Methods for purifying enveloped viruses

Abstract

The present disclosure relates generally to the manufacture of gene therapy products, and specifically to methods for purifying enveloped viruses from cell culture media involving endonucleases and / or anion exchange chromatography.

Description

[Technical field]

[0001] Related Application Data This application claims priority to U.S. Provisional Application No. 63 / 362,163, entitled "Methods of purifying an enveloped virus," filed March 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the manufacture of gene therapy products, and specifically to methods for purifying enveloped viruses from cell culture media involving endonucleases and / or anion exchange chromatography. [Background technology]

[0003] Retrovirus, for example lentivirus, is one of the most researched viral vectors for gene therapy.Generally, retrovirus is an RNA type virus that permanently integrates its genetic information into the chromosome of target cell.The advantages of retrovirus include long-term expression of transgene in target cell, low immunogenic potential, and transduction ability in dividing and non-dividing cells.

[0004] Lentiviruses are genetically engineered and are usually based on human immunodeficiency virus 1 (HIV-1). To increase safety, the latest vectors contain only the HIV genes necessary for infection and gene delivery, but genes necessary for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is exchanged with the VSV-G protein of another virus, such as Vesicular Stomatitis Indiana Virus (VSV), to allow infection of a broad range of target cells.

[0005] To generate lentivirus, cells such as human embryonic kidney cells HEK 293T are transfected with three to four plasmids. These include a transfer plasmid carrying the gene of interest as well as several packaging plasmids encoding the vesicular stomatitis G protein (VSV-G) and essential viral proteins involved in gene integration or self-assembly. These plasmids are either transiently transfected into the cells, or producer cell lines are created by stably integrating a plasmid carrying an inducible promoter that can drive lentivirus production.

[0006] When virus production is induced, successful assembly within the cell is followed by virus release by budding.

[0007] Lentivirus is harvested from producer cells, and then purified and concentrated in downstream processes.However, it is difficult to purify lentivirus on a commercial scale.The limiting barrier to purifying this type of virus is the impurities that occur in large-scale cell culture, and the instability of certain membrane glycoproteins when exposed to some purification conditions.Therefore, those skilled in the art need an efficient process for purifying lentivirus, for example for gene therapy. Summary of the Invention [Problem to be solved by the invention]

[0008] In the work leading to the present invention, the inventors have attempted to create a method for producing enveloped viruses for, for example, gene therapy on a commercial scale suitable to regulatory requirements. They have also attempted to develop a method that can be run in-line or in a continuous or semi-continuous mode. [Means for solving the problem]

[0009] In some examples, the downstream process for purifying and concentrating the viral vector produced by the present inventors includes a harvest filtration step to remove cell debris and components, and a purification step to reduce the total volume and separate the viral vector from host cell DNA, proteins and medium components.The downstream process for purifying and concentrating the viral vector produced by the present inventors can further include an ultrafiltration / diafiltration step to concentrate the viral vector in the final formulation buffer.In some examples, the downstream process further includes a filtration sterilization step to remove microorganisms from the final product.

[0010] In developing this method, the inventors determined that nucleic acids in the cell culture fluid (e.g., stable cell line supernatant) in which the virus was produced caused fouling of membranes, resins, and filters used in downstream processes (e.g., anion exchange chromatography purification and filtration sterilization steps). To solve this problem, the inventors added an endonuclease to the cell culture fluid. The inventors confirmed that the endonuclease can be added before purification, which improves purification and other downstream steps such as filtration sterilization. In one example, the inventors found that the endonuclease can be added just before harvest filtration and then proceeded to purification without the need for a separate incubation with the endonuclease. The addition of the endonuclease facilitated longer harvesting of the virus produced by both adherent and suspension cells as well as higher vector yields without clogging the chromatography column and / or filtration sterilization filter.

[0011] The inventors have further confirmed that it is desirable to increase the salt concentration in the cell culture fluid prior to anion exchange chromatography to reduce binding of impurities, thereby facilitating virus binding and passage of contaminants to the anion exchanger. However, increasing the salt concentration is complicated by several factors. For one, it is not desirable to add large volumes to the cell culture fluid prior to anion exchange, so a high concentration salt solution is preferred. However, hypertonic conditions destabilize virus particles. Furthermore, manual mixing on a commercial scale is cumbersome, time-consuming, and not suitable for in-line processing. If mixing is performed in a vessel, if the salt is not mixed properly, the more concentrated high salt solution will collect at the bottom of the vessel, and the higher local salt concentration may elute the virus during loading into the chromatography device. To solve these problems, the inventors add a high concentration salt solution to the cell culture fluid by running two fluid streams together just before chromatography. The high concentration salt solution and cell culture fluid are mixed when loaded onto the anion exchanger, or just before loading onto the anion exchanger, or run together within the anion exchanger. This makes it easy to mix the solutions without adding large volumes and without destabilizing the virus. Whether at a laboratory or commercial scale, this also allows for efficient mixing without the need for time-consuming mixing steps.

[0012] The findings by the present inventors have provided a method for purifying enveloped viruses.

[0013] In one example, the disclosure provides a method for purifying an enveloped virus from a cell culture fluid, the method comprising contacting the cell culture fluid with an endonuclease prior to harvest filtration.

[0014] The present disclosure further provides a method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, the method comprising contacting the cell culture fluid or the filtered cell culture fluid with an endonuclease prior to purifying the virus.

[0015] In an exemplary embodiment of the present disclosure, the cell culture medium or harvested cell culture medium is derived from stable producer cells, i.e., cells that have stably integrated the genetic material required to produce lentivirus. Such cells are distinguished from cells that have transiently integrated genetic elements therein.

[0016] An exemplary enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or its derivatives.

[0017] Exemplary nucleases are non-specific endonucleases, degrading both DNA and RNA without sequence specificity, hi one example, the endonuclease is a non-specific endonuclease, degrading DNA without sequence specificity.

[0018] Suitable endonucleases are known to those skilled in the art and include those derived from Serratia marcescens, Anabaena spp., Saccharomyces cerevisiae, Bos Taurus, Syncephalostrum racemosum and / or Borrelia burgdorferi. For example, the endonuclease is Serratia nuclease, NucA, Nuc1, endonuclease G, DNase I or micrococcal nuclease.

[0019] The endonuclease is added at any concentration suitable to prevent fouling of the membrane or anion exchanger after endonuclease treatment. Suitable concentrations are described herein. In one example, the endonuclease is added to the cell culture at a concentration of 0.001 to 100 units / mL of cell culture solution. In one example, the endonuclease is added to the cell culture at a concentration of 0.01 to 10 units / mL of cell culture solution. In one example, the endonuclease is added to the cell culture at a concentration of 0.1 to 1 unit / mL of cell culture solution. For example, the endonuclease is added to the cell culture at a concentration of 0.3 units / mL of cell culture solution.

[0020] The endonuclease is added before purification, for example, anion exchange. The present disclosure encompasses incubating the endonuclease and cell culture fluid before anion exchange. In some examples, the endonuclease and cell culture fluid are incubated for less than about 2 hours or less than about 1 hour before anion exchange.

[0021] The present inventors have shown that in certain examples, there is no need to include separate incubation of endonuclease and cell culture fluid.For example, the processing time of purification, for example, harvest filtration process itself before anion exchange is sufficient to prevent fouling of anion exchanger, so endonuclease is added to cell culture fluid before harvest filtration, and then cell culture fluid proceeds immediately to harvest filtration.However, the present disclosure also encompasses incubation of endonuclease and cell culture fluid before harvest filtration.In some examples, endonuclease and cell culture fluid are incubated for less than about 2 hours or less than about 1 hour before harvest filtration.

[0022] In some examples, the purification is performed less than about 30 hours after contacting the cell culture medium or filtered cell culture medium with the endonuclease, for example, less than about 6 hours after contacting the cell culture medium or filtered cell culture medium with the endonuclease, for example, less than about 4 hours after contacting the cell culture medium or filtered cell culture medium with the endonuclease, for example, less than about 2 hours after contacting the cell culture medium or filtered cell culture medium with the endonuclease, for example, less than about 1 hour after contacting the cell culture medium or filtered cell culture medium with the endonuclease, or less than about 22 hours after contacting the cell culture medium or filtered cell culture medium with the endonuclease, for example, less than about 30 minutes after contacting the cell culture medium or filtered cell culture medium with the endonuclease.

[0023] In one example, the method further comprises performing a harvest filtration immediately after contacting the cell culture fluid with the endonuclease to produce a filtered cell culture fluid.

[0024] In some examples, the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.3 units / mL of the cell culture medium or filtered cell culture medium, and purification is performed about 1 to about 2 hours after contacting.

[0025] In examples of the present disclosure, the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.01-0.3 units / mL of the cell culture medium or filtered cell culture medium, and purification is carried out for more than about 2 hours after contacting.

[0026] In additional examples of the present disclosure, the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.3-10 units / mL of the cell culture medium or filtered cell culture medium, and purification is performed less than about 1 hour after contacting.

[0027] In a further example of the present disclosure, the endonuclease is contacted with the cell culture fluid or filtered cell culture fluid at a concentration of about 0.3 units / mL of the cell culture fluid or filtered cell culture fluid, and purification is performed less than about 1 hour after contacting.

[0028] In one example, the method further comprises contacting the cell culture medium or the filtered cell culture medium with a magnesium salt prior to purifying the virus. In one example, the magnesium salt is a magnesium salt having a target magnesium concentration of less than 10 mM Mg 2+ For example, 0.001 mM to 10 mM, or 0.1 mM to 10 mM, or 1 mM to 10 mM. In one example, the magnesium salt is added in an amount to achieve a target magnesium concentration of less than 10 mM, such as 9 mM, or 8 mM, or 7 mM, or 6 mM. In one example, the magnesium salt is added in an amount to achieve a target magnesium concentration of less than 5 mM. For example, 5 mM, or 4 mM, or 3 mM, or 2 mM, or 1 mM. In one example, the magnesium salt is added in an amount to achieve a target magnesium concentration of about 2 mM.

[0029] Therefore, to achieve the target magnesium concentration, the ratio of the filtered cell culture fluid or cell culture fluid to the magnesium salt solution is determined by the concentration of the magnesium salt solution. In one example, the magnesium salt solution has a concentration of less than 500 mM, for example, 50 mM to 500 mM, or 100 mM to 400 mM, or 150 mM to 300 mM. For example, the magnesium salt solution has a concentration of 200 mM.

[0030] In one example, the magnesium salt is magnesium chloride. Other suitable forms of magnesium salts suitable for use in the present disclosure will be apparent to those of skill in the art and / or will be described herein.

[0031] In one example, the method further includes subjecting the filtered cell culture fluid to anion exchange chromatography, e.g., the filtered cell culture fluid is subjected to anion exchange chromatography immediately after harvest filtration.

[0032] As discussed above, the inventors have shown that contacting the cell culture fluid or filtered cell culture fluid with a high concentration salt solution prior to or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture fluid improves anion exchange purification, for example, by preventing impurities from binding to the anion exchanger, thereby enhancing virus binding and allowing contaminants to pass through.

[0033] Thus, in some cases, the filtered cell culture fluid is contacted with a high salt solution before or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture fluid. Such a mixed form facilitates in-line processing. For example, the cell culture fluid is contacted with an endonuclease, then filtered, and then directly loaded onto an anion exchange column and mixed with the high salt solution.

[0034] The present disclosure further provides a method of purifying an enveloped virus from a cell culture fluid using anion exchange chromatography, wherein the cell culture fluid is contacted with a high concentration salt solution prior to or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture fluid.

[0035] The anion exchanger may be a resin-based anion exchanger, an anion exchange membrane adsorber, or any other type of anion exchanger that includes a positively charged substrate to capture negatively charged particles. In the illustrated embodiment of the present disclosure, the anion exchanger is an anion exchange membrane adsorber.

[0036] In one example, the high salt solution and the filtered cell culture fluid or cell culture fluid are mixed in-line, for example, during loading onto an anion exchange chromatography column. For example, during loading onto an anion exchange column, the liquids are added separately and simultaneously to the column. In some examples, the liquids can contact each other before entering the anion exchange column, after entering the anion exchange column, or simultaneously entering the anion exchange column.

[0037] In one example, the combined filtered cell culture medium or cell culture medium and high salt solution has a target salt concentration of about 300 mM to 500 mM salt for loading onto an anion exchange chromatography column, for example, the target salt concentration is 400 mM salt.

[0038] Thus, to achieve a target salt concentration, the ratio of filtered cell culture fluid or cell culture fluid to high salt solution depends on the concentration of the salt solution. In one example, the high salt solution has a concentration of at least 1M, for example, 1M to 10M, or 2M to 9M, or 3M to 8M, or 4M to 6M. For example, the high salt solution has a concentration of 5M.

[0039] In one example, the filtered cell culture medium or cell culture fluid and the high salt solution are mixed in a ratio of about 70-99% (volume / volume) filtered cell culture medium or cell culture fluid and about 1-30% (volume / volume) high salt solution.

[0040] In one example, the high salt solution is provided at a concentration of 5M, and the filtered cell culture fluid or cell culture medium and the high salt solution are mixed in a ratio of 90-95% (volume / volume) of the filtered cell culture fluid or cell culture medium and 5-10% (volume / volume) of the high salt solution. For example, the filtered cell culture fluid or cell culture medium and the high salt solution are mixed in a ratio of 95% (volume / volume) of the filtered cell culture fluid or cell culture medium and 5% (volume / volume) of the high salt solution. For example, the filtered cell culture fluid or cell culture medium and the high salt solution are mixed in a ratio of 94% (volume / volume) of the filtered cell culture fluid or cell culture medium and 6% (volume / volume) of the high salt solution. For example, the filtered cell culture fluid or cell culture medium and the high salt solution are mixed in a ratio of 93% (volume / volume) of the filtered cell culture fluid or cell culture medium and 7% (volume / volume) of the high salt solution.

[0041] In another example, the high salt solution is provided at a concentration of 1 M, and the filtered cell culture medium or cell culture fluid and the high salt solution are mixed in a ratio of about 70% filtered cell culture medium or cell culture fluid (volume / volume) and about 30% high salt solution (volume / volume).

[0042] In another example, the high salt solution is provided at a concentration of 2M, and the filtered cell culture medium or cell culture fluid and the high salt solution are mixed in a ratio of about 85% filtered cell culture medium or cell culture fluid (volume / volume) and about 15% high salt solution (volume / volume).

[0043] In another example, the high salt solution is provided at a concentration of 10 M, and the filtered cell culture medium or cell culture fluid and the high salt solution are mixed in a ratio of about 97% filtered cell culture medium or cell culture fluid (volume / volume) and about 3% high salt solution (volume / volume).

[0044] In one example, the flow rate of the filtered cell culture fluid or the fluid stream containing the cell culture fluid is higher than the flow rate of the fluid stream containing the high salt solution. In another example, the flow rate of the filtered cell culture fluid or the fluid stream containing the cell culture fluid is lower than the flow rate of the fluid stream containing the high salt solution. In another example, the flow rate of the filtered cell culture fluid or the fluid stream containing the cell culture fluid is the same as the flow rate of the fluid stream containing the high salt solution.

[0045] In one example, the combined filtered cell culture medium or cell culture medium and high salt solution has a target conductivity of 35-45 mS / cm at 25° C. In one example, the combined filtered cell culture medium or cell culture medium and high salt solution has a target conductivity of 36-44 mS / cm at 25° C. For example, a target conductivity of about 40±4 mS / cm at 25° C.

[0046] In one example, the high salt solution includes a monovalent and / or divalent salt. For example, the high salt solution includes a monovalent salt, i.e., as the only salt. In one example, the monovalent salt is sodium chloride.

[0047] In one example, the high salt solution is sodium chloride at a concentration of at least about 1 M, for example, at a concentration of about 5 M.

[0048] In one example, the salt-spiked cell culture medium has a salt concentration of 300 mM to 500 mM, for example, about 400 mM.

[0049] In one example, the method further comprises washing the anion exchange chromatography column with one or more washing steps. For example, the method comprises a first washing step with a solution comprising 90-95% (volume / volume) buffer and 5-10% (volume / volume) high salt solution. For example, the solution comprises 10-100 mM Tris, 150 mM NaCl, pH 7.0-9.0. In one example, the solution comprises 50 mM Tris, 150 mM NaCl, pH 8. In another example, the solution comprises 5-50 mM histidine, 150 mM NaCl, pH 5.5-7.4. In another example, the solution comprises 10 mM histidine, 150 mM NaCl, pH 7. In another example, the solution comprises 5-50 mM HEPES, 150 mM NaCl, pH 6.8-8.2. In another example, the solution contains 10 mM HEPES, 150 mM NaCl, pH 7.5.

[0050] In one example, the method includes a second washing step with a second solution. For example, the second solution includes 10-100 mM Tris, 750 mM NaCl, pH 7.0-9.0. In one example, the second solution includes 50 mM Tris, 750 mM NaCl, pH 8. In another example, the second solution includes 5-50 mM histidine, 750 mM NaCl, pH 5.5-7.4. In another example, the second solution includes 10 mM histidine, 750 mM NaCl, pH 7. In another example, the second solution includes 5-50 mM HEPES, 750 mM NaCl, pH 6.8-8.2. In another example, the second solution includes 10 mM HEPES, 750 mM NaCl, pH 7.5.

[0051] In one example, the method further comprises eluting the bound virus from the anion exchange chromatography column with an elution solution. For example, the elution solution comprises 10-100 mM Tris, 1 M-2 M NaCl, pH 7.0-9.0. In one example, the elution solution comprises 50 mM Tris, 1.2 M NaCl, pH 8. In another example, the elution solution comprises 50 mM Tris, 1.5 M NaCl, pH 8. In another example, the elution solution comprises 5-50 mM histidine, 1 M-2 M NaCl, pH 5.5-7.4. In another example, the elution solution comprises 10 mM histidine, 1.2 M NaCl, pH 7. In another example, the elution solution comprises 10 mM histidine, 1.5 M NaCl, pH 7. In another example, the elution solution comprises 5-50 mM HEPES, 1 M-2 M NaCl, pH 6.8-8.2. In another example, the elution solution comprises 10 mM HEPES, 1.2 M NaCl, pH 7.5. In another example, the elution solution comprises 10 mM HEPES, 1.5 M NaCl, pH 7.5.

[0052] In one example, the method includes diluting the eluted virus to a 1:5 to 1:20 dilution. For example, the method includes diluting the eluted virus to a 1:10 dilution.

[0053] In one example, the method includes diluting the eluted virus with histidine, Tris, or HEPES. For example, the method includes diluting the eluted virus with HEPES. In one example, the method includes diluting the eluted virus with 10 mM HEPES, pH 7.5. For example, the virus is diluted immediately after elution.

[0054] In one example, the method further comprises incubating the eluted virus at room temperature for up to 15 minutes or at 2-8° C. for up to 60 minutes.

[0055] In one example, the method includes concentrating and / or diafiltering the eluted virus.

[0056] In one example, the method increases viral infectious titer yield by at least 2%, 3%, 4%, 5%, 10%, 15%, or 20%. In one example, the method increases viral infectious titer yield by at least 10%.

[0057] The present disclosure further provides a method for purifying an enveloped virus from a cell culture medium, comprising: (i) (optionally) providing a cell culture medium containing a viral vector produced from a stable producer cell line; (ii) contacting the cell culture with a recombinantly expressed Serratia endonuclease; (iii) contacting the endonuclease-treated cell culture medium with a filter to produce a filtered cell culture medium; (iv) loading the filtered cell culture fluid and a high salt solution containing 5 M sodium chloride onto an anion exchange chromatography membrane, such that the fluid and salt solution are loaded in a ratio of 94% (volume / volume) filtered cell culture fluid and 6% (volume / volume) high salt solution; (v) washing the membrane with one or more wash buffers; (vi) eluting the bound virus from the membrane with an elution buffer containing 1.2 M or 1.5 M sodium chloride; (vii) (optionally) diluting the eluted virus with a buffer; (viii) concentrating and diafiltering the eluted virus or the diluted eluted virus; Includes.

[0058] In one example, the method is performed in-line, or continuously or semi-continuously.

[0059] In one example, the method of the present disclosure further includes performing a sterile filtration. For example, the sterile filtration is performed before concentrating and diafiltering the eluted virus. In an alternative example, the sterile filtration is performed after concentrating and diafiltering the eluted virus.

[0060] In one example, the methods of the disclosure further provide for formulating the enveloped virus into a pharmaceutical preparation or into a solution suitable for infecting a cell.

[0061] The present disclosure further provides purified enveloped viruses produced by the methods described herein.

[0062] The present disclosure further provides a method for preventing fouling of an anion exchanger, the method comprising the same steps as described herein.

[0063] In one example, the chromatography method of the present disclosure is carried out on a substrate having a positively charged ligand immobilized thereon. As will be apparent to those skilled in the art, such a substrate is an anion exchange chromatography column. Such a substrate may also be a chromatographic column containing a positively charged ligand. Thus, the present disclosure further encompasses carrying out the method described herein, where a chromatographic column containing a positively charged ligand is used instead of an ion exchange chromatography column. [Brief description of the drawings]

[0064] [Figure 1] FIG. 1 is a graph showing that harvest stability is compromised by high salt conditions. [Diagram 2] FIG. 13 is a graphical representation showing RNA yield, infectious yield and sterile filter capacity of TFF eluates with and without benzonase treatment (as indicated). [Diagram 3] FIG. 1 is a graph showing the pressure on a Mustang Q anion chromatography column upon processing of harvested cell culture fluid without (A) or with (B) benzonase treatment (as indicated). [Figure 4] FIG. 1 is a graphical representation summarizing infectious titer and RNA content yields for harvests collected from planar and attached bioreactors. [Diagram 5]FIG. 1 is a graphical representation showing virus recovery from Mustang Q anion exchange run with or without in-line 5M NaCl salt spiking (as indicated). [Figure 6] FIG. 1 is a graph showing infectious titer yield during purification of a large-scale harvest of an attached bioreactor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] general Throughout this specification, unless otherwise stated or required by context, a reference to a single step, composition, group of steps, or group of compositions is intended to include one and the plurality (i.e., one or more) of that step, composition, group of steps, or group of compositions. Thus, in this specification, the singular forms "a," "an," and "the" include plural aspects unless the context clearly dictates otherwise. For example, a reference to "a" includes one and two or more; a reference to "an" includes one and two or more; a reference to "the" includes one and two or more, etc.

[0066] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described.It should be understood that the present disclosure includes all such variations and modifications.The present disclosure includes all of the steps, compositions, compositions and compounds mentioned or shown in this specification, individually or collectively, and also includes any combination of said steps or compositions or any combination of two or more of them.

[0067] The present disclosure should not be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0068] Unless otherwise stated, any example of the present disclosure herein is intended to apply mutatis mutandis to any other example of the present disclosure. In other words, any specific example of the present disclosure can be combined (except exclusively) with any other specific example of the present disclosure.

[0069] Any example of this disclosure disclosing a particular configuration or group of configurations, or method or method steps, shall provide explicit support for disclaiming the particular configuration or group of configurations, or method or method steps.

[0070] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, bacteriology, virology).

[0071] Unless otherwise indicated, the conventional techniques of molecular biology, bacteriology, virology, recombinant DNA techniques, peptide synthesis in solution, solid phase peptide synthesis and immunology utilized in this disclosure are standard procedures and well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, vols. 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, vols. 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions); Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998). Laboratory, (1988), and in JE Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current editions).

[0072] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall provide clear support for both meanings or for either meaning.

[0073] The term "about" refers to + / - 20% of the specified value, more for example + / - 10%, unless otherwise stated. For the avoidance of doubt, the specified value following the term "about" should be interpreted as including the exact specified value itself (e.g., "about 10" also includes exactly 10).

[0074] As used herein, the term "from" is intended to indicate that the specified integer is obtained from a particular source, but not necessarily directly from that source (i.e., including by recombinant means).

[0075] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0076] Selected Definitions The terms "purify" or "purifying" or "purification" are intended to mean the complete or partial removal of at least one impurity present in a cell culture medium, thereby improving the purity level of the enveloped virus in the solution.

[0077] The term "impurity" or "impurities" is intended to include one or more components in a cell culture medium other than enveloped viruses. For example, impurities include process-related impurities such as host cell DNA, host cell proteins, and media components (e.g., fetal bovine serum).

[0078] As used herein, the term "enveloped virus" refers to DNA and RNA viruses that have a viral envelope. The envelope is generally derived from host cell membrane (e.g., phospholipids and proteins), but may also contain viral glycoproteins on the envelope surface. Enveloped viruses also contain a "capsid", which is a protein layer between the envelope and the viral genome. In one example, the enveloped virus is a retrovirus. For example, the enveloped virus is a lentivirus, such as the human immunodeficiency virus.

[0079] As used herein, the term "cell culture medium" will be understood to encompass the liquid in which cells are grown to produce enveloped viruses. The liquid may contain the cells or the cells may have been removed, for example, by centrifugation and / or removal of the supernatant.

[0080] As used herein, "harvesting" refers to removing cell culture medium containing viral particles from producer cells for downstream processing, and "harvest" refers to cell culture medium containing viral particles that has been removed for downstream processing. The harvesting process includes collecting one or more harvests. "Harvest filtration" refers to either the filtered harvest or the cell culture medium containing viral particles that has been filtered to remove producer cells for downstream processing.

[0081] As used herein, the term "filtered cell culture fluid" will be understood to encompass cell culture fluid after it has been subjected to harvest filtration.

[0082] As used herein, the term "salt-spiked cell culture fluid" will be understood to encompass cell culture fluid or filtered cell culture fluid that has been mixed with a high concentration salt solution.

[0083] Those skilled in the art will understand that an "endonuclease" is an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. Endonucleases can cleave DNA or RNA or both DNA and RNA. Endonucleases can cleave in a sequence-nonspecific manner (also referred to as "nonspecific endonucleases") or at specific nucleotide sequences (also referred to as restriction enzymes).

[0084] References herein to "derived" from a source, e.g., Serratia marcescens derived endonuclease, include endonucleases purified from that source or produced by other means, e.g., recombinantly.

[0085] The term "anion exchange chromatography" specifically includes, but is not limited to, chromatography performed on anion exchange resins, matrices, absorbents, filters, and the like. For example, anion exchange chromatography is performed using a positively charged membrane. Those of skill in the art will understand that the terms "anion exchange chromatography" and "anion exchange purification" are used interchangeably herein, with each term providing clear support for the other term.

[0086] An "anion exchange chromatography column" is a device for separating compounds by anion exchange chromatography. A "column" is a container or tube used for anion exchange chromatography that contains a resin, substrate, adsorbent, filter, etc. to which a charged molecule is attached. Those skilled in the art will understand that the terms "anion exchange chromatography column", "anion exchange column" and "anion exchanger" are used interchangeably herein, with each term providing clear support for each of the other terms.

[0087] As used herein, in the context of performing anion exchange chromatography after harvest filtration, the term "immediately" means that there are no purification steps between harvest filtration and anion exchange, however, this term does not exclude additional steps such as adjusting the pH or adding salt to the cell culture fluid or filtered cell culture fluid between harvest filtration and anion exchange chromatography.

[0088] As used herein, the term "highly concentrated salt solution" will be understood to mean a salt concentration of 1 M or more, for example, greater than 500 mM, such as 1 M to 10 M.

[0089] As used herein, the term "in-line" in the context of a process step refers to a process step that is embedded or combined with one or more other process steps, or that flows directly from or to another process step without the need for manual intervention or handling.

[0090] As used herein, particularly in the context of a mixing step, "in-line mixing" or "in-line" refers to flowing a first fluid stream containing a first liquid in contact with a second fluid stream containing a second liquid such that the first and second liquids contact each other and are mixed together. In the context of loading an anion exchange chromatography column, in-line mixing includes contacting two fluid streams with each other before, after, or simultaneously with entering the anion exchange chromatography column.

[0091] Production of enveloped viruses Methods for producing enveloped viruses will be apparent to those skilled in the art and / or are described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48:362-377; Schweizer and Merten (2010) Curr. Gene Ther. 10:474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2:15-40.

[0092] In one example, the virus is a retrovirus, for example, a lentivirus.Exemplary retroviruses are derived from alpha retroviruses (such as avian leukemia virus (ALV)), beta retroviruses (such as mouse mammary tumor virus (MMTV)), gamma retroviruses (such as murine leukemia virus (MLV)), delta retroviruses (such as human T-lymphotropic virus (HTLV)), epsilon retroviruses (such as walleye dermal sarcoma virus (WDSV)), spumaviruses (such as human foamy virus (HFV) or simian foamy virus (SFV)), primate lentiviruses such as different types of human immunodeficiency virus (HIV), different types of simian immunodeficiency virus (SIV), or non-primate mammalian lentiviruses such as equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritis encephalitis virus (CAEV), or ovine visnamaedivirus (VMV).

[0093] In some instances, an enveloped virus, e.g., a retrovirus, is pseudotyped, i.e., the pseudotype comprises an envelope glycoprotein, a modified envelope glycoprotein, or a chimeric envelope glycoprotein derived from a virus different from the virus from which it is derived.

[0094] In some cases, the enveloped virus contains a transgene introduced into its genome. The transgene will depend on the specific use for which the enveloped viral vector is intended. Exemplary transgenes include transgenes that code for therapeutic RNA (e.g., that code for the antisense complementary RNA of a target RNA or DNA sequence), transgenes that code for proteins that are missing or absent in diseased subjects, or transgenes used for DNA vaccination, i.e., transgenes that code for proteins whose expression will induce vaccination of the recipient body against said proteins. In some cases, the transgene codes for a protein or nucleic acid that is useful for treating hemoglobinopathies, such as sickle cell disease or thalassemia. In some cases, the transgene codes for a protein or nucleic acid that is useful for treating primary immune deficiencies. In some cases, the transgene codes for a protein or nucleic acid that is useful for treating Wiskott-Aldrich syndrome. In some cases, the transgene codes for a protein or nucleic acid that is useful for treating X-linked agammaglobulinemia.

[0095] In some instances, enveloped viruses are generated by introducing into a host cell the following four elements: an expression cassette containing the lentiviral gene gagpol, an expression cassette containing the lentiviral gene rev, all transgenes located between the lentiviral LTR-5' and the lentiviral LTR-3', and an expression cassette encoding the envelope glycoprotein.

[0096] In some examples, enveloped viruses are produced from stable lines expressing one or several elements necessary to produce enveloped viruses (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra). In one example, enveloped viruses are produced from mammalian host cells transiently transfected with one or several plasmids encoding the elements necessary to produce viruses. According to another example, the elements are introduced into the cells using multiple plasmids: one plasmid carrying an expression cassette containing the lentiviral gagpol gene, one plasmid carrying an expression cassette containing the lentiviral rev gene, one plasmid carrying an expression cassette encoding an envelope glycoprotein, one plasmid carrying an expression cassette containing the tetracycline transactivator (tTA) gene, and / or one plasmid carrying an expression cassette containing the lentiviral tat gene. A transfer plasmid containing an expression cassette with a transgene contained between the LTR-5' and LTR-3' of a lentivirus can be introduced into producer cells as a concatemer together with a helper plasmid carrying an antibiotic resistance cassette conferring resistance.

[0097] The host cell is selected from any cell that allows the production of enveloped viruses. According to one example, the cell is selected from human cells (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), mouse cells (NIH-3T3), mustelid cells (Mpf), canine cells (D17). According to one example, the cells are selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0098] According to one example, the cell is selected from the GPR, GPRG, GPRT, GPRGT and GPRTG cell lines, hi another example, the cell is selected from a cell line derived from any of the above cell lines.

[0099] In one example, the enveloped virus is produced from a stable producer cell. The stable producer cell can be obtained from a packaging cell line, including any of the cell lines disclosed herein. In some embodiments, the packaging cell line is a GPRG or GPRTG cell line (Throm et al., (2009) Blood 113(21):5104-5110; and Bonner et al., (2015) Molecular Therapy, Vol. 23, Suppl. 1, S35). In one example, the stable producer cell line cells are generated by synthesizing a vector by cloning one or more genes into a recombinant plasmid; forming a concatemer array from an expression cassette excised from the synthesized vector and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfecting a packaging cell line cell with the formed concatemer array; and isolating a stable producer cell line cell. The virus is produced by inducing an inducible promoter in the stable producer cell line cells.

[0100] The cells are cultured in a medium suitable for mammalian cell culture and enveloped virus production. The cells can be cultured in an adherent environment, e.g., attached to a surface, or in a suspension environment, e.g., suspended in the medium. The medium is further supplemented with additives known in the art, such as antibiotics, serum (especially fetal bovine serum, etc.), added at appropriate concentrations. The medium is supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell Boost™ 5, and / or anti-aggregation agents. The medium used may be serum-containing or serum-free. Culture media for mammalian cells are known and include, for example, DMEM (Dulbecco's Modified Eagle's Medium) medium, RPMI 1640, or a mixture of various culture media including, for example, DMEM / F12, or serum-free media such as optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies) or Ex-Cell® 293 (Sigma-Aldrich).

[0101] In the process of using transiently transfected cells, any agent that allows transfection of plasmids can be used.Exemplary agents include calcium phosphate or polyethyleneimine.The conditions (e.g. amount of plasmid, ratio of plasmids to each other, ratio of plasmid to transfection agent, type of medium, etc.) and transfection time are adapted by those skilled in the art according to the characteristics of the transgene that is introduced into the virus produced and / or transfer plasmid.

[0102] According to some examples, the culture medium used has a neutral pH (for example comprised between 7 and 7.4, in particular 7, 7.1, 7.2, 7.3 or 7.4) that is conventionally used in the state of the art for culturing cells and producing viruses. In other examples, the production process used comprises the cultivation of producer cells in a moderately acidic medium. The expression "moderately acidic conditions" refers to a pH of the aqueous solution comprised between 5 and 6.8, for example between 5.5 and 6.5, such as between 5.8 and 6.2. The pH selected will depend on the buffering power of the culture medium used and can be easily determined by the skilled person taking into account general knowledge. The skilled person is capable of modifying the pH of the solution.

[0103] In one example, production of an enveloped virus includes: transient transfection of HEK293T cells or derivatives thereof with one or several plasmids encoding the elements required for production of the enveloped vector, or with stable producer cells that produce the vector constitutively or after induction, such as GPRG or GPRTG; culturing the cells in a suitable medium having a pH of about 6 or about 7; and harvesting the cell culture medium containing the enveloped virus.

[0104] Purification of enveloped viruses The present disclosure provides methods for improving the purity and / or recovery of enveloped viruses from cell culture fluid or filtered cell culture fluid.

[0105] The methods of the present disclosure are applicable to purifying enveloped viruses from small and large scale production, and are particularly useful for their ability to be scaled up for commercial scale pharmaceutical manufacturing.

[0106] In one example, the cells are grown in an attached or fixed bed environment. In one example, the cells are grown in a cell culture chamber such as the CellSTACK® (Corning). In one example, the cells are grown in an attached bioreactor such as the iCELLis® (Pall), scale-X™ or NevoLine™ (Univercells Technologies). The attached cell culture chamber or bioreactor has a volume of about 0.1 m. 2 Larger, about 1m 2 Larger, about 10m 2 Larger, about 30m 2 Larger, about 100m 2 Larger, about 200m 2 Larger, about 500m 2 Greater than or about 600m 2 It is possible to have a larger available growth surface.

[0107] In one example, the cells are grown in a suspension environment. In one example, the cells are grown in a stirred tank bioreactor. In one example, the cells are grown in a Biostat® or Univessel® bioreactor (Sartorius).

[0108] In an embodiment, the volume of the harvested cell culture medium is, for example, about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. For example, the volume of the harvested cell culture medium is about 5 L. In another embodiment, the volume of the harvested cell culture medium is about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the harvested product is about 35 to 150 L. For example, the volume of the harvested product is about 20 L. In one example, the volume of the harvested product is about 50 to 70 L. For example, the harvest volume is about 50L.

[0109] Downstream processes for purifying and concentrating viral vectors from cell culture fluid include harvest filtration steps (also known as "clarification filtration" or "harvest clarification filtration" or "biobium burden reduction") to remove cellular debris and components from the harvest, purification steps such as anion exchange chromatography to reduce the overall volume and separate the viral vector from host cell DNA, proteins and media components, and ultrafiltration / diafiltration steps to concentrate the viral vector in the final formulation buffer. In some cases, downstream processes further include a filter sterilization step to remove microorganisms from the final product.

[0110] Endonuclease treatment Endonuclease treatment of the cell culture fluid or filtered cell culture fluid has been added to downstream processes. Endonuclease treatment can occur during or after harvesting the cell culture fluid, but before anion exchange chromatography. For example, the endonuclease is added directly to the bag or other tube in which the harvested cell culture fluid is collected. In another example, the endonuclease is added to the cell culture fluid after harvesting but before harvest filtration. In another example, the endonuclease is added to the cell culture fluid after harvest filtration but before anion exchange chromatography. In another example, the endonuclease is mixed in-line with the cell culture fluid when loading onto an anion exchange chromatography column, such that the endonuclease contacts the cell culture fluid just before or after it enters the column.

[0111] In embodiments in which the endonuclease is contacted with the cell culture medium prior to loading onto the anion exchange chromatography column, the endonuclease can be incubated with the cell culture medium for up to about 30 hours. For example, the cell culture medium containing the endonuclease can be stored for about 22 hours, about 6 hours, about 4 hours, about 2 hours, or about 1 hour.

[0112] In embodiments in which the endonuclease is contacted with the cell culture fluid during harvest or prior to the harvest filtration step, in some cases, no additional incubation step is required, since incubation is effectively present during the intervening process step prior to loading the cell culture fluid onto the anion exchange chromatography column.

[0113] In one example, the harvested cell culture fluid is filtered after the production of enveloped virus. Before harvest filtration, the cell culture fluid is contacted with an endonuclease. The inventors have confirmed that contacting the cell culture fluid with an endonuclease before harvest filtration reduces clogging of the filter and subsequent purification processes, such as anion exchange chromatography. The inventors have further found that contacting the cell culture fluid with an endonuclease can filter-sterilize the purified enveloped virus without clogging the filter. The inventors have further found that treating the cell culture fluid with an endonuclease does not significantly reduce the infectivity or total RNA yield of the purified enveloped virus.

[0114] In one example, the cell culture fluid is contacted with the endonuclease before harvest filtration, i.e., filtration to remove cells and cell debris. In one example, harvest filtration is performed using membrane filtration. For example, harvest filtration is performed using a 0.8 μm filter and a 0.45 μm filter, which are contained in a single device.

[0115] In one example, the cell culture is contacted with the endonuclease for about 1-4 hours. For example, the cell culture is contacted with the endonuclease for about 1-3 hours. In one example, the cell culture is contacted with the endonuclease for about 1 hour. In some examples, the endonuclease is added to the cell culture medium and harvest filtration is initiated without any additional incubation time.

[0116] Suitable endonucleases will be clear to those skilled in the art based on the disclosure herein.In one example, the endonucleases cleave in a sequence-non-specific manner.For example, the endonucleases cleave DNA (and sometimes RNA) into short oligonucleotides, such as 3-7 bp in length, such as 3-5 bp in length, 2-10 bp in length, for example.

[0117] In one example, the endonuclease is from Serratia marcescens, Anabaena spp., Saccharomyces cerevisiae, Borrelia staurus, Syncephalastrum racemosum, and / or Borrelia burgdorferi.

[0118] In one example, the endonuclease is Serratia nuclease, NucA, Nuc1 and / or endonuclease G.

[0119] The endonuclease is isolated or purified from the sources listed. Alternatively, the endonuclease can be produced recombinantly.

[0120] Endonucleases may be obtained from suitable commercial sources as will be apparent to those of skill in the art and / or as described herein, for example, endonucleases are commercially available from NEW England Biolabs or c-LEcta GmbH.

[0121] In one example, the endonuclease is from Serratia marcescens. Such an endonuclease is also called Golden nuclease. This nuclease is sold under the trade name Benzonase® or Denarase®.

[0122] In one example, the method includes: 2+ Adjust the concentration to a maximum of 10 mM Mg 2+ In one example, the concentration of Mg 2+ The concentration of Mg is adjusted to about 1 to 2 mM. 2+In one example, the concentration of Mg in the cell culture medium is adjusted to 2 mM. 2+ The concentration of is approximately 0.8 mM and is not further adjusted.

[0123] In one example, the method includes adjusting the pH of the cell culture medium to between 6.0 and 10.0. In one example, the method includes adjusting the pH of the cell culture medium to between 8.0 and 9.2.

[0124] In one example, the cell culture medium is at a temperature of 0°C to 42°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of 2°C to 8°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of 4°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of 35°C to 40°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of about 37°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of 18°C ​​to 22°C while in contact with the endonuclease. In one example, the cell culture medium is at a temperature of 20°C while in contact with the endonuclease.

[0125] In one example, the concentration of dithiothreitol (DTT) in the cell culture medium is 0 to 100 mM. In one example, the concentration of 2-mercaptoethanol in the cell culture medium is 0 to 100 mM. In one example, the concentration of monovalent cations, such as Na + or K + In one example, the concentration of monovalent cations in the cell culture medium, such as Na + or K + The concentration of PO4 in the cell culture medium is 0 to 20 mM. 3- The concentration of PO4 in the cell culture medium is 0 to 100 mM. 3- The concentration is 0 to 10 mM.

[0126] In one example, the endonuclease is added to the cell culture medium at a concentration of 0.001 U / mL cell culture medium to 100 U / mL cell culture medium. For example, the endonuclease is added to the cell culture medium at a concentration of 0.01 U / mL cell culture medium to 10 U / mL cell culture medium. For example, the endonuclease is added to the cell culture medium at a concentration of 0.1 U / mL cell culture medium to 1 U / mL cell culture medium. For example, the endonuclease is added to the cell culture medium at a concentration of less than 0.5 U / mL cell culture medium. In one example, the endonuclease is added to the cell culture medium at a concentration of 0.3 U / mL cell culture medium.

[0127] In some cases, the pH of the cell culture medium is not adjusted prior to treatment with the endonuclease.

[0128] In some cases, the endonuclease is diluted before being added to cell culture medium. For example, the endonuclease is diluted in the medium in which the cells are grown. For example, the endonuclease is diluted in DMEM. In one example, the endonuclease is diluted in a buffer or a medium that does not contain fetal bovine serum (FBS). For example, the endonuclease is diluted in a buffer that contains HEPES.

[0129] After treatment with the endonuclease, the cell culture fluid is filtered.

[0130] Anion exchange purification After harvest filtration, the enveloped virus is purified using anion exchange.

[0131] In one example, anion exchange is performed in a bind-elute mode. In this case, enveloped viruses bind to the anion exchanger, while contaminants pass through. The viruses are then eluted from the anion exchanger. By performing anion exchange in this manner, the volume of liquid in which the viruses are suspended is reduced, and contaminants such as host cell DNA, host cell proteins, and medium components such as fetal bovine serum are removed.

[0132] Suitable anion exchangers will be apparent to those skilled in the art. An exemplary anion exchanger is a column containing a resin or membrane or another suitable substrate.

[0133] In one example, the anion exchanger is a membrane anion exchanger.

[0134] In one example, the anion exchanger is a weak anion exchanger, for example, comprising ion exchange groups selected from diethylaminoethyl (DEAE) or aminoethyl groups.

[0135] In another example, the anion exchanger is a strong anion exchanger, for example, containing an ion exchange group selected from quaternary ammonium (Q), diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE) or trimethylaminoethyl groups. Exemplary anion exchangers useful in the methods of the present disclosure include MUSTANG® E, MUSTANG® Q, SARTOBIND® Q, CHROMASORB®, POSSIDYNE®, CAPTO® Q, QSFF, POROS® Q, FRACTOGEL® Q, NATRIX® Q.

[0136] As exemplified herein, the anion exchanger comprises Q ion exchange groups.

[0137] As exemplified herein, the anion exchanger is a membrane anion exchanger that contains Q ion exchange groups. For example, the anion exchanger is MUSTANG® Q.

[0138] As described herein, the inventors determined that the salt concentration in the cell culture medium or filtered cell culture medium was too low for effective anion exchange chromatography. For example, the salt concentration of the harvested cell culture medium identified by the inventors was about 150 mM. The inventors determined that a final salt concentration of 300-500 mM, e.g., about 400 mM, in the cell culture medium or filtered cell culture medium was desirable for loading onto the anion exchange chromatography column. A salt concentration of about 400 mM reduced impurities that bound to the anion exchanger. Increasing the salt concentration required adding (or "spiking") a salt solution to the cell culture medium or filtered cell culture medium to produce a salt-spiked cell culture medium. The inventors wanted to avoid adding too much volume to the cell culture medium or filtered cell culture medium, so using a high concentration salt solution was desirable. However, they also recognized that the addition of salt to the cell culture medium or filtered cell culture medium could cause virus destabilization. Figure 1 shows that harvest stability is decreased under high salt conditions compared to low salt conditions. Immediately after salt addition, the titer is reduced by approximately 20-30%. Without wishing to be bound by theory, the inventors suggest that this initial loss in titer may be caused by the formation of localized regions of high salt upon mixing with the high salt solution.

[0139] This problem is exacerbated by the difficulty of mixing the cell culture medium or filtered cell culture medium with high salt solutions quickly enough to avoid prolonged contact of the virus with high salt concentrations without using more vigorous mixing techniques that would destabilize the virus by exposing it to high shear forces. Uneven or insufficient mixing not only damages the virus, but also impedes the anion exchange process itself by eluting the virus during loading.

[0140] Typical methods of mixing involve manual handling steps that are time consuming on a commercial scale. Achieving sufficient mixing using conventional methods on a commercial scale can add several hours to the process time.

[0141] Our solution to these problems is an in-line process in which the cell culture medium or filtered cell culture medium is contacted with a high concentration salt solution during loading onto an anion exchange chromatography column. This method provides improved mixing without manual handling steps or adding any process time and can be performed in-line, in a continuous or semi-continuous manner, thereby simplifying downstream processing.

[0142] For example, a high concentration salt solution and filtered cell culture medium or cell culture fluid are mixed while loading onto an anion exchange chromatography column to produce a salt-spiked cell culture medium.

[0143] For example, a highly concentrated salt solution is added to an anion exchange chromatography column and cell culture medium or filtered cell culture medium is loaded onto the column.

[0144] In one example, contacting the cell culture medium or filtered cell culture medium with a high concentration salt solution during loading includes flowing two fluid streams together (one containing the cell culture medium or filtered cell culture medium and the other containing the high concentration salt solution) into one fluid stream. By flowing the fluid streams together, they are mixed either when loaded onto the anion exchanger, just before loading onto the anion exchanger, or within the anion exchanger to produce the salt-spiked cell culture medium. Whether performed at a laboratory or commercial scale, the method does not add significantly to process time as it is performed in-line during the loading of the anion exchanger.

[0145] In one example, the salt in the high salt solution is monovalent or divalent. For example, the salt in the high salt solution is monovalent.

[0146] In one example, the salt in the high salt solution is NaCl or KCl. In the illustrated embodiment of the present disclosure, the salt in the high salt solution is NaCl.

[0147] In one example, the salt concentration in the high-concentration salt solution is 1 M to 10 M. For example, the salt concentration in the high-concentration salt solution is 2 M to 8 M. For example, the salt concentration in the high-concentration salt solution is 3 M to 7 M. For example, the salt concentration in the high-concentration salt solution is 5 M.

[0148] In one example, the high salt solution is 5M NaCl.

[0149] In one example, a high salt solution is added to achieve a final salt concentration of 300 mM to 500 mM in the salt-spiked cell culture medium for loading onto an anion exchange chromatography column. For example, a high salt solution is added to achieve a final salt concentration of 400 mM in the salt-spiked cell culture medium.

[0150] In one example, after loading, the anion exchange chromatography column is washed with a wash solution containing a buffer and a salt. For example, the buffer is histidine, HEPES or Tris. For example, the salt is a monovalent salt, such as NaCl.

[0151] In one example, the wash solution comprises 5-50 mM histidine, 150 mM NaCl, pH 5.5-7.4, such as 10 mM histidine buffer, 150 mM NaCl, pH 7. In another example, the wash solution comprises 10-100 mM Tris, 150 mM NaCl, pH 7.0-9.0, such as 50 mM Tris, 150 mM NaCl, pH 8. In another example, the wash solution comprises 5-50 mM HEPES, 150 mM NaCl, pH 6.8-8.2, such as 10 mM HEPES, 150 mM NaCl, pH 7.5.

[0152] In one example, the wash solution comprises 5-50 mM histidine, 750 mM NaCl, pH 5.5-7.4, for example, 10 mM histidine buffer, 750 mM NaCl, pH 7. In another example, the wash solution comprises 10-100 mM Tris, 750 mM NaCl, pH 7.0-9.0, for example, 50 mM Tris, 750 mM NaCl, pH 8. In another example, the wash solution comprises 5-50 mM HEPES, 750 mM NaCl, pH 6.8-8.2, for example, 10 mM HEPES, 750 mM NaCl, pH 7.5.

[0153] In one example, the conductivity of the cleaning solution is between 10 mS / cm and 20 mS / cm.

[0154] In one example, after loading, the anion exchange chromatography column is washed with a first washing solution containing a buffer and a salt and a second washing solution containing a buffer and a salt.For example, the buffer is histidine, HEPES or Tris.For example, the salt is a monovalent salt, such as NaCl.

[0155] In one example, the first and second wash solutions contain the same buffer and the same salt, but the second wash solution contains a higher concentration of salt than the first wash solution.

[0156] In one example, the conductivity of the second cleaning solution is between 60 mS / cm and 75 mS / cm.

[0157] In one example, the first wash solution comprises 5-50 mM histidine, 150 mM NaCl, pH 5.5-7.4, e.g., 10 mM histidine buffer and 150 mM NaCl, pH 7; the second wash solution comprises 5-50 mM histidine, 750 mM NaCl, pH 6.0-8.0, e.g., 10 mM histidine buffer and 750 mM NaCl, pH 7.

[0158] In one example, the first wash solution comprises 10-100 mM Tris, 150 mM NaCl, pH 7.0-9.0, e.g., 50 mM Tris and 150 mM NaCl, pH 8; the second wash solution comprises 10-100 mM Tris, 750 mM NaCl, pH 7.0-9.0, e.g., 50 mM Tris and 750 mM NaCl, pH 8.

[0159] In one example, the first wash solution comprises 5-50 mM HEPES, 150 mM NaCl, pH 6.8-8.2, e.g., 10 mM HEPES, 150 mM NaCl, pH 7.5; the second wash solution comprises 5-50 mM HEPES, 750 mM NaCl, pH 6.8-8.2, e.g., 10 mM HEPES, 750 mM NaCl, pH 7.5.

[0160] After washing, the method can include eluting the enveloped virus. In one example, the virus is eluted with a solution that includes a buffer and a salt. For example, the buffer is histidine, HEPES or Tris. For example, the salt is a monovalent salt, such as NaCl.

[0161] In one example, the first and second wash and elution solutions contain the same buffer and the same salt, but the elution solution contains a higher concentration of salt than the first and second (if used) wash solutions.

[0162] In one example, the elution solution comprises 5-50 mM histidine, 1 M-2 M NaCl, pH 5.5-7.4, such as 10 mM histidine buffer and 1200 or 1500 mM NaCl. In another example, the elution solution comprises 10-100 mM Tris, 1 M-2 M NaCl, pH 7.0-9.0, such as 50 mM Tris and 1200 or 1500 mM NaCl, pH 8. In another example, the elution solution comprises 5-50 mM HEPES, 1 M-2 M NaCl, pH 6.8-8.2, such as 10 mM HEPES, 1200 or 1500 mM NaCl, pH 7.5.

[0163] In one example, the conductivity of the elution solution is 110-130 ms / cm.

[0164] In one example, the pH of the histidine-containing solution is 7.

[0165] In one example, the pH of the Tris-containing solution is 8.

[0166] In one example, the pH of the HEPES-containing solution is 7.5.

[0167] In one example, anion exchange chromatography comprises: (i) loading cell culture medium or filtered cell culture medium and 5M NaCl; (ii) washing with 10 mM histidine and 150 mM NaCl; (iii) washing with 10 mM histidine and 750 mM NaCl; (iv) elution with 10 mM histidine and 1500 mM NaCl; Includes.

[0168] In another example, anion exchange chromatography comprises: (i) loading cell culture medium or filtered cell culture medium and 5M NaCl; (ii) washing with 50 mM Tris and 150 mM NaCl; (iii) washing with 50 mM Tris and 750 mM NaCl; (iv) elution with 50 mM Tris and 1500 mM NaCl; Includes.

[0169] In another example, anion exchange chromatography comprises: (i) loading cell culture medium or filtered cell culture medium and 5M NaCl; (ii) washing with 10 mM HEPES and 150 mM NaCl; (iii) washing with 10 mM HEPES and 750 mM NaCl; (iv) elution with 10 mM HEPES and 1500 mM NaCl; Includes.

[0170] In one example, after elution, the resulting eluate is diluted to reduce the salt concentration by either mixing the eluate in-line with a dilution buffer, or by directly eluting in a dilution buffer, or by eluting and diluting in separate steps. For example, the eluate is diluted with a solution that comprises or consists of a histidine buffer (e.g., containing 10 mM L-histidine) or Tris (e.g., containing 50 mM Tris) or HEPES (e.g., containing 10 mM HEPES). For example, the eluate is diluted with a solution that comprises the same buffer used to elute the virus. For example, if elution is performed with 1500 mM NaCl, the eluate is diluted 1:10 with that solution, or if elution is performed with 1200 mM NaCl, the eluate is diluted 1:8 with that solution. In one example, the eluate is diluted 1:10 with a solution that comprises 10 mM HEPES, pH 7.5.

[0171] Throughout the specification, reference is made to anion exchange chromatography. It should be understood that the methods described herein can also be applied to other ion exchange chromatography. For example, cation exchange chromatography can be used to bind impurities and pass viral vectors. Those skilled in the art can easily modify the methods disclosed herein to suit other ion exchangers as necessary.

[0172] Additional Process In one example, the enveloped virus eluted from the anion exchange column is further purified based on its size. In one example, the buffer in which the virus is eluted from the anion exchange column is exchanged approximately simultaneously. In the process of the present disclosure, tangential flow filtration is preferred. This method allows for almost simultaneous impurity removal and buffer exchange.

[0173] Tangential flow ultrafiltration / diafiltration is a method used to remove residual proteins and nucleic acids and exchange the working buffer into the final formulation buffer. Ultrafiltration using tangential flow is preferred, and different devices can be used [e.g., Proflux and LABSCALE (Ultrafiltration System) TFF systems, both from Millipore, or the KR2i system from Repligen]. The particular ultrafiltration membrane selected will have a filter pore size small enough to retain enveloped viruses, but large enough to allow impurities to pass through. Depending on the manufacturer and type of membrane, a nominal molecular weight cutoff of 100-1000 kDa is appropriate (e.g., UFP-750-E-5A, GE Healthcare; BIOMAX (Ultrafiltration Device) NMWC 1000, Millipore). In one example, the molecular weight cutoff is 500 kDa. The membrane composition can be, but is not limited to, regenerated cellulose, (modified) polyethersulfone, polysulfone. The membrane can be flat plate or hollow fiber type. The main parameters that must be optimized are the flux rate and the transmembrane pressure. In combination with the nominal molecular weight cut-off, these two parameters allow efficient purification and buffer exchange and high virus yields.

[0174] As an additional step, sterile filtration can be performed to eliminate the contaminating microbial load. The diluted eluate or final retentate from the ultrafiltration step is then filtered through a filter (e.g., a 0.22 μm filter). Filters are constructed from a variety of materials, including but not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose esters, cellulose acetate without wetting agents, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES, or any other material consistent with low non-specific influenza virus binding. Filters can have a single membrane layer or one or more layers, or can incorporate a pre-filter of the same or different material, e.g., a 0.45 μm pre-filter. Sterile filtered viruses can be frozen and kept for further manipulation.

[0175] In one example, the sterile filter is at least 15 cm 2 For example, a sterile filter has a filtration area of ​​about 17.8 cm 2 or about 20cm 2 In one example, the sterile filter has a filtration area of ​​at least 200 cm 2 For example, a sterile filter has a filtration area of ​​about 210 cm 2 Or about 220cm 2 The filtration area is .

[0176] In one example, the sterile filter has a flow rate of at least 2.5 mL / cm 2 For example, a sterile filter has a filtering capacity of at least 4.0 mL / cm 2 It has a filtering capacity of

[0177] The invention is further disclosed in the following numbered paragraphs: 1. A method for purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising contacting the cell culture fluid or the filtered cell culture fluid with an endonuclease prior to purifying the virus. 2. The method of paragraph 1, wherein the cell culture medium is harvested from the stable producer cells. 3. The method of paragraphs 1 or 2, wherein the endonuclease is a non-specific endonuclease, which has no sequence specificity and degrades both DNA and RNA. 4. The method of any of paragraphs 1-3, wherein the endonuclease is derived from Serratia marcescens, Anabaena spp., Saccharomyces cerevisiae, Borrelia staurus, Syncephalastrum racemosum, and / or Borrelia burgdorferi. 5. The method of any one of paragraphs 1-4, wherein the endonuclease is Serratia nuclease, NucA, Nuc1, endonuclease G, DNase I, or micrococcal nuclease. 6. The method of any one of paragraphs 1-5, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of 0.001-100 units / mL of the cell culture medium or filtered cell culture medium. 7. The method of paragraph 6, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of 0.01 to 10 units / mL of the cell culture medium or filtered cell culture medium. 8. The method of paragraph 7, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of 0.1 to 1 units / mL of the cell culture medium or filtered cell culture medium. 9. The method of paragraph 8, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.3 units / mL of the cell culture medium or filtered cell culture medium. 10. The method of any of paragraphs 1-9, wherein the purification is performed less than about 30 hours after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, less than about 22 hours after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, less than about 6 hours after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, less than about 4 hours after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, less than about 2 hours after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, less than about 1 hour after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease, or less than about 30 minutes after contacting the cell culture fluid or filtered cell culture fluid with the endonuclease. 11. The method of any of paragraphs 1-10, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.3 units / mL of the cell culture medium or filtered cell culture medium, and purification is performed about 1 to about 2 hours after contacting. 12. The method of any of paragraphs 1-10, wherein the endonuclease is contacted with the cell culture fluid or filtered cell culture fluid at a concentration of about 0.01-0.3 units / mL of the cell culture fluid or filtered cell culture fluid, and purification is performed for more than about 2 hours after contacting. 13. The method of any of paragraphs 1-10, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of about 0.3-10 units / mL of the cell culture medium or filtered cell culture medium, and purification is performed less than about 1 hour after contacting. 14. The method of any of paragraphs 1-10 or 13, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium immediately prior to purification. 15. The method of any one of paragraphs 1-14, comprising performing a harvest filtration on the cell culture fluid prior to contacting the filtered cell culture fluid with the endonuclease to produce a filtered cell culture fluid. 16. The method of any one of paragraphs 1-14, comprising performing a harvest filtration after contacting the cell culture fluid with an endonuclease to produce a filtered cell culture fluid. 17. The method of paragraph 16, wherein the harvest filtration is performed immediately after contacting the cell culture medium with the endonuclease. 18. The method of any one of paragraphs 1-17, wherein purifying comprises subjecting the filtered cell culture fluid to anion exchange chromatography. 19. The method of paragraph 18, wherein the filtered cell culture fluid is subjected to anion exchange chromatography immediately after harvest filtration. 20. The method of paragraph 19, wherein the filtered cell culture fluid is contacted with a high concentration salt solution prior to or during loading onto the anion exchange chromatography column to form a salt-spiked cell culture fluid. 21. A method for purifying an enveloped virus from a filtered cell culture fluid using anion exchange chromatography, comprising contacting the filtered cell culture fluid with a high concentration salt solution prior to or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture fluid. 22. The method of paragraphs 20 or 21, wherein the filtered cell culture fluid is contacted with a high concentration salt solution immediately prior to loading onto the anion exchange chromatography column to form a salt-spiked cell culture fluid. 23. The method of any one of paragraphs 20 or 22, wherein the concentrated salt solution and the filtered cell culture fluid are mixed in-line. 24. The method of any one of paragraphs 20-23, wherein the highly concentrated salt solution comprises monovalent and / or divalent salts. 25. The method of paragraph 24, wherein the monovalent salt is sodium chloride. 26. The method of any one of paragraphs 20-25, wherein the concentrated salt solution has a concentration of at least about 1M. 27. The method of any one of paragraphs 20-26, wherein the filtered cell culture fluid and the high concentration salt solution are mixed in a ratio of about 70-99% (volume / volume) filtered cell culture fluid and about 1-30% (volume / volume) high concentration salt solution. 28. The method of any one of paragraphs 20-27, wherein the concentrated salt solution has a concentration of about 1M. 29. The method of paragraph 28, wherein the filtered cell culture fluid and the high salt solution are mixed in a ratio of about 70% (volume / volume) filtered cell culture fluid and about 30% (volume / volume) high salt solution. 30. The method of any one of paragraphs 20 to 27, wherein the concentrated salt solution has a concentration of 2M. 31. The method of paragraph 30, wherein the filtered cell culture fluid and the high salt solution are mixed in a ratio of about 85% (volume / volume) filtered cell culture fluid and about 15% (volume / volume) high salt solution. 32. The method of any one of paragraphs 20 to 27, wherein the concentrated salt solution has a concentration of 5M. 33. The method of paragraph 32, wherein the filtered cell culture fluid and the high salt solution are mixed in a ratio of about 94% (volume / volume) filtered cell culture fluid and about 6% (volume / volume) high salt solution. 34. The method of any one of paragraphs 20 to 27, wherein the concentrated salt solution has a concentration of 10M. 35. The method of paragraph 34, wherein the filtered cell culture fluid and the high salt solution are mixed in a ratio of about 97% (volume / volume) filtered cell culture fluid and about 3% (volume / volume) high salt solution. 36. The method of any one of paragraphs 20-35, wherein the salt-spiked cell culture medium has a salt concentration of 300-500 mM. 37. The method of paragraph 36, wherein the salt-spiked cell culture medium has a salt concentration of about 400 mM. 38. The method of any one of paragraphs 20-37, wherein the salt-spiked cell culture medium has a target conductivity of 35-45 mS / cm at 25°C. 39. The method of any one of paragraphs 20-38, wherein the cell culture medium spiked with salt has a target conductivity of 36-44 mS / cm at 25°C. 40. The method of paragraphs 38 or 39, wherein the salt-spiked cell culture medium has a target conductivity of 40 mS / cm at 25°C. 41. The method of any one of paragraphs 18-40, further comprising washing the anion exchange chromatography column with one or more washing steps. 42. The method of paragraph 41, comprising a first wash step with a first wash solution comprising: 10-100 mM Tris, 150 mM NaCl, pH 7.0-9.0; 5-50 mM histidine, 150 mM NaCl, pH 5.5-7.4; or 5-50 mM HEPES, 150 mM NaCl, pH 6.8-8.2. 43. The method of paragraph 42, wherein the first wash solution comprises: 50 mM Tris, 150 mM NaCl, pH 8; 10 mM histidine, 150 mM NaCl, pH 7; or 10 mM HEPES, 150 mM NaCl, pH 7.5. 44. The method of any one of paragraphs 41-43, comprising a second wash step with a second wash solution comprising: 10-100 mM Tris, 750 mM NaCl, pH 7.0-9.0; 5-50 mM histidine, 750 mM NaCl, pH 5.5-7.4; or 5-50 mM HEPES, 750 mM NaCl, pH 6.8-8.2. 45. The method of paragraph 44, wherein the second wash solution comprises: 50 mM Tris, 750 mM NaCl, pH 8; 10 mM Tris, 750 mM NaCl, pH 7; or 10 mM HEPES, 750 mM NaCl, pH 7.5. 46. ​​The method of any one of paragraphs 18-45, further comprising eluting the bound virus from the anion exchange chromatography column with an elution solution. 47. The method of paragraph 46, wherein the elution solution comprises: 10-100 mM Tris, 1 M-2 M NaCl, pH 7.0-9.0; 5-50 mM histidine, 1 M-2 M NaCl, pH 5.5-7.4; or 5-50 mM HEPES, 1 M-2 M NaCl, pH 6.8-8.2. 48. The method of paragraph 47, wherein the elution solution comprises: 50 mM Tris, 1.2 M or 1.5 M NaCl, pH 8; 10 mM histidine, 1.2 M or 1.5 M NaCl, pH 7; or 10 mM HEPES, 1.2 or 1.5 M NaCl, pH 7.5. 49. The method of any one of paragraphs 46-48, further comprising diluting the eluted virus with histidine, Tris, or HEPES. 50. The method of any one of paragraphs 46-49, further comprising incubating the eluted virus or diluted eluted virus at room temperature for up to 15 minutes or at 2-8°C for up to 60 minutes. 51. The method of any one of paragraphs 46 to 50, further comprising concentrating and / or diafiltering the eluted virus or the diluted eluted virus. 52. The method of any one of paragraphs 18-51, wherein the anion exchange chromatography column is an anion exchange membrane adsorber. 53. The method of any one of paragraphs 1 to 52, increasing the viral infectious titer yield by at least 10%. 54. The method of any one of paragraphs 1 to 53, wherein the enveloped virus is a retrovirus. 55. The method of paragraph 54, wherein the retrovirus is a lentivirus. 56. A method for purifying an enveloped virus from a cell culture medium comprising: (i) providing a cell culture medium containing a viral vector produced from a stable producer cell line; (ii) contacting the cell culture with a recombinantly expressed Serratia endonuclease; (iii) contacting the endonuclease-treated cell culture medium with a filter to produce a filtered cell culture medium; (iv) loading the filtered cell culture fluid and a high salt solution containing 5 M sodium chloride onto an anion exchange chromatography membrane, such that the fluid and salt solution are loaded in a ratio of 94% (volume / volume) filtered cell culture fluid and 6% (volume / volume) high salt solution; (v) washing the membrane with one or more wash buffers; (vi) eluting the bound virus from the membrane with an elution buffer containing 1.2 M or 1.5 M sodium chloride; (vii) diluting the eluted virus with a buffer; (viii) concentrating and diafiltering the eluted virus; The method includes: 57. The method of any one of paragraphs 1-56, wherein the cell culture medium and / or the filtered cell culture medium has a volume of greater than about 1 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, or about 1000 L. 58. The method of any one of paragraphs 1 to 57, wherein the cell culture medium and / or the filtered cell culture medium has a volume of about 5 L. 59. The method of any one of paragraphs 1-57, wherein the cell culture medium and / or the filtered cell culture medium has a volume of about 20 L. 60. The method of any one of paragraphs 1-57, wherein the cell culture medium and / or the filtered cell culture medium has a volume of about 44 L. 61. The method of any one of paragraphs 1-57, wherein the cell culture medium and / or the filtered cell culture medium has a volume of about 60 L. 62. The method of any one of paragraphs 1-57, wherein the cell culture medium and / or the filtered cell culture medium has a volume of about 200 L. 63. The method of any one of paragraphs 18 to 62, wherein the anion exchange chromatography membrane has a capacity of at least about 1 mL per L of cell culture medium and / or filtered cell culture medium. 64. The method of any one of paragraphs 1-63, further comprising formulating the enveloped virus into a pharmaceutical preparation or solution suitable for infecting a cell. 65. Magnesium in cell culture medium or filtered cell culture medium 2+ 65. The method of any one of paragraphs 1-64, further comprising adjusting the concentration of to about 1-2 mM. 66. Magnesium in cell culture medium or filtered cell culture medium 2+ 66. The method of paragraph 65, further comprising adjusting the concentration of to about 2 mM. 67. The method of any one of paragraphs 1-64, further comprising adjusting the pH of the cell culture medium or filtered cell culture medium to between 6.0 and 10.0. 68. The method of paragraph 67, further comprising adjusting the pH of the cell culture medium or filtered cell culture medium to 8.0 to 9.2. 69. The method of any one of paragraphs 1 to 64, wherein the cell culture medium or filtered cell culture medium is at a temperature between 0°C and 42°C while in contact with the endonuclease. 70. The method of paragraph 69, wherein the cell culture medium or filtered cell culture medium is at a temperature of 2°C to 8°C while in contact with the endonuclease. 71. The method of paragraph 70, wherein the cell culture medium or filtered cell culture medium is at a temperature of 4° C. while in contact with the endonuclease. 72. The method of any one of paragraphs 1 to 71, wherein the cell culture medium is harvested from cells cultured in an adherent environment. 73. The method of any one of paragraphs 1 to 71, wherein the cell culture medium is harvested from cells cultured in a suspension environment. 74. A purified enveloped virus produced by a method according to any one of paragraphs 1 to 73.

[0178] The present disclosure is further described in the following non-limiting examples. EXAMPLES

[0179] method Cell culture and lentivirus production Cells were grown in "iCELLis Nano" bioreactors. After 4 days of successful cell growth, induction of virus production was started by changing the medium. After discarding the initial harvest volume, daily harvests were started for up to 10 days. Harvesting was done at 4°C.

[0180] Harvesting, clarification, filtration After transferring the harvest to the downstream department, a clarification filtration was performed using a Sartorius Sartopore 2 filter containing two membranes of 0.8 and 0.45 μm, respectively. The main objective of this step is to remove cells and cellular components / debris without affecting the function of the lentivirus or compromising its infectivity.

[0181] To study the effect of endonuclease treatment on the efficiency of downstream processing, Benzonase® was added to the harvest. Benzonase cleaves DNA into small fragments of 3-5 base pairs. Benzonase was added at 1 mL per litre of harvest prior to clarification filtration. Addition of endonuclease at this stage meant that the process time of the clarification filtration step was used for incubation and no additional process time had to be added.

[0182] Benzonase working solution was prepared by diluting the stock solution 1:1000 in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). 1 mL of Benzonase working solution was added per liter of harvest prior to the clarification filtration step.

[0183] The collection bag and the filter unit were connected using a tube with an inner diameter of 8–10 mm. Before starting the actual filtration process, the membrane was filled with a volume of 0.5–0.7 mL / cm. 2 The filter area was equilibrated by a washing step using equilibration buffer and then drained. Equilibration and subsequent filtration were performed at a flow rate of approximately 150 mL / min. The filtered harvest was then stored at +4° C. for up to 30 hours or directly processed and stored at room temperature for less than 1 hour.

[0184] Purification process using Mustang Q anion exchange membrane After filtration, the virus was captured using anion exchange chromatography. The role of this capture step is to reduce the volume and to remove process related contaminants such as host cell DNA, host cell proteins and media components like FBS. The chromatography step was carried out using an Akta Pure 150 system using a Mustang Q anion exchange membrane. Table 1 shows the buffers used during anion exchange purification.

[0185] [Table 1]

[0186] Prior to product application, the membrane was equilibrated with 5 MV of equilibration buffer at a flow rate of 10 MV / min. The filtered harvest was spiked (in-line) with 5 M NaCl solution using the built-in mixer of the Akta chromatography system to achieve a target conductivity of 40 mS / cm to reduce non-specific binding of cell culture medium components. Separately, anion exchange chromatography was performed without NaCl spiking. The harvest was then applied to the membrane at a flow rate of 10 MV / min. Membrane washing was performed with 20 MV of washing buffer at a flow rate of 10 MV / min to remove impurities such as host cell DNA. Lentivirus elution was performed with 11 MV of high-concentrated salt buffer at a flow rate of 2 MV / min. Eluate collection started after 1 MV and ended after 6 MV. The remaining elution volume was discarded. As lentivirus is unstable in highly concentrated salt buffers, the eluate was directly diluted 1:10 with cold (+4° C.) dilution buffer of either 10 mM L-histidine or 50 mM Tris. Elution directly into the dilution buffer reduces the time that the virus is in high salt concentration.

[0187] The collected and diluted Mustang Q eluate was in a volume of 500 mL and stored on ice with a maximum hold time of 30 min if a TFF step was performed as the immediate next step.

[0188] Concentration and diafiltration steps Tangential flow filtration (TFF) allows the diluted Mustang Q eluate to be concentrated and diafiltered into the final formulation buffer X-VIVO 10. This step was carried out on a Repligen KR2i system.

[0189] TFF also allowed for solution exchange of virus into X-VIVO 10 cell culture medium, ensuring direct addition of virus to target cells without diluting the growth medium.

[0190] Filter area 390cm 2 and Repligen Hollow Fiber PS membrane with a 500 kDa cutoff were used.

[0191] Equilibrate the membrane using TFF equilibration buffer at 2 mL / cm. 2 A run was performed at 1000 rpm. To concentrate the product, 500 mL of diluted Mustang Q eluate in the feed reservoir was connected to the backing pump. The backing pump was started at a flow rate of 20 mL / min to move the feed to the reservoir. The flow rate was adjusted so that the volume in the reservoir remained constant during the concentration step. For this, the KR2i pump flow rate was set to 50 mL / min, TMP 0.5 bar (upper limit 0.7 bar) and the back pressure valve was opened.

[0192] The concentration target was 25-30 times, and the ultrafiltration step was stopped when a retentate volume of 16-20 mL, including the hold-up volume, was reached.

[0193] The back pressure valve and filtrate line were opened to collect the product from the retentate side of the membrane. The flow rate of the main pump was set to 4 mL / min in reverse to collect the hold-up recirculation volume. After about 1 minute, the retentate line connecting the back pressure valve and the reservoir was shut off and supplied with air. This step allows for the removal of viruses that are stuck to the membrane by applying reverse low TMP to the membrane.

[0194] The objective of the TFF process was to achieve a 300-1000-fold concentration of the TFF retentate relative to the starting material (harvest). The TFF retentate was used to develop a sterilization filtration process.

[0195] Sterilization Filtration Research The sterile filtration step was performed using a Repligen KR2i system.

[0196] analysis Infectious titer assay In this assay, D1B and / or HEK293T cells were transduced with samples containing vectors. After a growth period of several days, the cells were stained with an antibody against human gamma globin to determine the infectious titer of the sample, measured in transducing units (TU) / mL.

[0197] ddPCR RNA content This assay uses digital droplet PCR to quantify RNA copy numbers in a sample. This assay measures the total number of RNA copies. Primers and probes were selected to ensure that primarily full-length RNA copies are counted.

[0198] p24 ELISA This ELISA assay measures the concentration of the viral capsid protein p24. An additional pull-down step has been established so that only p24 bound to the virus is detected, not free p24. From the ng / mL readout, an estimate of viral particles can be calculated. The assay also detects empty capsids or capsids with incomplete cargo RNA.

[0199] Total DNA This fluorescence-based assay detects total DNA within a sample. EXAMPLES

[0200] result Addition of benzonase improves lentiviral purification An initial preliminary study was performed to determine whether filtering the TFF retentate was in principle possible. Two solutions of untreated and benzonase-treated TFF retentate were filtered through Mini Kleenpak EKV filters. The results are shown in Figure 2. As shown, benzonase treatment increased the filterability by approximately 2.5-fold without affecting the infection yield (101.6% vs. 95.0%). In addition, the RNA yield for the benzonase-treated retentate was significantly higher at 89.5% when compared to 69.1% for the untreated retentate.

[0201] Benzonase treatment was clearly beneficial to filtration success and was investigated further.

[0202] Following the encouraging results of treating the TFF retentate with Benzonase, trials were conducted in which the harvest was treated with Benzonase. Treating the harvest with Benzonase allows for subsequent purification steps, such as anion exchange to remove the endonuclease. To determine the success of harvest treatment with Benzonase and to investigate the effect on the sterilization filtration process, the results of this treatment were compared to those of a preliminary study and are shown in Table 2.

[0203] [Table 2]

[0204] The infectivity and RNA yield of filter-sterilized benzonase-treated harvest was comparable to that of the benzonase-treated TFF retentate. However, filterability was reduced by 24% using benzonase treatment of the harvest. Despite this, all subsequent experiments used the TFF retentate resulting after benzonase treatment of the harvest.

[0205] As shown in Figure 3, the addition of Benzonase to the harvest also had a positive impact on anion exchange chromatography. Notably, without the addition of Benzonase, the Mustang Q filter fouled or clogged, causing an increase in pressure. This increase in pressure in turn caused slower flow rates and increased process times (50%-150% longer). Higher pressures also increased the risk of filter or fitting failure, which may result in the need to reprocess or discard the batch.

[0206] Thus, the addition of benzonase to the harvest improves the performance of both Mustang Q filters and sterile filtration.

[0207] Improving recovery from anion exchange chromatography Figure 4 is a graphical representation summarizing the yields of infectious titer (dark grey bars) and RNA content (light grey bars) for harvests from planar (left side of figure) and attached (right side of figure) bioreactors. The first column shows the yields from storage and filtration, the second column shows the yields from the anion exchange purification step, and the third column shows the yields from the TFF step. The bottom column shows the overall yields. As shown in Figure 4, the greatest virus loss during downstream purification occurred during the anion exchange purification, with recoveries between 43% and 63% observed. Therefore, any improvement in recovery from this step will substantially increase virus recovery.

[0208] Several strategies were tested to determine how to improve the recovery from the anion exchange chromatography step. One of these strategies was to increase the salt concentration in the harvest. Increasing the salt concentration to about 400 mM increased the binding of the virus to the anion exchanger while decreasing the binding of contaminants such as host cell DNA, host cell proteins, and media components such as fetal bovine serum. However, adding large volumes to the harvest is undesirable due to the difficulty of mixing, as well as adding processing time and cost. On the other hand, exposing the virus to high concentrations of salt (as would occur if salt were added directly to the harvest) would destabilize the virus, resulting in reduced recovery. To solve this problem, we spiked a 5M NaCl solution directly onto the anion exchange column itself rather than premixing it with the virus. This reduced the contact time between the virus and the high salt solution to seconds instead of minutes.

[0209] As shown in FIG. 5, the process developed by the inventors provided approximately a 10% improvement compared to the process without the NaCl spike. EXAMPLES

[0210] Large-scale sterilization To evaluate the scale-up of the sterilization process, Supor EKV (Pall, polypropylene; 20 cm 2 ), Sartopore Pt(Sartorius;220cm 2 , 220cm 2 ), OptiScale Durapore (Merck, Hydrophilic PVDF; 17.8cm 2 ) and Sartopore Pt (Sartorius, mPES; 210 cm 2 Several different filters were evaluated, including

[0211] As shown in Table 3, the Supor EKV 20cm 2Sterile filtration demonstrated infectious titer yields of >80% and RNA yields of approximately 80%. In comparison, OptiScale Durapore and Sartopore PT demonstrated infectious titer yields of approximately 60%. Further studies have demonstrated that 20 cm 2 The filter demonstrated that it was too small and the filtration pressure increased rapidly.

[0212] [Table 3]

[0213] Super EKV 20cm 2 To solve the filter filtration pressure problem, we used a larger Sartopore Pt 220cm 2 A filter was used. The loading capacity was 0.46 mL / cm 2 and 220cm 2 The filter capacity was not reached. 220cm 2 Sterilization by filtration using a filter resulted in an average infectious titer yield of 75.4% and RNA yield of 70.0% across the four sublots tested.

[0214] 220cm 2 Scale-up of the filter sterilization process was achieved with robust infection yields of approximately 80% in multiple runs, and all sublots passed safety testing with no detectable microbiological growth. EXAMPLES

[0215] Purification of 5L suspension harvest Cell culture and lentivirus production were carried out as described in Example 1, but the culture was carried out in a 5 L suspension bioreactor. Cells were grown in chemically defined medium without fetal bovine serum (FBS).

[0216] Clarification filtration of the harvest was performed as described in Example 1, except that the benzonase working solution was prepared by diluting the stock solution 1:1000 into equilibration buffer (i.e., 1 μL of benzonase per mL of equilibration buffer) rather than the FBS-containing medium described in Example 1. For each liter of harvest, 1 mL of diluted benzonase solution was added to each liter of harvest prior to the clarification filtration step to achieve a target concentration of 0.3 U / mL. In addition, 10 mL of 200 mM MgCl2 was added to achieve a target concentration of 2 mM MgCl2. 2+ The process parameters used for the 5 L suspension run are detailed in Table 4.

[0217] [Table 4]

[0218] Four bioreactors were run in parallel and harvests were collected and analyzed on days 5 and 12. Benzonase treatment of the harvests did not affect infectious titer yields, with an infectious yield of 86.1% achieved in benzonase-treated filtered harvests across seven harvests.

[0219] After filtration, the virus was captured using anion exchange chromatography with HEPES buffer similar to the method described in Example 1 above. After benzonase addition, the harvest was incubated for 50-55 minutes before loading onto the anion exchange chromatography membrane. The process parameters used for the chromatographic purification of the 5 L suspension run are detailed in Table 5. The membrane size used was determined based on a maximum of 1000 mL of harvest per mL of membrane volume. Thus, for harvests up to about 860 mL, use 0.86 mL Mustang Q membrane; for harvests from about 860 mL to about 1.72 L, use 1.72 mL Mustang Q membrane (or 2 x 0.86 mL Mustang Q membrane); For about 1.72 L to about 5 L of harvested material, 5 mL Mustang Q membrane was used; for about 5 L to about 10 L of harvested material, 10 mL Mustang Q membrane (or 2 x 5 mL Mustang Q membranes) was used; for about 10 L to about 20 L of harvested material, 2 x 10 mL Mustang Q membrane was used; for about 20 L to about 60 L of harvested material, 60 mL Mustang Q membrane was used; for about 60 L to about 120 L of harvested material, 140 mL Mustang Q membrane (or 2 x 60 mL Mustang Q membranes) was used; for about 120 L to about 140 L of harvested material, 140 mL Mustang Q membrane was used.

[0220] [Table 5]

[0221] The collected and diluted Mustang Q eluate was applied to the TFF step as described in Example 1, and the diluted Mustang Q eluate was concentrated and diafiltered into the final formulation buffer X-VIVO 10. The process parameters used for the TFF step are listed in Table 6.

[0222] [Table 6]

[0223] The infectious titer yield of benzonase treated TFF retentate across all seven harvests was 46.4%. The results obtained by us in the 5L suspension run were comparable to those achieved in Examples 1 and 2 described above and to the infectious titer yields in Figure 5. EXAMPLES

[0224] Large-scale purification of harvests from attached bioreactors Cell culture and lentivirus production were carried out as described in Example 1, except that the culture was carried out in a scale-X™ carbo bioreactor (Univercells Technologies). 22 L of harvest was collected daily for 8 days. Harvest clarification filtration was carried out (every 2 days) on 44 L sublots using the process described in Example 1, using the process parameters detailed in Table 7.

[0225] [Table 7]

[0226] Anion exchange chromatographic purification was also performed using HEPES buffer spiked in-line with 6% 5M NaCl as described in Example 1 to achieve a target conductivity of 40 mS / cm. The process parameters for the chromatographic purification step are provided in Table 8. As described in Example 4, the membrane size used was determined based on a maximum of 1000 mL of harvest per mL of membrane volume, therefore, 60 mL Mustang Q membranes were used for the 44 L sublot.

[0227] [Table 8]

[0228] The collected and diluted Mustang Q eluate was applied to the TFF step as described in Example 1, and the diluted Mustang Q eluate was concentrated and diafiltered into the final formulation buffer X-VIVO 10. The process parameters used for the TFF step are listed in Table 9.

[0229] [Table 9]

[0230] FIG. 6 shows the infectious titer yield at each process step of the processed 44 L sublots, with 96% infectious titer yield in the filtered harvest, 50% infectious titer yield in the Mustang Q eluate, 94% infectious titer yield in the TFF retentate, and 103% in the sterile filtered sublot (N=4 sublots for each process step).

Claims

1. A method for purifying enveloped viruses from cell culture medium or filtered cell culture medium, comprising contacting the cell culture medium or filtered cell culture medium with an endonuclease before purifying the virus.

2. The method according to claim 1, wherein the endonuclease is contacted with the cell culture medium or filtered cell culture medium at a concentration of 0.001 to 100 units / mL.

3. The method according to claim 1, wherein the purification is carried out for less than approximately 30 hours after contacting the cell culture medium or filtered cell culture medium with an endonuclease, less than approximately 22 hours after contacting the cell culture medium or filtered cell culture medium with an endonuclease, less than approximately 6 hours after contacting the cell culture medium or filtered cell culture medium with an endonuclease, less than approximately 4 hours after contacting the cell culture medium or filtered cell culture medium with an endonuclease, less than approximately 2 hours after contacting the cell culture medium or filtered cell culture medium with an endonuclease, less than approximately 1 hour after contacting the cell culture medium or filtered cell culture medium with an endonuclease, or less than approximately 30 minutes after contacting the cell culture medium or filtered cell culture medium with an endonuclease.

4. The method according to claim 1, comprising contacting the cell culture medium with an endonuclease and then performing collection and filtration to prepare a filtered cell culture medium.

5. The method according to claim 4, wherein the collection and filtration is performed immediately after contacting the cell culture medium with endonuclease.

6. The method according to claim 1, wherein the purification comprises subjecting the filtered cell culture medium to anion exchange chromatography.

7. The method according to claim 6, wherein the filtered cell culture medium is subjected to anion exchange chromatography immediately after collection and filtration.

8. The method according to claim 7, wherein the filtered cell culture medium is brought into contact with a high-concentration salt solution before or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture medium.

9. A method for purifying enveloped viruses from a cell culture medium filtered using anion exchange chromatography, wherein the filtered cell culture medium is brought into contact with a high-concentration salt solution before or during loading onto an anion exchange chromatography column to form a salt-spiked cell culture medium.

10. The method according to claim 9, wherein the filtered cell culture medium is brought into contact with a high-concentration salt solution immediately before being loaded onto an anion exchange chromatography column to form a salt-spiked cell culture medium.

11. The method according to claim 10, wherein a high-concentration salt solution and a filtered cell culture medium are mixed in line.

12. The method of claim 9, wherein the cell culture medium spiked with salt has a salt concentration of 300 to 500 mM.

13. The method according to claim 9, wherein the cell culture medium spiked with salt has a target conductivity of 35 to 45 mS / cm at 25°C.

14. The method according to claim 6, further comprising washing an anion exchange chromatography column by one or more washing steps.

15. The method according to claim 6, further comprising eluting the bound virus from an anion exchange chromatography column with an elution solution.

16. The method according to claim 15, wherein the elution solution comprises: 10-100 mM Tris, 1 M-2 M NaCl, pH 7.0-9.0; 5-50 mM histidine, 1 M-2 M NaCl, pH 5.5-7.4; or 5-50 mM HEPES, 1 M-2 M NaCl, pH 6.8-8.

2.

17. The method according to claim 15, further comprising diluting the eluted virus with histidine, Tris, or HEPES.

18. The method according to claim 15, further comprising concentrating and / or diafiltration of the eluted virus or diluted eluted virus.

19. A method for purifying enveloped viruses from cell culture medium: (i) Prepare a cell culture medium containing a viral vector produced from a stable production cell line; (ii) Contacting the cell culture medium with Serratia endonuclease expressed by recombinant means; (iii) Preparing a filtered cell culture medium by bringing a cell culture medium treated with endonuclease into contact with a filter; (iv) Load the filtered cell culture medium and a high-concentration salt solution containing 5M sodium chloride onto an anion exchange chromatography membrane, so that the liquid and salt solution are loaded in a ratio of 94% (volume / volume) of filtered cell culture medium and 6% (volume / volume) of high-concentration salt solution. ； (v) Washing the membrane with one or more wash buffers; (vi) Eluting the virus bound to the membrane with an elution buffer containing 1.2 M or 1.5 M sodium chloride; (vii) Diluting the eluted virus with a buffer solution; (viiii) Concentrating and dialysis-filtration the eluted virus and A method that includes this.

20. A purified enveloped virus produced by the method described in claim 1.

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