Method for removing endotoxin in protease

Abstract

The invention provides a method for removing endotoxin in TEV protease, and belongs to the field of bioengineering. The method comprises the following steps: firstly, expressing TEV protein by using a prokaryotic expression system, and carrying out ultrasonication and affinity chromatography to obtain a high-purity TEV protein solution containing endotoxin; then, removing residual thalli in the TEV protein solution by using a sterilization filter with the aperture of 0.22 mu m; and then, loading the sterilized sample solution into a chromatographic column with a medium of Purotoxin-Remote, flushing the protein solution out of the chromatographic column by a chromatographic buffer solution, collecting an effluent when a UV280 detection value is greater than or equal to 10mAu, and carrying out biochemical detection on the effluent. Detection results show that the removal efficiency of the endotoxin in the sample protein by the method is as high as 99.8%, and the recovery rate liquid of the TEV protein is as high as 96%. In conclusion, the chromatography method for removing the endotoxin in the TEV protein is simple in step, suitable for large-scale production and remarkable in overall effect.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a method for removing endotoxin from protease. Technical Background

[0002] TEV protease is a cysteine ​​protease derived from tobacco etch virus (TEV). It can specifically recognize and cleave the ENLYFQ▼G sequence, so that only one amino acid residue remains at the N-terminus of the target protein after enzyme cleavage, which can minimize the impact on the structure and function of the target protein. In addition, TEV protease has a wide range of pH and temperature tolerance, with a pH tolerance range of 4-8.0 and a temperature tolerance of 4-34°C. It also has varying degrees of tolerance to some common additives that increase protein solubility or stability (ethylene glycol, EGTA, detergents, and reducing agents). Therefore, TEV protease is an ideal tool enzyme for removing fusion tags from recombinant proteins.

[0003] Recombinant proteins produced in large quantities using biological culture technology are an indispensable part of public health. The recombinant protein is generally obtained by culturing bacteria upstream and inducing them to express the target protein, and then purifying it downstream. In the production process of biological drugs, when the bacteria die and dissolve or the bacterial cells are destroyed by artificial methods, endotoxins are released into the lysate. Endotoxins are components of the cell walls of Gram-negative bacteria, which can interact with biological molecules (including target therapeutic compounds) and form chemical bonds, making them difficult to remove. Moreover, residual endotoxins can cause fever, microcirculatory disorders, endotoxin shock, and disseminated intravascular coagulation, etc. Therefore, the content of microbial endotoxins in biological products becomes a key indicator of whether the recombinant protein can be used clinically. If the recombinant protein obtained after multiple experimental steps is contaminated when it is digested by TEV protease containing endotoxin, it not only wastes time, but also causes the product to fail to meet clinical standards, resulting in huge economic losses.

[0004] According to the structural properties of endotoxins, the commonly used endotoxin removal methods include ultrafiltration, adsorption, chromatography and phase separation, but each method has its limitations. For example, ultrafiltration produces a large amount of waste liquid, adsorption has poor removal effect, and phase separation has low protein recovery rate. In comparison, chromatography has a relative advantage in the above methods. Chinese patents "A method for removing endotoxins from recombinantly expressed porcine parvovirus VP2 protein in Escherichia coli" (application publication number CN111606978A) and "A process for removing endotoxins in the purification process of recombinant human serum albumin" (application publication number CN117088962A) respectively use chromatography to remove endotoxins from proteins, but the two methods require multiple pretreatments of the sample solution before removing endotoxins, which not only has cumbersome steps and increases time cost, but also loses the target protein. On the other hand, in the field of proteins, due to the different characteristics of proteins and production purposes, generally one protein has one solution, and there are few studies on removing endotoxins from TEV protease. Therefore, there is an urgent need for a method for removing endotoxin from TEV protease that is efficient, simple, and suitable for large-scale production. Summary of the invention

[0005] In response to the above needs, the present invention provides a method for removing endotoxins from TEV protease, which belongs to the field of bioengineering. The method steps are as follows: first, TEV protein is expressed using a prokaryotic expression system, and a high-purity but endotoxin-containing TEV protein solution is obtained after ultrasonic fragmentation and affinity chromatography; then, a filter with a pore size of 0.22 μm is used to remove the residual bacteria in the TEV protein solution; then, the sterilized sample solution is loaded into a chromatography column with a medium of Purose Endotoxin-Removal, and then the protein solution is flushed out of the chromatography column by the chromatography buffer, and the effluent is collected when the UV280 detection value is ≥10mAu, and the effluent is subjected to biochemical detection. The test results show that the endotoxin removal efficiency of the sample protein of the above method is as high as 99.8%, and the recovery rate of TEV protein is also as high as 96%. In summary, the chromatography method for removing endotoxins from TEV protein provided by the present invention has simple steps, is suitable for large-scale production, and has significant overall effect.

[0006] On the one hand, the present invention provides a method for removing endotoxin from TEV protease, the method comprising loading a TEV protein solution from which endotoxin needs to be removed onto a chromatography column whose filler is Purose Endotoxin-Removal, and then using a chromatography buffer to drive the protein solution to flow out of the chromatography column at a certain flow rate, collecting the effluent, and the resulting effluent is a TEV protein solution free of endotoxin; the molecular weight of the TEV protein is 26.96 kDa, the amino acid sequence is shown in the sequence listing SEQ ID NO: 1, and the nucleotide sequence is shown in the sequence listing SEQ ID NO: 2.

[0007] The present invention first optimizes the method for removing endotoxins from TEV protein solutions and tries three methods (chromatography, phase separation and ultrafiltration tube adsorption). Considering the endotoxin removal effect, protein purity and recovery rate, the chromatography method best meets the expected goal, that is, it ensures that endotoxins are completely removed and the protein recovery rate and purity are also very high.

[0008] Next, the present invention tried two chromatographic fillers, Purose Endotoxin-Removal and EzScreenHeparin FF, to further optimize the efficiency of chromatographic endotoxin removal. The results showed that the overall effect (protein recovery rate and endotoxin removal rate) of EzScreen Heparin FF was not as good as Purose Endotoxin-Removal. This may be because the cross-linked ligands of the filler are not suitable for binding with lipopolysaccharide. Therefore, Purose Endotoxin-Removal filler is preferred.

[0009] Furthermore, the components of the chromatography buffer are Tris-HCl, MgCl2 and NaCl.

[0010] The chromatography buffer plays the role of maintaining the activity of the target protein and reducing the non-specific adsorption of the target protein by the chromatography filler. Each component in the buffer is indispensable. The physicochemical properties (such as isoelectric point) of each protein are different. Some proteins are more stable in an acidic environment, while some proteins are more adapted to an alkaline environment. The preferred Tris-HCl of the present invention provides the most suitable pH environment for the TEV protein; MgCl2 is an activating factor for a commonly used protease, which can significantly improve the activity of the enzyme; on the one hand, similar to the effect of Tris-HCl, sodium chloride provides osmotic pressure and maintains the structure of the target protein. On the other hand, increasing the concentration of sodium chloride can reduce the non-specific adsorption of the target protein by the chromatography medium, because the chromatography filler can non-specifically adsorb other substances such as proteins in addition to adsorbing endotoxins, and competitive binding occurs when the sodium chloride content in the solution is high, reducing the non-specific adsorption of the target protein by the chromatography medium, thereby improving the recovery rate of the target protein. When the concentration of sodium chloride is too high, it will cause the target protein to denature. Therefore, the concentration of sodium chloride in the chromatography buffer also needs to be at a balance point, that is, the TEV protein will not precipitate due to denaturation, and its recovery rate can also be guaranteed.

[0011] Preferably, the concentration of NaCl is 25 mM to 2 M, more preferably, 25 to 200 mM, and even more preferably, 150 mM.

[0012] The present invention sets a concentration gradient of NaCl, proving that as the concentration of NaCl increases, the recovery rate of TEV protein also increases. However, when the concentration of NaCl exceeds a critical value (200 mM), the TEV protein denatures and then precipitates. Therefore, the optimal concentration of NaCl in the chromatography buffer is 150 mM.

[0013] Preferably, the concentration of MgCl2 is 1-100 mM, more preferably, 1-10 mM, and even more preferably, 2 mM.

[0014] Preferably, the concentration of Tris-HCl is 5 to 500 mM, more preferably, 10 to 50 mM, and even more preferably, 20 mM. At this time, the pH of Tris-HCl is 8.0.

[0015] Furthermore, the flow rate of the chromatography buffer is 0.01 to 1.0 mL / min, more preferably, 0.01 to 0.1 mL / min, and even more preferably, 0.1 mL / min.

[0016] It is understandable that the flow rate of the chromatography buffer for flushing the sample will affect the time the latter stays in the chromatography tube, thereby affecting the removal of endotoxins, that is, when the flow rate is too high, the chromatography medium has not yet fully contacted with the endotoxins, and the endotoxins are washed away by the newly added chromatography buffer, causing the endotoxins to continue to remain in the TEV protein solution; and when the flow rate of the chromatography buffer is too low, the chromatography purification time will be extended. At the same time, proteins are easily degraded substances. The longer the purification time, the lower the protein recovery rate. Therefore, the flow rate of the chromatography buffer needs to be at a balance point. After multiple experimental verifications, when the sample flow rate is 0.01-0.1 mL / min, the overall effect is better, and more preferably, when the chromatography buffer is 0.1 mL / min.

[0017] Furthermore, the TEV protein solution needs to be sterilized before loading, and the sterilization treatment refers to filtering the TEV protein solution using a sterilizing filter with a pore size of 0.2 to 0.45 μm; preferably, a sterilizing filter with a pore size of 0.22 μm is used.

[0018] It is understandable that because the filler in the chromatography column can only adsorb endotoxins but not bacteria, if the sample protein solution is not sterilized in advance, even if the chromatography method can remove the endotoxins in the original solution to the maximum extent, the bacteria in the solution will continue to produce new endotoxins, resulting in the final TEV protein solution having too high endotoxins, which cannot meet the safety standards of biological products and then needs to be reworked, resulting in time and economic losses.

[0019] The present invention adopts two methods, filtration sterilization and heating inactivation, to reduce the influence of live bacteria on the change of endotoxin content of TEV protein solution. The results show that filtration sterilization has better effect and is milder and will not cause structural changes of target protein.

[0020] Furthermore, the effluent refers to the liquid flowing out of the chromatography column when the UV280 detection value is ≥10mAu. When the 280nm wavelength absorption value of the UV detector is higher than 10mAU, it means that the protein has flowed out of the chromatography column, and collection begins at this time; when it drops to 10mAu again, it means that the protein liquid has completely flowed out of the chromatography column, and collection is terminated at this time; this can improve the protein recovery rate.

[0021] The experimental steps for removing endotoxin from TEV protease provided by the present invention are as follows:

[0022] A. Preparation of TEV protein solution: TEV protein was expressed by prokaryotic expression system, and TEV protein solution was obtained after ultrasonic disruption, high-speed centrifugation and affinity chromatography;

[0023] It is understandable that step A is a routine operation for obtaining a TEV protein solution. In addition to the method selected in the above steps, other protein purification methods can be used as needed to obtain a sample solution that needs to be treated with endotoxin, and subsequent operations (steps B and C) are also applicable to these sample solutions.

[0024] Furthermore, the protein buffer in the TEV protein solution described in step A is 20mM Tris-HCl (pH8.0), 2mM MgCl2 and 25mM NaCl. The function of the protein buffer is to maintain the target protein in the most stable state, that is, the protein structure does not change and the activity is not lost; the composition of the protein buffer is consistent with that of the chromatography buffer, and the difference lies in the NaCl concentration. 25mM NaCl is most suitable for TEV protein, but in the chromatography process, in order to take into account the nonspecific adsorption of the chromatography filler, the NaCl concentration needs to be increased to 150mM, which is high for TEV protein, but TEV protein can be in the NaCl solution of this concentration for a short time.

[0025] B. Sterilize the TEV protein solution in step A in the same manner as described above;

[0026] C. Chromatographic column separation: The sterilized protein solution in step B is loaded onto the chromatographic medium, and the effluent from the chromatography column is collected to obtain a TEV protein solution from which endotoxin has been removed. The specific operation is the same as described above.

[0027] In order to prepare endotoxin-free TEV protein that meets the standards of biological products, in addition to adopting a method to effectively remove endotoxins in the target protein solution, it is also necessary to prevent secondary contamination, that is, all instruments and reagents in the above-mentioned endotoxin removal process need to be treated with endotoxin removal before use. Ordinary instruments and reagents can be treated by high-temperature sterilization, while the chromatographic filler base ball is made of agarose and cannot be treated at high temperature. Therefore, an alkaline soaking method is used to remove endotoxins in the chromatographic column. Preferably, the chromatographic column is pretreated with a 0.5-2.0M NaOH solution, and a more preferred NaOH concentration is 1M.

[0028] On the other hand, the present invention provides a chromatography buffer for improving protein recovery and / or improving endotoxin removal efficiency, wherein the chromatography buffer has a formula of: 5-500mM Tris-HCl, 1-100mM MgCl2, 25mM-2M NaCl; preferably, 10-50mM Tris-HCl, 1-10mM MgCl2, 25-200mM NaCl, the chromatography medium is Purose Endotoxin-Removal, and the protein is TEV protease. Preferably, the components and concentrations of the chromatography buffer are: 20mM Tris-HCl (pH 8.0), 2mM MgCl2, and 150mM NaCl.

[0029] On the other hand, the present invention provides a use of a chromatography medium for improving protein recovery rate and / or improving endotoxin removal efficiency, wherein the chromatography medium is Purose Endotoxin-Removal, and the protein is TEV protease.

[0030] The present invention screens out a chromatographic filler capable of removing endotoxins, and proves through experiments that the filler has the advantages of high endotoxin removal rate, high protein recovery rate and simple operation.

[0031] The beneficial effects of the present invention include:

[0032] 1. The method provided by the present invention has a significant effect of removing endotoxin and a high protein recovery rate; the steps are simple, the technical requirements for operators are low, and it is suitable for scale-up production;

[0033] 2. The present invention provides a method for purifying TEV protein. First, the present invention adopts three different methods (chromatography, phase separation and ultrafiltration tube adsorption) to purify TEV protein, and screens out chromatography as the best method; then the chromatographic filler, chromatography buffer composition and flow rate and sterilization method are optimized to create a chromatography method that is most suitable for removing TEV protein endotoxins, with protein recovery rate and endotoxin removal rate reaching 96% and 99.8% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 :Changes in TEV protein purity before and after treatment with the Purose Endotoxin-Removal column method; M: Marker, lane 1: sample solution before treatment, lane 2: sample solution after treatment;

[0036] Figure 2 : Changes in TEV protein purity before and after treatment with Triton-114 phase separation method; M: Marker, lane 1: sample solution before treatment, lane 2: sample solution after treatment;

[0037] Figure 3 : Changes in TEV protein purity before and after treatment with High Capacity Endotoxin Removal centrifuge tube method; M: Marker, lane 1: sample solution before treatment, lane 2: sample solution after treatment;

[0038] Figure 4 :Changes in TEV protein purity before and after treatment with EzScreen Heparin FF column; M: Marker, lane 1: sample solution before treatment, lane 2: sample solution after treatment;

[0039] Figure 5 : Electrophoresis of TEV protein purified by 4 different methods; Lane 1: sample solution before treatment, M: Marker, Lane 2: sample solution treated by Purose Endotoxin-Removal column method, Lane 3: sample solution treated by Triton-114 phase separation method, Lane 4: Lane 5: Sample solution treated by High Capacity Endotoxin Removal centrifuge tube method, Lane 5: Sample solution treated by EzScreen Heparin FF column method. DETAILED DESCRIPTION

[0040] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] Example 1: Obtaining TEV recombinant protein

[0043] The preparation process of the recombinant TEV hydrolyzing protease to be detoxified is as follows:

[0044] 1. Use prokaryotic cells (preferably BL21 (DE3) strain in this embodiment) to express recombinant TEV protease (the amino acid sequence of TEV protease is shown in the sequence list SEQ ID NO: 1, and the nucleotide sequence is shown in the sequence list SEQ ID NO: 2), induce the expression of the target protein, and then collect the bacterial solution;

[0045] 2. After centrifugation at 6,000 g for 30 min, the cells were harvested and resuspended in buffer.

[0046] 3. After ultrasonic disruption of the cells, centrifuge at 12,000 g for 60 min and harvest the supernatant;

[0047] 4. Affinity chromatography purification to obtain high-purity recombinant TEV protease, the affinity chromatography operation steps are as follows:

[0048] 4.1. Fill the chromatographic column with Ni Sepharose 6FF (Cytiva) filler, with a column volume of 40-50 mL and a filling pressure of 0.1 MPa;

[0049] 4.2. Prepare equilibration and elution buffer:

[0050] Equilibration buffer: 20 mM Tris-HCl (pH 8.0), 25 mM imidazole, 0.5 M NaCl, 2 mM MgCl2;

[0051] Elution buffer: 20 mM Tris-HCl (pH 8.0), 500 mM imidazole, 0.5 M NaCl, 2 mM MgCl2;

[0052] (The above reagents are all from Sinopharm Group Shanghai Test)

[0053] 4.3. After the chromatography column is connected to the chromatography system (AKTA), rinse the chromatography column with 5 column volumes (5CV) of purified water, and then equilibrate the chromatography column with 5CV of equilibration buffer. After the data of the equipment are stable and the baseline is zeroed, prepare to load the protein solution supernatant and discard the flow-through;

[0054] 4.4. After loading, continue to rinse the column with 5CV equilibration buffer. After the A280 absorption value stabilizes, set 10CV 20% elution buffer to elute the impurities until the A280 absorption value stabilizes.

[0055] 4.5. Set 2CV 50% elution buffer to elute the target protein. Collection range: start collecting when the A280 absorbance value is greater than 100mAu, and stop collecting when the A280 absorbance value of the target peak drops to less than 120mAu.

[0056] It should be understood that this embodiment only provides a basic process for obtaining a TEV protein sample to be detoxified, and crude TEV protein obtained by other means is also applicable to the method for removing endotoxin from TEV protein provided by the present invention.

[0057] Example 2: Removal of endotoxin from TEV recombinant protein by column chromatography

[0058] This example provides a preferred Purose Endotoxin-Removal chromatography method to remove endotoxins from TEV recombinant protein.

[0059] The specific experimental steps for removing endotoxin from the supernatant containing the TEV recombinant protein after ultrasonic disruption and affinity chromatography purification in Example 1 are as follows:

[0060] 1. Filling the chromatographic column:

[0061] Take 25mL of Purose Endotoxin-Removal chromatographic filler (Jiaxing Qianchun), mix well and remove the preservation solution by suction; after washing three times with purified water, add 25mL of purified water to resuspend the filler, mix well and pour into a 10 / 20 empty chromatographic column (Borgron BXP10 / 20). Use the constant pressure column loading program of the chromatography equipment (AKTAPurifier 100) to control the column loading pressure to 0.1Mpa, and complete the column loading after the column bed is stable.

[0062] 2. Prepare sterile endotoxin-free buffer:

[0063] In an ultra-clean workbench, dry heat sterilized glassware and injection water were used to prepare chromatography buffer. The chromatography buffer formula was: 20 mM Tris-HCl (pH 8.0) (Sinopharm Group Shanghai Test), 2 mM MgCl2 (Sinopharm Group Shanghai Test), 150 mM NaCl (Sinopharm Group Shanghai Test).

[0064] 3. Chromatography system pipeline and chromatographic column pretreatment:

[0065] The chromatography system and chromatographic column were filled with 1 M NaOH solution (Sinopharm Shanghai Test) one day before use, with a flow rate of 1 mL / min;

[0066] 4. Inject water to flush the pipeline and chromatographic column:

[0067] Before performing the endotoxin removal operation, use injection water to flush the chromatography system pipeline and chromatographic column at a flow rate of 1 mL / min until the effluent is detected to be neutral by the terminal pH detector.

[0068] 5. TEV hydrolyzing protease pretreatment:

[0069] Before removing endotoxin, the protease-containing liquid was filtered using a 0.22 μm sterilizing filter (Millipore) before continuing with subsequent operations.

[0070] 6. Load the protein solution to be processed:

[0071] Use the prepared chromatography buffer to equilibrate the chromatographic column at a flow rate of 0.2 mL / min until the baseline of the UV280 detector (AKTAPurifier 100) is stable and the baseline is zeroed. This step is to make the environment inside the chromatographic column suitable for the target protein; the protein solution sample is then loaded into the 16 / 20 chromatographic column at a flow rate of 0.1 mL / min. After loading, the chromatographic column is rinsed with chromatography buffer at a flow rate of 0.1 mL / min.

[0072] 7. Harvest protein solution:

[0073] As the protein solution is loaded onto the chromatographic column, the effluent is collected when the UV280 detection value is ≥10mAu. The effluent must be collected in a sterile, endotoxin-free container and collection is stopped when the UV280 detection value is ≤10mAu.

[0074] 8. Detection of endotoxin removal effect, protein recovery rate and purity:

[0075] The endotoxin concentration (see the first method of 1143 of the third general rule of the Chinese Pharmacopoeia) and the protein concentration (see the fourth method of 0731 of the third general rule of the Chinese Pharmacopoeia) and purity (see the fifth method of 0541 of the third general rule of the Chinese Pharmacopoeia) in the sample protein before and after endotoxin removal were tested to evaluate the effect of the chromatographic column in removing endotoxin. The specific results are shown in Tables 1 to 2 and Figure 1 .

[0076] Table 1 Effect of Purose Endotoxin-Removal Column Method on Removal of Endotoxin

[0077]

[0078] Table 2 TEV protein recovery effect of Purose Endotoxin-Removal column method

[0079]

[0080] Based on the above test results, it can be seen that the chromatographic column method provided in this embodiment can not only remove 99.8% of endotoxin in the TEV protein solution, but also efficiently recover TEV protein with a recovery rate of up to 96%.

[0081] Comparative Example 1: Removal of endotoxin from TEV recombinant protein by phase separation

[0082] In this comparative example, a crude protein solution containing TEV protein was prepared according to the method described in Example 1, and then the endotoxin in the target protein was removed by using the Triton-114 phase separation method. The specific steps are as follows:

[0083] 1. Packaging of protein solution:

[0084] In the clean bench, use pyrogen-free 1 mL Tips to dispense the liquid to be detoxified into 1.5 mL EP tubes at 1 mL / tube. Then use pyrogen-free 0.2 mL Tips to add 1% (V / V) Triton X-114 (Sinopharm Shanghai Test) and vortex to mix.

[0085] 2. Triton-X114 phase separation:

[0086] After mixing, the liquid was transferred to a 4°C refrigerator, placed on ice for 5 min, and then transferred to a 37°C constant temperature water bath for 10 min. After the liquid was observed to have obvious stratification, it was centrifuged at room temperature at 1,2000 g / min using a centrifuge (Thermo Scientific) for 5 min.

[0087] 3. Collect the supernatant:

[0088] After centrifugation, the EP tube is transferred to the clean bench. Under the light condition, the liquid in the EP tube can be seen to be clearly layered. If there is endotoxin in the liquid, white flocculent precipitation can be seen at the bottom of the EP tube. Use 1mL Tips to remove pyrogen to absorb the upper clear liquid, and discard the lower clear liquid and EP tube;

[0089] 4. Reprocessing:

[0090] Repeat the above steps twice.

[0091] The method for detecting endotoxin removal effect, protein recovery rate and purity is the same as that described in Example 2. The specific results are shown in Tables 3 to 4 and Figure 2 .

[0092] Table 3 Effect of Triton-114 phase separation method on endotoxin removal

[0093]

[0094]

[0095] Table 4 TEV protein recovery effect of Triton-114 phase separation method

[0096]

[0097] Based on the above test results, it can be seen that although the phase separation method provided in this example can effectively remove endotoxins and concentrate the volume of protein solution in the sample, its protein recovery effect is far inferior to that of the column chromatography method (Example 2).

[0098] Comparative Example 2: Removal of endotoxin from TEV recombinant protein by centrifuge tube method

[0099] In this comparative example, a crude protein solution containing TEV protein was prepared according to the method described in Example 1, and then High Capacity Endotoxin Removal centrifuge tube method is used to remove endotoxins from the target protein. The specific steps are as follows:

[0100] 1. Solution preparation

[0101] 0.2 M NaOH (Sinopharm Shanghai test);

[0102] 1 M Tris-HCl (Sinopharm Shanghai Trial);

[0103] 2 M NaCl (Sinopharm Shanghai Trial);

[0104] 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl (Sinopharm Shanghai Trial);

[0105] All the above liquids must be sterile and endotoxin-free;

[0106] 2. Pretreatment of endotoxin-free centrifuge tubes:

[0107] The materials to be used were placed in a clean bench in advance and exposed to ultraviolet light for 30 min. The centrifugal column (Thermo Scientific, Pierce High Capacity Endotoxin Removal) was soaked in 0.2 M NaOH overnight in advance. When used, the stopper was removed and centrifuged at 4°C, 500 g for 1 min, and the NaOH solution was discarded.

[0108] 3. Rinse:

[0109] Add 5 mL of pyrogen-free H2O, centrifuge at 4°C and 500 g for 1 min, and rinse repeatedly until the pH in the column is neutral;

[0110] 4. Activation:

[0111] Add 5 mL of 2 M NaCl, centrifuge for 1 min as above, and discard the flow-through;

[0112] 5. Balance:

[0113] Add 5 mL of buffer (50 mM Tris-HCl (pH 8.0), 0.2 M NaCl), centrifuge at 4°C, 500 g for 1 min, repeat three times, and discard the flow-through;

[0114] 6. Binding endotoxin:

[0115] Add 5 mL of sample, seal with sealing film, place on a rotary shaker at room temperature, and rotate slowly for 1-2 h;

[0116] 7. Collection:

[0117] Remove the lower plug in the clean bench, replace the outer centrifuge tube with a sterile, endotoxin-free centrifuge tube, centrifuge at 4°C, 500 g for 1 min, and collect the sample;

[0118] The method for detecting endotoxin removal effect, protein recovery rate and purity is the same as that described in Example 2. The specific results are shown in Tables 5 to 6 and Figure 3 .

[0119] Table 5 Effect of High Capacity Endotoxin Removal Centrifuge Tube Method on Removal of Endotoxin

[0120]

[0121] Table 6 TEV protein recovery effect of High Capacity Endotoxin Removal centrifuge tube method

[0122]

[0123] Based on the above test results, although the centrifuge tube method provided in this example is as effective as column chromatography (Example 2) in removing endotoxins and recovering the target protein, the amount of protein recovered is far less than that of the column chromatography method (Example 2), which is only 1 / 3 to 1 / 4 of the latter, indicating that the centrifugation method is not suitable for large-scale production of endotoxin-free TEV protein.

[0124] In summary, compared with the phase separation method and the centrifuge tube adsorption method, the column chromatography method has the best effect in removing endotoxin from TEV protein and the highest efficiency in recovering the target protein, and is most suitable for large-scale production of endotoxin-free TEV protein.

[0125] Example 3: Optimization of chromatographic column packing

[0126] It can be seen from Example 2 and Comparative Examples 1 to 2 that the endotoxin removal effect of TEV protein by chromatography is the best. Therefore, the filler of the chromatographic column is optimized in this embodiment, that is, Purose Endotoxin-Removal filler and EzScreen Heparin FF filler (Bogelon) are used to remove endotoxins to screen out the best chromatographic column filler. The acquisition of TEV protein is the same as described in Example 1; except for the filler as described above, the remaining column chromatography experimental steps are the same as described in Example 2, and the method for detecting endotoxin removal effect, protein recovery rate and purity is the same as described in Example 2. The detection results of Purose Endotoxin-Removal filler are shown in Tables 1 to 2 and Figure 1 , EzScreen Heparin FF filler is shown in Tables 7-8 and Figure 4 .

[0127] Table 7 The effect of EzScreen Heparin FF column method on the removal of endotoxin

[0128]

[0129] Table 8 TEV protein recovery effect of EzScreen Heparin FF column method

[0130]

[0131] Combined with the above results, it can be seen that the endotoxin removal effect and protein recovery rate of Purose Endotoxin-Removal filler are better than those of EzScreen Heparin FF filler, so Purose Endotoxin-Removal filler is preferred.

[0132] The effects of all the above methods on removing endotoxin from TEV protein and recovering target protein are summarized in Tables 9 and Figure 5 .

[0133] Table 9 Summary of endotoxin removal rate and protein recovery rate of each method

[0134]

[0135] Considering the protein recovery effect and endotoxin removal effect, the Purose Endotoxin-Removal column method is preferred for removing endotoxin from TEV protein.

[0136] Example 4: Optimization of chromatography buffer

[0137] While the filler of the chromatographic column effectively adsorbs endotoxin, it also non-specifically adsorbs protein, resulting in a decrease in protein recovery rate. Sodium chloride reduces the non-specific adsorption of the filler, that is, the filler first adsorbs sodium chloride and then adsorbs the target protein. However, when the sodium chloride concentration in the chromatography buffer is too high, it will cause protein denaturation. Therefore, the sodium chloride concentration in the chromatography buffer needs to be at a balance point, that is, it can both improve the protein recovery rate and keep the protein in a stable state; on the other hand, magnesium chloride is also a common component for maintaining protein stability in purification experiments. In summary, this embodiment optimizes the components of the chromatography buffer and the concentration of sodium chloride. Among them, the protein stability is evaluated by whether there is a precipitate visible to the naked eye in the solution, because the protein solution in a normal state is a colorless, transparent and clear liquid, and the protein will precipitate after denaturation.

[0138] The preparation method of TEV protein is the same as that described in Example 1, and the experimental steps of column chromatography, protein recovery rate and purity method are the same as those described in Example 2. The specific optimized scheme and corresponding effects are shown in Table 10.

[0139] Table 10 Effect of chromatography buffer on protein recovery and endotoxin removal rate

[0140]

[0141]

[0142] As shown in Table 10, the presence of MgCl2 can increase the protein yield, although it only increases by 2%, but when the protein is produced in large quantities, the difference will be magnified, that is, the amount of protein loss will be greatly reduced; consistent with expectations, with the increase of NaCl concentration, the recovery rate of TEV protein increases, and when the NaCl concentration is greater than 150mM, the solution begins to become turbid, indicating that this salt concentration is not conducive to maintaining the stability of the TEV protein structure, so the applicable range of NaCl is 50-150mM, more preferably, 100-150mM. In summary, the chromatography buffer of 20mM Tris-HCl (pH8.0), 2mM MgCl2, and 150mM NaCl is most suitable for purifying TEV protein.

[0143] Example 5: Optimization of Chromatography Buffer Flow Rate

[0144] After all the protein sample solutions enter the chromatography column, new chromatography buffer needs to be added to the chromatography column to drive the protein solution in the column to flow. Otherwise, the TEV protein solution will be retained in the chromatography column for a long time and be in a high-salt environment for a long time, resulting in protein denaturation. At the same time, the protein is exposed to the outside for a long time, increasing the risk of degradation. Therefore, this example explores the effect of the flow rate of the chromatography buffer on the removal of endotoxins. The preparation method of TEV protein is the same as that described in Example 1, and the experimental steps of column chromatography and the method for determining the endotoxin removal effect are the same as those described in Example 2; the specific results are shown in Table 11.

[0145] Table 11 Effect of chromatography buffer flow rate on endotoxin removal

[0146]

[0147] It can be seen from Table 11 that when the chromatography buffer is higher than 0.1 mL / min, the endotoxin removal effect becomes worse. This may be because the sample solution has already flowed away before the chromatographic filler has time to adsorb the endotoxin in the protein solution. Therefore, when the protein solution flow rate is 0.01-0.1 mL / min, it is more suitable for removing endotoxin in TEV protein. When the liquid flow rate in the chromatographic column is faster, the time for purifying the protein can be shortened. In summary, 0.1 mL / min is preferred.

[0148] Example 6: Determination of sterilization method

[0149] Although the chromatographic column can efficiently adsorb lipopolysaccharide, it cannot adsorb bacteria. If the protein solution is not subjected to additional sterilization treatment, the bacteria in the solution will continue to produce lipopolysaccharide (endotoxin), resulting in endotoxins in the protein solution after chromatography, and the endotoxin content will increase. Therefore, this example explores the effects of two sterilization methods, filtration and heat inactivation, on the endotoxin content in the solution. The preparation method of TEV protein is the same as that described in Example 1, the method of filtration sterilization is the same as that described in Example 2, and the experimental steps of heat inactivation are to place the sample in boiling water and heat for 2-3 minutes; the experimental steps of column chromatography and the method of determining the endotoxin removal effect are the same as those described in Example 2. The specific sterilization scheme and the corresponding effects are shown in Table 12.

[0150] Table 12 Effect of bacterial treatment methods on the removal of endotoxins from TEV protein

[0151]

[0152] As can be seen from Table 12, the heating inactivation method is not suitable for TEV protein solution, so this method is not considered; compared with no sterilization treatment, filtration sterilization can significantly improve the removal efficiency of endotoxin in TEV protein, so the TEV protein solution needs to be filtered and sterilized.

[0153] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

Claims

1. A method for removing endotoxin from TEV protease, characterized in that: The method is to load the TEV protein solution that needs to remove endotoxin into a chromatography column filled with Purose Endotoxin-Removal, then use a chromatography buffer to drive the protein solution to flow out of the chromatography column at a certain flow rate, collect the effluent, and the obtained effluent is the TEV protein solution without endotoxin.

2. The method according to claim 1, characterized in that The components of the chromatography buffer are Tris-HCl, MgCl2 and NaCl.

3. The method according to claim 2, characterized in that The concentration of NaCl is 25mM to 2M.

4. The method according to claim 2, characterized in that The concentration of MgCl2 is 1-100 mM.

5. The method according to claim 2, characterized in that The concentration of the Tris-HCl is 5-500 mM.

6. The method according to claim 2, characterized in that The flow rate of the chromatography buffer is 0.01-1 mL / min.

7. The method according to claim 6, characterized in that The TEV protein solution needs to be sterilized before loading, and the sterilization treatment refers to filtering the TEV protein solution using a sterilizing filter with a pore size of 0.2 to 0.45 μm.

8. The method according to claim 7, characterized in that The effluent refers to the liquid flowing out of the chromatography column when the UV280 detection value is ≥10mAu.

9. A chromatography buffer for improving protein recovery rate and / or improving endotoxin removal efficiency, wherein the chromatography buffer has a formula of: 5-500 mM Tris-HCl, 1-100 mM MgCl2, 25 mM-2 M NaCl, the chromatography medium is Purose Endotoxin-Removal, and the protein is TEV protease.

10. Use of a chromatography medium for improving protein recovery rate and / or improving endotoxin removal efficiency, wherein the chromatography medium is Purose Endotoxin-Removal and the protein is TEV protease.

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