present invention is not limited to the specific embodiments disclosed below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Firstly, this application provides a method for treating lysate to reduce endotoxin levels. In one embodiment, the method includes the following steps S100 and S200, as detailed below: S100: The proteolytic enzyme is dissolved in a buffer solution to form a lysis stock solution; wherein the buffer solution contains phosphate ions; By dissolving proteolytic enzymes in a buffer solution to form a lysis stock solution, subsequent endotoxin removal treatment can be facilitated. The presence of phosphate ions in the buffer solution helps improve the stability of subsequent proteases.

[0024] For example, the proteolytic enzyme is selected from one or more of collagenase, caseinase, neutral protease, papain, trypsin, and metalloproteinases. Single or complex enzyme systems can be flexibly combined according to the gelatin microcarrier material and cell culture conditions to adapt to the lysis requirements of gelatin microcarriers with different densities and pore structures. For example, the proteolytic enzyme is commercially available. For example, the endotoxin content of commercially available proteolytic enzymes is above 50 EU / mg, above 100 EU / mg, above 150 EU / mg, above 200 EU / mg, above 250 EU / mg, or even as high as 300 EU / mg. For example, commercially available proteolytic enzymes contain added protective agents. It should be understood that the proteolytic enzymes used in this invention are commercially available industrial crude products / raw materials that have not been deeply depyrogened and biochemically processed. These raw materials generally have high endotoxin levels: the endotoxin content of conventional unpyrogenated bacterial fermentation-type neutral proteases, metalloproteinases, caseinases, crude collagenases extracted from animal organs, and ordinary porcine trypsin is usually ≥50 EU / mg, with typical batches reaching 100 EU / mg, 150 EU / mg, 200 EU / mg, and 250 EU / mg, and some crude raw materials reaching up to 300 EU / mg. The endotoxin content of plant-derived papain is relatively low, but the overall endotoxin content of the lysate will still exceed the standard after being compounded with industrial crude enzymes, making it unsuitable for direct use in clinical-grade cell preparation. Optional commercially available crude enzyme raw material suppliers include, but are not limited to: Novozymes industrial enzyme product line, DSM industrial fermentation protease, DuPont Danisco food-grade protease, Amano microbial crude protease, Advanced Enzymes industrial protease, Worthington general biochemical grade crude collagenase, and FUJIFILM Wako non-purified animal-derived protease. These manufacturers also sell GMP-purified enzymes that have undergone deep pyrogen removal. However, these low-endotoxin finished enzymes have high procurement costs and are not suitable for large-scale industrial production, and are not the preferred raw materials for this invention. Commercially available crude proteolytic enzymes typically contain preservatives to maintain storage stability. It should be understood that the endotoxin reduction treatment method for the lysis buffer of this application is designed to address the industry pain point of high endotoxin content in existing low-cost industrial crude enzyme raw materials. By pre-treating the lysis buffer to remove pyrogens, the overall endotoxin level of the lysis system can be significantly reduced, avoiding the introduction of large amounts of pyrogens during cell harvesting from gelatin microcarriers, reducing downstream cellular inflammatory responses and the risk of fever during human infusion, and making it suitable for the production of clinical-grade biopharmaceuticals such as cell therapy and viral vaccines.

[0025] For example, the buffer solution can be any one of phosphate-buffered saline (PBS), Hanks' balanced salt solution, or serum-free cell culture medium. Thus, these three types of suitable buffer solutions are limited. These systems inherently contain phosphate groups, which can stabilize proteases. The applicant's research has shown that using such buffer solutions, enzyme activity retention reaches over 75%. For example, when the proteolytic enzyme is a non-metal-dependent protease, the buffer solution does not contain calcium or magnesium ions. Buffer solutions without calcium or magnesium ions are suitable for papain, trypsin, and neutral proteases. Buffer solutions containing calcium and / or magnesium ions are suitable for collagenases and metalloproteinases. The buffer system of this invention can selectively add calcium and magnesium ions: for metal-dependent hydrolases such as collagenases and metalloproteinases, low concentrations of calcium and magnesium ions can stabilize the enzyme's active site; simultaneously, during ultrafiltration, the buffer solution is dynamically diluted to ≤0.25× to reduce the phosphate concentration in the system and prevent calcium and magnesium phosphate precipitation from clogging the ultrafiltration membrane. For non-metal-dependent proteases such as trypsin and papain, the buffer solution can be free of calcium and magnesium ions, completely eliminating the risk of precipitation and reducing membrane adsorption loss.

[0026] In one embodiment, the concentration of proteolytic enzyme in the lysis stock solution is 1-320 g / L. For example, the concentration of proteolytic enzyme is 20-80 g / L, or 20-80 mg / mL. Thus, single / compound proteases and suitable balanced salt ions are dissolved together in the corresponding aqueous solvent and stirred at room temperature until completely dissolved to prepare a homogeneous lysis stock solution for ultrafiltration. The concentration range is suitable for the ultrafiltration percolation operation conditions, which will not reduce the processing efficiency due to excessively dilute feed solution, nor will it aggravate membrane concentration polarization and damage enzyme activity due to excessively high feed solution. The phosphate buffer is used to weaken the destruction of proteases during the ultrafiltration process, thus balancing process efficiency and enzyme activity retention stability.

[0027] S200: The lysate is subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane. During the ultrafiltration process, the buffer is dynamically diluted to reduce its concentration. Endotoxins are removed by utilizing the molecular weight difference of the materials, and the purified feed solution containing protease is collected to obtain the lysate.

[0028] Ultrafiltration removes or reduces the endotoxin content in the lysis feed solution. Tangential flow ultrafiltration, also known as cross-flow ultrafiltration, differs from dead-end filtration where the liquid passes vertically through the membrane. The system relies on a pump to drive the lysis feed solution to flow parallel (tangentially) through the inner wall of the hollow fiber ultrafiltration membrane. Transmembrane pressure drives small molecule solvents, endotoxin aggregates, and other impurities to penetrate vertically through the membrane wall, forming permeate that is separated and removed. Larger protease molecules are retained by the membrane. The retained concentrate carrying the target protease is not directly discharged but is returned to the feed tank through pipelines for continuous closed-loop circulation. The high-speed tangential fluid on the membrane surface continuously generates transverse scouring shear force, which can significantly reduce the amount of protease and impurities present. Adsorption and buildup on the membrane pore surface prevents rapid membrane blockage and protein denaturation and loss. During the circulation process, sterile water for injection can be continuously added to the feed system to complete gradient percolation dilution. This simultaneously achieves two major objectives: buffer concentration replacement and continuous sieving and removal of small molecule endotoxin impurities. The entire process can be stably controlled under low pressure, ambient temperature, and ambient temperature conditions. It is suitable for ambient temperature purification of heat-sensitive protease lysis buffers. It can selectively collect retentate or permeate based on the molecular weight difference of the protease, completely preserving the original enzyme components and balanced salt ion system, and realizing continuous and scalable protein purification and endotoxin removal operations.

[0029] Please see Figure 1The attached diagram is a flow chart of a complete tangential flow circulation ultrafiltration system using an internal pressure hollow fiber membrane column. The feed solution is stored in the inlet tank and, after being pressurized by a transfer pump, is fed into the lower inlet of the hollow fiber membrane column. The feed solution flows continuously tangentially upwards along the longitudinal direction of the membrane fibers. Pressure gauges are installed before and after the pipeline to monitor the feed pressure and return pressure in real time. The transmembrane pressure is regulated by the upper return valve. Small molecule solvents and endotoxin aggregates can vertically penetrate the hollow fiber membrane wall under the action of the transmembrane pressure difference and are discharged from the permeate outlet on the side of the membrane column, collected in the waste liquid / permeate tank, thus achieving impurity separation. Large molecule proteases that cannot pass through the membrane pores are trapped by the membrane fibers and flow back to the inlet tank along the pipeline with the mainstream liquid from the upper outlet of the membrane column, forming a closed loop circulation. The continuous high-speed tangential flow can constantly flush the inner wall of the membrane, effectively alleviating the concentration polarization and membrane blockage caused by the accumulation of proteases and impurities on the membrane surface. During the circulation process, sterile water for injection can be continuously added to the inlet tank to complete dynamic dilution and percolation, continuously reducing the system buffer concentration and continuously sieving to remove endotoxins. The entire circulation process operates at room temperature and low pressure throughout, relying on physical particle size sieving to separate endotoxins and proteases. At the same time, the circulation reflux mode completely retains the target protease, and the combination of a phosphate buffer system stabilizes the enzyme conformation, greatly improving the enzyme activity retention rate after treatment. Moreover, the membrane retention specifications can be adjusted according to the molecular weight of the protease to selectively retain and collect the target protease, completing the purification of the lysate at room temperature and removing endotoxins.For example, an ultrafiltration device is used to perform tangential flow circulation ultrafiltration on the pyrolysis feedstock. This ultrafiltration device is an internal pressure hollow fiber ultrafiltration membrane column device, which includes an inlet tank, a delivery pump, pressure gauges, a reflux regulating valve, an internal pressure hollow fiber membrane column, a permeate-side control valve, and a waste collection tank. An ultrafiltration membrane is installed in the internal pressure hollow fiber membrane column. The pyrolysis feedstock is stored in the inlet tank and, after being pressurized by the delivery pump, is introduced into the membrane cavity through the feed inlet at the lower end of the hollow fiber membrane column and flows tangentially upwards along the membrane cavity. Pressure gauges are installed on the feed and reflux lines to monitor the feed and reflux pressures in real time. A regulating valve is installed on the reflux line to control the transmembrane pressure difference. Small molecule solvents and endotoxin aggregates in the system vertically penetrate the hollow fiber membrane wall under the drive of the transmembrane pressure difference, are discharged through the permeate outlet on the side of the membrane column and the permeate-side control valve, and are collected in the waste collection tank. Large molecule protease components are absorbed by the hollow fiber membrane. The purified protein solution is collected at the outlet, while the mainstream feed solution flows out from the top of the membrane column and returns to the feed tank via the reflux pipeline, forming a closed-loop tangential flow circulation. The continuously flowing tangential feed solution in the membrane fiber lumen can flush the inner wall of the membrane, reducing concentration polarization and pore blockage caused by the accumulation of protease and impurities on the membrane surface. During the circulation process, sterile water for injection can be continuously added to the feed tank to achieve dynamic percolation dilution, gradually reducing the system buffer concentration and continuously sieving to remove endotoxins. The entire device operates at room temperature, relying solely on membrane pore size sieving to separate endotoxins and proteases. The closed-loop circulation structure can completely retain the target protease. Combined with a phosphate-containing buffer system, the spatial conformation of the protease is stabilized, improving the enzyme activity retention rate after treatment. Hollow fiber membrane columns with corresponding molecular weight cutoff specifications can also be selected according to the apparent molecular weight of the protease to achieve selective recovery of the target protease and complete the gentle endotoxin removal purification treatment of the lysate.

[0030] For example, the ultrafiltration membrane has a molecular weight cutoff of 10-1000 kDa, preferably 20-500 kDa; for example, the ultrafiltration membrane is a hydrophilic modified polyethersulfone membrane or a polyvinylidene fluoride membrane; by selecting the membrane material: using a hydrophilic synthetic polymer membrane with inherently low protein adsorption properties for sieving, the overall molecular weight cutoff of the membrane is in the range of 10-1000 kDa, preferably in the purified range of 20-500 kDa; thus, limiting the molecular weight cutoff of the hydrophilic modified PES / PVDF ultrafiltration membrane to 10-1000 kDa, preferably 20-500 kDa, the low protein adsorption of the membrane material can reduce protease loss and improve enzyme activity retention.

[0031] For example, in the tangential flow ultrafiltration, the ultrafiltration temperature is ambient temperature, preferably 15-30℃, between 4-35℃. The operating pressure is controlled at 0.5-1.5 bar, preferably 0.5-1 bar. This ambient temperature (4-35℃) and low pressure (0.5-1.5 bar) operating condition avoids enzyme denaturation caused by high temperature and high pressure shearing. The entire process operates under a constant ambient temperature environment of 4-35℃, preferably 15-30℃, with no heating, high-pressure high-temperature sterilization, or heat treatment modification processes.

[0032] In one embodiment, the dynamic dilution method is any one of one-time feed dilution, continuous closed-loop percolation, or segmented gradient dilution; the diluent used is sterile water for injection; preferably, the stock solution buffer concentration is ≤1×, and the finished buffer concentration after dynamic dilution is ≤1×; preferably, the stock solution buffer concentration is 0.1× to 1×, and the finished buffer concentration after dynamic dilution is 0.1× to 1×; preferably, the stock solution buffer concentration is ≤1×, and the finished buffer concentration after dynamic dilution is ≤0.25×. Thus, by using three sterile water for injection dilution methods—one-time feed, closed-loop percolation, and gradient dilution—the stock solution buffer concentration of ≤1× can be controlled to be diluted to a finished concentration of ≤0.25×. The dilution process continuously maintains the phosphate ion environment of the system, continuously stabilizing the protease structure. Multiple dilution modes are suitable for production lines of different scales. The combined effects of ambient temperature and low pressure, hydrophilic ultrafiltration membrane, and phosphate buffer further enhance the enzyme activity retention effect, while accurately trapping and separating endotoxin aggregates. In this application, the formulation liquid phase control involves dynamically adjusting the composition ratio of the lysis buffer formulation liquid phase during the ultrafiltration closed-loop reflux operation, and progressively adjusting the solvent concentration to the target low value of the finished product formulation. The control mode can be selected from any one of the following: one-time replenishment dilution, continuous closed-loop percolation, or segmented gradient dilution. The control process retains beneficial functional salt ions in the system, only diluting the solvent osmotic pressure, which helps improve the ultrafiltration effect on endotoxin removal and enhances enzyme and enzyme activity retention. It should be noted that the stock solution buffer concentration is ≤1×, meaning that the buffer concentration can be diluted, down to a minimum of 0.125×, to reduce the addition of salt ions from the buffer salt. For example, if the stock solution buffer concentration is 0.125× to 1×, the water for injection should be dynamically diluted at least four times.

[0033] In one embodiment, in the ultrafiltration operation, the ultrafiltration device is an inner-pressure type hydrophilic hollow fiber membrane column with an ultrafiltration membrane built therein; preferably, before subjecting the crude lysate to the ultrafiltration operation, the treatment method further comprises the following step of pre-treating the ultrafiltration device: the pre-treatment step sequentially includes the processes of alkaline washing, water washing, membrane column integrity detection, and buffer solution equilibration; the alkaline washing step: the hollow fiber membrane column is circularly washed with 0.2 to 0.5 M sterile sodium hydroxide solution for 1 h, the alkaline washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and after the alkaline washing is completed, the residual alkaline solution in the hollow inner cavity and the permeate side of the membrane column is drained; the water washing step: the membrane column is continuously rinsed with sterile water for injection for 1.5 to 4 h, the water washing pressure is controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and after the water washing is completed, the residual water for injection in the hollow inner cavity and the permeate side of the membrane column is drained; the membrane column integrity detection step: the liquid outlet pipeline of the membrane column is closed, sterile gas is introduced from the liquid inlet pipeline, the pressure is increased to no more than 2 bar, and the pressure drop is observed under the maintained pressure. If the pressure drop per minute is less than 0.1 to 0.2 bar, the membrane column is judged to be qualified in integrity; if not qualified, the alkaline washing, water washing and integrity detection steps are repeated, and if the re-test is still unqualified, the membrane column is replaced; the membrane column integrity detection can be performed before alkaline washing and water washing, but the integrity detection must be performed both before the ultrafiltration feeding and after the ultrafiltration is completed; the buffer solution equilibration process: the retentate side and the permeate side of the membrane column are both connected to a buffer solution storage tank, the membrane column is circularly rinsed with the buffer solution for 5 to 10 min, and the pH and conductivity of the filtrate are detected in real time until the pH and conductivity of the filtrate are consistent with those of the buffer solution, so as to complete the pre-equilibration of the membrane column. In this way, the inner-pressure type hydrophilic hollow fiber membrane column is used as the ultrafiltration device, the ultrafiltration membrane built in the membrane column is adapted to the tangential flow circulation working condition, and the supporting standardized pre-treatment processes of alkaline washing, water washing, integrity detection and buffer solution equilibration are adopted. The alkaline washing and water washing can completely remove residual impurities and exogenous endotoxin in the membrane column, the integrity test can investigate the membrane leakage defect in advance, and the buffer solution equilibration makes the pH and salt ion environment in the membrane match with the crude lysate, so as to reduce membrane adsorption and stress denaturation of protease; it is beneficial to give consideration to both the endotoxin removal effect and the reduction of enzyme loss and activity inactivation.

[0034] In one embodiment, after ultrafiltration, the treatment method further includes a membrane column recovery and preservation step for the ultrafiltration equipment; the membrane column recovery and preservation step includes: after ultrafiltration, firstly, the membrane column is cleaned with sterile water for injection for 0.5 to 1 hour, then replaced with fresh sterile water for injection and cleaned again for 0.5 to 1 hour, with the cleaning pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar; after cleaning, the hollow inner cavity of the membrane column and the residual water for injection at the permeate end are drained; then, the membrane column is circulated and alkaline washed or soaked with 0.5 to 1 M sodium hydroxide solution for 0.5 to 1 hour, with the alkaline washing pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar; finally, the membrane column is soaked and preserved in 0.1 to 0.2 M sodium hydroxide solution. Thus, the membrane column can be reused multiple times by segmented water washing and gradient alkaline washing and soaking after ultrafiltration, reducing the cost of industrial production. The entire standardized pretreatment and regeneration process maintains the low adsorption characteristics of the membrane column. Combined with the phosphate buffer system, it ensures the stability of protease and meets the standards for endotoxin control throughout the process, making it suitable for large-scale continuous production.

[0035] In one embodiment, the process of separating and removing endotoxins and collecting the purified liquid containing protease by utilizing the molecular weight difference of the materials is as follows: When the apparent molecular weight of the proteolytic enzyme is lower than the first retention threshold of 10-100 kDa, an ultrafiltration membrane with a molecular weight cutoff not higher than the first retention threshold is selected, and the membrane module retention concentrate is collected as the purification feed solution. When the apparent molecular weight of the proteolytic enzyme is higher than the second retention threshold of 100-500 kDa, an ultrafiltration membrane with a molecular weight cutoff not lower than the second retention threshold is selected, and the permeate from the membrane module is collected as the purification feed.

[0036] Thus, based on the molecular weight of the protease, two differentiated collection schemes are used: small molecule proteases are collected using membranes with corresponding low molecular weight cutoffs, while large molecule proteases are collected using membranes with high molecular weight cutoffs. Combined with phosphate buffer to stabilize the enzyme molecular structure, proteases and endotoxin aggregates can be accurately distinguished, preventing the target protease from being lost with the permeate or trapped and enriched by endotoxin impurities. This ensures that proteases of different molecular weights can be completely recovered, maximizing the retention of the original enzyme composition ratio while efficiently removing endotoxins, without changing the original lysis activity of the lysis buffer, and is suitable for ultrafiltration purification of all types of proteases.

[0037] Differential fraction collection: Based on the difference in apparent monomer molecular weight of proteases and steady-state aggregate particle size of endotoxins within the formulation, effective active fractions are collected separately, preserving the original enzyme ratio and functional salt ion ratio of the formulation, and targeted removal / reduction of endotoxin impurities in the system. ① Small molecule protease formulation system: The apparent molecular weight of the protease is lower than the first threshold of the molecular weight cutoff. It is matched with an ultrafiltration membrane with a cutoff specification not higher than the first threshold. After closed-loop operation, the membrane module retains the concentrate.

[0038] ② Macromolecular protease formulation system: The apparent molecular weight of the protease is higher than the second threshold of the molecular weight cutoff. It is matched with an ultrafiltration membrane with a cutoff specification not lower than the second threshold. After closed-loop operation, the permeate of the membrane module is collected.

[0039] The first threshold for molecular weight cutoff is defined as the range of 10–100 kDa, and the second threshold for molecular weight cutoff is defined as the range of 100–500 kDa.

[0040] After processing, the enzyme composition and beneficial salt ion formulation of the lysate remain completely unchanged, the overall endotoxin removal rate is ≥98%, the endotoxin index of the finished lysate is ≤5 EU / mg, and the functional protease activity retention rate is ≥75%.

[0041] In one embodiment, before ultrafiltration of the pyrolysis feed solution, the treatment method further includes the following step: pre-filtering the pyrolysis feed solution through a 0.2-0.45 μm filter; thus, the 0.2-0.45 μm pre-filtration before ultrafiltration can remove large molecular impurities and microparticles in the feed solution in advance, prevent impurities from clogging the ultrafiltration membrane pores and aggravating membrane fouling, protect the membrane material and reduce ultrafiltration shear loss.

[0042] In one embodiment, after collecting the purified liquid, the processing method further includes a finished product preparation step: the collected purified liquid is aseptically filtered through a 0.22μm terminal and then dispensed to prepare a liquid lysis buffer; or the purified liquid is freeze-dried at low temperature under vacuum to prepare a freeze-dried lysis buffer product. Thus, by using a 0.22μm terminal for aseptic dispensing after purification to prepare a liquid finished product, or by low-temperature vacuum freeze-drying to prepare a freeze-dried powder, both finished product forms can fully retain the enzyme stabilizing effect provided by the phosphate buffer system. The liquid product can be used directly, while the freeze-dried product is convenient for long-term storage and transportation, and for supplying to different locations, thus broadening the storage, transportation, and use scenarios of the finished product. Furthermore, the aseptic processing meets the aseptic access standards for biological cell preparations.

[0043] In one embodiment, after collecting the refined feed liquid, the processing method further includes desalting the liquid pyrolysis solution, thereby further removing salt ions or salts from the refined feed liquid as needed. For example, the desalting process can be performed using a desalting column. The above-mentioned method for reducing endotoxins in lysate involves dissolving proteolytic enzymes in a phosphate-containing buffer solution to prepare the lysate stock solution. This is combined with tangential flow circulating ultrafiltration, dynamically diluting the buffer solution during ultrafiltration to lower the system concentration. Endotoxins are removed by sieving based on the molecular weight difference of the materials. Phosphate ions synergistically stabilize the protease hydration membrane and spatial conformation with the equilibrium salt ions in the buffer system, reducing the damage to the heat-sensitive protease structure caused by fluid shear forces during ultrafiltration. Simultaneously, the phosphate buffer system optimizes the osmotic pressure of the feed solution, preventing protease denaturation due to sudden changes in osmotic pressure. Combined with a room-temperature ultrafiltration physical separation mode relying solely on particle size sieving, the entire process operates under a mild 4-35℃ environment with no high temperatures, no resin adsorption, and no added exogenous surfactants. This avoids activity loss due to non-specific binding of the adsorption medium to the protease, and allows for precise differentiation of protease molecules from high-molecular-weight endotoxin aggregates through the ultrafiltration membrane. The endotoxin removal rate is high, stabilizing the endotoxin background of 300 EU / mg in the stock solution to 5 EU / mg in the finished lysate. The following method utilizes the synergistic effect of the phosphate buffer system and ultrafiltration sieving process to maintain a stable protease activity retention rate of over 75% after treatment. The dynamic dilution step simultaneously adjusts the buffer concentration and removes endotoxins. The process is streamlined and can be scaled up to GMP standards. The resulting lysate has no harmful additive residues, excellent cell biocompatibility, and can effectively lyse gelatin microcarriers and release and recover live cells without damage. This synergistic approach achieves the core technical effects of significantly reducing endotoxins, providing mild treatment conditions, and maintaining high enzyme activity.

[0044] In one specific embodiment, the method for treating endotoxins with lysis buffer includes the following steps: 1. Preparation of stock solution: Weigh the single / compound protease and dissolve it in a suitable aqueous solvent with a concentration range of 1-320 g / L, preferably 10-320 g / L, and mix thoroughly. Filter the stock solution through a 0.2-0.45 μm pore size filter and set aside for use.

[0045] 2. Column preparation: Select hollow fiber columns with appropriate pore size. After ensuring that there are no leaks in the system pipeline, wash with 0.2-0.5M sodium hydroxide alkali for 1 hour, drain the hollow part and the perforated end part of the alkali solution, and then wash with water for injection for 1.5-4 hours. Sterile alkali solution and sterile water for injection are preferred. The alkali washing and water washing pressure is controlled at 0.1-1 bar, preferably 0.1-0.5 bar.

[0046] 3. Column Integrity Test: After water washing, drain the hollow portion and the permeate end, inject water, close the outlet line, and slowly pump sterile gas into the inlet line. Observe the inlet pressure and stop gas injection when it reaches a suitable pressure, with a maximum of about 2 bar. Optimize the ultrafiltration pressure for the sample. If the pressure drop is less than 0.1-0.2 bar / min, the column integrity is considered good, and the pressure is released. Otherwise, repeat the steps once; if the abnormality persists, the column has an integrity defect and needs to be replaced. This step can optionally be performed before water washing or alkaline washing, but it must be performed before and after sample ultrafiltration.

[0047] 4. Optional pre-process buffer equilibration: The hollow fiber column is rinsed with buffer to ensure that the membrane's pH and salt ion state are close to the initial state of the sample to be treated, thereby reducing membrane fouling and product loss. In this step, both the reflux and permeate ends are connected to a container filled with sufficient buffer solution. The solution is circulated for 5-10 minutes, and the pH or conductivity of the filtrate is checked to ensure it is similar in properties to the buffer solution.

[0048] 5. Remove endotoxins: 1) When collecting the percolation end: Connect the filtered stock solution bottle to the inlet and outlet of the column reflux end, and connect an empty bottle to the percolation end to collect the endotoxin-removed solution. At least a peristaltic pump and pressure detection device should be installed at the inlet tube, with the pressure controlled at 0.5-1.5 bar, preferably 0.5-1 bar. Optionally, if the liquid level in the stock solution bottle does not decrease, after draining the liquid from the percolation end, add 0-2 times the initial stock solution volume of injection water to the stock solution bottle and continue the ultrafiltration operation, repeating this process at least once.

[0049] 2) When collecting the reflux end: Connect the filtered stock solution bottle to the inlet and outlet of the column reflux end, and collect the waste through the empty bottle connected to the end. At least a peristaltic pump and pressure detection device should be installed at the inlet pipe, with the pressure controlled at 0.5-1.5 bar, preferably 0.5-1 bar. Optionally, when the liquid in the stock solution bottle is reduced to 1 / 2-1 / 10, drain the liquid from the reflux end and add 0-2 times the initial stock solution volume of water for injection to continue the concentration operation. Repeat this process at least once.

[0050] 3) Column integrity test is the same as S3. 4) Low endotoxin liquid lysis buffer: The low endotoxin target liquid sample obtained in step 1) or 2) is aliquoted. Optionally, a filter with a pore size of 0.2 μm or less is used for terminal filtration before aliquoting. The liquid lysis buffer is then frozen for storage.

[0051] 5) Low endotoxin powder lysis solution: The low endotoxin target liquid sample obtained in step 1) or 2) is aliquoted into a freeze-drying box for freeze-drying, and the freeze-dried powder is aliquoted and stored at low temperature.

[0052] 6. Column recovery: After rinsing with water for 0.5-1 hour, replace with new water and continue rinsing for another 0.5-1 hour. Then drain the hollow part and the perforated end part for injection. Afterward, wash or soak with 0.5-1M sodium hydroxide solution for 0.5-1 hour, and then replace with 0.1-0.2M sodium hydroxide solution for storage. The pressure of alkaline washing and water washing should be controlled at 0.1-1 bar, preferably 0.1-0.5 bar.

[0053] The lysis stock solution prepared according to the proprietary enzyme and balanced salt ion formula of this invention has a steady-state background of native endotoxin of 300 EU / mg. After purification using the proprietary process of the matching formula, the measured endotoxin content of the finished lysis solution is 0.125-2 EU / mg, with a batch-wide batch stability of ≤5 EU / mg, meeting the pharmacopoeia limit requirements for biological agents. The endotoxin content of the finished lysis solution is ≤5 EU / mg; the enzyme activity retention rate after purification is 75-90%; there is no significant difference in the enzyme components, functional salt ions, and lysis kinetic parameters between the formulated enzyme solution and the unrefined stock solution; the effective shelf life of the liquid product is ≥6 months when stored frozen, protected from light, and sealed; the freeze-dried product, when stored at low temperature and protected from light, shows no decrease in formulation performance, enzyme activity, or lysis performance after reconstitution. It is suitable for large-scale cell culture in GMP workshop bioreactors, industrial mass production of human vaccines, preparation of mesenchymal stem cells / immune cells, and degradation and dissociation of medical-grade gelatin solid carriers, and is used for controlled lysis of gelatin microcarriers at the end of the culture period and non-destructive release and recovery of live cells. This application utilizes a proprietary balanced salt ion composition to stabilize the protease's spatial conformation and withstand ultrafiltration shear forces. Unlike commercially available salt-free, single-solvent, crude lysis buffers, this formulation offers greater originality. The proprietary lysis buffer formulation is compatible with a room-temperature ultrafiltration endotoxin reduction process. This process removes endotoxins without altering the enzyme composition or beneficial salt ion ratio, requiring no external modifying reagents or subsequent recombining of the lysis buffer. The entire process is physically purified at room temperature (4-35℃), with zero high-temperature treatment, zero heat treatment, and zero high-pressure sterilization, preventing protease denaturation and inactivation from the source. It can reliably refine the stock solution from a high background concentration of 300 EU / mg to a finished lysis buffer of ≤5%. With an EU / mg concentration and an endotoxin removal rate ≥98%, meeting the GMP excipient access standards for cell therapy, the formulation utilizes three optimal solvent formulations: PBS, Hanks' solution, and MSC culture medium, combined with suitable salt ions. After ultrafiltration, enzyme activity is stably retained at 75-90%, far superior to the 30%-40% enzyme activity retention rate of physiological saline and pure water systems. Based on molecular weight sieving logic, it can adapt to small molecule, large molecule, and compound protease lysis buffer formulations, offering strong process versatility. Ultrafiltration simultaneously completes endotoxin removal and lysis buffer solvent concentration adjustment, retaining functional salt ions to directly obtain a usable finished product formulation, simplifying production processes, reducing membrane material costs, and facilitating GMP industrial scale-up. High biosafety: the enzyme and balanced salt ion composition of the lysis buffer formulation is controllable, with no exogenous adjuvants or toxic additives. The purification process does not introduce impurities, resulting in excellent cell compatibility of the finished product and no damage to lysed ex vivo cells. Versatile product storage and transportation: the formulation can be prepared in liquid and lyophilized forms, suitable for on-site use, remote storage and transportation, and long-term stockpiling scenarios, with stable shelf life.

[0054] Secondly, this application also provides a low-endotoxin gelatin microcarrier lysis buffer, prepared by the endotoxin reduction treatment method described in any of the above embodiments; the finished product is in sterile liquid or vacuum lyophilized solid form; the endotoxin content of the lysis buffer is ≤5 EU / mg, and the proteolytic enzyme activity retention rate is 75% to 90% or higher. Thus, the low-endotoxin gelatin microcarrier lysis buffer product is prepared by the aforementioned phosphate buffer-assisted room temperature ultrafiltration process, and is available in sterile liquid and vacuum lyophilized forms. The finished product has a stable endotoxin content of ≤5 EU / mg, and the protease activity retention rate can reach 75% to 90%. The formulation contains no surfactants or harmful additives, the phosphate balance salt system is completely preserved, and it exhibits excellent cell biocompatibility, without damaging the live cells released after the gelatin microcarrier lysis. Simultaneously, the liquid and lyophilized finished products have stable shelf lives, meet the excipient limit requirements for cell preparations in the Chinese Pharmacopoeia, and can be directly used in large-scale cell lysis processes in bioreactors.

[0055] Thirdly, the application of the low-endotoxin gelatin microcarrier lysis buffer described in any of the above embodiments in the preparation of cell harvesting reagents after cell culture. Thus, applying this low-endotoxin gelatin microcarrier lysis buffer to the cell harvesting stage after cell culture, relying on the product's low endotoxin, high enzyme activity, and cell-friendly properties, can gently degrade the gelatin microcarrier framework, completely detaching stem cells, somatic cells, and vaccine culture cells adhering to the carrier. The harvesting process results in a low apoptosis rate and high cell viability. Simultaneously, the low endotoxin level avoids endotoxin-induced cellular inflammatory stress, meeting the compliance and quality control requirements for cell therapy and vaccine industrial production.

[0056] Fourthly, this application provides a cell-scale culture kit comprising the low-endotoxin gelatin microcarrier lysis buffer described in any of the above embodiments, for gelatin microcarrier lysis and non-destructive release and recovery of live cells. Thus, the cell-scale culture kit comprising the low-endotoxin gelatin microcarrier lysis buffer of this invention integrates a core lysis reagent with low endotoxin and high lysis activity, and can be directly used with a bioreactor gelatin microcarrier culture system. Microcarrier lysis and non-destructive recovery of live cells can be completed on-site without the need for additional lysis buffer preparation or endotoxin removal equipment. The operation is convenient, and the standardized kit is suitable for laboratory pilot-scale, GMP pilot-scale, and industrial mass production scenarios, significantly reducing the operational threshold and quality control risks for large-scale cell expansion.

[0057] The following specific embodiments further illustrate this application.

[0058] Example 1: Small molecule compound enzyme + balanced salt ion lysis buffer formulation + endotoxin reduction process The stock lysis buffer formulation concentration is 40 mg / ml: collagenase, trypsin, neutral protease, and clostridium lysin complex enzyme, in 1× calcium- and magnesium-free PBS (Note: standard PBS, 1×PBS indicates undiluted, 0.25×PBS indicates that the previous 1×PBS was diluted four times to become 0.25×PBS). Ultrafiltration consumables: Hydrophilic modified PES hollow fiber membrane with a molecular weight cutoff of 20 kDa; Endotoxin removal process: Closed-loop tangential flow ultrafiltration at room temperature (15-30℃), continuous addition of sterile water for injection during operation, gradient perfiltration, diluting the buffer to 0.25× PBS product system, retaining the beneficial sodium / potassium / calcium balance salts of the system throughout the process; Collection method: For proteases with a molecular weight below the 100 kDa threshold, collect the membrane retrieval concentrate; Finished product specifications: endotoxin 0.125 EU / mg, protease activity retention rate 86%, and lysis rate not significantly different from the untreated stock solution.

[0059] Example 2: Macromolecular complex enzyme + balanced salt ion lysis buffer formulation + endotoxin reduction process The stock solution of the lysis buffer was formulated with a concentration of 20 mg / ml: collagenase monoenzyme, in 1× Hanks balanced salt solution; Ultrafiltration consumables: Hydrophilic modified PES membranes with a molecular weight cutoff of 100~300 kDa; Endotoxin removal process: Closed-loop ultrafiltration at room temperature, followed by staged addition of sterile water for injection to dilute to 0.25 × 10⁻⁵ Hanks; Collection method: For proteases with a molecular weight higher than the 100 kDa threshold, collect the membrane permeate filtrate; Finished product specifications: endotoxin 0.125 EU / mg, enzyme activity retention rate 83%, and stable lysis performance.

[0060] Example 3: Medium molecular weight single enzyme + balanced salt ion lysis buffer formulation + endotoxin reduction process The stock lysis buffer formulation concentration was 80 mg / ml: papain single enzyme, solvent 0.5× MSC serum-free basal medium; Ultrafiltration consumables: 50 kDa hydrophilic PES ultrafiltration membrane; Process: Continuous closed-loop percolation dilution to 0.25× MSC medium; Material collection method: Collect permeate; Finished product specifications: endotoxin 0.125 EU / mg, enzyme activity retention rate 88%.

[0061] Example 4: Compound protease + balanced salt ion lysis buffer formulation + endotoxin reduction process The stock lysis buffer formulation concentration was 20 mg / ml: a mixture of casein and neutral protease, in 0.5× PBS as solvent; Ultrafiltration consumables: 30 kDa hydrophilic PES membrane; Process: Dilute with water for injection to 0.25× PBS in stages; Material collection method: Collect the retained concentrate; Finished product specifications: endotoxin 0.125 EU / mg, enzyme activity retention rate 85%.

[0062] Comparative Example 1: Physiological saline-based lysis buffer formulation control group The solvent for the lysis buffer was replaced with medical saline, and the enzyme components, functional salt ions, and endotoxin reduction process were completely matched in Example 1; Test results: after purification, only 25.5% of the enzyme activity was retained, the lysis time doubled, and the formula was not suitable for ultrafiltration purification.

[0063] Comparative Example 2: Control Group with Calcium-Magnesium Tris-HCl Base Lysis Buffer Formulation The solvent for the lysis buffer was replaced with Tris-HCl buffer modified with calcium and magnesium ions, pH 7.4, and the rest matched Example 1; Detection results: 30% of the enzyme activity was retained after purification, and the protease activity was irreversibly damaged.

[0064] Comparative Example 3: Control Group of Pure Water-Based Lysis Buffer Formulation The solvent for the lysis buffer was replaced with sterile water for injection. There were no suitable balanced salt ions. The rest were matched in Example 1. Test results: Pure water destroyed the hydration membrane of the protease. After purification, only 30% of the enzyme activity was retained, and the formula was unqualified.

[0065] As can be seen from the above, this application uses PBS, Hanks, and serum-free culture medium as buffers or solvents, which can effectively remove endotoxins while maintaining a high enzyme activity retention rate of over 75%.

[0066] To visually verify the combined advantages of the phosphate buffer system and the room-temperature dynamic dilution ultrafiltration process of this invention, parallel comparative experiments were conducted using the same detection standards: collagenase activity was detected according to GB / T 47329-2026 "Methods for Determination of Collagenase Activity and Purity"; the microcarrier lysis time was determined by taking 3 mL of 1 mg / mL lysis buffer, degrading 20 mg of 3D TableTrix® (W01) gelatin microcarriers at 37℃, and recording the time required for complete dissolution; endotoxin was detected using the gel permeation method with Limulus amebocyte lysate (LAL) reagent. Sterile water for injection, calcium-magnesium water for injection, calcium-magnesium Tris-HCl, and physiological saline were used as control solvents, while calcium-magnesium-free PBS, Hanks' balanced salt, and serum-free basal culture medium were used as the phosphate buffer systems specified in this invention. All groups were treated using the unified room-temperature ultrafiltration process of this invention, and the changes in enzyme activity, endotoxin, and lysis time before and after ultrafiltration were tested. The experimental data are shown in Table 1.

[0067] Table 1 Enzyme activity and degradation time before and after endotoxin treatment.

[0068] As can be seen from the data in Table 1: Common defects in the control group: After treatment with the same ultrafiltration process, the protease activity recovery rate of phosphate-free injection water, physiological saline, and calcium-magnesium Tris-HCl buffer was only 25.5%~30.7%, and the protease underwent irreversible denaturation; the microcarrier lysis time was extended from 5~6 min to 20 min, and the lysis efficiency was significantly reduced, which could not meet the requirements of rapid cell harvesting in industrial applications. The reason for this is that this type of system lacks phosphate ions, which prevents the formation of a stable hydration membrane to resist ultrafiltration shear forces and osmotic pressure fluctuations, thus exacerbating the adsorption and loss of protease.

[0069] Advantages of this invention: Using phosphate-containing PBS, Hanks, and serum-free basal medium as lysis buffers, after gradient dilution and ultrafiltration at room temperature, the protease activity recovery rate is stably maintained at 75%~88%, and the lysis time is close to that of the original solution, without significant prolongation. Specifically, calcium- and magnesium-free PBS is suitable for non-metal-dependent enzymes such as papain and trypsin, with optimal enzyme activity retention of up to 88%; Hanks naturally contains low concentrations of calcium and magnesium, suitable for collagenase and metalloproteinases; after buffer dilution to ≤0.25×, the phosphate concentration decreases, eliminating the risk of phosphate precipitation, and enzyme activity is retained at 83%; the serum-free basal medium ensures cell compatibility, with enzyme activity retention exceeding 75%.

[0070] Endotoxin removal effect: The original endotoxin of all raw materials in the system was >300 EU / mg. After treatment by the ultrafiltration sieving process of this invention, the endotoxin of the finished product can be reduced to <0.25 EU / mg, with a removal rate of over 98%, which meets the pharmacopoeia limit standard of ≤5 EU / mg for cell preparations.

[0071] To further verify the endotoxin removal of all protease types applicable to this invention, specific ultrafiltration purification processes were applied to the stock solutions of collagenase, papain, trypsin, and neutral protease, and the endotoxin levels before and after treatment were compared. The results are shown in Table 2.

[0072] Table 2. Effects of compound enzymes and collagenase on endotoxin removal.

[0073] Table 2 shows that the differentiated molecular weight cutoff sieving scheme of the present invention can be adapted to all target proteases: small molecule proteases use low molecular weight cutoff membranes to collect the retentate, and large molecule proteases use high molecular weight cutoff membranes to collect the permeate. This can completely separate high molecular weight endotoxin aggregates. The endotoxin levels of all types of proteases are far below the pharmacopoeia control threshold, and the process does not destroy the original cleavage activity of the proteases.

[0074] To demonstrate that the refined liquid lysis buffer and lyophilized lysis buffer do not interfere with the gel-method endotoxin detection specified in the Chinese Pharmacopoeia, and to ensure the authenticity and reliability of the quality control test results of the finished product, Limulus amebocyte lysate (LAL) reagent interference verification tests were conducted on four batches of small-scale and pilot-scale liquid samples and corresponding lyophilized powders. The test grouping and parallel judgment results are shown in Tables 3 and 4.

[0075] Table 3. Results of interference test on lyophilized powder of lysate after treatment

[0076] Table 4. Results of Interference Tests on Treated Lysis Buffer (Liquid)

[0077] Note: All parallel tubes of the negative control and experimental negative control of the test sample showed negative results; the series of endotoxin standard solution test results met the requirements of the Limulus Amebocyte Lysate (LAL) reagent sensitivity verification test, and the interference test was generally valid. Es values ​​fell within the range of 0.5λ to 2λ (inclusive); the Es values ​​of the four batches of liquid lysis buffer and the four batches of lyophilized lysis buffer were all within the range of 0.5λ to 2λ (inclusive), indicating that the test sample did not interfere with endotoxin detection, and the interference test was deemed qualified. The endotoxin content of the four batches of treated liquid lysis buffer (20× dilution system) was <5 EU / mL; the endotoxin content of the four batches of treated lyophilized lysis buffer was <0.25 EU / mg.

[0078] As shown in Tables 3 and 4, the validity of this interference test and the interference of the test sample were determined according to the bacterial endotoxin test method in Part IV of the Pharmacopoeia of the People's Republic of China as follows: Test validity: All negative control parallel tubes showed negative results; the reaction results of the endotoxin standard series Es2, Es1, and Es0.5 met the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification, the entire set of interference tests was effective, and the detection data were reliable.

[0079] Interference determination: The Et values ​​of the four batches of liquid lysis buffer and the four batches of lyophilized powder test samples all fell within the qualified range of 0.5λ~2λ, and the recovery rate met the pharmacopoeia standard of 50%~200%, proving that the purified lysis buffer of this invention, whether in liquid or lyophilized form, will not enhance or inhibit the detection of endotoxins by gel electrophoresis, and the factory quality control test results are true and valid.

[0080] Finished product internal control indicators: Quantitative testing showed that the endotoxin content of the 20-fold diluted liquid lysis buffer was <5 EU / mL; the endotoxin content of the lyophilized lysis powder was <0.25 EU / mg, which is far below the statutory limit of ≤5 EU / mg for cell therapy excipients, and GMP production release is controllable.

[0081] The comprehensive experimental data in Tables 1-4 fully validate the integrated technical solution of this invention: using phosphate-containing buffer as the lysis substrate, combined with a room-temperature tangential gradient dilution ultrafiltration process, three core technical effects are achieved simultaneously: 1. No heat treatment is required throughout the process at room temperature (4-35℃), completely avoiding high-temperature inactivation of heat-sensitive proteases; 2. Relying on phosphate to stabilize the enzyme conformation, the enzyme activity is stably retained at over 75% after ultrafiltration, with no significant decrease in lysis rate; 3. Utilizing molecular weight difference sieving to efficiently remove endotoxins, and the finished product indicators meet the compliance requirements for medical cell preparations; the entire process requires no addition of exogenous reagents such as surfactants and adsorption resins, leaves no cytotoxic residues, and is adaptable to both liquid and lyophilized finished product formulations. The process is streamlined and can be mass-produced under GMP standards.

[0082] This invention employs a synergistic design of a proprietary lysis buffer formulation and an integrated endotoxin-reducing ultrafiltration process. First, the enzyme components, buffer solvent, and osmotic pressure concentration are optimized to obtain a basic formulation of a gelatin microcarrier lysis buffer with low protein adsorption, enzyme structure protection, and ultrafiltration compatibility. Then, based on the physicochemical properties of this formulation, a hydrophilic synthetic polymer ultrafiltration membrane is selected. The solvent concentration of the formulation system is dynamically controlled throughout the process at room temperature. Utilizing the differential sieving principle of protease and endotoxin aggregate particle size, endotoxin impurities are separated in stages. No external adsorbents or surfactants are needed to modify the formulation components. Endotoxin removal is achieved without altering the functional ratio of the lysis buffer, ultimately yielding a dual-form low-endotoxin finished lysis buffer. The entire process complies with GMP production standards. The overall process flow diagram of this invention is as follows: Preparation of a proprietary lysis buffer stock solution containing enzymes and balanced salt ions → Room temperature closed-loop ultrafiltration + adjustment of formulation solvent concentration and retention of functional salt ions → Differential removal of endotoxins and collection of qualified lysis buffer fractions → Aseptic dispensing / lyophilization → Low-endotoxin finished lysis buffer.

[0083] This invention possesses several outstanding advantages: The formulation incorporates a built-in salt ion stabilization system, which stabilizes the protease's spatial conformation and makes it resistant to ultrafiltration shear forces, demonstrating greater originality compared to commercially available salt-free, single-solvent-based coarse lysis buffers; This invention employs an integrated formulation and process design approach, with a dedicated lysis buffer that can be matched with a room-temperature ultrafiltration endotoxin reduction process. This process only removes endotoxins without altering the ratio of enzyme components and beneficial salt ions within the system, eliminating the need for additional modifying reagents and subsequent lysis buffer preparation; The entire process utilizes physical purification at 4-35℃, without high-temperature, heat treatment, or autoclaving operations, fundamentally preventing the denaturation and inactivation of heat-sensitive proteases and achieving comprehensive protection of the protease; High endotoxin control standards are maintained, stabilizing and purifying lysis stock solutions with initial endotoxin levels as high as 300 EU / mg to a finished product with endotoxin levels ≤5 EU / mg, achieving an endotoxin removal rate of no less than 98%, meeting the GMP excipient access requirements for cell therapy; PBS and Hanks were screened and obtained. The process utilizes three optimal solvents—liquid, MSC culture medium, and MSC culture medium—and corresponding balanced salts to achieve stable enzyme activity retention of 75%–90% after ultrafiltration, significantly superior to the 30%–40% retention in physiological saline and pure water systems. Based on molecular weight sieving principles, this process is compatible with all types of proteases, including small molecules, large molecules, and complex formulations, demonstrating strong versatility. The ultrafiltration process simultaneously removes endotoxins and adjusts buffer solvent concentration, retaining functional salt ions throughout the process, resulting in a qualified finished product in one step. This significantly simplifies the production process, reduces membrane material costs, and facilitates GMP-compliant industrial-scale production. The overall composition is controllable, consisting only of proteases and balanced salt ions, free of exogenous auxiliaries and toxic additives. The purification process introduces no impurities, resulting in good cell compatibility. The lysis process does not damage ex vivo living cells, ensuring excellent biosafety. Furthermore, the finished product can be processed into sterile liquid and vacuum freeze-dried forms, suitable for various scenarios such as on-site use, long-distance transportation, and long-term storage, with a stable product shelf life.

[0084] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that the terms "in one embodiment," "for example," and "again," etc., in this application are intended to illustrate the application and not to limit it. The embodiments described above only illustrate several implementations of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. A method for treating lysate to reduce endotoxin levels, characterized in that, Includes the following steps: The proteolytic enzyme is dissolved in a buffer solution to form a lysis stock solution; wherein the buffer solution contains phosphate ions; The lysate was subjected to tangential flow circulation ultrafiltration using an ultrafiltration membrane. During the ultrafiltration process, the buffer was dynamically diluted to reduce its concentration. Endotoxins were removed by utilizing the molecular weight difference of the materials, and the purified feed containing protease was collected to obtain the lysate.

2. The processing method according to claim 1, characterized in that, The proteolytic enzyme is selected from one or more of collagenase, caseinase, neutral protease, papain, trypsin, and metalloproteinase.

3. The processing method according to claim 1, characterized in that, The buffer solution is at least one of phosphate buffer, Hanks balanced salt solution, and serum-free cell basal culture medium. And / or, the concentration of proteolytic enzyme in the lysis stock solution is 1-320 g / L.

4. The processing method according to claim 1, characterized in that, The ultrafiltration membrane has a molecular weight cutoff of 10-1000 kDa, preferably 20-500 kDa. And / or, the ultrafiltration membrane is a hydrophilic modified polyethersulfone membrane or a polyvinylidene fluoride membrane; And / or, in the tangential flow circulation ultrafiltration, the ultrafiltration temperature is room temperature, which is 4-35℃, preferably 15-30℃; And / or, the operating pressure of tangential flow ultrafiltration is controlled at 0.5 to 1.5 bar, preferably 0.5 to 1 bar; And / or, the dynamic dilution method is any one of one-time replenishment dilution, continuous closed-loop percolation, or segmented gradient dilution; the diluent used for dilution is sterile water for injection; preferably, the concentration of the stock solution buffer is ≤1×, and the concentration of the finished buffer after dynamic dilution is ≤1×; preferably, the concentration of the stock solution buffer is 0.1× to 1×, and the concentration of the finished buffer after dynamic dilution is 0.1× to 1×.

5. The processing method according to claim 4, characterized in that, The process involves separating and removing endotoxins based on differences in the molecular weight of the materials and collecting the purified liquid containing protease. When the apparent molecular weight of the proteolytic enzyme is lower than the first retention threshold of 10-100 kDa, an ultrafiltration membrane with a molecular weight cutoff not higher than the first retention threshold is selected, and the membrane module retention concentrate is collected as the purification feed solution. When the apparent molecular weight of the proteolytic enzyme is higher than the second retention threshold of 100-500 kDa, an ultrafiltration membrane with a molecular weight cutoff not lower than the second retention threshold is selected, and the permeate from the membrane module is collected as the purification feed.

6. The processing method according to claim 1, characterized in that, Before performing ultrafiltration on the lysate, the treatment method further includes the following step: pre-filtering the lysate through a 0.2–0.45 μm filter; And / or, after collecting the refined liquid, the processing method further includes a finished product preparation step: the collected refined liquid is aseptically filtered through a 0.22μm terminal filter and then dispensed to prepare a liquid lysis solution; or the refined liquid is freeze-dried at low temperature under vacuum to prepare a freeze-dried lysis solution product; preferably, after collecting the refined liquid, the processing method further includes desalting the liquid lysis solution.

7. The processing method according to claim 1, characterized in that, In the described ultrafiltration operation, the ultrafiltration device is an inner-pressure hydrophilic hollow fiber membrane column with an ultrafiltration membrane built therein; preferably, before subjecting the crude lysate to the ultrafiltration operation, the treatment method further comprises the following step: a pretreatment step of the ultrafiltration device, which sequentially includes the processes of alkaline washing, water washing, membrane column integrity testing, and buffer solution equilibration; the alkaline washing step: circularly washing the hollow fiber membrane column for 1 h using a 0.2 to 0.5 M sterile sodium hydroxide solution, with the alkaline washing pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and draining the residual alkaline solution in the hollow inner cavity and the permeate end of the membrane column after the alkaline washing is completed; the water washing step: continuously flushing the membrane column for 1.5 to 4 h using sterile water for injection, with the water washing pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and draining the residual water for injection in the hollow inner cavity and the permeate end of the membrane column after the water washing is completed; the membrane column integrity testing step: closing the liquid outlet pipeline of the membrane column, introducing sterile gas from the liquid inlet pipeline, raising the pressure to no more than 2 bar, maintaining the pressure to observe the pressure drop, and determining that the membrane column has qualified integrity if the pressure drop per minute is less than 0.1 to 0.2 bar; if the integrity is unqualified, repeating the alkaline washing, water washing and integrity testing steps, and replacing the membrane column if the retest is still unqualified; the membrane column integrity testing can be performed before alkaline washing and water washing, but the integrity testing must be performed both before the ultrafiltration feeding and after the ultrafiltration is completed; the buffer solution equilibration process: connecting both the retentate end and the permeate end of the membrane column to the buffer solution storage tank, circularly rinsing the membrane column with the buffer solution for 5 to 10 min, and detecting the pH and conductivity of the filtrate in real time until the pH and conductivity of the filtrate are consistent with those of the buffer solution to complete the pre-equilibration of the membrane column; and / or, after the ultrafiltration is completed, the treatment method further comprises a membrane column recovery and preservation step for the ultrafiltration device; the membrane column recovery and preservation step comprises: after the ultrafiltration is completed, first washing the membrane column with sterile water for injection for 0.5 to 1 h, replacing with new sterile water for injection and washing again for 0.5 to 1 h, with the washing pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar, and draining the residual water for injection in the hollow inner cavity and the permeate end of the membrane column after the washing is completed; then circularly alkaline washing or soaking the membrane column with a 0.5 to 1 M sodium hydroxide solution for 0.5 to 1 h, with the alkaline washing pressure controlled at 0.1 to 1 bar, preferably 0.1 to 0.5 bar; finally, replacing the solution with a 0.1 to 0.2 M sodium hydroxide solution for soaking and preserving the membrane column.

8. A low-endotoxin gelatin microcarrier lysis buffer, characterized in that, Prepared by the method for reducing endotoxin in a lysate according to any one of claims 1 to 7; the finished product is in a sterile liquid form or a vacuum freeze-dried solid form; the endotoxin content in the lysate is ≤5 EU / mg, and the retention rate of proteolytic enzyme activity is 75% to 90%.

9. The use of the low-endotoxin gelatin microcarrier lysate according to claim 8 in the preparation of a reagent for harvesting cells after cell culture.

10. A cell-scale culture kit, characterized in that, Comprising the low-endotoxin gelatin microcarrier lysate according to claim 8, which is used for lysing gelatin microcarriers and non-destructively releasing and recovering viable cells.

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