Eluent for improving anion exchange chromatography recovery rate of lentivirus and application thereof

Abstract

The invention discloses an eluent for improving the anion exchange chromatography recovery rate of lentivirus and application of the eluent. The eluent is prepared from a basic buffer solution, 50 to 500 mM of NaCl and 0.2 to 2 M of L-arginine; the pH (Potential of Hydrogen) value of the eluent is 6.5 to 8.0. In order to solve the problems that in the prior art, high-salt elution is adopted, so that the lentivirus activity damage is large, and the recovery rate is low, the concentration of a main elution component NaCl in an eluent is reduced, then the main elution component NaCl is compounded with L-arginine, the lentivirus can be effectively eluted, meanwhile, the damage of a high-salt environment to a virus envelope structure is avoided, and the yield of the lentivirus is improved. And the elution efficiency is improved by utilizing the characteristic that L-arginine inhibits protein interaction. When the eluent is used for chromatographic purification, the recovery rate and activity of the lentivirus can be remarkably improved, impurities such as host cell protein and the like are effectively removed, and a key purification solution is provided for preparing a high-purity and high-activity lentiviral vector for gene therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an eluent for improving the recovery rate of lentivirus anion exchange chromatography and its application. Background Technology

[0002] In gene therapy, lentiviral vectors have become a key tool due to their unique advantages. Lentivirals, belonging to the retroviridae family, can stably integrate foreign genes into the host cell's genome, achieving long-term expression of the target gene. Based on lentiviral delivery technology, the US FDA has approved more than ten products, including well-known CAR-T therapies such as YESCARTA, TECARTUS, KYMRIAH, and CARVYKTI. These successfully marketed products not only provide patients with new treatment options but also fully demonstrate the enormous potential and important value of lentiviral vectors in gene therapy.

[0003] In recent years, the number of clinical trials related to the in vivo application of lentiviruses globally has increased by an average of 35% annually. In vivo application places far higher standards on the purity, activity, and safety of lentiviruses than on in vitro applications. For example, in vivo injectable formulations must strictly control host cell protein (HCP) residues to below 100 ng / mg, and viral particles must maintain an intact envelope structure to avoid in vivo immunogenic reactions. This presents entirely new challenges to lentivirus purification technology. Whether it's basic research conducted by scientific research institutions or clinical trials and drug development carried out by pharmaceutical companies, a large number of high-quality lentiviral vectors are needed. Market research institutions predict that the global lentiviral vector market will continue to expand at a high growth rate in the coming years, and its market size will continue to grow. Against this market backdrop, how to efficiently and stably produce high-quality lentiviral vectors to meet the ever-increasing market demand has become a critical issue that urgently needs to be addressed in the field of gene therapy.

[0004] The Challenges of Traditional Lentiviral Purification Technology: The purification of lentiviral vectors is a crucial step in obtaining highly active and pure products, directly impacting the safety and efficacy of gene therapy. However, current traditional lentiviral purification technologies face numerous challenges in practical applications, failing to meet the ever-growing market demand and stringent quality standards.

[0005] (a) Limitations of ultracentrifugation sedimentation Ultracentrifugation precipitation is one of the more commonly used methods for lentivirus purification. Its principle is to separate lentiviruses from complex systems such as cell culture media by utilizing the difference in sedimentation rates of different substances in a high-speed centrifugal force field. In practice, the supernatant containing lentiviruses needs to be centrifuged at ultra-high speed for several hours to allow the lentivirus particles to precipitate to the bottom of the centrifuge tube. Although this method can obtain lentiviruses of a certain purity in small-scale laboratory preparations, its limitations are also quite obvious.

[0006] On the one hand, ultracentrifugation precipitation requires highly sophisticated equipment, including expensive ultracentrifuges, and the high maintenance and operating costs significantly increase the economic burden of production, limiting its application in large-scale manufacturing. On the other hand, this method is complex to operate, requiring specialized technicians. Furthermore, during centrifugation, lentiviral particles are susceptible to high shear forces and temperatures, leading to viral inactivation and reduced infectivity and titer. Moreover, ultracentrifugation precipitation is difficult to implement for continuous production, resulting in low efficiency and failing to meet the demands of large-scale industrial production. Studies have shown that when using ultracentrifugation precipitation for lentiviral purification, the virus recovery rate is typically only 30%-50%, with significant loss of activity. This represents a substantial waste of valuable lentiviral resources.

[0007] (II) Shortcomings of conventional ion exchange chromatography Ion exchange chromatography is another commonly used lentivirus purification technique. It is based on the electrostatic interaction between the lentivirus surface charge and the ion exchange medium. By adjusting the pH and ionic strength of the buffer solution, lentiviruses are separated from impurities. In conventional ion exchange chromatography, high-salt elution is typically used to elute the lentiviruses bound to the medium. However, this method has serious drawbacks.

[0008] High-salt elution conditions significantly damage lentiviral activity, leading to the inactivation of a large number of lentiviruses and reducing product quality and efficacy. Simultaneously, conventional ion-exchange chromatography yields low concentrations, making it difficult to obtain high-purity, high-activity lentiviral products. This is because the binding characteristics of lentiviruses on ion-exchange media are complex; some lentiviruses may be difficult to elute effectively, or they may aggregate or denature during elution, thus affecting the final purification effect. Research data shows that the lentiviral yield of conventional ion-exchange chromatography is generally between 40% and 60%, and the activity of eluted lentiviruses often only retains 50% to 70% of the initial activity, failing to meet the stringent requirements of gene therapy for high-quality lentiviral vectors.

[0009] Furthermore, the characteristics of lentiviruses present the following challenges for purification: (a) Structural complexity and sensitivity The outermost layer of a lentiviral particle is an envelope, followed by matrix proteins and a capsid. The innermost layer consists of two identical positive-stranded RNA chains and enzymes (including reverse transcriptase, integrase, and protease). This complex structure makes lentiviruses extremely sensitive to environmental factors. Changes in conditions such as low pH, high salinity, temperature, and shear stress can significantly affect their structure and activity. Studies have shown that when the pH is below 6.0, the lentiviral envelope structure is disrupted, leading to viral inactivation. In high-salt environments, such as when the sodium chloride concentration exceeds 0.5 M, the stability of lentiviruses decreases sharply, and their infectivity is significantly reduced. Furthermore, lentiviruses are also highly sensitive to temperature changes; their half-life at 37°C is only 7-8 hours, and the inactivation rate accelerates further with increasing temperature. In addition, shear stress generated during purification processes, such as centrifugation and tangential flow filtration, can also cause the lentiviral envelope to rupture, rendering the virus infective.

[0010] (II) The challenge of impurity removal In the production process of lentiviruses, the production fluid often contains various impurities, among which host cell proteins (HCPs) are an important type of impurity. These impurities can bind to lentiviruses, affecting their purity and activity. Furthermore, in gene therapy applications, residual HCPs may trigger immune responses, posing a threat to patient safety. Studies have reported that the HCP content in unpurified lentivirus production fluids can be as high as 2E5 ng / mL (i.e., 2 × 10⁻⁵ ng / mL). 5 (ng / mL), severely impacting the quality of lentiviral products. Host cell DNA (HCD) is also a common impurity. During cell culture, HCD may be released into the culture medium and mix with the lentivirus. If residual HCD enters the final lentiviral product, it may integrate into the host genome, leading to potential risks such as gene mutations; therefore, it needs to be removed as much as possible during purification. In addition, the production medium may also contain unassembled viral proteins, culture medium components, endotoxins, and other impurities. These impurities not only affect the purity of the lentivirus but may also interfere with its biological function and safety assessment.

[0011] Removing impurities while avoiding damage to lentiviral activity presents a significant challenge in lentiviral purification. Traditional purification methods have limited effectiveness in impurity removal, failing to meet increasingly stringent quality and safety standards. Therefore, there is an urgent need to develop new purification technologies to effectively address impurity removal and improve the quality and safety of lentiviral products.

[0012] Anion exchange chromatography is a separation technique based on electrostatic interactions. Its basic principle is to achieve separation by utilizing the electrostatic interaction between anions (negatively charged ions) on the surface of the substance to be separated and positively charged ligands on the stationary phase. In lentivirus purification, lentivirus particles carry a negative charge on their surface, which, under specific buffer conditions, can electrostatically adsorb onto the positively charged ligands on the anion exchange medium. Increasing the pH of the mobile phase (elution buffer) alters the charge state of the substance to be separated, weakening its electrostatic interaction with the stationary phase. Increasing the salt concentration introduces more ions, which compete with the substance for binding sites on the stationary phase, causing the substance to desorb from the stationary phase. The eluted substances elute from the column in order of decreasing binding strength to the stationary phase, thus achieving separation.

[0013] Anion exchange chromatography has unique advantages in lentivirus purification, including high selectivity, good scalability, and strong operational controllability.

[0014] High selectivity: Anion exchange chromatography can separate lentiviruses and impurities based on the differences in the properties and quantities of their surface charges, exhibiting high selectivity. The charge distribution on the surface of lentiviruses differs from that of host cell proteins, host cell DNA, and other impurities. By appropriately adjusting the pH and ionic strength of the buffer solution, lentiviruses can specifically bind to the anion exchange medium, while most impurities do not bind or bind weakly. This allows for effective removal in subsequent washing and elution steps, achieving efficient separation of lentiviruses from impurities and improving the purity of the lentivirus product.

[0015] Excellent scalability: This technology boasts excellent scalability, easily scaling up from laboratory to industrial production. Whether using small chromatography columns for laboratory research or large-scale chromatography equipment for mass production, the principle and operation of anion exchange chromatography remain essentially the same. Only the column size, packing material quantity, and relevant process parameters need to be adjusted according to the production scale. This allows for the scaling up of anion exchange chromatography in the industrial production of lentiviral vectors to meet the ever-growing market demand.

[0016] High operational controllability: In anion exchange chromatography, precise control of operating parameters such as buffer pH, salt concentration, and flow rate allows for accurate regulation of the lentivirus purification process. For example, gradient elution can be used to gradually change the salt concentration of the eluent, allowing lentiviruses and impurities with different binding forces to be eluted at different times, thus achieving finer separation. Furthermore, operating conditions can be flexibly adjusted according to the characteristics and quality requirements of the lentivirus to improve its recovery rate and activity. This high operational controllability allows anion exchange chromatography to better adapt to different lentivirus purification needs, ensuring the stability and consistency of product quality.

[0017] Although anion exchange chromatography has demonstrated numerous advantages and gained some application in the field of lentivirus purification, certain conditions during the process can significantly impair lentiviral activity. High-salt elution is a commonly used method in anion exchange chromatography, but a high-salt environment can damage the lentiviral envelope structure, leading to viral inactivation. Studies have shown that when the salt concentration in the eluent exceeds a certain threshold, the infectivity of lentiviruses decreases sharply. For example, when using sodium chloride as the eluent, a concentration above 0.5 M can result in a loss of more than 50% of lentiviral activity. Furthermore, the interaction between lentiviruses and the media surface, as well as shear forces during elution, can also negatively impact viral activity. How to ensure effective impurity removal while maximizing the preservation of lentiviral activity remains a major challenge for anion exchange chromatography.

[0018] In summary, while existing anion exchange chromatography techniques have been applied in lentivirus purification, they have several shortcomings. Developing more efficient anion exchange chromatography processes is of great significance for improving lentivirus purification efficiency and product quality, and for promoting the development of the gene therapy industry. Summary of the Invention

[0019] To address at least one of the aforementioned deficiencies in the prior art, the present invention provides an eluent for improving the recovery rate of lentivirus anion exchange chromatography. The eluent comprises a basal buffer, 50-500 mM NaCl, and 0.2-2 M L-arginine; the pH of the eluent is 6.5 to 8.0.

[0020] In some embodiments, the concentration of L-arginine is 0.5 M to 1 M, preferably 0.6 M to 0.75 M, and more preferably 0.75 M.

[0021] In some embodiments, the concentration of NaCl is 150 mM.

[0022] In some embodiments, the base buffer is selected from phosphate buffer or Tris-HCl buffer; the pH of the elution buffer is 6.5 to 7.5.

[0023] In some embodiments, the eluent further comprises a protectant selected from sucrose or lactose at a concentration of 2%-4% m / V, preferably 2% m / V sucrose.

[0024] In some embodiments, the eluent also contains 2 mM MgCl2.

[0025] In some embodiments, the eluent is primarily composed of 20 mM phosphate buffer, 150 mM NaCl, 0.5 M to 0.75 M L-arginine, 2% m / V sucrose, and 2 mM MgCl2, with a pH of 6.5.

[0026] In some embodiments, the eluent is primarily composed of 20 mM phosphate buffer, 150 mM NaCl, 0.75 mL arginine, 2% m / V sucrose, and 2 mM MgCl2, with a pH of 6.5.

[0027] In another invention, the present invention also provides the use of the eluent in lentiviral anion exchange chromatography. This application includes the following steps: S1. Loading: Load the sample containing lentivirus onto an anion exchange chromatography column that has been equilibrated with equilibration buffer. S2. Washing: Rinse the anion exchange chromatography column with the equilibrium solution to remove impurities; S3. Elution: Elute the lentivirus bound to the anion exchange chromatography column using the elution buffer and collect the elution peak.

[0028] In some embodiments, the turbidity of the lentivirus-containing solution is below 20 NTU; the packing ligand of the anion exchange chromatography column is DEAE; the equilibration buffer consists of 20 mM phosphate buffer, 150 mM NaCl, 2% m / V sucrose and 2 mM MgCl2, with a pH of 6.5; the loading retention time is 3-6 min; and the elution retention time is 3-10 min.

[0029] In some implementations, the application also includes: S4. Adjusting Osmotic Pressure: Dilute the eluent with 20mM phosphate buffer at pH 6.5 to adjust the osmotic pressure to 280-350 Osm / kg. For the critical need in the large-scale, high-purity preparation of lentiviral vectors in gene therapy, efficient, mild, and economical downstream purification processes are of significant practical importance. However, the widely used anion exchange chromatography method relies on high-salt elution, resulting in severe damage to viral activity and low recovery rates, hindering the stable production of high-quality products. Therefore, this invention develops a novel eluent and its application. The purification of lentiviruses is optimized by applying this eluent. This invention uses L-arginine as the core functional component, eluting lentiviruses under optimized buffer systems, ionic strength, and pH conditions, effectively replacing the traditional single high-salt elution mode. This invention systematically studies and optimizes the concentration of L-arginine in the eluent, the concentration of auxiliary salts, the type of protective agent, elution kinetic parameters, and post-treatment conditions, forming a purification process that is controllable, mild, has high recovery rates, and maintains good product activity. This method significantly reduces the damage to the viral envelope structure caused by high-salt environments. Utilizing the competitive elution and stabilizing effects of L-arginine, it simultaneously improves lentiviral recovery and activity while effectively removing key impurities such as host cell proteins. To our knowledge, no complete method has been disclosed that achieves high recovery and high-activity purification of lentiviruses using the same optimized L-arginine elution buffer system. This invention provides a practical and excellent process route for the large-scale, high-quality purification of lentiviral vectors for gene therapy.

[0030] The present invention has the following beneficial effects: The eluent and its application (i.e., lentivirus purification method) provided by the present invention have mild elution conditions, strong process controllability, and significantly improved recovery rate and viral activity, which is conducive to stable and efficient large-scale production; when using the eluent of the present invention for chromatography, the chromatographic recovery rate of lentivirus is high, nearly twice that of traditional high-salt elution, while the host cell protein removal rate is as high as 97%; the entire process optimizes the elution components, simplifies the operation process, and reduces process damage to viral activity. From a production perspective, the cost is controllable and it is easy to scale up production.

[0031] The above description is merely an overview of the technical solutions of the embodiments of the present invention. In order to better understand the technical methods of the embodiments of the present invention and to implement them in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the embodiments of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the invention will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is the anion exchange chromatography spectrum of Example 1. The horizontal axis represents the elution volume (ml), and the vertical axis is divided into a left side (mAU, absorbance units) and a right side (percentage of solution B). The four curves—blue, red, brown, and cyan—correspond to UV values, respectively. 260nm Detection channel, UV 280nm The detection channels, conductivity, and the proportion of solution B.

[0034] Figure 2 This is the anion exchange chromatography spectrum of Example 2. The horizontal axis represents the elution volume (ml), and the vertical axis is divided into a left side (mAU, absorbance units) and a right side (mS / cm, conductivity units). The four curves—blue, red, brown, and purple—correspond to UV values, respectively. 280nm Detection channel, UV 260nm Detection channels, conductivity, and pH value. Detailed Implementation

[0035] To make the technical means, inventive features, achieved objectives, and effects of the invention readily understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific illustrations. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] It should be noted that the structures, proportions, sizes, etc., described in the embodiments of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modification of the structure, change of the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed in the present invention, provided that it does not affect the effects and objectives that the present invention can produce.

[0037] The synthesis method of the present invention will be further described in detail below with reference to specific embodiments. These embodiments are used to more specifically illustrate the implementation process and effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Unless otherwise expressly stated, all raw materials and reagents used in each embodiment are commercially available, and the lentivirus transfection, packaging, and preparation of the lentivirus-containing solution for anion exchange chromatography loading are all routine experiments well known to those skilled in the art.

[0038] In some embodiments, the eluent comprises a basal buffer, 50-500 mM NaCl, and 0.2-2 M L-arginine; the pH of the eluent is 6.5 to 8.0.

[0039] In some implementations, the use of the eluent in lentiviral anion exchange chromatography includes the following steps: S1. Loading: Load the sample containing lentivirus onto an anion exchange chromatography column that has been equilibrated with equilibration buffer. S2. Washing: Rinse the anion exchange chromatography column with the equilibrium solution to remove impurities; S3, Elution: Elute the lentivirus bound to the anion exchange chromatography column using the elution buffer and collect the elution peak; S4. Adjust osmotic pressure: Dilute the eluent with 20mM phosphate buffer at pH 6.5 to adjust the osmotic pressure to 280-350 Osm / kg.

[0040] The following details some implementation schemes of the eluent and its application involved in this invention.

[0041] Lentiviral cells were packaged using 293 cells that had been domesticated by transient suspension transfer of four plasmids to obtain a lentiviral harvest fluid of a certain titer.

[0042] Add 50 U / mL of pluripotent nuclease and 2 mM magnesium chloride to the lentivirus harvest fluid, and place it in a constant temperature shaker at 37°C for nucleic acid digestion for 2 hours (nuclease digestion step).

[0043] The digested viral sample was filtered using a depth filter, and the turbidity was controlled below 20 NTU after filtration (clarification filtration process). The resulting sample containing lentivirus was used for loading onto the subsequent chromatography column.

[0044] The anion exchange chromatography column should be thoroughly equilibrated with equilibration buffer. DEAE is the preferred ligand for the column packing material; Capto DEAE, Diamond DEAE Mustang, DEAE HP, and DEAE 650C are also suitable options. The equilibration buffer system is preferably phosphate buffer (PB) and Tris-HCl buffer, containing 50-200 mM NaCl. 2%-4% m / V sucrose or lactose can be selectively added. The pH of the equilibration buffer should be 6.5-8.0, preferably 6.5-7.5.

[0045] The clarified lentivirus-containing solution was loaded onto an anion exchange chromatography column, with a retention time of 3-6 minutes. After loading, the column was flushed with equilibration buffer to remove impurities until UV chromatography was completed. 280nm Approaching 0. Elution is then performed using the eluent of this invention. When UV... 280nm Start collecting the eluent at the peak, UV 280nm The collection should be stopped when the temperature drops to a plateau. The eluent contains basal buffer, 50-500 mM NaCl, and 0.2-2 M L-arginine (preferably 0.6-1 M), with a pH of 6.5-8.0 (preferably 6.5-7.5). The basal buffer is either phosphate buffer or Tris-HCl buffer. The elution retention time is 3-10 min, and the elution direction can be either forward or reverse. After elution, the eluent is diluted with 20 mM PPB at pH 6.5 to an osmotic pressure of 280-350 Osm / kg.

[0046] Example 1 The inventors optimized the elution process to find the minimum arginine concentration that could completely elute lentivirus. In this embodiment, 20 mM PB + 2 mM MgCl2 + 150 mM NaCl + 2% m / V sucrose, pH 6.5, was used as the equilibration buffer. After fully equilibrating the DEAE chromatography column (in this embodiment, the chromatographic packing material was Diamond DEAE mustang), the clarified lentivirus-containing solution was loaded onto the anion exchange chromatography column. The column was then rinsed with the equilibration buffer, and a gradient elution method was used to find the minimum arginine concentration. During elution, solution A was the aforementioned equilibration buffer, and solution B was the eluent, composed of 20 mM + 150 mM NaCl + 1 M L-arginine + 2% m / V sucrose + 2 mM MgCl2, pH 6.5, with an elution gradient of 0-100% B, 20 CV. The results were obtained by analyzing the anion exchange chromatography chromatogram (…). Figure 1It was found that once the gradient reached 50%, there were no UV absorption peaks other than arginine itself, indicating that most of the effective components bound to the anion exchange chromatography column could be eluted completely with the eluent containing 0.5M arginine. To further ensure the yield, the inventors set the actual elution concentration of arginine to 0.75M arginine.

[0047] Example 2 The anion exchange chromatography column was packed with Diamond DEAE mustang. The equilibration buffer was 20 mM PB + 150 mM NaCl + 2% m / V sucrose + 2 mM MgCl2, pH 6.5. The clarified lentivirus-containing solution was loaded onto the anion exchange chromatography column, with a retention time of 3-6 min. After loading, the column was continuously rinsed with the equilibration buffer until UV light was applied. 280nm The pH value approaches 0. Elution is then performed using the eluent of this invention. The eluent is 20 mM PB + 150 mM NaCl + 0.75 M L-arginine + 2% m / V sucrose + 2 mM MgCl2, pH 6.5. When UV... 280nm Start collecting the eluent at the peak, UV 280nm The collection was stopped when the osmotic pressure level stabilized. After elution, the eluent was diluted with 20 mM PB at pH 6.5 to an osmotic pressure of 280 Osm / kg to complete the anion exchange chromatography step. The anion exchange chromatography chromatogram is shown below. Figure 2 .

[0048] Table 1. Lentiviral yield and HCP removal rate Note: Yield = Total TU after process / Total TU before process × 100%. Since the titer detection is performed by flow cytometry, there is a certain coefficient of variation, so the yield may exceed 100%.

[0049] The results are shown in Table 1. Using the eluent of this invention, the anion exchange chromatography recovery rate can reach nearly 100%, which is almost double that of traditional NaCl elution. Furthermore, the HCP removal rate is as high as 97%, indicating that the eluent components of this invention effectively improve the purification efficiency of lentivirus anion exchange chromatography. Combined with other purification processes such as multimode chromatography and tangential flow filtration, the HCP level of the final lentivirus product can be reduced to below 100 ppm, thus meeting the standards for in vivo lentivirus preparations.

[0050] This invention achieves 100% elution yield using a low arginine concentration, while also maintaining a high HCP removal rate of 97%. Since lentiviruses are enveloped viruses, they are sensitive to high-salt, high-osmotic-pressure environments. The unique eluent dilution step (step S4, adjusting osmotic pressure) of this invention effectively protects the lentivirus's activity titer, preventing titer loss under high arginine concentrations and high osmotic pressure conditions, which is crucial for achieving 100% yield. Arginine is the most expensive component in the buffer solution of anion exchange chromatography. This invention uses a low arginine concentration, which controls the cost of eluent preparation and reduces the dilution factor of the eluted sample during osmotic pressure adjustment, saving time and steps in subsequent concentration of lentivirus to the usable concentration (e.g., 1E8TU / ml).

[0051] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the detailed process of the specific operation steps in the above embodiments has been specifically described in the method description, and will not be repeated here.

[0052] In the embodiments provided by this invention, it should be understood that the disclosed eluent components and purification process can be implemented in other appropriate ways. For example, the process parameters given in the embodiments, such as L-arginine concentration, NaCl concentration, pH value, elution retention time, sample loading retention time, and osmotic pressure range, are illustrative and can be reasonably adjusted according to the characteristics of the virus sample, the specifications of the chromatography column, and the purity and activity requirements of the target product in practical applications. For example, the buffer system (such as phosphate, Tris-HCl, etc.), protective agent (such as sucrose, lactose, etc.), and chromatography packing material (such as DEAE packing material with different matrices) used in each step can all be selected and replaced within the categories listed in the claims of this invention. Furthermore, within the core method framework defined by this invention, reasonable variations and combinations of the specific implementation methods of each step, including equilibration, sample loading, washing, elution, and post-treatment, are also extensions based on the core concept of this invention. All such equivalent substitutions or adaptive changes based on the technical solutions of this invention should be covered within the substance and protection scope of this invention.

[0053] The operational steps involved in the above embodiments can be implemented individually, or they can be combined or separated according to experimental needs. Some or all of the steps can be selected to achieve the technical effects of the present invention based on actual sample processing requirements.

[0054] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions such as parameter adjustments and equivalent equipment replacements that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An eluent for improving the recovery rate of lentiviruses in anion exchange chromatography, characterized in that, The eluent contains a basal buffer, 50-500 mM NaCl and 0.2-2 M L-arginine; the pH of the eluent is 6.5 to 8.

0.

2. The eluent according to claim 1, characterized in that, The concentration of L-arginine is 0.5 M to 1 M, preferably 0.6 M to 0.75 M.

3. The eluent according to claim 1, characterized in that, The concentration of NaCl is 150 mM.

4. The eluent according to claim 1, characterized in that, The base buffer is selected from phosphate buffer or Tris-HCl buffer; the pH of the elution buffer is 6.5 to 7.

5.

5. The eluent according to claim 1, characterized in that, The eluent also contains a protectant selected from sucrose or lactose, at a concentration of 2%-4% m / V, preferably 2% m / V sucrose.

6. The eluent according to claim 1, characterized in that, The eluent also contains 2 mM MgCl2.

7. The eluent according to claim 1, characterized in that, The eluent mainly consists of 20 mM phosphate buffer, 150 mM NaCl, 0.5 M to 0.75 M L-arginine, 2% m / V sucrose and 2 mM MgCl2, with a pH of 6.

5.

8. The eluent according to claim 7, characterized in that, The eluent mainly consists of 20 mM phosphate buffer, 150 mM NaCl, 0.75 M L-arginine, 2% m / V sucrose and 2 mM MgCl2, with a pH of 6.

5.

9. The application of the eluent according to any one of claims 1-8 in lentiviral anion exchange chromatography, characterized in that, Includes the following steps: S1. Loading: Load the sample containing lentivirus onto an anion exchange chromatography column that has been equilibrated with equilibration buffer. S2. Washing: Rinse the anion exchange chromatography column with the equilibrium solution to remove impurities; S3. Elution: Elute the lentivirus bound to the anion exchange chromatography column using the elution buffer and collect the elution peak.

10. The application according to claim 9, characterized in that, The turbidity of the lentivirus-containing solution is below 20 NTU; the packing material of the anion exchange chromatography column is DEAE; the equilibration solution consists of 20 mM phosphate buffer, 150 mM NaCl, 2% m / V sucrose and 2 mM MgCl2, and its pH is 6.

5. The retention time for loading the sample is 3-6 minutes; The elution retention time is 3-10 min; The application also includes: S4. Adjust osmotic pressure: Dilute the eluent with 20mM phosphate buffer at pH 6.5 to adjust the osmotic pressure to 280-350 Osm / kg.

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