A method for purifying vaccinia virus capping enzymes

Abstract

This invention provides a method for purifying vaccinia virus capped enzymes, relating to the field of vaccinia virus capped enzyme processing technology, comprising the following steps: Step A: The recombinant expression cells are disrupted, centrifuged, and filtered to collect the supernatant; Step B: The supernatant from Step A is captured by NiN affinity chromatography to obtain an eluted sample; Step C: The eluted sample from Step B is further purified by cation exchange chromatography; Step D: The eluted sample from Step C is further purified by hydrophobic chromatography, and the SEC-HPLC purity meets expectations; Step E: The eluted sample from Step D is ultrafiltered and stored in a storage solution after ultrafiltration. Compared to commercially available, complex, and costly vaccinia virus capped enzyme purification methods, this invention optimizes the purification steps, achieving a sample SEC-HPLC purity greater than 90%, significantly improving the overall purification recovery rate, and reducing preparation costs. Furthermore, the ultrafiltration device of this invention has been adapted, resulting in high SEC-HPLC purity of the vaccinia virus capped enzyme and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of vaccinia virus capping enzyme processing technology, specifically to a method for purifying vaccinia virus capping enzymes. Background Technology

[0002] In eukaryotes, mRNA undergoes post-transcriptional modification to form a special structure at its 5′ end, known as a cap structure, such as... Figure 1 As shown, this structure plays a crucial role in the stability, transport, and translation of mRNA.

[0003] Vaccinia virus capping enzyme is an efficient enzyme that catalyzes the formation of a cap structure. It consists of two subunits, D1 and D12, and possesses activities combining RNA triphosphatase, guanylate transferase, and guanine methyltransferase. Vaccinia virus capping enzyme can be used in mRNA production. Adding certain basic structural elements to in vitro synthesized mRNA can achieve similar effects to eukaryotic mRNA. For example, adding an m7GPPPN structure (7-methylguanosine cap) and a 3' poly(A) tail to the 5' end of the protein-coding open reading frame (ORF) can promote mRNA stability.

[0004] like Figure 1 As shown, the vaccinia virus capping enzyme consists of two subunits (D1 and D12), with protein molecular weights of 98 kDa for D1 and 33 kDa for D12. The D1 subunit functions as an RNA triphosphatase and guanylate transferase, while the D12 subunit functions as a guanine methyltransferase. Both are essential for the addition of a complete Cap0 structure m7Gppp5´N.

[0005] In existing technologies, recombinant vaccinia virus capping enzyme refers to the introduction of the vaccinia virus capping enzyme gene into host cells through genetic engineering, enabling its expression and synthesis within the host cells. This process aims to utilize the host cell's biosynthetic mechanisms to produce large quantities of vaccinia virus capping enzyme protein.

[0006] Recombinant vaccinia virus capping enzymes are typically expressed using E. coli fermentation, and the protein is expressed via a non-covalently linked form where both subunits are co-expressed. This recombination process has the following drawbacks: Firstly, the large molecular weight and easy degradation of the vaccinia virus capping enzyme protein make it difficult for E. coli to express in a soluble manner, resulting in low expression levels after fermentation, increased difficulty in purification and production, low final product yield, and increased mRNA production costs.

[0007] Secondly, the post-fermentation purification process, involving cell disruption, chromatography, and ultrafiltration, yields products meeting quality requirements. Commercially available methods for vaccinia virus purification using capped enzymes typically include: cell disruption and clarification, Nitrogen affinity chromatography, ultrafiltration with liquid exchange, cation exchange chromatography, anion exchange chromatography, cation exchange chromatography, and molecular sieve chromatography. These methods are highly complex, resulting in low purification yields and long production cycles. The complexity of the process and the product's susceptibility to degradation can lead to very low purification recovery rates, thereby increasing production costs.

[0008] Therefore, there are areas for improvement. This invention provides a method for purifying vaccinia virus by adding a capping enzyme. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to propose a method for purifying vaccinia virus by adding a capping enzyme, the specific scheme of which is as follows: A method for purifying vaccinia virus by adding a capping enzyme, the method comprising the following steps: Step A: The recombinant expression cells are broken, centrifuged, filtered, and the supernatant is collected; Step B: Capture the filtered supernatant from Step A using Ni affinity chromatography to obtain the eluted sample; Step C: The eluted sample from step B is further purified by cation exchange chromatography; Step D: The eluted sample from step C was further purified by hydrophobic chromatography, and the purity obtained by SEC-HPLC met expectations. Step E: After ultrafiltration and liquid exchange using an ultrafiltration liquid exchanger, the eluted sample from step D is stored in a storage solution.

[0010] The specific process of step A is as follows: (1) Resuspend the recombinant expression cells in a 1:7 ratio with the lysis buffer at room temperature for 1-3 hours; (2) The resuspended bacterial cells were crushed by a high-pressure homogenizer. The high-pressure homogenizer crushing parameters were: 800 bar, 3 times. (3) Centrifuge the broken bacterial cells at 8000 rpm, 4℃, and 1 h, and centrifuge twice. (4) The supernatant from centrifugation was filtered using a 0.45μm filter.

[0011] Specifically, the components of the lysis buffer are: 20mM PB, 50mM NaCl, 1mM EDTA·2Na·2H2O, 10% glycerol, pH 8.0.

[0012] It should be noted that step A involves lysing the recombinant expression cells to release their internal organelles and solutes, thereby obtaining the target protein or other cellular components. The 20mM phosphate buffer (PB) provides a buffer environment for the lysed target protein. The 50mM sodium chloride (NaCl) prevents non-specific adsorption of impurities onto the NI affinity chromatography column during loading. The 1mM EDTA·2Na·2H2O is a chelating agent that chelates metal ions, helping to prevent the activation of some metal-dependent nucleases (such as nucleases) during cell lysis, thus protecting intracellular nucleic acid molecules. The presence of 10% glycerol in the lysis buffer acts as a protectant, slowing protein degradation and maintaining protein integrity. Glycerol also increases solubility after cell lysis, aiding in the separation and purification of the target protein.

[0013] The specific process of step B is as follows: The chromatography system and column were cleaned with purified water and 0.5M NaOH solution, and then the column was treated with 0.1M NiSO4 solution to coat it with nickel. The column was then equilibrated with NI affinity chromatography binding buffer, and the sample was loaded. After the sample loading was completed, the column was first washed with NI affinity chromatography binding buffer, then washed with NI affinity chromatography washing buffer, and finally eluted isocratically with NI affinity chromatography binding buffer and NI affinity chromatography elution buffer, with gradients set to 5%, 10%, 25%, 50%, and 100%.

[0014] Specifically, in step B, the chromatography column used is a NI-NTA-HP chromatography column; The components of the NI affinity chromatography binding buffer are 20 mM PB, 50 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, pH 8.0; The NI affinity chromatography wash buffer consisted of 20 mM PB, 500 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, and pH 8.0. The NI affinity chromatography elution buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M imidazole, 10% glycerol, and pH 8.0.

[0015] It should be noted that step B mainly involves NI affinity chromatography capture. Affinity chromatography is a technique for separating and purifying biomolecules, in which separation is achieved through the selective interaction between the target molecule and a specific affinity ligand. This step uses nickel (Ni) ions as an affinity ligand. The chromatography system and column are cleaned with purified water and 0.5M NaOH solution to prepare the system and ensure the column surface is free of impurities. Next, the column is coated with nickel using 0.1M NiSO4 solution to introduce nickel ions, facilitating affinity interactions with the target molecule. The column is then equilibrated with Ni affinity chromatography binding buffer to ensure the nickel ions are ready to bind to the target molecule. Sample loading is then performed to add the sample to be separated into the column. The column is then cleaned, first with Ni affinity chromatography binding buffer and then with Ni affinity chromatography washing buffer to remove weakly bound impurities. Finally, isocratic elution with binding buffer and elution buffer elutes the target molecule from the column. This step is crucial for the separation and purification of the target molecule.

[0016] Unlike the lysis buffer in step A, the NI affinity chromatography elution buffer also uses imidazole. The main role of imidazole in NI affinity chromatography is to competitively bind to the chromatography medium (usually a nickel column) with the target protein, thus competitively dissociating it. A high concentration of 1M imidazole can also effectively elute proteins bound to the chromatography medium. This elution condition is achieved by adjusting the imidazole concentration, thereby affecting the interaction between the protein and the nickel column, allowing the protein to desorb and elute from the column.

[0017] The addition of imidazole can also be used to adjust the conditions of the elution buffer to ensure that elution is specific and efficient. Optimal elution results can be achieved by optimizing the concentration of imidazole and the composition of other elution buffers.

[0018] The specific process of step C is as follows: The pH of the eluted sample in step B was adjusted to 7.0±0.1 using a cationic chromatography acidic adjustment buffer, while the sample conductivity was monitored. The sample was filtered through a 0.45 μm filter, and the chromatography system and column were washed with purified water and 0.5 M NaOH solution. The column was then equilibrated with cationic chromatography binding buffer, the sample was loaded, and the column was equilibrated again with cationic chromatography binding buffer. Finally, the sample was eluted with cationic chromatography elution buffer at 0-100%.

[0019] Specifically, cation chromatography uses a Rigose-SP-HP chromatography column; The acid conditioning solution for cation chromatography consisted of 1M citric acid. The cation chromatography binding buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, and pH 7.0. The elution buffer for cation chromatography consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M NaCl, and pH 7.0.

[0020] It should be noted that cation chromatography is based on the interaction between the target protein and the negatively charged chromatographic medium. Elution conditions are controlled by adjusting pH and ionic strength to achieve the separation and purification of the target protein. This chromatographic technique uses a cation chromatography acidic adjusting buffer to adjust the pH of the elution sample to 7.0 ± 0.1. This ensures the sample is within an appropriate pH range during loading, maximizing the chromatographic loading of the target protein. Monitoring the sample conductivity during pH adjustment and filtering the sample using a 0.45-micron filter are all done to prevent sample flow-through during cation chromatography loading. The chromatography system and column are cleaned with purified water and 0.5M NaOH solution to remove any impurities and buffer residues that may have remained from previous operations, preparing them for the next step. Equilibrating the column with a cation chromatography binding buffer ensures the medium within the column is at a suitable buffering condition, creating appropriate conditions for sample loading and separation. After loading the sample, the column is reequilibrated with cationic chromatography binding buffer to ensure that the column remains under appropriate conditions after loading, thus ensuring efficient separation. Elution is then performed using cationic chromatography elution buffer to elute the target protein from the column. In this step, the concentration of the elution buffer is usually gradually increased from 0% to 100% to gradually reduce the interaction between the target protein and the chromatography medium, thereby achieving elution.

[0021] The specific process of step D is as follows: First, dilute the (NH4)2SO4 concentration of the eluted sample in step C to 0.75M using hydrophobic chromatography stock solution. Filter through a 0.45μm filter. Clean the chromatography system and column with purified water and 0.5M NaOH solution. Then, equilibrate the column with hydrophobic chromatography binding buffer, load the sample, equilibrate the column again with hydrophobic chromatography binding buffer, and finally elute isocratically with hydrophobic chromatography elution buffer at gradients of 20%, 40%, 60%, 80%, and 100%.

[0022] Specifically, in step D, hydrophobic chromatography uses a Capto Butyl ImpRes chromatography column; The hydrophobic chromatography mother liquor consisted of 20 mM PB, 3 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.5. The hydrophobic chromatography binding buffer consisted of 20 mM PB, 0.75 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.5. The hydrophobic chromatography elution buffer was 20 mM PB, 1 mM EDTA·2Na·2H2O, pH 7.5.

[0023] It should be noted that hydrophobic chromatography is based on the difference in surface hydrophobicity of proteins under different solvent polarity conditions, and uses gradient elution to achieve the separation of target proteins. Diluting the mother liquor can increase the salt concentration (ammonium sulfate) in the sample, exposing the hydrophobic groups of the target protein and preparing it for subsequent chromatographic steps; the sample is filtered using a 0.45-micron filter. The purpose of this step is to remove particulate matter and suspended solids, ensuring sample clarity and protecting the chromatography column and instrument. Cleaning the chromatography system and column with purified water and 0.5M NaOH solution removes any impurities and buffer residues that may have remained from previous steps, preparing them for the next step. Equilibrating the column with hydrophobic chromatography binding buffer ensures the medium within the column is under suitable buffering conditions, creating appropriate conditions for sample loading and separation. Sample loading is then performed. The column is then equilibrated again with hydrophobic chromatography binding buffer to ensure it remains under appropriate conditions after sample loading, ensuring efficient separation. Elution is then performed using hydrophobic chromatography elution buffer with a gradient. By changing the salt concentration of the elution buffer, the binding affinity between the target protein and the chromatography medium is gradually reduced, achieving elution of the target protein.

[0024] In step E, the ultrafiltration fluid exchange device includes an ultrafiltration replacement fluid storage tank, a water for injection storage tank, a NaOH solution storage tank, a diaphragm pump, an ultrafiltration replacement bag, an ultrafiltration membrane pack, a peristaltic pump, and a waste liquid storage tank. The outlets of the ultrafiltration replacement fluid storage tank, the water for injection storage tank, the NaOH solution storage tank, and the ultrafiltration replacement bag are all connected to the inlet of the diaphragm pump. The outlet of the diaphragm pump is connected to the inlet of the ultrafiltration membrane pack. The waste liquid end and the reflux end of the ultrafiltration membrane pack are connected to the waste liquid storage tank and the inlet of the ultrafiltration replacement bag, respectively. The ultrafiltration replacement fluid storage tank is also connected to the inlet end of the peristaltic pump, and the outlet end of the peristaltic pump is connected to the inlet end of the ultrafiltration replacement bag.

[0025] Therefore, this ultrafiltration device uses a peristaltic pump and a diaphragm pump connected to the ultrafiltration membrane pack and the ultrafiltration replacement bag. The diaphragm pump is mainly connected to different buffer solutions and the ultrafiltration replacement bag containing the sample. The diaphragm pump is mainly connected to the ultrafiltration buffer solution for replenishing the ultrafiltration replacement solution. The inlet end of the ultrafiltration membrane pack is connected to the diaphragm pump, and the outlet end is divided into a reflux end and a waste liquid end. The reflux end is connected to the ultrafiltration replacement bag, and the waste liquid end is connected to the waste liquid storage tank. The ultrafiltration membrane pack needs to be matched with ultrafiltration clamps. Ultrafiltration clamps of different sizes can be matched with membrane packs of various membrane areas to meet the production needs of different specifications.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The purification method developed in this invention includes: cell disruption and clarification, NI affinity chromatography, cation chromatography, hydrophobic chromatography, and ultrafiltration with medium exchange. Its purification steps are simpler than the commercially available vaccinia virus capped enzyme purification method. Most importantly, it does not require separation methods such as molecular sieves, which have high scale-up costs and require highly skilled personnel. Moreover, the purification method of this invention also has a significantly higher yield than commercially available purification methods.

[0027] (2) The purification method of the present invention has a relatively short process, which reduces the complexity of the steps and makes it easier to operate and implement. Moreover, NI affinity chromatography can efficiently enrich target proteins with specific affinity tags and improve the purity of the target proteins. Hydrophobic chromatography is used to further improve the purity, remove overexpressed small subunits and some non-specifically bound impurities, and ultrafiltration can be used to concentrate the target proteins while removing low molecular weight solvents and salts.

[0028] (3) Among them, compared with the ultrafiltration liquid exchange device on the market, the ultrafiltration liquid exchange device set in this invention, through the adjustment of the components and the improvement of the cooperation between the components, makes the ultrafiltration liquid exchange process stable and efficient, and will not damage the sample. This results in the high purity of the vaccinia virus capped enzyme obtained in the end, with no impurities generated, and thus has a wider range of application prospects.

[0029] (4) In summary, when the purification method and ultrafiltration liquid exchange device of the present invention are applied to the field of vaccinia virus capped enzyme purification, the purification method can be simplified, and a higher recovery rate can be obtained, thereby increasing product yield and product quality and reducing commercial production costs. Attached Figure Description

[0030] Figure 1 A schematic diagram of the protein-simulated crystal structure of the capping enzyme for vaccinia virus.

[0031] Figure 2 This is a diagram of the 5' cap structure of mRNA.

[0032] Figure 3 A schematic diagram illustrating the protein expression of the vaccinia virus capping enzyme (VCE).

[0033] Figure 4 SEC-HPLC chromatogram of vaccinia virus after purification by capping enzyme (VCE).

[0034] Figure 5 This is a schematic diagram of an ultrafiltration liquid exchange device.

[0035] Figure 6 This is an electrophoretic pattern of vaccinia virus capping enzyme (VCE) purified by the ultrafiltration liquid exchange device of the present invention.

[0036] Figure 7 This is an electrophoretic pattern of vaccinia virus capping enzyme (VCE) purified using commercially available ultrafiltration exchange devices. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] In existing technologies, affinity chromatography is a technique for separating and purifying biomolecules. Its basic principle is to utilize the selective interaction between the target molecule and specific affinity ligands in an affinity column. The affinity ligands in the affinity column are typically domains specific to the target molecule or specific tags, which allows the target molecule to bind to the affinity ligands on the affinity column and subsequently be separated by an elution step.

[0039] As shown in the figure, the present invention provides a method for purifying vaccinia virus by capping enzyme and an ultrafiltration device. The present invention will be described below with reference to specific embodiments. Experimental methods in the following embodiments without specific conditions are generally carried out under conventional conditions.

[0040] Example 1: A method for purifying vaccinia virus by adding a capping enzyme, specifically including the following steps: Step A: The recombinant expression cells are broken, centrifuged, filtered, and the supernatant is collected; Step B: Capture the filtered supernatant from Step A using Ni affinity chromatography to obtain the eluted sample; Step C: The eluted sample from step B is further purified by cation exchange chromatography; Step D: The eluted sample from step C was further purified by hydrophobic chromatography, and the purity obtained by SEC-HPLC met expectations. Step E: After ultrafiltration and liquid exchange using an ultrafiltration liquid exchanger, the eluted sample from step D is stored in a storage solution.

[0041] It should be noted that the recombinant expression cells of the vaccinia virus capping enzyme are Escherichia coli cells.

[0042] The specific process of step A is as follows: The recombinant expressed bacterial cells were resuspended in lysis buffer at room temperature for 1-3 hours. The amount of lysis buffer used was 7 mL per gram of bacterial cells. The bacterial cells were then homogenized using a high-pressure homogenizer, and the supernatant after centrifugation was filtered to obtain the filtrate.

[0043] The preferred high-pressure homogenizer is 800 bar, with 3 cycles of sterilization; the preferred centrifugation conditions are centrifugation at 8000 rpm for 1 hour at 4°C, followed by 2 cycles; the preferred filtration conditions are filtration using a 0.45 μm filter at a pressure less than 1.5 bar; the components of the sterilization buffer are: 20 mM PB, 50 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, pH 8.0.

[0044] The supernatant and precipitate after lysis were subjected to electrophoresis to characterize protein expression.

[0045] When performing electrophoresis on the supernatant and precipitate after bacterial lysis, whole bacteria and markers were used for comparison. Samples were taken at 0h, 12h, and 24h during induction, and corresponding electrophoresis analyses were performed at different induction times. The electrophoresis results are as follows: Figure 3 As shown, the band patterns of the whole bacteria and supernatant after induction for 0h, 12h, and 24h were not significantly different. The precipitates induced for 12h and 24h showed corresponding bands at relative molecular weights of 35KD and 95KD, respectively, possibly indicating inclusion body expression. The precipitate induced for 0h only showed a corresponding band at a relative molecular weight of 35KD. This indicates that as the induction time increases, the greater the weight of the bacterial precipitate, the higher the final protein yield. Therefore, the supernatant obtained after lysis in step A can be used for subsequent steps.

[0046] The specific process of step B is as follows: The chromatography system and column were cleaned with purified water and 0.5M NaOH solution. The column was then treated with 0.1M NiSO4 solution to deposit nickel. The column was then equilibrated with NI affinity chromatography-binding buffer, rinsed with purified water until pH neutral, and equilibrated again with NI affinity chromatography-binding buffer. The column was zeroed at 280nm UV light, and the sample was loaded (at a loading capacity not exceeding 6 mg / mL) for 5 min. After loading, the column was first washed with NI affinity chromatography-binding buffer until the UV light reached the baseline, then washed with NI affinity chromatography washing buffer. Finally, isocratic elution was performed with NI affinity chromatography-binding buffer and NI affinity chromatography elution buffer at gradients of 5%, 10%, 25%, 50%, and 100%.

[0047] The chromatographic retention time was set to 5 min, and the eluted sample was subjected to electrophoresis detection to finally obtain the purified vaccinia virus capping enzyme.

[0048] It should be noted that the chromatography column used is a NI-NTA-HP chromatography column (purchased from Qianchun Biotechnology), and the chromatography column filled with NI-NTA-HP packing material is connected to the column position valve of the chromatography system. The components of the NI affinity chromatography binding buffer are 20 mM PB, 50 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, pH 8.0; The NI affinity chromatography wash buffer consisted of 20 mM PB, 500 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, and pH 8.0. The NI affinity chromatography elution buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M imidazole, 10% glycerol, and pH 8.0.

[0049] The specific process of step C is as follows: The pH of the eluted sample in step B was adjusted to 7.0±0.1 using cation chromatography acidic adjustment buffer, while the sample conductivity was monitored. The sample was filtered through a 0.45 μm filter, and the chromatography system and column were cleaned with purified water and 0.5 M NaOH solution. The column was then equilibrated with cation chromatography system binding buffer. After zeroing the column with UV A280 nm, the sample was loaded (the loading amount was no more than 5 mg / mL). The column was then equilibrated again with cation chromatography binding buffer, and finally eluted with cation chromatography elution buffer at 0-100% concentration for a retention time of 2 min.

[0050] It should be noted that the cation chromatography uses a Rigose-SP-HP chromatography column (purchased from Qianchun Biotechnology), and the chromatography column packed with Rigose-SP-HP packing material is connected to the column position valve of the chromatography system. The acid conditioning solution for cation chromatography consisted of 1M citric acid. The cation chromatography binding buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, and pH 7.0. The elution buffer for cation chromatography consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M NaCl, and pH 7.0.

[0051] The specific process of step D is as follows: First, dilute the (NH4)2SO4 concentration of the eluted sample in step C to 0.75M using hydrophobic chromatography stock solution. Filter through a 0.45μm filter. Clean the chromatography system and column with purified water and 0.5M NaOH solution. Then, equilibrate the column with hydrophobic chromatography binding buffer, load the sample, equilibrate the column again with hydrophobic chromatography binding buffer, and finally elute isocratically with hydrophobic chromatography elution buffer at gradients of 20%, 40%, 60%, 80%, and 100%, with a retention time of 2 min.

[0052] It should be noted that in step D, hydrophobic chromatography uses a Capto Butyl ImpRes chromatography column (purchased from Cytiva), and the chromatography column packed with Capto Butyl ImpRes packing material is connected to the column position valve of the chromatography system. The hydrophobic chromatography mother liquor consisted of 20 mM PB, 3 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.5. The hydrophobic chromatography binding buffer consisted of 20 mM PB, 0.75 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.5. The hydrophobic chromatography elution buffer was 20 mM PB, 1 mM EDTA·2Na·2H2O, pH 7.5.

[0053] The eluted sample purified by hydrophobic chromatography was analyzed by SEC-HPLC, and the results are as follows: Figure 4 As shown.

[0054] It should be noted that SEC-HPLC is commonly used to analyze proteins, peptides, nucleic acids, and other biomolecules. By measuring the residence time of molecules in the column, their relative molecular mass, molecular size distribution, or purity can be determined.

[0055] When SEC-HPLC is used to assess sample purity, if the column contains only the target molecule, other impurities or small molecules in the sample will be eluted later at positions with longer residence times on the column, while the target molecule will be eluted earlier at positions with shorter residence times, thus achieving the separation of the target molecule. Figure 4 As can be seen, a peak appears at a residence time of 13.591 min, which represents the target molecule (vaccinia virus capping enzyme). The residence time is basically consistent with that of the standard, indicating that the purity of SEC-HPLC meets expectations.

[0056] In step E, the ultrafiltration fluid exchange device includes an ultrafiltration replacement fluid storage tank, a water for injection storage tank, a NaOH solution storage tank, a diaphragm pump, an ultrafiltration replacement bag, an ultrafiltration membrane pack, a peristaltic pump, and a waste liquid storage tank. The outlets of the ultrafiltration replacement fluid storage tank, the water for injection storage tank, the NaOH solution storage tank, and the ultrafiltration replacement bag are all connected to the inlet of the diaphragm pump. The outlet of the diaphragm pump is connected to the inlet of the ultrafiltration membrane pack. The waste liquid end and the reflux end of the ultrafiltration membrane pack are connected to the waste liquid storage tank and the inlet of the ultrafiltration replacement bag, respectively. The ultrafiltration replacement fluid storage tank is also connected to the inlet end of the peristaltic pump, and the outlet end of the peristaltic pump is connected to the inlet end of the ultrafiltration replacement bag.

[0057] The ultrafiltration liquid exchange device of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the ultrafiltration liquid exchange device can be understood by those skilled in the art, thereby making a clearer definition of the scope of protection of the ultrafiltration liquid exchange device.

[0058] like Figure 5 As shown, this ultrafiltration fluid exchange device uses a peristaltic pump and a diaphragm pump connected to the ultrafiltration membrane pack and the ultrafiltration replacement bag. The diaphragm pump is mainly connected to different buffer solutions and the ultrafiltration replacement bag containing the sample. The diaphragm pump is mainly connected to the ultrafiltration buffer solution for replenishing the ultrafiltration replacement solution. The inlet end of the ultrafiltration membrane pack is connected to the diaphragm pump, and the outlet end is divided into a reflux end and a waste liquid end. The reflux end is connected to the ultrafiltration replacement bag, and the waste liquid end is connected to the waste liquid storage tank.

[0059] The specific usage process is as follows: (1) First, use a torque wrench to assemble the ultrafiltration membrane pack and ultrafiltration fixture. Then, clean the ultrafiltration membrane pack with 0.5M NaOH and purified water until the pH is neutral. Then, perform a water flux test on the ultrafiltration membrane pack and compare the test result with the initial water flux of the membrane pack. If the water flux result is less than 90% of the initial water flux, it means that the ultrafiltration membrane pack may not have been cleaned thoroughly and needs to be rinsed repeatedly with 0.5M NaOH or 0.1M citric acid until the water flux test is qualified.

[0060] (2) Next, perform an integrity test on the ultrafiltration membrane pack that has passed the water flux test. The integrity test parameters shall be based on those provided in the instruction manual for the ultrafiltration membrane pack (diffusion flow or bubble point method). Only after passing the test can the next step be carried out. If the test fails, the cause needs to be investigated. It may be that the membrane pack was not cleaned properly, which caused the surface tension of the liquid in the membrane pack pores to change and thus fail the test, or that the ultrafiltration membrane pack was not clamped tightly to the ultrafiltration fixture. It is necessary to reassemble it with a torque wrench. Only after passing the test can the next step be carried out.

[0061] (3) Next, the ultrafiltration membrane pack is cleaned. Different buffer solutions are selected by controlling the inlet pipeline of the diaphragm pump. First, purified water is selected to rinse the membrane surface and pores of the ultrafiltration membrane pack. Then, NaOH solution is selected to clean the membrane surface and pores of the membrane pack. Next, purified water is selected to rinse the membrane surface and pores of the membrane pack until the pH is neutral. Finally, ultrafiltration replacement solution is selected to rinse the membrane surface and pores of the membrane pack until the pH of the reflux end and the permeate end is consistent with that of the replacement buffer.

[0062] (4) Next, perform ultrafiltration concentration and liquid replacement of the sample. Lock the peristaltic pump end, start the diaphragm pump, and pressurize after the flow rate stabilizes so that the transmembrane pressure reaches the set value. After concentration to the set volume, open the peristaltic pump end and start the peristaltic pump. Adjust the flow rate of the peristaltic pump so that the volume of the ultrafiltration replacement bag remains unchanged. Replace the liquid to the specified number of liquid replacements and empty the membrane bag.

[0063] (5) Top washing: Take a new ultrafiltration replacement bag, add an appropriate amount of ultrafiltration replacement solution to the ultrafiltration replacement bag, clamp the permeate end of the ultrafiltration membrane pack, circulate for 5-10 minutes, drain, and combine the top washing sample with the ultrafiltration replacement solution sample.

[0064] (6) Finally, the ultrafiltration membrane pack is cleaned and regenerated with purified water and NaOH solution. After cleaning, the membrane pack is rinsed until the pH is neutral and a water flux test is performed. If the test is qualified, it is stored in the storage solution.

[0065] It should be noted that this ultrafiltration fluid exchange device can replace diaphragm pumps, peristaltic pumps and ultrafiltration membrane packs of different sizes at any time to meet the needs. Most importantly, the ultrafiltration fluid exchange device has a small dead volume and the ultrafiltration system is emptied very thoroughly. After the sample is emptied, it is washed and combined with ultrafiltration buffer 2-3 times, which has almost no effect on the concentration of the final product and the recovery rate is close to 100%.

[0066] Regarding Example 1 above, the present invention performed electrophoretic detection on the vaccinia virus capping enzyme purified in Example 1 and the vaccinia virus capping enzyme obtained by commercially available vaccinia virus capping enzyme purification methods. The results are as follows: Figure 6 and Figure 7 The recovery rate of vaccinia virus capping enzyme obtained by commercially available vaccinia virus capping enzyme purification methods was also compared with that obtained by commercially available vaccinia virus capping enzyme purification methods. See Table 1 below.

[0067] Figure 6 The electrophoretic detection results of this invention, Figure 7 Electrophoretic detection results of capped enzymes for vaccinia virus on the market, combined with Figure 6 and Figure 7 It can be seen that, Figure 6 Only at relative molecular weights of 35 KD and 95 KD did corresponding bands appear; however, in Figure 7In addition to the corresponding bands at relative molecular weights of 35KD and 95KD, a corresponding band appeared at a relative molecular weight of 65KD, which belongs to the impurities generated after ultrafiltration.

[0068] from Figure 6 and Figure 7 The electrophoresis results show that because the ultrafiltration liquid exchange device of the present invention uses a combination of diaphragm pump and ultrafiltration membrane pack, the shear force generated by the diaphragm pump during use is small, so no corresponding band appears at the relative molecular weight of 65KD, that is, no impurities are generated.

[0069] Table 1 As shown in Table 1, the vaccinia virus capped enzyme purification method of the present invention has a better recovery rate than commercially available vaccinia virus capped enzyme purification methods, both in terms of the recovery rate in a single step and the overall recovery rate.

[0070] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for purifying vaccinia virus by adding a capping enzyme, characterized in that, The method includes the following steps: Step A: The recombinant bacterial cells are broken, centrifuged, filtered, and the supernatant is collected; Step B: Capture the supernatant from step A using Ni affinity chromatography to obtain the eluted sample; Step C: The eluted sample from Step B is further purified by cation exchange chromatography. The specific process is as follows: The pH of the eluted sample in step B was adjusted to 7.0±0.1 using a cationic chromatography acidic adjustment buffer while monitoring the sample conductivity. The sample was filtered through a 0.45 μm filter, and the chromatography system and column were washed with purified water and 0.5 M NaOH solution. The column was then equilibrated with cationic chromatography binding buffer, the sample was loaded, and the column was equilibrated again with cationic chromatography binding buffer. Finally, the sample was eluted with cationic chromatography elution buffer at 0-100%. Cation chromatography was performed using a Rigose-SP-HP column; The acid conditioning solution for cation chromatography consisted of 1M citric acid. The cation chromatography binding buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, and pH 7.

0. The elution buffer for cation chromatography consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M NaCl, and pH 7.

0. Step D: The eluted sample from Step C is further purified by hydrophobic chromatography. The specific process is as follows: First, the concentration of (NH4)2SO4 in the eluted sample from step C was diluted to 0.75M using hydrophobic chromatography stock solution. The solution was then filtered through a 0.45μm filter. The chromatography system and column were washed with purified water and 0.5M NaOH solution. The column was then equilibrated with hydrophobic chromatography binding buffer, and the sample was loaded. The column was then equilibrated again with hydrophobic chromatography binding buffer. Finally, isocratic elution was performed using hydrophobic chromatography elution buffer with gradients of 20%, 40%, 60%, 80%, and 100%. Hydrophobic chromatography was performed using a Capto Butyl ImpRes chromatography column; The hydrophobic chromatography mother liquor consisted of 20 mM PB, 3 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.

5. The hydrophobic chromatography binding buffer consisted of 20 mM PB, 0.75 M (NH4)2SO4, 1 mM EDTA·2Na·2H2O, and pH 7.

5. The hydrophobic chromatography elution buffer was 20 mM PB, 1 mM EDTA·2Na·2H2O, pH 7.5; Step E: After ultrafiltration and liquid exchange of the eluted sample from Step D, the sample is stored in the storage solution. The ultrafiltration and liquid exchange device includes an ultrafiltration replacement solution storage tank, a water for injection storage tank, a NaOH solution storage tank, a diaphragm pump, an ultrafiltration replacement bag, an ultrafiltration membrane pack, a peristaltic pump, and a waste liquid storage tank. The outlets of the ultrafiltration replacement fluid storage tank, the water for injection storage tank, the NaOH solution storage tank, and the ultrafiltration replacement bag are all connected to the inlet of the diaphragm pump. The outlet of the diaphragm pump is connected to the inlet of the ultrafiltration membrane pack. The waste liquid end and the reflux end of the ultrafiltration membrane pack are connected to the waste liquid storage tank and the inlet of the ultrafiltration replacement bag, respectively. The ultrafiltration replacement fluid storage tank is also connected to the inlet end of the peristaltic pump, and the outlet end of the peristaltic pump is connected to the inlet end of the ultrafiltration replacement bag.

2. The method for purifying vaccinia virus by capping enzyme according to claim 1, characterized in that, The specific process of step A is as follows: (1) Resuspend the recombinant expression cells in a 1:7 ratio with the lysis buffer at room temperature for 1-3 hours; (2) The resuspended bacterial cells were crushed by a high-pressure homogenizer. The high-pressure homogenizer crushing parameters were: 800 bar, 3 times. (3) Centrifuge the broken bacterial cells at 8000 rpm, 4℃, and 1 h, and centrifuge twice. (4) The supernatant from centrifugation was filtered using a 0.45μm filter.

3. The method for purifying vaccinia virus by capping enzyme according to claim 2, characterized in that, The components of the lysis buffer are: 20mM PB, 50mM NaCl, 1mM EDTA·2Na·2H2O, 10% glycerol, pH 8.

0.

4. The method for purifying vaccinia virus by capping enzyme according to claim 3, characterized in that, The specific process of step B is as follows: The chromatography system and column were cleaned with purified water and 0.5M NaOH solution, and then the column was treated with 0.1M NiSO4 solution to coat it with nickel. The column was then equilibrated with NI affinity chromatography binding buffer, and the sample was loaded. After the sample loading was completed, the column was first washed with NI affinity chromatography binding buffer, then washed with NI affinity chromatography washing buffer, and finally eluted isocratically with NI affinity chromatography binding buffer and NI affinity chromatography elution buffer, with gradients set to 5%, 10%, 25%, 50%, and 100%.

5. The method for purifying vaccinia virus by capping enzyme according to claim 2, characterized in that, In step B, a NI-NTA-HP chromatography column is used; The components of the NI affinity chromatography binding buffer are 20 mM PB, 50 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, pH 8.0; The NI affinity chromatography wash buffer consisted of 20 mM PB, 500 mM NaCl, 1 mM EDTA·2Na·2H2O, 10% glycerol, and pH 8.

0. The NI affinity chromatography elution buffer consisted of 20 mM PB, 1 mM EDTA·2Na·2H2O, 1 M imidazole, 10% glycerol, and pH 8.0.

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