A method for removing endotoxin from recombinant protein

Abstract

The present invention provides a method for removing endotoxin from a recombinant protein, comprising step S1, performing a first pretreatment on a first packing column; step S2, loading a sample; step S3, removing endotoxins in a sample to be processed, first using a second equilibrium liquid to rinse the first packing column after loading, until the ultraviolet absorption of the flow-through liquid at 280nm returns to the baseline, and then using isopropanol with a volume fraction of 10%-60% to rinse the first packing column after loading, until the ultraviolet absorption of the flow-through liquid at 280nm returns to the baseline, so as to remove endotoxins in the sample to be processed, and obtain a crude protein bound to the first packing column; the second equilibrium liquid comprises Triton X-114 with a volume fraction of 0.5%-1.25%. The present invention uses Triton X-114 in combination with isopropanol to improve the removal rate of endotoxins and reduce the removal cost.

Description

Technical Field

[0001] The invention relates to the technical field of protein purification, and in particular to a method for removing endotoxin from a recombinant protein. Background Art

[0002] Endotoxin is a component of the cell wall of Gram-negative bacteria, called lipopolysaccharide (LPS). Endotoxin is released only when the bacteria die and dissolve or when the cells are destroyed by artificial methods. Endotoxin is toxic to the host, and its toxic component is mainly lipid A. Even a trace amount of endotoxin entering an animal or human body can cause inflammation, ranging from fever to shock. Endotoxin contamination is common in proteins. For example, proteins expressed by E. coli will produce a large amount of endotoxin when the E. coli is broken. Therefore, it is necessary to remove endotoxin to ensure the safety of the protein.

[0003] Endotoxins have a complex structure and can combine with proteins in the solution to form a complex, which increases the difficulty of removal. At present, the methods for removing endotoxins from solutions mainly include liquid phase separation, molecular sieves, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, ultrafiltration, activated carbon adsorption, and distillation. Although these methods can remove some endotoxins, there are problems such as low removal rate or high cost, making it difficult to achieve industrialization. Summary of the invention

[0004] The present invention aims to provide a method for removing endotoxin from recombinant proteins by combining TritonX-114 with isopropanol to improve the removal rate of endotoxin and reduce the removal cost. The specific technical scheme is as follows:

[0005] A method for removing endotoxin from a recombinant protein comprises the following steps:

[0006] Step S1: Performing a first pretreatment on the first packing column

[0007] The first packing column is first rinsed with water, and then rinsed with a first balancing solution, so that the pH value and conductivity of the first packing column are the same as those of the first balancing solution; the packing in the first packing column is a nickel-containing packing; the first balancing solution includes components with the following concentrations: 20-50 mM tris(hydroxymethyl)aminomethane, 200-500 mM sodium chloride and 5-10 mM imidazole; the pH value of the first balancing solution is 7.3-7.5, and the conductivity is 10-25 mS / cm;

[0008] Step S2: Sample loading

[0009] First, the pH value and conductivity of the sample to be processed are adjusted to be the same as those of the first equilibrium solution, and then the adjusted sample to be processed flows to the first filler column to achieve the combination between the two; the sample to be processed is a recombinant protein solution containing histidine;

[0010] Step S3: Removal of endotoxins from samples to be processed

[0011] The first packing column after the sample is firstly rinsed with a second equilibrium liquid until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, and then the first packing column after the sample is rinsed with isopropanol with a volume fraction of 10%-60% until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, so as to remove endotoxins in the sample to be processed and obtain crude protein bound to the first packing column; the second equilibrium liquid comprises the following components in concentration and volume fraction: 20-50mM tris(hydroxymethyl)aminomethane, 200-500mM sodium chloride, 5-10mM imidazole and 0.5%-1.25% Triton X-114 by volume fraction.

[0012] In some embodiments, the method for removing endotoxin from a recombinant protein further comprises step S4, obtaining the target protein; step S4 comprises the following steps:

[0013] Step S4.1: Obtaining the first preformed protein by intermediate washing

[0014] Firstly, the crude protein bound to the first filler column is washed with a first equilibrium solution, and then the crude protein bound to the first filler column is washed with an eluent having a volume fraction of 10%-20% to remove impurity proteins and obtain a first preformed protein bound to the first filler column;

[0015] Step S4.2: Obtaining the first flow-through liquid

[0016] The first preformed protein in step S4.1 is washed with an eluent, and a first flow-through at a 280 nm ultraviolet absorption peak is collected, wherein the first flow-through contains the first preformed protein;

[0017] Step S4.3, post-processing the first flow-through to obtain the target protein.

[0018] In some embodiments, step S4.3 includes the following steps:

[0019] Step S4.3.1: Performing a second pretreatment on the second packing column

[0020] The second packing column is first rinsed with water, and then rinsed with the third balancing liquid, so that the pH value and conductivity of the second packing column are the same as those of the third balancing liquid; the packing in the second packing column is UniGel-80Q packing or QSEPHAROSE Fast Flow packing or UniGel-80DEAE; the third balancing liquid includes the following components at the following concentrations: 20-50 mM tris(hydroxymethyl)aminomethane and 200-500 mM sodium chloride; the pH value of the third balancing liquid is 7.3-7.5, and the conductivity is 10-25 mS / cm;

[0021] Step S4.3.2: Obtaining the second flow-through liquid

[0022] The pH value and conductivity of the first flow-through liquid are adjusted to be the same as those of the third equilibrium liquid, and the adjusted first flow-through liquid is flowed to the second filler column, and the second flow-through liquid at the 280nm ultraviolet absorption peak is collected; the second flow-through liquid contains a second preformed protein, and the second preformed protein is a protein that absorbs residual endotoxin in the first preformed protein through the second filler column;

[0023] Step S4.3.3, obtaining the third flow-through liquid

[0024] The second packing column is rinsed with the third equilibrium solution, and the third flow-through liquid at the 280nm ultraviolet absorption peak is collected; the third flow-through liquid contains the second preformed protein that has not been completely rinsed in step S4.3.2;

[0025] Step S4.3.4: Obtain target protein

[0026] The second flow-through liquid and the third flow-through liquid are mixed and then post-treated to obtain the target protein.

[0027] In some embodiments, the volume fraction of Triton X-114 in the second balanced solution is 0.75%-1.25%.

[0028] In some embodiments, the volume fraction of Triton X-114 in the second balanced solution is 0.9%-1.25%.

[0029] In some embodiments, the volume fraction of isopropyl alcohol used in step S3 is 20%-60%.

[0030] In some embodiments, the volume fraction of isopropyl alcohol used in step S3 is 30%-50%.

[0031] In some embodiments, before step S1, the first packing column is further subjected to a first pretreatment, specifically, the first packing column is firstly rinsed with a 0.1-0.5M sodium hydroxide solution; then the first packing column is rinsed with a 2M sodium chloride solution; finally, the first packing column is rinsed with 30% by volume isopropanol;

[0032] When performing the first pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 3-5 times the volume of the first packing column;

[0033] Before step S4.3.1, the second packing column is also subjected to a second pretreatment, specifically, the second packing column is firstly rinsed with a 1M sodium hydroxide solution; then the second packing column is rinsed with a 2M sodium chloride solution; finally, the second packing column is rinsed with 30% by volume isopropanol;

[0034] When performing the second pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 3-5 times the volume of the second packing column.

[0035] In some embodiments, the eluent comprises components at the following concentrations: 20-50 mM Tris, 200-500 mM sodium chloride and 200-500 mM imidazole; and the pH value of the eluent is 7.3-7.5.

[0036] In some embodiments, when performing the first pretreatment, the volume of water, the volume of the first equilibrated solution, the volume of the second equilibrated solution, the volume of isopropanol and the volume of the eluent are all 3-5 times the volume of the first filler column;

[0037] When performing the second pretreatment, the volume of water and the volume of the third equilibrium liquid are both 3-5 times the volume of the second packing column.

[0038] The application of the technical solution of the present invention has at least the following beneficial effects:

[0039] The method for removing endotoxin from recombinant protein described in the present invention, in step S3, the first filler column is washed successively with the second equilibrium liquid and the isopropanol with a volume fraction of 10%-60%. First, the amphoteric property of Triton X-114 in the second equilibrium liquid is used to remove the endotoxin in the recombinant protein, that is, Triton X-114 is a non-ionic surfactant with a low micelle concentration. At 4°C, it can bind to endotoxin and be miscible with water, and at 25°C, it and the endotoxin are precipitated from the aqueous phase together. However, the single use of Triton X-114 cannot reduce the endotoxin to the injection preparation standard (generally 0.50EU / mL) and Triton X-114 residue is prone to occur; secondly, the first filler column is washed with isopropanol with a volume fraction of 10%-60%, which can wash the residual Triton X-114 and the endotoxin remaining in the recombinant protein on the one hand, ensuring that the endotoxin is reduced to the injection preparation standard; on the other hand, it is friendly to the recombinant protein and will not cause the recombinant protein to denature. Therefore, the present invention uses TritonX-114 in combination with isopropanol to improve the removal rate of endotoxin and reduce the removal cost.

[0040] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0042] Figure 1 is the standard curve of endotoxin standard and absorbance value;

[0043] Figure 2 is the SDS-PAGE electrophoresis diagram of the target protein in Example 1 of the present invention;

[0044] Figure 3 is the SDS-PAGE electrophoresis diagram of the target protein in Example 2 of the present invention;

[0045] Figure 4 is the SDS-PAGE electrophoresis diagram of the target protein in Example 3 of the present invention;

[0046] Figure 5 is the SDS-PAGE electrophoresis diagram of the target protein in Example 4 of the present invention;

[0047] Figure 6 is the SDS-PAGE electrophoresis diagram of the target protein in Example 5 of the present invention;

[0048] Figure 7 is the SDS-PAGE electrophoresis diagram of the target protein in Example 6 of the present invention;

[0049] Figure 8 is the SDS-PAGE electrophoresis diagram of the target protein in Comparative Example 1 of the present invention;

[0050] Fig. 9 is the SDS-PAGE electrophoresis diagram of the target protein in Comparative Example 2 of the present invention;

[0051] Fig.10 is the SDS-PAGE electrophoresis diagram of the target protein in Comparative Example 4 of the present invention;

[0052] Where M is the molecular weight standard, the value "1" represents the target protein, and the value "2" represents 1 mg / mL BSA. DETAILED DESCRIPTION

[0053] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0054] Embodiment 1:

[0055] A method for removing endotoxin from a recombinant protein comprises the following steps:

[0056] Step S1: Performing a first pretreatment on the first packing column

[0057] The first packing column is first rinsed with water, and then rinsed with the first balancing solution, so that the pH value and conductivity of the first packing column are the same as those of the first balancing solution; the packing in the first packing column is UniNTA-80Ni packing of Nano Micro; the first balancing solution includes components of the following concentrations: 50 mM tris(hydroxymethyl)aminomethane, 200 mM sodium chloride and 10 mM imidazole; the pH value of the first balancing solution is 7.37, and the conductivity is 23.45 mS / cm;

[0058] Step S2: Sample loading

[0059] First, the pH value and conductivity of the sample to be processed are adjusted to be the same as those of the first equilibrium solution, and then the adjusted sample to be processed flows to the first filler column to achieve the combination between the two; the sample to be processed is a recombinant protein solution containing histidine;

[0060] Step S3: Removal of endotoxins from samples to be processed

[0061] The first packing column after the sample is first rinsed with a second equilibrium liquid until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, and then the first packing column after the sample is rinsed with isopropanol with a volume fraction of 35% until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, so as to remove endotoxins in the sample to be processed and obtain crude protein bound to the first packing column; the second equilibrium liquid includes the following components in concentration and volume fraction: 50mM tris(hydroxymethyl)aminomethane, 200mM sodium chloride, 10mM imidazole and 0.9% volume fraction of Triton X-114.

[0062] Step S4, obtaining the target protein;

[0063] The step S4 comprises the following steps:

[0064] Step S4.1: Obtaining the first preformed protein by intermediate washing

[0065] Firstly, the crude protein bound to the first filler column is washed with the first equilibrium solution, and then the crude protein bound to the first filler column is washed with the eluent having a volume fraction of 10% to remove impurity proteins and obtain the first preformed protein bound to the first filler column;

[0066] Step S4.2: Obtaining the first flow-through liquid

[0067] The first preformed protein in step S4.1 is washed with an eluent, and a first flow-through at a 280 nm ultraviolet absorption peak is collected, wherein the first flow-through contains the first preformed protein;

[0068] Step S4.3, post-processing the first flow-through to obtain the target protein.

[0069] Step S4.3 includes the following steps:

[0070] Step S4.3.1: Performing a second pretreatment on the second packing column

[0071] The second packing column is first rinsed with water, and then rinsed with the third balancing solution, so that the pH value and conductivity of the second packing column are the same as those of the third balancing solution; the packing in the second packing column is QSEPHAROSE Fast Flow packing of Cytiva; the third balancing solution includes components with the following concentrations: 50 mM tris(hydroxymethyl)aminomethane and 200 mM sodium chloride; the pH value of the third balancing solution is 7.37, and the conductivity is 23.45 mS / cm;

[0072] Step S4.3.2: Obtaining the second flow-through liquid

[0073] The pH value and conductivity of the first flow-through liquid are adjusted to be the same as those of the third equilibrium liquid, and the adjusted first flow-through liquid is flowed to the second filler column, and the second flow-through liquid at the 280nm ultraviolet absorption peak is collected; the second flow-through liquid contains a second preformed protein, and the second preformed protein is a protein that absorbs residual endotoxin in the first preformed protein through the second filler column;

[0074] Step S4.3.3, obtaining the third flow-through liquid

[0075] The second packing column is rinsed with the third equilibrium solution, and the third flow-through liquid at the 280nm ultraviolet absorption peak is collected; the third flow-through liquid contains the second preformed protein that has not been completely rinsed in step S4.3.2;

[0076] Step S4.3.4: Obtain target protein

[0077] The second flow-through liquid and the third flow-through liquid are mixed and then post-processed to obtain the target protein, wherein the post-processing is to ultrafilter and concentrate the second flow-through liquid and the third flow-through liquid and then freeze-dry them to obtain freeze-dried powder of the protein.

[0078] Before step S1, the first packing column is also subjected to a first pretreatment to minimize the content of endotoxin introduced from the exogenous source. Specifically, the first packing column is first rinsed with a 0.3M sodium hydroxide solution; then the first packing column is rinsed with a 2M sodium chloride solution; finally, the first packing column is rinsed with 30% by volume isopropanol;

[0079] When performing the first pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 5 times the volume of the first packing column;

[0080] Before step S4.3.1, the second packing column is also subjected to a second pretreatment, specifically, the second packing column is firstly rinsed with a 1M sodium hydroxide solution; then the second packing column is rinsed with a 2M sodium chloride solution; finally, the second packing column is rinsed with 30% by volume isopropanol;

[0081] When performing the second pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 5 times the volume of the second packing column.

[0082] The eluent includes components with the following concentrations: 50 mM Tris, 200 mM sodium chloride and 200 mM imidazole; the pH value of the eluent is 7.37.

[0083] When performing the first pretreatment, the volume of water, the volume of the first equilibrium liquid, the volume of the second equilibrium liquid, the volume of isopropanol and the volume of the eluent are all 5 times the volume of the first packing column;

[0084] When performing the second pretreatment, the volume of water and the volume of the third equilibrium liquid are both 5 times the volume of the second packing column.

[0085] When flushing the first packing column and the second packing column, the chromatography flow rate is controlled to be 50-200 cm / h.

[0086] In Example 1, the method for obtaining the sample to be processed (which is a conventional method) is as follows:

[0087] Firstly, the Escherichia coli expression system BL21 (DE3) and pet-24a vector were used to construct a recombinant protein expression platform.

[0088] Specifically, the gene sequence of the target protein (the target protein is recombinant collagen, with a theoretical molecular weight of approximately 25 KDa and an apparent molecular weight of 23 KDa) is synthesized in one step by chemical synthesis, and then subcloned into an expression vector and transferred to a DH5α competent strain and a BL21 (DE3) competent strain, and the strains that are positive in antibiotic plate identification, colony PCR identification, and sequencing identification are stored in a -80°C refrigerator for use.

[0089] Secondly, bacterial shake flask fermentation

[0090] Specifically, LB medium was used for culture and IPTG was used for induction to obtain bacterial culture solution.

[0091] Finally, bacterial cells are broken

[0092] Specifically, the cells are disrupted by an ultrasonic disruptor, the disrupted liquid is centrifuged to collect the supernatant, and the supernatant is filtered using a 0.45 μm filter membrane to obtain the sample to be processed.

[0093] Embodiment 2:

[0094] Different from Example 1, in step S1, the filler in the first filler column is UniIDA-80Ni filler produced by Nano Micro Corporation; in step S3, the volume fraction of Triton X-114 in the second equilibrium solution is 0.75%, and the volume fraction of isopropanol is 25%.

[0095] In Example 2, the method for obtaining the sample to be processed (which is a conventional method) is as follows:

[0096] Firstly, the Escherichia coli expression system BL21 (DE3) and pet-24a vector were used to construct a recombinant protein expression platform.

[0097] The specific process is the same as in Example 1.

[0098] Secondly, bacterial shake flask fermentation

[0099] Specifically, LB medium was used for culture and lactose was used for induction to obtain bacterial culture fluid.

[0100] Finally, bacterial cells are broken

[0101] Specifically, the cells are disrupted by an ultrasonic disruptor, the disrupted liquid is centrifuged to collect the supernatant, and the supernatant is filtered using a 0.45 μm filter membrane to obtain the sample to be processed.

[0102] Embodiment 3:

[0103] Different from Example 1, in step S1, the filler in the first filler column is UniIDA-80Ni filler produced by Nanovitamin Corporation; in step S3, the volume fraction of Triton X-114 in the second equilibrium solution is 1.25%, and the volume fraction of isopropanol is 60%; in step S4.3.1, the filler in the second filler column is UniGel-80Q filler produced by Nanovitamin Corporation.

[0104] In Example 3, the method for obtaining the sample to be processed (which is a conventional method) is as follows:

[0105] Firstly, the Escherichia coli expression system BL21 (DE3) and pet-24a vector were used to construct a recombinant protein expression platform.

[0106] The specific process is the same as in Example 1.

[0107] Secondly, high-density bacterial fermentation

[0108] Specifically, the bacterial culture solution is obtained by adopting batch feeding.

[0109] Finally, bacterial cells are broken

[0110] Specifically, the cells are broken by using an ATS homogenizer, the broken liquid is centrifuged to collect the supernatant, the supernatant is ultrafiltered by a three-stage membrane separation system to collect the permeate, and the sample to be processed is obtained.

[0111] Embodiment 4:

[0112] Different from Example 1, in step S3, the volume fraction of Triton X-114 in the second equilibrium solution is 0.5%.

[0113] Embodiment 5:

[0114] Different from Example 1, in step S3, the volume fraction of isopropanol is 10%.

[0115] Embodiment 6:

[0116] Different from Example 1, in step S3, the volume fraction of isopropanol is 60%.

[0117] Comparative Example 1:

[0118] Different from Example 1, in step S1, the filler in the first filler column is UniIDA-80Ni filler produced by Nano Micro Corporation; in step S3, the volume fraction of Triton X-114 in the second equilibrium solution is 1%, and isopropanol is not used.

[0119] In Comparative Example 1, the method for obtaining the sample to be processed is the same as that in Example 1.

[0120] Comparative Example 2:

[0121] Different from Example 1, in step S3, the second balancing liquid is not used, and the volume fraction of isopropanol is 30%.

[0122] In Comparative Example 2, the method for obtaining the sample to be processed is the same as that in Example 1.

[0123] Comparative Example 3:

[0124] Different from Example 1, in step S3, the volume fraction of Triton X-114 in the second equilibrium solution is 1.5%, and the volume fraction of isopropanol is 80%; steps S4.3.1 to S4.3.4 are not set.

[0125] In Comparative Example 3, the method for obtaining the sample to be processed is the same as that in Example 1.

[0126] Comparative Example 4: Different from Example 1, in step S3, isopropanol is replaced with ethanol with a volume fraction of 30%. In Comparative Example 4, the method for obtaining the sample to be processed is the same as that in Example 1.

[0127] The endotoxin content in the target protein in Examples 1-3 and Comparative Examples 1-4 was detected. The specific detection method is as follows: horseshoe crab reagent (dynamic colorimetric method) is carried out in accordance with Section 1143 of the fourth volume of the 2020 edition of the Pharmacopoeia of the People's Republic of China "Bacterial Endotoxin Test Method (Photometric Method)". Among them, the standard curve is Figure 1 .Depend on Figure 1 The linear calculation formula of the endotoxin standard and the absorbance value is obtained. Then, the specific content of endotoxin in Table 1 can be calculated by measuring the absorbance value of the sample. The measurement results are shown in Table 1.

[0128] The protein purity and yield of the target protein in Examples 1-3 and Comparative Examples 1-4 were tested. The specific testing method is as follows: SDS-PAGE gel electrophoresis was used to verify the protein purity and yield of the purified flow-through. The results are shown in Table 1 and Figure 2-10 .

[0129] Table 1

[0130]

[0131] From the data in Table 1, we know that:

[0132] Compared with Comparative Examples 1-4, the present invention can reduce the endotoxin content in the treated sample to below 0.5EU / mL by using Examples 1-6. This shows that the present invention can effectively reduce the endotoxin content in the sample by combining isopropanol and Triton X-114 and controlling the appropriate dosage.

[0133] In addition, according to the purity and yield data of the target protein in Table 1 and Figure 2-10 It is known that the method for removing endotoxin from recombinant protein of the present invention will not affect the purity and yield of the target protein while ensuring effective removal of endotoxin.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for removing endotoxin from a recombinant protein, characterized in that: The steps include: Step S1: Performing a first pretreatment on the first packing column The first packing column is first rinsed with water, and then rinsed with a first balancing solution, so that the pH value and conductivity of the first packing column are the same as those of the first balancing solution; the packing in the first packing column is a nickel-containing packing; the first balancing solution includes components with the following concentrations: 20-50 mM tris(hydroxymethyl)aminomethane, 200-500 mM sodium chloride and 5-10 mM imidazole; the pH value of the first balancing solution is 7.3-7.5, and the conductivity is 10-25 mS / cm; Step S2: Sample loading First, the pH value and conductivity of the sample to be processed are adjusted to be the same as those of the first equilibrium solution, and then the adjusted sample to be processed flows to the first filler column to achieve the combination between the two; the sample to be processed is a recombinant protein solution containing histidine; Step S3: Removal of endotoxins from samples to be processed The first packing column after the sample is first rinsed with a second equilibrium solution until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, and then the first packing column after the sample is rinsed with isopropanol with a volume fraction of 10%-60% until the ultraviolet absorption of the flow-through liquid at 280 nm returns to the baseline, so as to remove endotoxins in the sample to be processed and obtain crude protein bound to the first packing column; the second equilibrium solution comprises the following components in concentration and volume fraction: 20-50 mM tris(hydroxymethyl)aminomethane, 200-500 mM sodium chloride, 5-10 mM imidazole and 0.5%-1.25% Triton X-114 by volume fraction; The method also includes step S4, obtaining the target protein; step S4 includes the following steps: Step S4.1: Obtaining the first preformed protein by intermediate washing Firstly, the crude protein bound to the first filler column is washed with a first equilibrium solution, and then the crude protein bound to the first filler column is washed with an eluent having a volume fraction of 10%-20% to remove impurity proteins and obtain a first preformed protein bound to the first filler column; Step S4.2: Obtaining the first flow-through liquid The first preformed protein in step S4.1 is washed with an eluent, and a first flow-through at a 280 nm ultraviolet absorption peak is collected, wherein the first flow-through contains the first preformed protein; Step S4.3, post-processing the first flow-through to obtain the target protein.

2. The method for removing endotoxin from recombinant protein according to claim 1, characterized in that: Step S4.3 includes the following steps: Step S4.3.1: Performing a second pretreatment on the second packing column The second packing column is first rinsed with water, and then rinsed with the third balancing liquid, so that the pH value and conductivity of the second packing column are the same as those of the third balancing liquid; the packing in the second packing column is UniGel-80Q packing or QSEPHAROSE Fast Flow packing or UniGel-80DEAE; the third balancing liquid includes the following components at the following concentrations: 20-50 mM tris(hydroxymethyl)aminomethane and 200-500 mM sodium chloride; the pH value of the third balancing liquid is 7.3-7.5, and the conductivity is 10-25 mS / cm; Step S4.3.2: Obtaining the second flow-through liquid The pH value and conductivity of the first flow-through liquid are adjusted to be the same as those of the third equilibrium liquid, and the adjusted first flow-through liquid is flowed to the second filler column, and the second flow-through liquid at the 280nm ultraviolet absorption peak is collected; the second flow-through liquid contains a second preformed protein, and the second preformed protein is a protein that absorbs residual endotoxin in the first preformed protein through the second filler column; Step S4.3.3, obtaining the third flow-through liquid The second packing column is rinsed with the third equilibrium solution, and the third flow-through liquid at the 280nm ultraviolet absorption peak is collected; the third flow-through liquid contains the second preformed protein that has not been completely rinsed in step S4.3.2; Step S4.3.4: Obtain target protein The second flow-through liquid and the third flow-through liquid are mixed and then post-treated to obtain the target protein.

3. The method for removing endotoxin from recombinant protein according to claim 1, characterized in that: The volume fraction of Triton X-114 in the second equilibrium solution is 0.75%-1.25%.

4. The method for removing endotoxin from recombinant protein according to claim 3, characterized in that: The volume fraction of Triton X-114 in the second equilibrium solution is 0.9%-1.25%.

5. The method for removing endotoxin from recombinant protein according to claim 1, characterized in that: The volume fraction of isopropyl alcohol used in step S3 is 20%-60%.

6. The method for removing endotoxin from recombinant protein according to claim 5, characterized in that: The volume fraction of isopropyl alcohol used in step S3 is 30%-50%.

7. The method for removing endotoxin from recombinant protein according to claim 2, characterized in that: Before step S1, the first packing column is subjected to a first pretreatment, specifically, the first packing column is firstly rinsed with a 0.1-0.5M sodium hydroxide solution; then the first packing column is rinsed with a 2M sodium chloride solution; finally, the first packing column is rinsed with 30% by volume isopropanol; When performing the first pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 3-5 times the volume of the first packing column; Before step S4.3.1, the second packing column is also subjected to a second pretreatment, specifically, the second packing column is firstly rinsed with a 1M sodium hydroxide solution; then the second packing column is rinsed with a 2M sodium chloride solution; finally, the second packing column is rinsed with 30% by volume isopropanol; When performing the second pretreatment, the volume of the sodium hydroxide solution, the volume of the sodium chloride solution and the volume of the isopropanol are all 3-5 times the volume of the second packing column.

8. The method for removing endotoxin from recombinant protein according to claim 1, characterized in that: The eluent comprises components with the following concentrations: 20-50 mM tris(hydroxymethyl)aminomethane, 200-500 mM sodium chloride and 200-500 mM imidazole; the pH value of the eluent is 7.3-7.

5.

9. The method for removing endotoxin from recombinant protein according to any one of claims 1 to 8, characterized in that: When performing the first pretreatment, the volume of water, the volume of the first equilibrium liquid, the volume of the second equilibrium liquid, the volume of isopropanol and the eluent are all 3-5 times the volume of the first filler column; When performing the second pretreatment, the volume of water and the volume of the third equilibrium liquid are both 3-5 times the volume of the second filler column.

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