Kit and method for purifying retroviruses

Abstract

The present disclosure relates to a kit and a method for purifying retrovirus, which comprises a reagent combination for purifying retrovirus, comprising a first eluent, a second eluent, an elution liquid and a polyanion compound. By using the purification chromatography combination and method of the present disclosure, the viral load can be increased, the chromatography recovery rate of retrovirus is increased by 2-3 times, reaching 80% and above; at the same time, the impurity level in the virus elution liquid is significantly reduced, such as the residual amount of HCP, which can be reduced by 2-3 times; the eluted retrovirus elution liquid can be stored for 24 hours without losing the infection activity under high salt conditions without adding other special protection reagents; after elution, the next step of ultrafiltration dialysis can be directly carried out without the step of dilution and then concentration, effectively simplifying the operation steps and shortening the processing time.

Description

Technical Field

[0001] This disclosure relates to the biological field, and more particularly to a kit and method for purifying retroviruses. Background Technology

[0002] Retroviruses, also known as retrotome viruses, are a type of RNA virus whose genetic information is RNA. There are three subfamilies of retroviruses: the tumor virus subfamily, the lentivirus subfamily, and the foamy virus subfamily. Retroviral vectors are a class of viral vectors capable of carrying exogenous genes, developed based on the retroviral genome and through modifications to its components. After the genome of the retroviral vector enters the recipient cell, it is reverse transcribed into DNA in the cytoplasm of the recipient cell, forming a pre-integration DNA complex, which then enters the cell nucleus and integrates into the recipient cell's genome. The integrated DNA is transcribed into mRNA, returns to the cytoplasm, and expresses the target protein or produces small RNA. Retroviral-derived vectors have become one of the most widely used vectors in gene therapy clinical trials. Currently, the preparation and purification processes of retroviruses still face significant challenges.

[0003] Lentiviruses are a class of viruses modified from the human immunodeficiency virus (HIV). They primarily infect lymphocytes and macrophages. Early lentivirus infection is difficult to observe, often involving a latent period of several years before the disease develops slowly, hence the name "lentivirus." Compared to other viral vectors, lentiviruses have unique advantages:

[0004] (1) Wider host range: It has the ability to infect both dividing and non-dividing cells. For some cells that are difficult to transfect, such as primary cells, stem cells, and undifferentiated cells, it can greatly improve the transduction efficiency of the target gene.

[0005] (2) Stable expression: Lentiviral genomes can integrate into the host genome, thus allowing for long-term and stable expression of exogenous genes;

[0006] (3) Capable of carrying exogenous gene fragments of approximately 5kb or longer: Based on the advantages of lentiviral vectors, they are widely used in research on RNAi, gene therapy, and transgenic animals. Lentiviral vectors capable of expressing siRNA can be constructed in vitro, and then transfected into cells to transcribe siRNA intracellularly, thus exerting a long-term blocking effect on gene expression. Meanwhile, various gene therapies using lentiviruses as gene vectors have been clinically studied both domestically and internationally, showing broad application prospects in gene therapy.

[0007] When using retroviral vectors to infect cells, it is usually necessary to concentrate the viral particles to obtain high-titer and high-purity retroviruses. Existing large-scale retrovirus production processes include:

[0008] (1) Packaging retroviruses: adherent culture or suspension culture;

[0009] (2) Harvest and clarify the retrovirus supernatant: After centrifugation or filtration to remove cells in suspension culture, harvest the retrovirus supernatant. For adherent culture, harvest the retrovirus supernatant directly and then filter it with a 0.45μm membrane to further remove cell debris. The collected clarified liquid is used for the next step of ultrafiltration or chromatography purification.

[0010] (3) Two-step chromatography to purify retroviruses: anion exchange chromatography combined with molecular sieve chromatography or composite mode chromatography; ultrafiltration steps may also be added before and after the chromatography process; the anion chromatography buffer is generally Tris-HCl or PBS, the elution buffer and salt concentration are generally the same as the equilibrium buffer (salt concentration is generally ≤0.3M NaCl), and the elution salt concentration is ≤2M NaCl.

[0011] (4) Nuclease treatment can be performed during the virus packaging stage, before or after clarification, or after anion chromatography.

[0012] (5) 300-750 kDa tangential flow ultrafiltration (UF / DF) yields the finished retrovirus product, followed by sterile filtration and cryopreservation.

[0013] However, the chromatography solvent used in this method has the following drawbacks:

[0014] (1) The recovery rate of retroviruses in ion exchange chromatography is about 50% or lower, and the recovery rate is unstable.

[0015] (2) Two-step chromatography is required to ensure that impurities remain at an acceptable level. Molecular sieve technology cannot be scaled up for large-scale production. Composite mode chromatography, such as Capto Core 700 packing material, generally uses flow-through mode to purify viruses. The sample volume increases during the process, making it impossible to achieve concentration through chromatography. Ultrafiltration concentration and liquid exchange processes are required.

[0016] (3) In the current buffer system, retroviruses have a short tolerance time under high-salt conditions, generally only about half an hour. After high-salt elution by anion exchange chromatography, the sample needs to be diluted immediately to a concentration of ≤0.3M NaCl (approximately 4-10 times dilution), which increases the sample handling volume and subsequent processing time. In addition, the above-mentioned ultrafiltration process requires two steps: concentration and dialysis.

[0017] Therefore, there is an urgent need to provide a kit for purifying retroviruses that has a high chromatographic yield, fewer steps, simple operation, and short time consumption. Summary of the Invention

[0018] To address the problems existing in the prior art, the purpose of this disclosure is to provide a new reagent kit and purification method for purifying retroviruses. Using the reagent combination or kit described above can improve the recovery rate of retroviruses, and the purification process involves fewer steps, is simple to operate, and is less time-consuming.

[0019] To achieve the above objectives, the present disclosure adopts the following specific technical solutions:

[0020] In one aspect, this disclosure provides a composition for the purification chromatography of retroviruses, comprising a first eluent, a second eluent, and an elution buffer, specifically:

[0021] The first rinsing solution comprises the following components: citric acid, sodium chloride, and water;

[0022] The second elution solution comprises the following components: buffer solution, sodium chloride, and water;

[0023] The eluent comprises the following components: buffer solution, sodium chloride, and water.

[0024] Optionally, the reagent combination further includes a polyanionic compound; preferably, the polyanionic compound is selected from polyanionic compounds of dextran, and more preferably, a sodium salt of dextran sulfate.

[0025] In another aspect, this disclosure provides a kit for purifying retroviruses, which comprises the aforementioned reagent combination.

[0026] On the other hand, this disclosure provides a method for purifying retroviruses using the aforementioned reagent combination or the aforementioned kit, comprising the following steps:

[0027] (1) Obtain the retrovirus supernatant;

[0028] (2) Clarify the retrovirus supernatant:

[0029] The retrovirus supernatant obtained in step (1) is mixed with a polyanionic compound and filtered to obtain a clarified retrovirus supernatant; preferably, nuclease is added before or after filtration.

[0030] (3) Anion chromatography and elution:

[0031] The clarified retrovirus supernatant obtained in step (2) is loaded onto anion exchange membrane chromatography or anion exchange chromatography column. After loading, the sample is washed with the first elution buffer and the second elution buffer in sequence, and then eluted with elution buffer. The elution buffer containing retrovirus is collected.

[0032] (4) Purify the eluent;

[0033] Optionally, the method further includes dialysis and / or concentration.

[0034] In another aspect, this disclosure provides the use of the aforementioned reagent combination, the aforementioned kit and / or the aforementioned method in the preparation of purified retroviruses.

[0035] The beneficial effects achieved by this disclosure are at least as follows:

[0036] During the application of the purification chromatography composition disclosed herein in the examples, the inventors discovered for the first time that adding citric acid to the eluent has the following function:

[0037] (1) To ensure that the retrovirus is stably bound to the ligand of the chromatography packing material, that is, to fix the retrovirus on the chromatography column, so as to ensure that only impurities are eluted and not the virus is eluted during the high-salt washing process of the second elution buffer in the next step.

[0038] (2) It helps to wash away some impurities such as HCP, so that with the combination of the first and second eluents, impurities can be effectively eluted without eluting the virus, greatly improving the recovery rate of retroviruses. Without the addition of citric acid, a concentration of only 0.5M NaCl can elute the virus, resulting in a lower recovery rate of retroviruses. At the same time, the inventors of this disclosure also unexpectedly discovered that the virus eluent obtained by elution can be stored for 20 hours under high-salt conditions without losing its infectivity, overcoming the defect of short tolerance time of retroviruses under high-salt conditions in existing buffer systems. The obtained eluent can be directly used for the next step of ultrafiltration dialysis without the need for dilution and concentration, effectively simplifying the operation steps and shortening the processing time.

[0039] Furthermore, the retrovirus purification method disclosed in this paper also reveals for the first time that adjusting the pH of the clarified retrovirus supernatant to 5-6.5 can promptly remove nucleases. The pH of the clarified retrovirus supernatant is lower than the pI of the nuclease (around 6.8), causing the nuclease to carry a positive charge and not bind to the positively charged anion exchange chromatography material, while the retrovirus carries a negative charge and can bind to the anion exchange chromatography material. Therefore, the nuclease can be removed promptly.

[0040] Furthermore, in the retrovirus purification method disclosed herein, it was discovered for the first time that adding polyanionic compounds during the virus clarification process can reduce the non-specific adsorption of the virus by the filter and effectively improve the recovery rate of the retrovirus in the clarification step.

[0041] Finally, by using the reagent combination (first eluent, second eluent, and elution buffer), kit, and purification method disclosed herein for purifying retroviruses, the viral load can be increased, and the chromatographic recovery rate of retroviruses can be improved by 2-3 times, reaching 80% or higher. Simultaneously, the level of impurities in the retrovirus eluent is significantly reduced; for example, the residual amount of host cell protein (HCP) can be reduced by 2-3 times. Furthermore, the eluted retrovirus eluent can be stored for 24 hours under high-salt conditions without losing its infectivity without the addition of other special protective reagents. After elution, it can be directly subjected to the next step of ultrafiltration dialysis without the need for dilution and concentration, effectively simplifying the operation and shortening the processing time. Attached Figure Description

[0042] Figure 1 The lentivirus yield after clarification process with the addition of sodium dextran sulfate.

[0043] Figure 2 Yields of different types of lentiviruses and different batches of anion exchange chromatography lentiviruses.

[0044] Figure 3 Lentiviral 1-lot #1 anion chromatography chromatogram.

[0045] Figure 4 HCP removal rates by anion exchange chromatography for different types of lentiviruses and different batches.

[0046] Figure 5 The effect of NaCl concentration in the first eluent on the yield of anion exchange chromatography lentivirus.

[0047] Figure 6 The effect of NaCl concentration in the first eluent on the residual amount of HCP in anion exchange chromatography.

[0048] Figure 7 The effect of citric acid concentration in the first eluent on the yield of anion exchange chromatography lentivirus.

[0049] Figure 8 The effect of citric acid concentration in the first eluent on the removal of anion exchange chromatography impurities.

[0050] Figure 9 The effect of NaCl concentration in the eluent and second elution buffer on the yield of anion exchange chromatography lentiviruses.

[0051] Figure 10 The effect of NaCl concentration in the eluent and second elution buffer on the residual amount of HCP in anion chromatography.

[0052] Figure 11 The effect of different polyanionic compounds on the yield of lentiviruses in the clarification process.

[0053] Figure 12The effect of different buffer solutions on the virus yield of anion exchange chromatography.

[0054] Figure 13 Stability of lentiviruses in anion exchange chromatography eluent. Detailed Implementation

[0055] Experimental methods in the following embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0056] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0057] The terms “comprising” and “having”, and any variations thereof, in this disclosure are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0058] In this disclosure, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. Furthermore, to better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0059] The term "reagent combination for purifying retroviruses" as used in this disclosure includes at least the combination of elution and eluent used in the anion exchange chromatography process during retrovirus purification.

[0060] As used in this disclosure, the term "polyanionic compound" refers to a compound with a large number of negative charges, which can reduce the non-specific adsorption of viruses by the filter during chromatography and effectively improve the recovery rate of retroviruses in the clarification step.

[0061] As used in this disclosure, the term "nuclease" refers to an enzyme that catalyzes the hydrolysis of phosphodiester bonds in ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA). An enzyme that only hydrolyzes ribonucleic acid (RNA) is called a ribonuclease; an enzyme that only hydrolyzes deoxyribonucleic acid (DNA) is called a deoxyribonuclease.

[0062] As used in this disclosure, "siRNA" refers to a small, double-stranded RNA molecule, approximately 22–24 nucleotides long, produced intracellularly upon induction by endogenous or exogenous (e.g., viral) double-stranded RNA. SiRNAs can induce the degradation of specific target messenger RNAs to maintain genomic stability, protect the genome from exogenous nucleic acid invasion, and regulate gene expression.

[0063] As used in this disclosure, "microRNA" refers to a type of ribonucleic acid molecule that is about 22 nt (21-24 nt) long and is widely found in eukaryotes. Mature miRNA is a single-stranded RNA that regulates the expression of target genes by degrading target mRNA or inhibiting protein translation.

[0064] As used in this disclosure, the term "shRNA" refers to short hairpin RNA, a short double-stranded RNA structure (19-25 nt) produced in dependence of stem-loop sequences. It can be introduced into cells using vectors, where it is digested with enzymes to form siRNA, which regulates target genes through the RNA interference pathway.

[0065] As used in this disclosure, the term "sodium dextran sulfate" refers to a polyanionic derivative of dextran, formed by the esterification reaction of dextran and chlorosulfonic acid, wherein the sulfur content is approximately 17%, equivalent to an average of 1.9 sulfate groups per glucose residue in each dextran molecule. As used in this disclosure, "dextran sulfate" and "sodium dextran sulfate" both refer to the same substance and are two alternative names for the same substance.

[0066] In one aspect, this disclosure provides a reagent combination for purifying retroviruses, comprising a first eluent, a second eluent, and an elution buffer, wherein,

[0067] The first rinsing solution comprises the following components: citric acid, sodium chloride, and water;

[0068] The second elution solution comprises the following components: buffer solution, sodium chloride, and water;

[0069] The eluent comprises the following components: buffer solution, sodium chloride, and water.

[0070] Optionally, the reagent combination also includes a polyanionic compound.

[0071] In some embodiments of this disclosure, the polyanionic compound is selected from one or more of the following: polyanionic compounds of dextran, sodium heparin, heparin sulfate, dermatan sulfate, chondroitin sulfate, polysulfated pentosan esters, cellulose sulfate, cellulose acetate sulfate, naphthalene sulfonic acid derivatives, polystyrene sulfonic acid, polycarboxylic acid or polyvinylpyrrolidone and / or anti-clustering agents containing any of the foregoing compounds.

[0072] In some embodiments of this disclosure, the polyanionic compound is selected from polyanionic compounds of dextran, preferably sodium dextran sulfate.

[0073] In some embodiments of this disclosure, the retrovirus is selected from one or more of tumor viruses, lentiviruses, and / or foam viruses.

[0074] In some embodiments of this disclosure, the retrovirus is a lentivirus.

[0075] In some embodiments of this disclosure, the concentration of citric acid in the first rinsing solution is 0.01M-0.3M, preferably 0.02M-0.15M.

[0076] In some embodiments of this disclosure, the concentration of citric acid in the first rinsing solution is 0.1M.

[0077] In some embodiments of this disclosure, the concentration of sodium chloride in the first rinsing solution is 0.025M-0.5M, preferably 0.05M-0.25M.

[0078] In some embodiments of this disclosure, the concentration of sodium chloride in the first rinsing solution is 0.15M.

[0079] In some embodiments of this disclosure, the pH of the first rinsing solution is 5.0-6.5.

[0080] In some embodiments of this disclosure, the pH of the first rinsing solution is 6.0.

[0081] In some embodiments of this disclosure, the buffer solution in the second elution solution is a PB buffer or a Tris buffer.

[0082] In some embodiments of this disclosure, the concentration of the PB buffer or Tris buffer in the second elution solution is 5-150 mM, preferably 10-100 mM.

[0083] In some embodiments of this disclosure, the concentration of the PB buffer or Tris buffer in the second elution solution is 50 mM.

[0084] In some embodiments of this disclosure, the concentration of sodium chloride in the second rinsing solution is 0.25M-2.5M, preferably 0.5M-1.2M.

[0085] In some embodiments of this disclosure, the concentration of sodium chloride in the second rinsing solution is 0.8M.

[0086] In some embodiments of this disclosure, the pH of the second rinsing solution is 5.0-6.5;

[0087] In some embodiments of this disclosure, the pH of the second rinsing solution is 6.0.

[0088] In some embodiments of this disclosure, the elution buffer is a PB buffer or a Tris buffer.

[0089] In some embodiments of this disclosure, the concentration of the PB buffer or Tris buffer in the eluent is 5-150 mM, preferably 10-100 mM.

[0090] In some embodiments of this disclosure, the concentration of the PB buffer or Tris buffer in the eluent is 50 mM.

[0091] In some embodiments of this disclosure, the concentration of sodium chloride in the eluent is 2.0-5M, preferably 2.5-4M.

[0092] In some embodiments of this disclosure, the concentration of sodium chloride in the eluent is 2.8M.

[0093] In some embodiments of this disclosure, the pH of the eluent is 5.0-6.5.

[0094] In some embodiments of this disclosure, the pH of the eluent is 6.0.

[0095] In some embodiments of this disclosure, the concentration of the polyanionic compound is 0.05 g / L to 0.75 g / L.

[0096] In some embodiments of this disclosure, the concentration of the polyanionic compound is 0.1 g / L to 0.5 g / L.

[0097] In some embodiments of this disclosure, the concentration of the polyanionic compound is 0.1 g / L or 0.5 g / L.

[0098] In another aspect, this disclosure provides a kit for purifying retroviruses, which comprises the aforementioned reagent combination.

[0099] On the other hand, this disclosure provides a method for purifying retroviruses using the aforementioned reagent combination or the aforementioned kit, comprising the following steps:

[0100] (1) Obtain the retrovirus supernatant;

[0101] (2) Clarify the retrovirus supernatant:

[0102] The retrovirus supernatant obtained in step (1) is mixed with a polyanionic compound and filtered to obtain a clarified retrovirus supernatant; preferably, nuclease is added before or after filtration.

[0103] (3) Anion chromatography and elution:

[0104] The clarified retrovirus supernatant obtained in step (2) is loaded onto anion exchange membrane chromatography or anion exchange chromatography column. After loading, the sample is washed with the first elution buffer and the second elution buffer in sequence, and then eluted with elution buffer. The elution buffer containing retrovirus is collected.

[0105] (4) Purify the eluent;

[0106] Optionally, the method further includes dialysis and / or concentration.

[0107] In some embodiments of this disclosure, the retrovirus is selected from one or more of tumor viruses, lentiviruses, and / or foam viruses.

[0108] In some embodiments of this disclosure, the retrovirus is a lentivirus.

[0109] In some embodiments of this disclosure, the concentration of the polyanionic compound in step (2) is 0.05 g / L to 0.75 g / L.

[0110] In some embodiments of this disclosure, the concentration of the polyanionic compound is 0.1 g / L to 0.5 g / L.

[0111] In some embodiments of this disclosure, the concentration of the polyanionic compound is 0.1 g / L or 0.5 g / L.

[0112] In some embodiments of this disclosure, the polyanionic compound comprises one or more of the following: a polyanionic compound of dextran, sodium heparin, heparin sulfate, dermatan sulfate, chondroitin sulfate, pentosan polysulfate, cellulose sulfate, cellulose acetate sulfate, naphthalene sulfonic acid derivatives, polystyrene sulfonic acid, polycarboxylic acid or polyvinylpyrrolidone and / or an anti-clustering agent containing any of the foregoing compounds.

[0113] In some embodiments of this disclosure, the polyanionic compound of the dextran is sodium dextran sulfate.

[0114] In some embodiments of this disclosure, in step (2), the concentration of the nuclease is 10-50 U / ml.

[0115] In some embodiments of this disclosure, the concentration of the nuclease in step (2) is 20 U / ml.

[0116] In some embodiments of this disclosure, step (3) further includes adjusting the salt concentration and pH of the clarified retrovirus supernatant before chromatographic loading, so that the sodium chloride concentration is 0.1M-0.5M and the pH is 5.0-6.5.

[0117] In some embodiments of this disclosure, the NaCl concentration in the retrovirus supernatant is adjusted to 0.3M, and the pH is adjusted to 5.0.

[0118] In some embodiments of this disclosure, in step (4), the eluent is purified by ion exchange or filtration.

[0119] In some embodiments of this disclosure, the filtration is selected from one or more of gel filtration, ultrafiltration, tangential flow filtration, and membrane filtration.

[0120] In another aspect, this disclosure provides the use of the aforementioned reagent combinations, the aforementioned kits, and / or the aforementioned methods in the preparation of purified retroviruses.

[0121] In some embodiments of this disclosure, the retrovirus is selected from one or more of tumor viruses, lentiviruses, and / or foam viruses.

[0122] In some embodiments of this disclosure, the retrovirus is a lentivirus.

[0123] Using the method disclosed herein, the chromatographic yield of retroviruses can be increased by 2-3 times, reaching 80% or more; at the same time, the level of impurities in the retrovirus eluent can be significantly reduced, such as the residual amount of HCP, which can be reduced by 2-3 times; in addition, the retrovirus eluent obtained by elution can be stored for 24 hours under high-salt conditions without the addition of other special protective reagents without losing its infectious activity; after elution, it can be directly subjected to the next step of ultrafiltration dialysis without the need for dilution and concentration, which effectively simplifies the operation steps and shortens the processing time.

[0124] For the purpose of clarity and concise description, features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of the invention may include some embodiments having a combination of all or some of the features described.

[0125] The present disclosure will now be described in more detail with reference to specific embodiments; however, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0126] Example

[0127] Example 1 Lentiviral Purification

[0128] This embodiment provides a method for purifying lentiviruses, which includes the following steps:

[0129] (1) Obtaining lentivirus supernatant

[0130] HEK 293T cells (purchased from ATCC; catalog number CRL-3216) were resuscitated and cultured in DMEM + 10% FBS (purchased from Gibco; catalog number 10091148) at 37°C and 5% CO2. When the cell confluence was ≥70%, the cells were passaged at a ratio of 1:10. After expanding to a sufficient number of cells, the cells were plated at a density of 1E+05 cells / cm². 2 After one day of inoculation and culture, the medium was changed to DMEM before transfection. Then, four plasmids (vector plasmid, Gag-Pol, Rev, and VSV-G) were co-transfected into 293T cells using the PEI transient transfection method. The DNA:PEI ratio was 1:2. After 4-6 hours of transfection, the supernatant was discarded, and packaging medium DMEM + 2% FBS was added. The cells were cultured for another 48 hours after transfection, and the lentivirus supernatant was harvested.

[0131] (2) Clarification of lentivirus supernatant

[0132] Before clarification, 20 U / ml nuclease (purchased from Merck Millipore; catalog number 1.01697.0001) and 0.1 g / L sodium dextran sulfate (purchased from WAKO; catalog number 196-13401, molecular weight 5 kDa) were added to the lentivirus supernatant obtained in step (1). The lentivirus supernatant was then filtered through a 0.45 μm filter membrane to further remove cell debris, yielding a clear solution. This clear solution was used for the next step, anion exchange chromatography. The average yield of lentivirus in the clear solution was 97.4% (e.g., ...). Figure 1 As shown, N=3).

[0133] (3) Anion chromatography and elution

[0134] Adjust the NaCl concentration of the clarified solution obtained in step (2) to 0.3M and the pH to 5.5. Load the solution onto a pre-equilibrated anion exchange membrane chromatography or anion exchange chromatography column. After loading, first wash with citrate buffer (first eluent: containing 0.1M citric acid, 0.15M NaCl, pH 6.0) for 50CV (column volume), then wash with high-salt PB buffer (second eluent: containing 50mM PB, 0.8M NaCl, pH 6.0) for 50CV. The second eluent must be applied after the first eluent is applied. Finally, elute with high-salt PB buffer (elution buffer: containing 50mM PB, 2.8M NaCl, pH 6.0) for 10CV and collect the eluent containing lentivirus.

[0135] The average yield of lentiviruses in the chromatography solvent was 96.7% (e.g., ...). Figure 2 As shown, N=4); by Figure 3The chromatogram of lentivirus 1-lot #1 shows that both the first and second eluents produced obvious peaks, while the binding... Figure 2 The lack of a significant decrease in virus yield indicates that most of the eluted material consists of process- or product-related impurities, with an average HCP removal rate of 99.4% (e.g., Figure 4 As shown, N=4). Lentiviral virus can be stored for 24 hours without losing its infectious activity under high-salt conditions in anionic elution buffer (elution solution: 50mM PB, 2.8M NaCl, pH 6.0) without the need for additional special protective reagents.

[0136] (4) Ultrafiltration dialysis and preservation

[0137] The solution was directly dialyzed into low-salt PB buffer (50 mM PB, 0.15 M NaCl, pH 6.0-7.3) using anion exchange eluent and 750 kDa tangential flow ultrafiltration. 2 wt% HSA and 1 wt% sucrose were added, and the solution was aliquoted into cryovials and stored at -80°C.

[0138] Example 2

[0139] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the first eluent in Example 1 is changed from 0.15M to 0.05M.

[0140] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0141] Example 3

[0142] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the first eluent in Example 1 is changed from 0.15M to 0.075M.

[0143] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0144] Example 4

[0145] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the first eluent in Example 1 is changed from 0.15M to 0.25M.

[0146] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0147] Experimental results show that, taking the lentivirus yield in the anion exchange chromatography step of Example 1 as 100%, the lentivirus yields in Examples 2-3 were slightly lower than those in Example 1, but the change was less than 10%. The lentivirus yield in Example 4 was relatively reduced by 19.2% (see [link to example]). Figure 5 Using the residual HCP from anion exchange chromatography in Example 1 as 100%, the residual HCP from anion exchange chromatography in Examples 2-3 was slightly higher, but the change was less than 10%. The residual HCP from anion exchange chromatography in Example 4 was relatively lower by 11.9% (see [link to example 4]). Figure 6 ).

[0148] Example 5

[0149] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the concentration of citric acid in the first eluent in Example 1 is changed from 0.1M to 0.02M.

[0150] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0151] Example 6

[0152] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the concentration of citric acid in the first eluent in Example 1 is changed from 0.1M to 0.15M.

[0153] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0154] Example 7

[0155] Referring to the purification method of Example 1, the difference between this example and Example 1 is that citric acid is not added to the first eluent of Example 1.

[0156] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0157] Experimental results show that, taking the lentivirus yield in the anion exchange chromatography step of Example 1 as 100%, the lentivirus yield changes in Examples 5 and 6 were less than 10%, and in Example 7, the lentivirus yield in anion exchange chromatography decreased by more than 3 times without the addition of citric acid. Figure 7 Using the residual HCP in anion exchange chromatography of Example 1 as 100%, the changes in residual HCP in anion exchange chromatography of Examples 5 and 6 were less than 10%, while the residual HCP in anion exchange chromatography of Example 7 without the addition of citric acid increased by 2.7 times. Figure 8 The results showed that adding citrate buffer rinsing during lentiviral anion exchange chromatography effectively improved virus yield and enhanced impurity removal.

[0158] Example 8

[0159] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the eluent in Example 1 is changed from 2.8M to 3M.

[0160] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0161] Example 9

[0162] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the eluent in Example 1 is changed from 2.8M to 2M.

[0163] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0164] Example 10

[0165] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the second eluent in Example 1 is changed from 0.8M to 0.5M.

[0166] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0167] Example 11

[0168] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sodium chloride concentration of the second eluent in Example 1 is changed from 0.8M to 1.2M.

[0169] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0170] Experimental results show that, taking the virus yield in the anion exchange chromatography step of Example 1 as 100%, the decrease in lentivirus yield in Examples 8 and 11 was less than 10%, while the decrease in lentivirus yield in Examples 9 and 10 was greater than 20%. Figure 9 Using the residual HCP from anion exchange chromatography in Example 1 as 100%, the relative variation in residual HCP from anion exchange chromatography in Examples 8-11 was less than 15%, see [reference needed]. Figure 10 .

[0171] Example 12

[0172] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the 5 kDa sodium dextran sulfate in Example 1 is replaced with sodium dextran sulfate with a molecular weight of 40 kDa.

[0173] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0174] Example 13

[0175] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the 5 kDa sodium dextran sulfate in Example 1 is replaced with sodium heparin (average molecular weight of 3.8-5 kDa).

[0176] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0177] Example 14

[0178] Anti-clustering agents can reduce cell clumping, which helps to obtain higher viable cell densities in suspension culture. Essentially, their main component is also a polyanionic compound. Referring to the purification method of Example 1, the difference between this example and Example 1 is that the 5 kDa sodium dextran sulfate in Example 1 is replaced with an anti-clustering agent (brand: Gibco, catalog number 0010057DG).

[0179] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0180] Example 15

[0181] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the concentration of 5 kDa sodium dextran sulfate in Example 1 is changed from 0.1 g / L to 0.5 g / L.

[0182] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0183] Example 16

[0184] Referring to the purification method of Example 1, the difference between this example and Example 1 is that sodium dextran sulfate is not added in Example 1.

[0185] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0186] Experimental results show that, compared with Example 16 without the addition of polyanionic compounds, the lentivirus yield of the clarification process in Examples 12-15 can be increased by 100%. Figure 11 The results showed that adding polyanionic compounds can effectively reduce the non-specific adsorption of lentiviruses by the filter in the clarification process.

[0187] Example 17

[0188] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the PB buffer in the second eluent and elution buffer in step (3) of Example 1 anion chromatography and elution is replaced with Tris buffer, and the concentration of Tris buffer is 50mM.

[0189] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0190] Example 18

[0191] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the concentration of PB buffer in the second eluent and the elution in step (3) of anion chromatography and elution in Example 1 is changed from 50mM to 10mM.

[0192] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0193] Example 19

[0194] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the concentration of PB buffer in the second eluent and the elution in step (3) of Example 1 (anion chromatography and elution) is changed from 50mM to 100mM.

[0195] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0196] The yield of lentivirus in the chromatographic eluent of Examples 17-19 and the yield of virus after storage in high-salt eluent for 24 hours were tested.

[0197] Experimental results showed that, compared with Example 1, the yields of Examples 17-19 did not decrease significantly, and the yields were all greater than 80%. Figure 12 Furthermore, lentiviruses did not lose their infectivity after being stored in high-salt elution buffer for 24 hours. Figure 13 .

[0198] Example 20

[0199] Referring to the purification method of Example 1, the difference between this example and Example 1 is that the sample loading conditions in step (3) are different (i.e., adjusting the NaCl concentration to 0.3M and the pH to 5.5 in Example 1).

[0200] The other components, dosages, and purification steps in the purification method are the same as in Example 1.

[0201] To detect lentivirus in the flow-through solution during sample loading, sample loading was stopped if the chromatography column exceeded the pressure. The maximum sample loading amount was investigated under different sample loading conditions when there was neither overpressure nor flow-through of lentivirus.

[0202] Table 1. Effect of different loading conditions on chromatography loading

[0203]

[0204] Experimental results show that increasing the salt concentration of the sample or decreasing the pH of the sample can appropriately increase the chromatography loading, as shown in Table 1 above.

[0205] Comparative Example 1

[0206] (1) Obtaining lentivirus supernatant

[0207] Lentiviral supernatant was harvested after packaging lentivirus into HEK 293T cells.

[0208] (2) Clarification of lentivirus supernatant

[0209] Before clarification, 20 U / ml nuclease was added to the lentivirus supernatant obtained in step (1), and then the lentivirus supernatant was filtered through a 0.45 μm filter membrane to further remove cell debris, resulting in a clear solution. The yield of lentivirus in the clear solution was 43.1%.

[0210] (3) Anion chromatography and elution

[0211] The equilibration buffer was Tris buffer (20 mM Tris, 0.3 M NaCl), the elution buffer and salt concentration were the same as the equilibration buffer, and the elution salt concentration was 2.8 M NaCl;

[0212] The anion exchange chromatography yield was 17.7%. Testing of the viral activity in the eluent showed that the viral infection titer decreased by 95% after 20 hours of storage, indicating that the virus needs to be diluted 9-10 times with buffer solution as soon as possible after elution.

[0213] (4) Ultrafiltration dialysis and preservation

[0214] Lentiviral products are obtained by 300-750 kDa tangential flow ultrafiltration (UF / DF). The ultrafiltration steps include concentration and dialysis, aseptic filtration and cryopreservation.

[0215] Lentivirals are a type of retrovirus. Theoretically, the lentivirus purification chromatography composition and purification method provided in this disclosure are applicable to any other type of retrovirus.

[0216] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0217] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of this disclosure. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A reagent combination for purifying retroviruses, comprising a first elution buffer, a second elution buffer and an elution buffer, wherein: the first elution buffer comprises the following components: 0.02 M-0.15 M citric acid, 0.05 M-0.15 M sodium chloride and water; the second elution buffer comprises the following components: a buffer, 0.8 M-1.2 M sodium chloride and water; the elution buffer comprises the following components: a buffer, 2.8 M-5 M sodium chloride and water; the reagent combination further comprises a polyanionic compound; the polyanionic compound is selected from one or more of the following: dextran sulfate sodium salt, heparin sodium and / or an anti-cell clumping agent comprising dextran sulfate sodium salt or heparin sodium; the concentration of the polyanionic compound is 0.1 g / L-0.5 g / L; and the pH of the first elution buffer, the second elution buffer and the elution buffer is 5-6.

5. 2.The retroviruses are selected from one or more of the following: oncoviruses, lentiviruses and / or spumaviruses. 3.The retroviruses are lentiviruses. 4.In the first elution buffer, the concentration of citric acid is 0.1 M. 5.In the first elution buffer, the concentration of sodium chloride is 0.15 M. 6.The buffer is a PB buffer or a Tris buffer. 7.The concentration of the PB buffer or the Tris buffer is 50 mM. 8.In the second elution buffer, the concentration of sodium chloride is 0.8 M.

2. The reagent combination according to claim 1, wherein, 9.In the elution buffer, the concentration of sodium chloride is 2.8 M.

3. The combination of agents according to claim 2, wherein, 10.The concentration of the polyanionic compound is 0.1 g / L or 0.5 g / L.

4. The combination of agents of claim 1, wherein, 11.A kit for purifying retroviruses, comprising the reagent combination of any one of claims 1-9.

5. The combination of agents of claim 1, wherein, 12.A method for purifying retroviruses using the reagent combination of any one of claims 1-10 or the kit of claim 11, comprising the following steps: (1) obtaining a retrovirus supernatant; (2) clarifying the retrovirus supernatant: mixing the retrovirus supernatant obtained in step (1) with a polyanionic compound, filtering and obtaining a clarified retrovirus supernatant; adding a nuclease before filtering; (3) anion chromatography and elution: loading the clarified retrovirus supernatant obtained in step (2) onto an anion membrane chromatography or an anion chromatography column, sequentially washing with the first elution buffer, the second elution buffer and the elution buffer after loading is completed, and collecting the elution buffer containing the retroviruses; and (4) purifying the elution buffer.

6. The combination of agents of claim 1, wherein, 13.The method further comprises dialysis and / or concentration.

7. The combination of agents according to claim 6, wherein, 14.The retroviruses are selected from one or more of the following: oncoviruses, lentiviruses and / or spumaviruses.

8. The combination of agents of claim 1, wherein, 15.The retroviruses are lentiviruses.

9. The combination of agents of claim 1, wherein, 16.In step (2), the concentration of the polyanionic compound is 0.1 g / L-0.5 g / L.

10. The combination of agents of claim 1, wherein, 17.The polyanionic compound is selected from one or more of the following: dextran sulfate sodium salt, heparin sodium and / or an anti-cell clumping agent comprising dextran sulfate sodium salt or heparin sodium. 18.In step (2), the concentration of the nuclease is 10-50 U / ml. 19.In step (2), the concentration of the nuclease is 20 U / ml. ​ ​ ​ ​ ​ ​ 13. The method of claim 12, wherein, ​ 14. The method of claim 12, wherein, ​ 15. The method of claim 14, wherein, ​ 16. The method of claim 12, wherein, ​ 17. The method of claim 12, wherein, ​ 18. The method of claim 12, wherein, ​ 19. The method of claim 18, wherein, ​ 20. The method of claim 12, wherein, In step (3), the sodium chloride concentration and pH of the clarified retroviral supernatant are adjusted prior to chromatography loading to a sodium chloride concentration of 0.1 M to 0.5 M and a pH of 5.0 to 6.

5.

21. The method of claim 12, wherein, The sodium chloride concentration of the retroviral supernatant is adjusted to 0.3 M and the pH is 5.

5.

22. The method of claim 12, wherein, In step (4), the eluate is purified using ion exchange or filtration.

23. The method of claim 22, wherein, The filtration is selected from one or more of gel filtration, ultrafiltration, tangential flow filtration, and membrane filtration.

24. Use of the reagent combination of any one of claims 1 to 10 or the kit of claim 11 for the preparation of purified retrovirus.

25. The use of claim 24, wherein, The retrovirus is selected from one or more of oncovirus, lentivirus, and / or spumavirus.

26. The use of claim 25, wherein, The retrovirus is a lentivirus.

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