Direct purification method of inclusion somatic cell culture

Abstract

The invention provides a direct purification method of an inclusion somatic cell culture. The direct purification method comprises the following steps: S1, collecting and crushing zymophyte; s2, purification of inclusion body re-suspension: re-suspending the thallus crushed substance in a first buffer solution, wherein the first buffer solution at least contains a cosolvent of the inclusion body so as to dissolve the inclusion body in the first buffer solution; combining the inclusion body protein dissolved in the first buffer solution by adopting a purification medium; washing the purification medium with a second buffer solution; and eluting and renaturating the inclusion body protein combined with the purification medium from the purification medium by adopting a third buffer solution to obtain the refolded inclusion body protein with activity. According to the method, the complex centrifugal separation step of extracting the inclusion body from the cell disruption substance is successfully omitted, the complex system of the cell disruption substance is directly incubated with the chromatography medium for purification, the operation time is greatly shortened, the operation efficiency is improved, and the degree of dependence on high-price equipment such as large and medium-sized centrifuges is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of biological separation, and in particular to a method for the direct purification of inclusion body homogenates in the downstream purification process of biopharmaceuticals and biochemical products. Background Technology

[0002] Inclusion bodies are insoluble aggregates formed when exogenous genes are expressed in host cells due to protein misfolding. Inclusion body purification is a key and challenging step in the biopharmaceutical field, aiming to extract biologically active target proteins from inclusion bodies. Compared to supernatant expression, inclusion body expression offers advantages such as higher expression levels, less susceptibility to intracellular protease hydrolysis, and better product storage. However, the purification process for proteins expressed in inclusion bodies is generally more complex and cumbersome.

[0003] Traditional inclusion body purification methods mainly include the following steps: (1) cell collection and disruption, (2) centrifugation to obtain crude inclusion bodies, inclusion body washing and dissolution, and (3) protein refolding and target protein purification (as disclosed in Chinese Patent CN117362382A). In the first two steps, the main purpose of cell collection and disruption, and inclusion body washing and dissolution is to obtain a clear solution. This process requires high-quality centrifugation and filtration equipment. The refolding step can usually be achieved by reducing the denaturant concentration through dialysis or stepwise dilution, while the purification step is generally achieved through ion exchange or affinity chromatography. The order of refolding and purification can also be achieved by dissolution followed by on-column refolding during chromatography (as disclosed in Chinese Patent CN102807599A). These methods have disadvantages such as low efficiency, high cost, and easy denaturation and inactivation of proteins. For example, the operation is complex and cumbersome, and the requirements for professional chromatography equipment, centrifugation equipment, filtration equipment, etc. are very high. Large-scale purification of inclusion bodies still faces significant challenges.

[0004] Key technological breakthroughs in inclusion body purification include: using novel dissolving reagents that differ from traditional denaturants (e.g., urea, guanidine hydrochloride) to dissolve inclusion bodies; developing novel chromatographic media for chromatographic purification; and employing novel refolding methods for protein refolding. International patent WO2013068603A2 discloses the use of N-lauroyl sarcosinate sodium as a solubilizer to dissolve recombinant granulocyte colony-stimulating factor inclusion bodies; the remaining processes are consistent with traditional methods, resulting in a lower dilution factor during refolding (2-fold, compared to 50-200 times when using guanidine hydrochloride / urea). Chinese patent CN115181160A uses a magnetic bead method (a novel chromatographic medium) to purify refolded inclusion body proteins, which improves purification and refolding efficiency. However, it still uses conventional processes such as centrifugation after disruption to obtain crude inclusion bodies, inclusion body washing, and dissolution, and is not easily scaled up for production due to limitations in the binding method.

[0005] On the other hand, suitable chromatographic media, such as large-diameter microspheres or magnetic beads, can be used to purify unclarified biological samples. International patent WO2018122089A1 discloses a method for preparing magnetic beads and its use in purifying unclarified supernatant expression products, simplifying the purification steps for biological samples. Therefore, simplifying and improving the purification process of inclusion bodies using suitable chromatographic media, eliminating the need for centrifugation to separate crude inclusion bodies after cell lysis, and directly purifying cell lysates containing inclusion bodies could become a new strategy for inclusion body purification. Summary of the Invention

[0006] The purpose of this invention is to provide a method for direct purification of inclusion body cell cultures after disruption, integrating processes such as inclusion body dissolution, purification, and refolding, thereby solving engineering problems related to large-scale inclusion body purification.

[0007] Therefore, the above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] A method for direct purification of inclusion body cell cultures includes the following steps:

[0009] S1. Collection and disruption of fermentation cells;

[0010] After fermentation, the bacterial cells were collected and broken using a common method.

[0011] Methods for collecting bacterial cells include centrifugation, filtrate collection, and ultrafiltration, which are suitable for bacterial cell collection in laboratories or industrial production.

[0012] Methods for disrupting bacterial cells include mechanical methods such as high-pressure homogenization, vibrating bead crushing, high-speed stirring bead grinding, and ultrasonic crushing, as well as non-mechanical methods such as osmotic pressure impact crushing, freeze-thaw crushing, enzyme dissolution crushing, chemical crushing, and detergent crushing, which are suitable for laboratory or industrial production processes.

[0013] S2. Purification of the weight suspension containing the sample:

[0014] The bacterial cell fragments are resuspended in a first buffer solution, which contains at least a solubilizer for inclusion bodies to dissolve the inclusion bodies in the first buffer solution, wherein the volume concentration of inclusion bodies in the first buffer solution is 0.1% to 10%.

[0015] A suitable purification medium is used to bind inclusion body proteins dissolved in the first buffer. The binding modes of the purification medium to the target product include affinity (e.g., metal chelate affinity chromatography), ion exchange (e.g., anion exchange chromatography, cation exchange chromatography), hydrophobic interaction, and multiple modes of action.

[0016] The purification medium was washed with a second buffer solution;

[0017] The inclusion body proteins bound to the purification medium were eluted and refolded using a third buffer to obtain refolded, active inclusion body proteins.

[0018] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0019] As a preferred embodiment of the present invention, the first buffer solution is a buffer solution having one of the following: Tris-HCl, phosphate (PB), PBS, 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a concentration preferably of 20-100 mM.

[0020] As a preferred embodiment of the present invention: the first buffer solution further comprises at least one of an ionic strength regulator, a reducing agent, and a chelating agent;

[0021] The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM;

[0022] The reducing agent is at least one of disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione, with a preferred concentration of 1-20 mM.

[0023] The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), preferably with a concentration of 0-5 mM and a pH of 4.0-10.0.

[0024] As a preferred embodiment of the present invention: the co-solvent in the first buffer solution is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea;

[0025] The concentration of the anionic surfactant is 0.2% to 2% (w / v), and the anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate, and is used in a weakly alkaline or near-neutral system.

[0026] The concentration of the cationic surfactant is 0.2% to 2% (w / v);

[0027] The concentration of guanidine hydrochloride is preferably 4–6 M;

[0028] The concentration of the urea is preferably 4–8 M;

[0029] In addition, 2-4M urea can be added and the mixture can be rapidly frozen and thawed to dissolve the inclusion bodies.

[0030] As a preferred embodiment of the present invention, the second buffer solution is a buffer solution having one of the following: Tris-HCl, phosphate (PB), PBS, 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a preferred concentration of 20-50 mM.

[0031] As a preferred embodiment of the present invention: the second buffer solution further comprises at least one of an ionic strength regulator, a solubilizer, a reducing agent, and a chelating agent;

[0032] The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM;

[0033] The co-solvent is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea;

[0034] The concentration of the anionic surfactant is 0.2% to 2% (w / v), and the anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate.

[0035] The concentration of the cationic surfactant is 0.2% to 2% (w / v);

[0036] The concentration of guanidine hydrochloride is preferably 4–6 M;

[0037] The concentration of the urea is preferably 4–8 M;

[0038] The reducing agent is at least one of disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione, with a preferred concentration of 1-20 mM.

[0039] The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), preferably with a concentration of 0-5 mM and a pH of 4.0-10.0.

[0040] As a preferred embodiment of the present invention, the third buffer solution is a buffer solution having one of the following: Tris-HCl, phosphate (PB), PBS, 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a concentration preferably of 20-50 mM.

[0041] As a preferred embodiment of the present invention: the third buffer solution further comprises at least one of imidazole or sodium chloride;

[0042] The concentration of the imidazole is preferably 100-500 mM, which is suitable for metal chelation chromatography;

[0043] The concentration of sodium chloride is preferably 100–1000 mM, which is suitable for ion exchange chromatography.

[0044] As a preferred embodiment of the present invention: the third buffer solution further comprises at least one of a solubilizer, a reducing agent, and a chelating agent;

[0045] The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM;

[0046] The co-solvent is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea;

[0047] The concentration of the anionic surfactant is 0.2% to 2% (w / v), and the anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate.

[0048] The concentration of the cationic surfactant is 0.2% to 2% (w / v);

[0049] The concentration of guanidine hydrochloride is preferably 4–6 M;

[0050] The concentration of the urea is preferably 4–8 M;

[0051] The reducing agent is at least one of disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione, with a preferred concentration of 1-20 mM.

[0052] The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), preferably with a concentration of 0-5 mM and a pH of 4.0-10.0.

[0053] As a preferred embodiment of the present invention, the purification medium is column chromatography microspheres (e.g., NiNTANUPharose BB), magnetic beads (e.g., Ni-NTANUPharose Mag), chromatography membrane, monolithic column, or expanded column bed medium.

[0054] This invention provides a direct purification method for inclusion body cell cultures, which has the following beneficial effects:

[0055] 1) By cleverly using chromatography media (or chromatography packing material) with large-diameter microspheres as the base spheres, the complex centrifugation separation step of extracting inclusion bodies from cell fragments was successfully eliminated, and the complex system of cell fragments was directly incubated with chromatography media for purification, which greatly reduced the operation time, improved the operation efficiency, and greatly reduced the dependence on high-priced equipment such as large and medium-sized centrifuges.

[0056] 2) It enriches the selection space of inclusion body cosolvents or denaturants, and has adjusted the economical and efficient use concentration, temperature and time, bypassing the problems of high system viscosity, large volume of solution and container, large area and long time required for dissolution and refolding processes caused by using traditional denaturants alone, and greatly reducing the dependence on ultrafiltration membrane packs.

[0057] 3) The processes of inclusion body dissolution, purification, and refolding have been integrated, simplified, and optimized, solving engineering problems related to large-scale inclusion body purification. This achieves advantages such as high efficiency, low cost, reduced operation time, and reduced protein denaturation and inactivation. Attached Figure Description

[0058] Figure 1 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) images of inclusion bodies E3(a) and E4(b) were obtained to compare the ability of Tris solution (blank control), sodium lauroyl amino acid, guanidine hydrochloride, and sodium lauroyl amino acid + guanidine hydrochloride to dissolve inclusion bodies.

[0059] Figure 2 Comparative study of the dissolution of inclusion bodies E3 and E4 by sodium lauroyl sarcosinate and urea, and their incubation and binding with Ni TED NUPharose FF and Ni NTANUPharose FF fillers. SDS-PAGE images.

[0060] Figure 3 SDS-PAGE images are used to study the effects of different concentrations of sodium lauroyl amino acids during the incubation process, taking E4 inclusion bodies as an example, and the influence of the presence or concentration of sodium lauroyl amino acids during the rinsing or impurity washing process.

[0061] Figure 4 This is an SDS-PAGE image of E3 inclusion bodies incubated and purified using NiNTANUPharose BB (batch 1 purification).

[0062] Figure 5 The image shows the SDS-PAGE of the first batch of purified flow-through buffer (the first batch was not completely bound, and the target band was still present in the flow-through buffer, so the sample can be loaded repeatedly) after the second incubation and purification of E3 inclusion bodies using NiNTANUPharose BB incubator.

[0063] Figure 6 This is an SDS-PAGE image of E3 inclusion bodies purified by incubation with NiNTANUPharose BB (second batch purification). The following elution regimen was used: 5 CV rinsing, 5 CV washing, and 7 CV elution with 300 mM.

[0064] Figure 7 This is an SDS-PAGE image of E3 inclusion bodies purified by incubation with NiNTANUPharose BB (batch 3 purification). The following elution regimen was used: 5 CV rinsing, 5 CV washing, and 7 CV elution with 300 mM.

[0065] Figure 8 This is an SDS-PAGE image of E3 inclusion bodies purified by incubation with NiNTANUPharose BB (batch 4 purification). The following elution regimen was used: 5 CV rinsing, 5 CV washing, and 7 CV elution with 300 mM.

[0066] Figure 9 This is an SDS-PAGE image of the second batch of E3 inclusion body flow-through solution (still containing the target protein) that was incubated and purified again with large-sphere Ni NTANUPharose BB (referred to as the fifth batch of purification).

[0067] Figure 10 The image shows SDS-PAGE images of the third and fourth batches of E3 inclusion bodies purified by re-incubation with NiNTANUPharose BB (referred to as the sixth and seventh batch purifications).

[0068] Figure 11 This is an SDS-PAGE image of E4 inclusion bodies incubated and purified using NiNTANUPharose BB with large spheres.

[0069] Figure 12 The image shows the SDS-PAGE of E3 homogenate and E4 solution after incubation and purification using conventional NiNTANUPharose FF spheres (90 μm in diameter) for Comparative Example 1.

[0070] Figure 13 The image shows the SDS-PAGE of the supernatant of the purified E4 inclusion bodies after incubation with conventional NiNTANUPharose FF beads (90 μm in diameter) for Comparative Example 2.

[0071] Figure 14 SDS-PAGE and AKTA chromatography chromatograms for the purification of E4 inclusion bodies using multi-mode salt-tolerant NuPerleyAdhere packing material. Detailed Implementation

[0072] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0073] Example 1: Dissolution of inclusion bodies E3 and E4

[0074] It should be noted that in this embodiment, inclusion bodies are used directly as an example, and the collection and disruption of fermentation cells are not included.

[0075] 1) Inclusion body washing

[0076] After thawing, the inclusion bodies were mixed with 20 mM Tris-HCl, pH 8.0 buffer for 15 min, and then centrifuged at 15000 rpm for 15 min to collect the supernatant and precipitate.

[0077] 2) Inclusion body dissolution

[0078] The above precipitate was resuspended in buffer solution for 16 h, and then centrifuged at 15000 rpm for 30 min to obtain the inclusion body dissolution product and the inclusion body precipitate.

[0079] The buffer solution is a mixture of 20 mM Tris-HCl, pH 8.0, 1% (w / v) sodium lauroyl sarcosinate, 6 M guanidine hydrochloride, 1% (w / v) sodium lauroyl sarcosinate and 6 M guanidine hydrochloride.

[0080] 3) The effect of buffer solution on inclusion body dissolution

[0081] Three types of samples (40 μL each) were prepared: precipitate before and after inclusion body washing, product after inclusion body dissolution, and solution of inclusion body dissolved precipitate washed with 20 mM Tris, pH 8.0 solution and then resuspended and reconstituted. 20 μL of 3× protein loading buffer was added, and the mixture was heated at 100℃ for 5 min. Polyacrylamide gel electrophoresis was then performed. The gels were removed, stained, destained, and the results observed. Figure 1 As shown in the figure (indicating reduced sample, 12% separation gel; 5 μL of marker loading, 10 μL of other samples loading; sample dilution factor of 1), comparing inclusion body samples before and after washing, it can be seen that inclusion bodies are mainly present in the precipitate. Therefore, washing and dissolving the inclusion body precipitate with washing solution can yield more inclusion body proteins.

[0082] After resuspending inclusion bodies in four different buffers, the supernatant containing 20 mM Tris-HCl at pH 8.0 showed the worst solubility. 1% (w / v) sodium lauroyl sarcosinate showed the best solubility. Therefore, adding 1% (w / v) sodium lauroyl sarcosinate to the buffer significantly improves the solubility of inclusion bodies and can increase the yield of inclusion body proteins.

[0083] Example 2: Effects of sodium lauroyl sarcosinate and urea on the dissolution and incubation binding of inclusion bodies E3

[0084] 1) Comparative study on the solubility and incubation binding of inclusion bodies E3 by sodium lauroyl sarcosinate and urea.

[0085] Ni TED NUPharose FF packing material (metal chelate affinity packing material with tricarboxymethyl ethylenediamine group as ligand and nickel ion as coordinating metal ion) or Ni NTANUPharose FF packing material (metal chelate packing material containing nitrotriacetic acid (NTA) group, which has chelated nickel ions) was placed in a gravity column, washed with ultrapure water for 10 CV, equilibrated with 20 mM Tris, 150 mM NaCl, and pH 8.0 solution for 10 CV; a certain amount of packing material was weighed into three conical flasks, 40 mL of E3 feed solution (flow-through solution containing 300 mM NaCl) was added, and incubated at 35℃ for a certain period of time.

[0086] Collect the flow-through solution, rinse with the eluent for 10 CV, collect the eluent A, and elute with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 500 mM imidazole, pH 8.0 solution. Collect the eluent. Perform SDS-PAGE analysis on the collected sample solution. The incubation conditions are shown in Table 1.

[0087] Table 1. Incubation conditions for NiTEDNUPharoseFF and NiNTANUPharoseFF at E3

[0088]

[0089]

[0090] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0091] Ni TED NUPharose FF and Ni NTANUPharose FF were incubated with inclusion bodies E3 and then run as gels. Figure 2 (In the figure: reduced sample, 12% separation gel; marker loading 5 μL, other samples loading 10 μL; sample dilution factor is 1).

[0092] Figure 2 In (a), the flow bands of NTA (lanes 4, 5, 6) are significantly shallower than those of other swimmers. Figure 2 (b) shows the flow-through bands of TED (lanes 7 and 8), indicating that Ni NTANUPharose FF and E3 are more bound. The condensation layer of band 6 has no band, which may be due to protein denaturation caused by urea.

[0093] Ni NTANUPharose FF after incubation E3 Figure 2As shown in (b), the target protein in the 4g flow-through band (lanes 2, 5, 6) was lighter than that in the 2g band, indicating that 10x E3 incubation can bind more protein; the 4h flow-through band (lanes 7, 8) and the 2h supernatant (lanes 5, 6) were not significantly different; the 4h eluent (lane 9) also contained the target protein, possibly due to the presence of 10mM imidazole in the eluent.

[0094] Example 3: Sodium lauroyl sarcosinate was added during the incubation of inclusion bodies E4.

[0095] 1) Purification conditions

[0096] Take inclusion body E4 feed solution (inclusion body suspension), dilute with different buffers, incubate at 25°C for 4 hours, transfer to a 10 mL packed column to collect flow-through, wash for about 15 CV, elute for 7 CV, and the purification conditions are shown in Table 2.

[0097] Table 2 Purification conditions for inclusion bodies E4

[0098]

[0099] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0100] Ni NTANUPharose FF was incubated overnight at 25°C; the gel image is shown below. Figure 3 (In the image: reduced form, 13.5% separation gel, 0.75 mm thick).

[0101] Adding 0.5%–1% (w / v) sodium lauroyl sarcosinate has a dissolving effect on E4 solution, thereby promoting the binding of inclusion bodies to the target protein and filler ligands. 1% (w / v) sodium lauroyl sarcosinate has a better dissolving effect. Lauroyl sarcosinate can be omitted during washing / rinsing (experimental group 1-B) to save raw materials.

[0102] Example 4: Incubation of inclusion bodies E3 with Ni NTANUPharose BB (large spheres, approximately 200 μm in diameter)

[0103] 1) Purification process

[0104] Batch I: Weigh approximately 5 L of equilibrated packing material into a 100 L glass reactor, add approximately 80 mL of E3 feed solution (1% (w / v) sodium lauroyl sarcosinate, pH adjusted to 7.76 with 2 M Tris); incubate overnight at room temperature. The next day, collect the flow-through and elute with the eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0) for 10 CV, and collect the eluent; wash with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0 solution for 2 CV; elute with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 200 mM imidazole, pH 8.0 solution for 5 CV, and collect the eluent; elute with 20 mM Tris, 150 mM... Elute with NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 500 mM imidazole, pH 8.0 solution for 2 CV. Collect the eluent, wash with water 0.5 CV, regenerate with 0.1 M NaOH for 1 CV, and wash with water 7 CV until pH neutral. Perform SDS-PAGE analysis on the collected sample.

[0105] Batch II: Weigh approximately 6 L of packing material into a 100 L glass reactor, add approximately 90 mL of E3 feed solution (1% (w / v) sodium lauroyl sarcosinate, pH adjusted to 8.0 with 2 M Tris); incubate overnight at room temperature. The next day, collect the flow-through and wash with eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0) for 5 CV, and collect the eluent; wash with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0 solution for 5 CV; elute with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 300 mM imidazole, pH 8.0 solution for 7 CV, and collect the eluent; regenerate as in Batch I. Perform SDS-PAGE analysis on the collected sample solution.

[0106] Batch III: Weigh approximately 6 L of packing material into a 100 L glass reactor, add approximately 90 mL of E3 feed solution (1% (w / v) sodium lauroyl sarcosinate, pH adjusted to 8.0 with 2 M Tris); incubate overnight at room temperature, collect the flow-through the next day, wash with eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0) for 2 CV, collect the eluent; wash with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0 solution for 5 CV; elute with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 300 mM imidazole, pH 8.0 solution for 7 CV, collect the eluent; regenerate as in Batch I.

[0107] Batch IV: The flow-through of the Batch I homogenate was recovered. Approximately 4 L of NiNTANUPharose BB packing material was weighed into a 100 L glass reactor, and approximately 40 mL of E3 feed solution (Batch I flow-through) was added. The mixture was incubated overnight at room temperature. The next day, the flow-through was collected and washed with eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0) for 5 CV, and the eluent was collected. The mixture was then washed with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0 solution for 3 CV; eluted with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 300 mM imidazole, pH 8.0 solution for 7 CV, and the eluent was collected. The mixture was then washed with 0.5 CV of water and 0.1 M... Regeneration with NaOH (1 CV), followed by washing with water (7 CV) until pH neutral. The collected sample solution was then analyzed by SDS-PAGE.

[0108] Batch V: The inclusion bodies from Batch II were recovered via flow-through. Approximately 4 L of NiNTANUPharose BB packing material was weighed into a 100 L glass reactor, and approximately 60 mL of E3 feed solution (Batch II flow-through solution) was added. The mixture was incubated overnight at room temperature. The next day, the flow-through solution was collected and washed with eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0) for 2 CV, and the eluent was collected. The mixture was then washed with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0 solution for 5 CV. The mixture was then eluted with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 300 mM imidazole, pH 8.0 solution for 7 CV, and the eluent was collected. The mixture was then washed with 0.5 CV of water and 0.1 M... Regeneration with NaOH (1 CV), followed by washing with water (7 CV) until pH neutral. The collected sample solution was then analyzed by SDS-PAGE.

[0109] Batch VI and VII: Inclusion bodies from batches II and III were recovered via flowthrough. Approximately 6 L of NiNTANUPharose BB (Lot 20241105) was weighed into a 100 L glass reactor, and approximately 60 mL of E3 feed solution (inclusion body flowthrough solution) was added. The reactor was incubated overnight at room temperature. The next day, the flowthrough solution was collected and washed for 2 cycles with eluent (20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0). The eluent was collected. The reactor was then washed for 5 cycles with a solution of 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 10 mM imidazole, pH 8.0. Elute with NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 300 mM imidazole, pH 8.0 solution for 7 CV, collect the eluent; wash with water 0.5 CV, regenerate with 0.1 M NaOH for 1 CV, wash with water 7 CV until pH neutral. Perform SDS-PAGE analysis on the collected sample.

[0110] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0111] Batch I: After overnight incubation, take 16 mL of the mixture and pass it through a 10 mL gravity column. Wash and elute as shown in the table below. Figure 4 (In the figure: reduced form, 12% separating gel; 5 μL of marker sample, 10 μL of sample sample; stock solution and flow-through diluted 10 times, other samples not diluted), the target protein is present in the eluent, which indicates that the packing material has bound to the protein and confirms the protocol of 10 mM imidazole washing and 200 mM imidazole elution.

[0112] The first batch of incubation solution was collected and flowed through, washed with 10 CV, washed with 2 CV to remove impurities, eluted with 200 mM 5 CV, and eluted with 500 mM imidazole 2 CV. The resulting gel image is shown below. Figure 5 (In the figure: reduced sample, 12% separating gel; 5 μL for marker loading, 10 μL for sample loading; stock solution and flow-through solution diluted 10-fold, other samples not diluted). A small amount of protein was still eluted in the first batch of solution with 500 mM imidazole.

[0113] Batch II: The following regimen was used: rinsing with 5 CV, washing with impurities with 5 CV, and eluting with 300 mM for 7 CV. See gel image below. Figure 6 (In the figure: reduced form, 12% separating gel; 5 μL of marker sample, 10 μL of sample sample; stock solution and flow-through solution diluted 10 times, other samples not diluted).

[0114] Batch III: The incubation solution from batch III was collected and flowed through, rinsed 2 CV, washed for impurities 5 CV, and eluted with 300 mM for 7 CV. See the gel image below. Figure 7(In the figure: reduced form, 12% separating gel; 5 μL of marker sample, 10 μL of sample sample; stock solution and flow-through solution diluted 10 times, other samples not diluted).

[0115] Batch IV: The incubation solution for batch IV was collected and flowed through, rinsed with 5 CV, washed with impurities with 3 CV, and eluted with 300 mM for 7 CV. The resulting gel image is shown below. Figure 8 (In the figure: reduced form, 12% separating gel; 5 μL of marker sample, 10 μL of sample sample; stock solution and flow-through solution diluted 10 times, other samples not diluted).

[0116] Batch V: The second batch of inclusion body flow-through solution was incubated again at a 1:10 ratio for recovery, followed by 2 CV of rinsing, 5 CV of washing to remove impurities, and 7 CV of elution with 300 mM imidazole. See the gel image below. Figure 9 (In the figure: reduced form, 12% separating gel; 5 μL of marker sample loaded, 10 μL of sample sample loaded; stock solution and flow-through diluted 10 times, other samples not diluted), the flow-through supernatant is relatively light, which indicates that the protein is basically completely bound to the filler.

[0117] Lots VI and VII: E3 inclusion bodies were recovered from lots VI and VII. The eluent was washed with 2 CV of elution, followed by 5 CV of impurity washing and 7 CV of elution with 300 mM. See the gel image below. Figure 10 (In the figure: reduced form, 12% separating gel; 5 μL of marker sample loaded, 10 μL of sample sample loaded; stock solution and flow-through diluted 10 times, other samples not diluted), the target bands (band 2 and band 8) in the flow-through solution are very light, which indicates that the target protein in the inclusion body is basically completely bound to the filler.

[0118] Example 5: Ni NTABB incubation of inclusion bodies E4

[0119] 1) Purification process

[0120] Weigh 50g of naturally filtered Ni NTABB packing material into a 1L Erlenmeyer flask, add 500mL of E4 feed solution (E4 resuspension diluted 12 times with 20mM Tris, 150mM NaCl, 1% (w / v) sodium lauroyl sarcosinate, 10mM imidazole, pH 8.0), incubate overnight in a shaker at 25°C, transfer to a 300mL packing column to collect flowthrough, elute (20mM Tris, 150mM NaCl, 10mM imidazole, pH 8.0) for approximately 15CV, and elute (20mM Tris, 1% (w / v) sodium lauroyl sarcosinate, 500mM imidazole, pH 8.0) for 7CV.

[0121] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0122] The gel image after incubating 50g NiNTABB filler and 500mL E4 solution (diluted 12 times) overnight under 1% (w / v) lauryl incubation conditions is shown in the image. Figure 11 (In the figure: reduced form, 12% separating gel, 1.5 mm thick; 5 μL of marker sample loaded, 10 μL of sample sample loaded, undiluted). It can be seen that the eluent is not significantly different before and after filtration. The reduction and boiling conditions cause the polymers to accumulate on the top layer of the stacking gel (bands 4 and 6), while the reduction without boiling causes them to aggregate in the middle layer of the stacking and separating gels. Furthermore, the target bands are deeper in the reduction without boiling conditions (bands 5 and 7).

[0123] Comparative Example 1: Ni NTANUPharose FF (small spheres, 90 μm in diameter) was incubated with E3 homogenate and E4 feed solution.

[0124] 1) Purification of E3 and E4

[0125] After equilibrating NiNTANUPharose FF packing material with 20 mM Tris, 150 mM NaCl, pH 8.0 solution, a certain amount of NiNTANUPharose FF packing material was weighed into three conical flasks, and 40 mL of E3 homogenate or E4 solution was added to each flask. The mixture was incubated overnight, and the flow-through was collected. The next day, the mixture was washed for 10 CV with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, pH 8.0, and 10 mM imidazole solution. The eluent was collected and eluted with 20 mM Tris, 150 mM NaCl, 0.5% (w / v) sodium lauroyl sarcosinate, 500 mM imidazole, pH 8.0 solution. The eluent was collected.

[0126] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0127] Ni NTANUPharose FF was used to incubate the E3 homogenate and E4 homogenate. Figure 12 (In the figure: reduced form, no boiling, 12% separating gel, 1.5 mm thick; 5 μL of marker sample loaded, 10 μL of sample sample loaded, undiluted), the elution buffer of E3 contains obvious impurity bands (see bands 4-7); the elution band of E4 does not contain the target protein, possibly because it has not been bound.

[0128] Comparative Example 2: Small-scale incubation of small balls with E4

[0129] 1) Purification process

[0130] TED and NTA incubation: Weigh about 2g of Ni TED NUPharose FF packing material or Ni NTANUPharose FF that has been naturally filtered and balanced into a 50mL centrifuge tube, add E4 inclusion body supernatant and incubate overnight, rinse 5CV, wash impurities 5CV, elute with 300mM imidazole 7CV, and elute with 500mM imidazole 2CV.

[0131] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0132] Ni TED NUPharose FF filler or Ni NTANUPharose FF and E4 inclusion body supernatant incubated overnight at 25°C. The resulting gel image is shown below. Figure 13 (In the figure: reduced form, not boiled, 13.5% separating gel, 0.75 mm thick, 5 μL for marker loading, 10 μL for sample loading, undiluted), Ni NTANUPharose FF has a purification effect, but the purity is not high.

[0133] Example 6: Adhere column purification of E4

[0134] 1) Purification process

[0135] The chromatography column used in this embodiment is an Adhere chromatography column with a column volume of 1 mL.

[0136] The inclusion body sample was obtained from the E4 eluent obtained in Example 5.

[0137] Equilibration and loading buffer (mobile phase I): 20 mM Tris, 1% (w / v) sodium lauroyl sarcosinate, pH 8.0; equilibration flow rate 1 mL / min, equilibration 10 CV, loading flow rate 0.5 mL / min, loading 20 mL of E4 inclusion body protein.

[0138] Elution buffer (mobile phase II): 20 mM Tris, 1% (w / v) sodium lauroyl sarcosinate, 1 M NaCl, pH 8.0; elution flow rate 1 mL / min, elution 25 mL.

[0139] 2) SDS-PAGE polyacrylamide gel electrophoresis experiment

[0140] The chromatogram of 20 mL of E4 eluent purified using Adhere packing material is shown below. Figure 14 (In the figure: reduced form, no boiling, 12% separating gel, 1.5 mm thickness; 5 μL marker loading, 20 μL sample loading, undiluted). The bands A4, A5, A6, and A7 in the Adhere eluent are darker (compared to band E4), and there are fewer bands below 28 kDa in the flow-through buffers A1-A3, indicating that Adhere has a good purification effect.

[0141] The above specific embodiments are used to explain and illustrate the present invention, and are only preferred embodiments of the present invention, not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A method for direct purification of inclusion body cell cultures, characterized in that: Includes the following steps: S1. Collection and disruption of fermentation cells; S2. Purification of the weight suspension containing the sample: The bacterial cell fragments are resuspended in a first buffer solution, which contains at least a solubilizer for inclusion bodies to dissolve the inclusion bodies in the first buffer solution, wherein the volume concentration of inclusion bodies in the first buffer solution is 0.1% to 10%. Use a suitable purification medium to bind inclusion body proteins dissolved in the first buffer solution; The purification medium was washed with a second buffer solution; The inclusion body proteins bound to the purification medium were eluted and refolded using a third buffer to obtain refolded, active inclusion body proteins.

2. The method according to claim 1, characterized in that: The first buffer is a buffer containing one of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), phosphate (PB), phosphate (PBS), 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a preferred concentration of 20-100 mM.

3. The method according to claim 1, characterized in that: The first buffer solution also contains at least one of an ionic strength modifier, a reducing agent, and a chelating agent; The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM; The reducing agent is at least one of the following: disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione; The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA).

4. The method according to claim 1, 2, or 3, characterized in that: The co-solvent in the first buffer solution is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea. The anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate.

5. The method according to claim 1, characterized in that: The second buffer is a buffer containing one of the following: tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), phosphate (PB), phosphate (PBS), 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a preferred concentration of 20-50 mM.

6. The method according to claim 5, characterized in that: The second buffer solution also contains at least one of an ionic strength modifier, a solubilizer, a reducing agent, and a chelating agent; The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM; The co-solvent is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea; The anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate. The reducing agent is at least one of the following: disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione; The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA).

7. The method according to claim 1, characterized in that: The third buffer solution is a buffer solution containing one of the following: Tris-HCl, phosphate (PB), PBS, 3-(N-morpholino)propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), with a preferred concentration of 20-50 mM.

8. The method according to claim 7, characterized in that: The third buffer solution also contains at least one of imidazole or sodium chloride; The concentration of the imidazole is preferably 100–500 mM; The concentration of sodium chloride is preferably 100–1000 mM.

9. The method according to claim 7 or 8, characterized in that: The third buffer solution also contains at least one of a solubilizer, a reducing agent, and a chelating agent; The ionic strength modifier is one of sodium chloride and potassium chloride, and the concentration is preferably 50-500 mM; The co-solvent is at least one of anionic surfactant, cationic surfactant, guanidine hydrochloride, and urea; The anionic surfactant is preferably one of sodium lauroyl amino acid, n-hexadecyltrimethylammonium chloride, sodium dodecyl sulfate, and sodium deoxycholate. The reducing agent is at least one of the following: disulfide reducing agent (DTT), tris(2-carboxyethyl)phosphamide (TCEP), β-mercaptoethanol, dithioerythritol, cysteine, and reduced glutathione; The chelating agent is at least one of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA).

10. The method according to claim 1, characterized in that: The purification medium is column chromatography microspheres, magnetic beads, chromatography membranes, monolithic columns, or expanded column bed media.

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