Methods of purifying an enveloped virus

Abstract

The present disclosure relates generally to the manufacturing of gene therapy products, and specifically to methods of purifying an enveloped virus from a cell culture fluid, comprising a filtration medium.

Description

[0001] METHODS OF PURIFYING AN ENVELOPED VIRUS

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from United States patent Application No. 63 / 670,242 filed 12 July 2024 entitled “Methods of purifying an enveloped virus”. The entire contents of this application are hereby incorporated by reference.

[0004] FIELD

[0005] The present disclosure relates generally to the manufacturing of gene therapy products, and specifically to methods of purifying an enveloped virus from a cell culture fluid, comprising a filtration medium.

[0006] BACKGROUND

[0007] Retroviruses, e.g., lentiviruses are one of the most studied viral vectors for gene therapy. Retroviruses in general are RNA-based viruses which integrate their genetic information into the target cell chromosomes permanently. The advantages of retroviruses include long-term transgene expression in target cells, a low immunogenic potential, and the ability to transduce into dividing and non-dividing cells.

[0008] Lentiviruses are genetically engineered and usually based on human immunodeficiency virus 1 (HIV-1). To increase safety, modern vectors contain only those HIV genes which are necessary for infection and gene delivery, but the genes necessary for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is exchanged with that of another virus to allow infection of a wide range of target cells, e.g., VSV-G protein from Vesicular stomatitis Indiana virus NSV .

[0009] To produce lentiviruses, cells such as human embryonic kidney cells HEK 293T are transfected with 3 or more plasmids. These include the transfer plasmid with the gene of interest and several packaging plasmids encoding, vesicular stomatitis G protein (VSV-G), and essential viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of the plasmids with inducible promoters, in which lentivirus production can be induced.

[0010] Once the virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells.

[0011] The lentivirus is harvested from the producer cells and subsequently purified and concentrated in the downstream process. However, purification of lentiviruses at commercial scale is difficult. Limiting obstacles for the purification of this type of virus are the impurities that are produced with large scale cell culture and the instability of certain membrane glycoproteins when exposed to some purification conditions. Thus, there is a need in the art for an efficient process for purifying lentiviruses, e.g., for gene therapy.

[0012] SUMMARY

[0013] In work leading up to the present invention, the inventors sought to produce a method for producing enveloped viruses, e.g., for gene therapy, at commercial scale and suitable for regulatory requirements.

[0014] In some examples, the downstream process for purifying and concentrating viral vector produced by the inventors includes a harvest filtration step to remove cellular debris and components and a purification step to reduce overall volume and to separate viral vector from host cell DNA, proteins, and media components. The downstream process for purifying and concentrating viral vector produced by the inventors can additionally include an ultrafiltration / diafiltration step to concentrate the viral vector into a final formulation buffer. In some examples, the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.

[0015] In developing this method, the inventors identified that the purification step was the step with the largest losses in virus yield. In addition, existing purification methods include capture chromatography columns which require high salt content, and increasing the salt concentration is complicated by several factors, including the salt is harmful to virus and the high-salt elution must then be diluted. This results in larger volume, creating an additional burden on downstream processing.

[0016] To address this problem, the inventors used a charged depth filtration purification step. The inventors identified that they could use an anionic charged depth filtration medium as the purification step, which would improve purification as well as other downstream steps such as sterile filtration. In particular, the inventors showed that purification with a charged depth filtration medium comprising a non-woven substrate and a plurality of quaternary ammonium monomers resulted in increased viral yields and improved purity compared to other media. In particular, the inventors showed that virus could bind to and be effectively eluted from a charged depth filtration medium.

[0017] Accordingly, the findings by the inventors provide a method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a charged depth filtration medium. In one example, the method further comprises eluting the enveloped virus from the charged depth filtration medium. For example, the enveloped virus is bound to the charged depth filtration medium and subsequently eluted from the medium (e.g., bind- and-elute format).

[0018] In one example, the filtration medium is an anionic charged depth filtration medium.

[0019] In one example, the filtration medium comprises a non-woven substrate and a plurality of quaternary ammonium monomers.

[0020] The present disclosure also provides a method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a filtration medium comprising a nonwoven substrate and a plurality of quaternary ammonium monomers.

[0021] In one example, the plurality of quaternary ammonium monomers is grafted to the surface of the non-woven substrate as a co-polymer comprising interpolymerized monomer units of the quaternary ammonium monomers, thereby producing a co-polymer grafted non-woven substrate.

[0022] In one example, the grafted co-polymer further comprises an amide monomer and / or an oxy monomer. For example, the grafted co-polymer further comprises an amide monomer. In another example, the grafted co-polymer further comprises an oxy monomer.

[0023] In one example, the grafted copolymer comprises interpolymerized monomer units of: a) 10 to 50 parts by weight of the quaternary ammonium monomer; b) 10 to 80 parts by weight of the amide monomer; and c) 10 to 40 parts by weight of the oxy monomer; wherein the sum of a) to c) is 100 parts by weight.

[0024] In one example, the grafted copolymer comprises interpolymerized monomer units of: a) 20 to 40 parts by weight of the quaternary ammonium monomer; b) 30 to 60 parts by weight of the amide monomer; and c) 15 to 35 parts by weight of the oxy monomer; wherein the sum of a) to c) is 100 parts by weight.

[0025] In one example, the quaternary ammonium monomers comprise methacrylamidopropyltrimethylammonium chloride (MAPTAC).

[0026] In one example, the amide monomer is N-vinyl pyrrolidone (NVP) and / or wherein the oxy monomer is glycidyl methacrylate (GMA). For example, the amide monomer is N-vinyl pyrrolidone (NVP). In another example, the oxy monomer is glycidyl methacrylate (GMA).

[0027] In one example, the grafted copolymer comprises interpolymerized monomer units of: a) 10 to 50 parts by weight of MAPT AC; b) 10 to 80 parts by weight of NVP; and c) 10 to 40 parts by weight of GMA; wherein the sum of a) to c) is 100 parts by weight.

[0028] In one example, the grafted copolymer comprises interpolymerized monomer units of: a) 20 to 40 parts by weight of MAPT AC; b) 30 to 60 parts by weight of NVP; and c) 15 to 35 parts by weight of GMA; wherein the sum of a) to c) is 100 parts by weight.

[0029] In one example, the grafted co-polymer comprises 10 to 50 parts by weight of the quaternary ammonium monomer relative to 100 parts by weight total grafting monomers. For example, the grafted co-polymer comprises 10 to 50 parts by weight of MAPTAC relative to 100 parts by weight total grafting monomers. In one example, the grafted copolymer comprises 20 to 40 parts by weight of the quaternary ammonium monomer relative to 100 parts by weight total grafting monomers. For example, the grafted copolymer comprises 20 to 40 parts by weight of MAPTAC relative to 100 parts by weight total grafting monomers.

[0030] In one example, the grafted co-polymer comprises 10 to 80 parts by weight of the amide monomer relative to 100 parts by weight total grafting monomers. For example, the grafted co-polymer comprises 10 to 80 parts by weight of NVP relative to 100 parts by weight total grafting monomers. In one example, the grafted co-polymer comprises 30 to 60 parts by weight of the amide monomer relative to 100 parts by weight total grafting monomers. For example, the grafted co-polymer comprises 30 to 60 parts by weight of NVP relative to 100 parts by weight total grafting monomers.

[0031] In one example, the grafted co-polymer comprises 10 to 40 parts by weight of the oxy monomer relative to 100 parts by weight total grafting monomers. For example, the grafted co-polymer comprises 10 to 40 parts by weight of GMA relative to 100 parts by weight total grafting monomers. In one example, the grafted co-polymer comprises 15 to 35 parts by weight of the oxy monomer relative to 100 parts by weight total grafting monomers. For example, the grafted co-polymer comprises 15 to 35 parts by weight of GMA relative to 100 parts by weight total grafting monomers. In one example, the non-woven substrate comprises a thermoplastic polymeric material. In one example, the thermoplastic polymeric material is a hydrophobic thermoplastic polyolefin. For example, the polyolefin is poly(propylene).

[0032] In one example, the non-woven substrate has one or more of the following properties: a) a tensile strength of at least 4.0 newtons prior to grafting of the co-polymer; b) a surface area of 15 to 50 m2per square meter of nonwoven substrate; c) a mean pore size of 1-40 microns; d) a solidity of less than 20%; and e) an effective fiber diameter of about 3 to about 10 mm.

[0033] In one example, the non-woven substrate has a tensile strength of at least 4.0 newtons prior to grafting of the co-polymer.

[0034] In one example, the non-woven substrate has a surface area of 15 to 50 m2per square meter of nonwoven substrate.

[0035] In one example, the non-woven substrate has a mean pore size of 1-40 microns. For example, the non-woven substrate has a mean pore size of 2 to 20 microns. Methods of determining mean pore size will be apparent to the skilled person and / or described herein.

[0036] In one example, the non-woven substrate has a solidity of less than 20%.

[0037] In one example, the non-woven substrate has an effective fiber diameter of about 3 to about 10 mm. For example, the non-woven substrate has an effective fiber diameter of about 4 to 10 mm, or about 4 to 6 mm.

[0038] In one example, the non-woven substrate has a basis weight in the range of about 10 to 400 g / m2For example, the non-woven substrate has a basis weight of about 60 to 150 g / m2.

[0039] In one example, the non-woven substrate has an average thickness of about 0.1 to 10 mm. For example, the average thickness of the non-woven substrate is about 0.25 to 5 mm.

[0040] It will be apparent to the skilled person that the above recited properties of the non-woven substrate relate to the non-functionalized (i.e., non-grafted) non-woven substrate.

[0041] In one example, the grafted co-polymer is 0.5 to 3 times the weight of the nonwoven substrate. For example, the grafted co-polymer is 1 to 3 times the weight of the non-woven substrate.

[0042] In one example, the filtration medium comprises at least four layers of the copolymer grafted non-woven substrate. In one example, the filtration membrane comprises at least four layers of the co-polymer grafted non-woven substrate, wherein each layer may be the same or different. For example, each of which layers may have the same, or different average fiber size, void volume, degree of polymer grafting, monomer composition of grafted polymer, porosity, tensile strength and / or surface area.

[0043] In one example, each downstream layer of the co-polymer grafted non-woven substrate has a smaller effective fiber diameter. It will be apparent to the skilled person that each subsequent layer may have a smaller effective pore size or smaller average fiber size so that finer contaminants may be filtered.

[0044] In one example, the filtration media further comprises a non-functionalized substrate downstream of the non-woven substrate.

[0045] In one example, the non-functionalized substrate comprises a microporous, nonfunctionalized size-exclusion membrane.

[0046] In one example, the non-functionalized size-exclusion membrane comprises an asymmetric pore structure.

[0047] In one example, the non-functionalized size exclusion membrane comprises a gradient or a multizone pore morophology, wherein the pore size decreases from the upstream surface toward the downstream surface.

[0048] In one example, the non-functionalized size-exclusion membrane comprises a 0.2-micron rated, asymmetric, non- functionalized polyamide size-exclusion membrane.

[0049] In one example, the filtration medium is configured in a filter unit as a planar or lenticular disk.

[0050] In one example, the filtration medium is pleated.

[0051] In one example, the filtration medium has a volume of at least about 1 mL per L of the cell culture fluid and / or the filtered cell culture fluid.

[0052] In one example, the cell culture fluid is harvested from stable producer cells, i.e., cells having stably incorporated therein the genetic material required to produce the lentivirus. Such cells are distinguished from cells having the genetic elements transiently incorporated therein.

[0053] In one example, the cell culture fluid or the filtered cell culture fluid is contacted with an endonuclease prior to purifying the virus.

[0054] In one example, the endonuclease is a non-specific endonuclease and degrades both DNA and RNA without sequence specificity. In one example, the endonuclease is a non-specific endonuclease and degrades DNA without sequence specificity.

[0055] Suitable endonucleases are known in the art and include those from Serratia marcescens. Anabaena sp., Saccharomyces cerevisiae. Bos Taurus, Syncephalostrum racemosum and / or Borrelia burgdorferi . For example, the endonuclease is a Serratia nuclease, NucA, Nucl, endonuclease G, DNase I, or micrococcal nuclease.

[0056] In one example, the endonuclease is added to the cell culture fluid at a concentration of 0.001 to 100 units / mL of cell culture fluid. In one example, the endonuclease is added to the cell culture fluid at a concentration of 10 to 50 units / mL of cell culture fluid. For example, the endonuclease is added to the cell culture fluid at a concentration of about 30 units / mL of cell culture fluid. In one example, the endonuclease is added to the cell culture fluid at a concentration of 0.01 to 10 units / mL of cell culture fluid. In one example, the endonuclease is added to the cell culture fluid at a concentration of 0.1 to 1 unit / mL of cell culture fluid. For example, the endonuclease is added to the cell culture fluid at a concentration of about 0.1 units / mL of cell culture fluid, or about 0.2 units / mL of cell culture fluid, or about 0.3 units / mL of cell culture fluid, or about 0.4 units / mL of cell culture fluid, or about 0.5 units / mL of cell culture fluid, or about 0.6 units / mL of cell culture fluid, or about 0.7 units / mL of cell culture fluid, or about 0.8 units / mL of cell culture fluid, or about 0.9 units / mL or cell culture fluid, or about 1 unit / mL of cell culture fluid. In another example, the endonuclease is added to the cell culture fluid at a concentration of 1 to 10 units / mL of cell culture fluid.

[0057] For example, the endonuclease is added to the cell culture fluid at a concentration of about 1 unit / mL of cell culture fluid, or about 2 units / mL of cell culture fluid, or about 3 units / mL of cell culture fluid, or about 4 units / mL of cell culture fluid, or about 5 units / mL of cell culture fluid, or about 6 units / mL of cell culture fluid, or about 7 units / mL of cell culture fluid, or about 8 units / mL of cell culture fluid, or about 9 units / mL of cell culture fluid, or about 10 units / mL of cell culture fluid.

[0058] In one example, the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of about 1 unit / mL of cell culture fluid or filtered cell culture fluid, and purification is performed between about 1 and about 2 hours after contact.

[0059] In one example, the method further comprises contacting the cell culture fluid or the filtered cell culture fluid with a magnesium salt prior to purifying the virus. In one example, the magnesium salt is added in an amount to achieve a target magnesium concentration of less than 10 mM Mg2+. 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.

[0060] It will apparent to the skilled person that to achieve the target magnesium concentration, the ratio of filtered cell culture fluid or cell culture fluid to the magnesium salt solution is dependent on the concentration of the magnesium salt solution. In one example, the magnesium salt solution is at 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 is at a concentration of 200 mM.

[0061] 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 the skilled person and / or described herein.

[0062] In one example, the cell culture fluid is harvested from cells cultivated in an adherent environment or from cells cultivated in a suspension environment. In one example, the cell culture fluid is harvested from cells cultivated in an adherent environment. In another example, the cell culture fluid is harvested from cells in a suspension environment.

[0063] In one example, the method comprises performing a harvest filtration on the cell culture fluid to produce the filtered cell culture fluid before subjecting the filtered cell culture fluid to the filtration medium. For example, the filtered cell culture fluid is subjected to the filtration medium immediately after harvest filtration.

[0064] In one example, the filtered cell culture fluid is contacted with a high concentration salt solution to form a salt-spiked cell culture fluid prior to or during loading on to the filtration medium.

[0065] In one example, the high concentration salt solution and the filtered cell culture fluid or the cell culture fluid are mixed, e.g., in-line during loading on to the filter unit. For example, during loading onto the filter unit (i.e., the filter unit comprising the filtration medium), the fluids are separately added to the filter unit at the same time. In some examples, the fluids may contact each other prior to entering the filter unit, after entering the filter unit, or at the same time as entering the filter unit.

[0066] In one example, the mixed filtered cell culture fluid or the cell culture fluid and the high concentration salt solution have a target salt concentration of about 300 mM to 500 mM salt for loading onto the filter unit. For example, the target salt concentration is 400 mM salt.

[0067] Therefore to achieve the target salt concentration, the ratio of filtered cell culture fluid or cell culture fluid to high concentration salt solution is dependent on the concentration of the salt solution. In one example, the high concentration salt solution is at a concentration of at least IM, for example, IM to 10M or 2M to 9M or 3M to 8M or 4M to 6M. For example, the high concentration salt solution is at a concentration of 5M.

[0068] In one example, the mixed filtered cell culture fluid or the cell culture fluid and the high concentration salt solution have a target conductivity of 30 to 45 mS / cm at 25°C. In one example, the mixed filtered cell culture fluid or the cell culture fluid and the high concentration salt solution have a target conductivity of 38 to 44 mS / cm at 25°C. For example, a target conductivity of about 40 ± 4 mS / cm at 25°C.

[0069] In one example, the high concentration salt solution comprises a monovalent and / or a divalent salt. For example, the high concentration salt solution comprises a monovalent salt, i.e., as the only salt. In one example, the monovalent salt is sodium chloride.

[0070] In one example, the high concentration salt solution is sodium chloride at a concentration of at least about IM, e.g., at a concentration of about 5M.

[0071] In one example, the method further comprises washing the filtration medium with one or more wash solutions. In another example, the method optionally comprises washing the filtration medium with one or more wash solutions.

[0072] In one example, the one or more wash solutions comprises a salt solution comprising a monovalent and / or a divalent salt. For example, the one or more wash solutions comprises a salt solution comprising a monovalent salt (i.e., as the only salt).

[0073] In one example, the monovalent salt is at a concentration of between O. IM and IM. For example, the monovalent salt is at a concentration of between 0.4M and 0.6M. For example, the monovalent salt is at a concentration of about 0.4M. In one example, the monovalent salt is at a concentration of about 0.5M. In another example, the monovalent salt is at a concentration of about 0.6M.

[0074] In one example, the monovalent salt is sodium chloride. For example, one or more wash solutions comprises a salt solution comprising sodium chloride.

[0075] In one example, the method further comprises washing the filtration medium with one or more wash solutions comprising sodium chloride at a concentration of about 0.4M. For example, the method optionally comprises washing the filtration medium with one or more wash solutions comprising sodium chloride at a concentration of about 0.4M.

[0076] In one example, the method further comprises washing the filtration medium with one or more wash solutions comprising sodium chloride at a concentration of about 0.6M. For example, the method optionally comprises washing the filtration medium with one or more wash solutions comprising sodium chloride at a concentration of about 0.6M. In one example, the method further comprises eluting bound enveloped virus from the filtration medium with an elution solution.

[0077] In one example, the elution solution comprises a buffer selected from the group consisting of Tris, histidine and HEPES. In one example, the elution solution comprises a Tris buffer. In one example, the elution solution comprises a histidine buffer. In one example, the elution solution comprises a HEPES buffer.

[0078] In one example, the elution solution comprises a buffer at a concentration of 10 mM to 50 mM. For example, the elution solution comprises a buffer at a concentration of 10 mM.

[0079] In one example, the elution solution comprises 10 mM HEPES.

[0080] In one example, the elution solution has a pH of between 5 and 10.

[0081] In one example, the elution solution comprises 10-50 mM Tris buffer at pH 5-10. For example, the elution solution comprises 50 mM Tris buffer at pH 8.0.

[0082] In one example, the elution solution comprises 10-50 mM histidine buffer at pH 5-10. For example, the elution solution comprises 10 mM histidine buffer at pH7.0.

[0083] In one example, the elution solution comprises 10-50 mM HEPES buffer at pH 5- 10. For example, the elution solution comprises 10 mM HEPES buffer at pH7.5.

[0084] In one example, the elution solution further comprises a monovalent and / or a divalent salt. In one example, the elution solution further comprises a monovalent salt. For example, the monovalent salt is sodium chloride. In another example, the elution solution further comprises a divalent salt.

[0085] In one example, the elution solution further comprises a monovalent and / or a divalent salt at a concentration of between 0.5M and 2M. For example, the salt is at a concentration of about 0.5 M, 0.6 M, 0.7 M, 0.8M, or 0.9 M. In one example, the salt is at a concentration of 0.5 M. In another example, the salt is at a concentration of 0.6 M. In a further example, the salt is at a concentration of 0.7 M. In one example, the salt is at a concentration of 0.8 M. In a further example, the salt is at a concentration of 0.9M. In another example, the salt is at a concentration of between about 1 M and 2 M. For example, the salt is at a concentration of 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M or 2 M. In one example, the salt is at a concentration of 1.2 M. In another example, the salt is at a concentration of 1.5 M.

[0086] In one example, the elution solution further comprises a monovalent and / or a divalent salt at a concentration of 1.2 M. For example, the elution solution further comprises 1.2 M sodium chloride. In one example, the elution of the bound virus is achieved using a stepwise increase of salt concentration. For example, the bound virus is eluted in a stepwise manner by increasing the concentration of salt in the elution solution. In one example, the salt concentration in the elution solution is increased from a concentration of about 0.5 M to a concentration of about 1 ,5M. For example, the salt concentration in the elution solution is increased from a concentration of about 0.6 M to a concentration of about 1.5M. In one example, the salt concentration in the elution solution is increased from a concentration of about 0.6 M to a concentration of about 1.2M. For example, the salt concentration in the elution solution is increased from a concentration of about 0.6 M, to a concentration of about 0.9 M, to a concentration of about 1.2 M.

[0087] In one example, the method increases the virus infectious titer yield by at least 2% or 3% or 4% or 5% or 10% or 15% or 20%. In one example, the method increases the virus infectious titer yield by at least 10%.

[0088] In one example, the method results in a viral infectious titer yield of at least 1 x 107transducing units (TU) / mL of eluted virus. For example, the method results in a viral infectious titer yield of 1 x 107TU / mL, 2 x 107TU / mL, 3 x 107TU / mL, 4 x 107TU / mL, 5 x 107TU / mL, 6 x 107TU / mL of eluted virus, 7 x 107TU / mL, 8 x 107TU / mL, 9 x 107TU / mL, or 10 x 107TU / mL of eluted virus. In one example, the method results in a viral infectious titer yield of at least 1 x 108transducing units (TU) / mL of eluted virus. For example, the method results in a viral infectious titer yield of 1 x 108TU / mL, 2 x 108TU / mL, 3 x 108TU / mL, 4 x 108TU / mL, 5 x 108TU / mL, 6 x 108TU / mL of eluted virus, 7 x 108TU / mL, 8 x 108TU / mL, 9 x 108TU / mL, or 10 x 108TU / mL of eluted virus. In one example, the method results in a viral infectious titer yield of at least 1 x 109transducing units (TU) / mL of eluted virus. For example, the method results in a viral infectious titer yield of 1 x 109TU / mL, 2 x 109TU / mL, 3 x 109TU / mL, 4 x 109TU / mL, 5 x 109TU / mL, 6 x 109TU / mL of eluted virus, 7 x 109TU / mL, 8 x 109TU / mL, 9 x 109TU / mL, or 10 x 109TU / mL of eluted virus.

[0089] In one example, the method results in a residual dsDNA content of less than 100 pg / mL of eluted virus. For example, the method results in a residual dsDNA content of 100 pg / mL of eluted virus or less, or 90 pg / mL of eluted virus or less, or 80 pg / mL of eluted virus or less, or 70 pg / mL of eluted virus or less, or 60 pg / mL of eluted virus or less, or 50 pg / mL of eluted virus or less, or 40 pg / mL of eluted virus or less, or 30 pg / mL of eluted virus or less, or 20 pg / mL of eluted virus or less, or 10 pg / mL of eluted virus or less. For example, the method results in a residual dsDNA content of 9 pg / mL of eluted virus or less, or 8 pg / mL of eluted virus or less, or 7 pg / mL of eluted virus or less, or 6 pg / mL of eluted virus or less, or 5 pg / mL of eluted virus or less, or 4 pg / mL of eluted virus or less, or 3 pg / mL of eluted virus or less, or 2 pg / mL of eluted virus or less, or 1 pg / mL of eluted virus or less.

[0090] In one example, the method further comprises diluting the eluted virus with histidine, Tris or HEPES. For example, the method further comprises diluting the eluted virus with histidine buffer. In another example, the method further comprises diluting the eluted virus with Tris buffer. In a further example, the method further comprises diluting the eluted virus with HEPES buffer.

[0091] In one example, the method comprises diluting the eluted virus at a dilution of 1 :5 to 1 :20. For example, the method comprises diluting the eluted virus at a dilution of 1 :5. In another example, the method comprises diluting the eluted virus at a dilution of 1 :6.

[0092] In another example, the method comprises diluting the eluted virus at a dilution of 1 :7.

[0093] In another example, the method comprises diluting the eluted virus at a dilution of 1 :8.

[0094] In another example, the method comprises diluting the eluted virus at a dilution of 1 :9.

[0095] In another example, the method comprises diluting the eluted virus at a dilution of 1: 10.

[0096] In one example, the method further comprises incubating the eluted virus for up to 15 minutes at room temperature or up to 60 minutes at 2-8 °C.

[0097] In one example, the method comprises concentrating and / or diafiltering the eluted virus.

[0098] In one example, a method of the disclosure additionally comprises performing sterile filtration. For example, the sterile filtration is performed prior to concentrating and diafiltering the eluted virus. In an alternative example, the sterile filtration is performed after concentrating and diafiltering the eluted virus.

[0099] In one example, the cell culture fluid and / or the filtered cell culture fluid has a volume of greater than about 1 mL, about 5 mL, about 10 mL, about 50 mL, about 100 mL, about 500 mL, about 1 L, about 5 L, about 10 L, about 50 L, about 100 L, about 500 L, or about 1000 L.

[0100] An exemplary enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0101] In one example, the method is performed in-line or continuously or semi- continuously.

[0102] In one example, a method of the disclosure additionally provides formulating the enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0103] The disclosure additionally provides a purified enveloped virus produced by a method described herein. The present disclosure further provides a method of purifying an enveloped virus from a cell culture fluid, comprising:

[0104] (i) providing a cell culture fluid comprising viral vector produced from a stable producer cell line;

[0105] (ii) contacting the cell culture fluid with an endonuclease;

[0106] (iii) contacting the endonuclease treated cell culture fluid to a filter to produce a filtered cell culture fluid;

[0107] (iv) loading the filtered cell culture fluid and a high concentration salt solution to a charged depth filtration medium;

[0108] (v) optionally, washing the filtration medium with one or more wash solutions;

[0109] (vi) eluting the bound enveloped virus from the filtration medium with an elution solution;

[0110] (vii) diluting the eluted virus with a buffer; and

[0111] (viii) concentrating and diafiltering the eluted virus.

[0112] BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure l is a graphical representation showing infectious titer (top), p24 (middle) and DNA (bottom) data for Mustang Q, Sartobind Q, CIM QA, CIM DEAE, Sartobind D, and Emphaze. Shown is the sum of the main elution fractions.

[0114] Figure l is a series of graphical representations showing chromatograms for the elution parts of (A) Emphaze and (B) Mustang Q. Traces: UV214 (pink), UV280 (blue), conductivity (brown), delta column pressure (green).

[0115] Figure 3 is a graphical representation showing infectious particles recoveries from Mustang Q, Emphaze and Sartobind Q membranes using different buffer systems: 50 mM Tris, pH 8.0; 10 mM Histidine, pH 7.0; 10 mM Histidine, 3% Sucrose, pH 7.0; and 10 mM Hepes, pH 7.5. Number within each bar corresponds to the run number (see Table 3).

[0116] Figure 4 is a graphical representation showing infectious particles recoveries from TFF step runs using different buffer systems: 50 mM Tris, pH 8.0; 10 mM Histidine, pH 7.0; 10 mM Histidine, 3 % Sucrose, pH 7.0; 10 mM Hepes, pH 7.5. Hollow fibers from Repligen (solid bars) or Cytiva (striped bars) were run at 1200, 4000 or 8000 s-1 shear rates. Numbers within each bar correspond to the run number (see Table 3).

[0117] Figure 5 is a graphical representation showing infectious particles recoveries from TFF step runs using EKV or Millex-GP syringe filters. Numbers within each bar correspond to the run number (see Table 3). Figure 6 is a series of graphical representations showing continuous chromatography of WASp using Emphaze in RC mode. Thirteen cycles were performed. (A) WASp elution (fraction 1.2 M NaCl) and full chromatogram in the insert of RC cycles. Each line of the chromatogram represents a different cycle. (B) Membrane differential pressure for the different cycles from RC1 (bottom line) to RC3 (top line). (C) WASp infectious particles recoveries and (D) RNA copies recovery from RC1 to RC10.

[0118] Figure 7 is a series of graphical representations showing continuous chromatography of WASp using Mustang Q in PCC mode with 3 membranes. Two cycles were corresponding to 6 switches (A) WASp elution (fraction 1.5 M NaCl) and full chromatogram in the insert of PCC switches. (B) System pressure for switches 1 (bottom) to 7 (top). (C) WASp infectious particles recoveries and (D) RNA copies recovery from different switches.

[0119] Figure 8 is a graphical representation showing total infectivity (by ddPCR; top panel), volume-adjusted infectivity (by ddPCR; middle panel), and RNA content yield (bottom panel) from 60mL Emphaze membrane (with and without a wash step) compared with 60mL Mustang Q and the Emphaze wash step alone.

[0120] DETAILED DESCRIPTION

[0121] General

[0122] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0123] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0124] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0125] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0126] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, molecular biology, microbiology, virology).

[0127] Unless otherwise indicated, the conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan etal. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0128] 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 be taken to provide explicit support for both meanings or for either meaning.

[0129] The term “about”, unless stated to the contrary, refers to + / - 20%, more for example + / - 10%, of the designated value. For the avoidance of doubt, the term “about” followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, “about 10” also encompasses 10 exactly). As used herein the term “from” in the shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source (i.e., includes recombinantly obtained).

[0130] 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.

[0131] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0132] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0133] Selected Definitions

[0134] The term “purify” or “purifying” or “purification” shall be taken to mean the removal, whether completely or partially, of at least one impurity present in the cell culture fluid, which thereby improves the level of purity of enveloped virus in solution.

[0135] The term “impurity” or “impurities” shall be taken to include one or more components in the cell culture fluid other than the enveloped virus. For example, impurities may include process related impurities such as host cell DNA, host cell proteins, and media components (e.g., fetal bovine serum).

[0136] As used herein, the term “enveloped virus” refers to DNA and RNA viruses that have a viral envelope. Envelopes are typically derived from host cell membranes (e.g., phospholipids and proteins), but may include viral glycoproteins on the surface of the envelope. Enveloped viruses also comprise a “capsid”, which is a protein layer between the envelope and viral genome. In one example, the enveloped virus is a retrovirus. For example, the enveloped virus is a lentivirus, e.g., human immunodeficiency virus.

[0137] As used herein, the term “cell culture fluid” will be understood to encompass the fluid in which cells are grown for the purpose of producing an enveloped virus. The fluid may comprise the cells or the cells may have been removed, e.g., by centrifugation and / or removal of supernatant.

[0138] As used herein, “harvesting” refers to removal of the cell culture fluid containing virus particles from the producer cells for downstream processing, and “harvest” refers to the cell culture fluid containing virus particles that has been removed for the purpose of downstream processing. A harvesting process may include collecting one or more harvests. “Harvest filtration” refers to either a harvest that has been filtered or cell culture fluid containing virus particles that has been filtered to remove the producer cells for downstream processing.

[0139] As used herein, the term “filtered cell culture fluid” will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.

[0140] As used herein, the term “salt-spiked cell culture fluid” will be understood to encompass a cell culture fluid or a filtered cell culture fluid that has been mixed with a high concentration salt solution.

[0141] As used herein, the term “in-line” in the context of a process step refers to a process step that is integrated into or combined with one or more other process steps, or that flows directly from or to another process step without requiring manual intervention or handling.

[0142] The skilled artisan will understand that an “endonuclease” is an enzyme that cleaves the phosphodiester bond within a polynucleotide chain. Endonucleases can cleave DNA or RNA or both DNA and RNA. Endonucleases can cleave in a sequence non-specific manner (also referred to as a “non-specific endonuclease”) or can cleave at specific nucleotide sequences (also referred to as “restriction endonucleases”).

[0143] Reference herein to an endonuclease “from” a source, e.g., from Serratia marcescens encompasses the endonuclease purified from that source or produced by other means, e.g., by recombinant techniques.

[0144] As used herein, the term “immediately after” in the context of performing charged depth filtration after harvest filtration means that there are no intervening purification steps between the harvest filtration and the charged depth filtration. However, this term does not exclude additional steps such as adjusting the pH or adding a salt to the cell culture fluid or filtered cell culture fluid between the harvest filtration and the charged depth filtration.

[0145] As used herein, the term “in-line” in the context of a process step refers to a process step that is integrated into or combined with one or more other process steps, or that flows directly from or to another process step without requiring manual intervention or handling. As used herein, the term “non-functionalized” refers to a substrate that is substantially devoid (i.e., less than 10, 1, or even 0.1 pmol per gram of nonfunctionalized size exclusion membrane) of ion exchange functional chemical groups.

[0146] Production of Enveloped Viruses

[0147] Methods for the production of enveloped viruses will be apparent to the skilled artisan and / or described, for example, in Ansorge etal., (2010) Biochem. Eng. J. 48: 362- 377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474-486 ; and Rodrigues etal., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40.

[0148] In one example, the virus is a retrovirus, for example, a lentivirus. Exemplary retroviruses are from alpha retroviruses (such avian leukosis virus (ALV)), from beta retroviruses (such as mouse mammary tumor virus (MMTV)), from gamma retroviruses (such as murine leukemia virus (MLV)), from delta retroviruses (such as human T- lymphotropic virus (HTLV)), from epsilon retroviruses (such as Walleye dermal sarcoma virus (WDSV)), from spumavirus (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses such as the different types of human immunodeficiency viruses (HIV), the different types of simian immunodeficiency viruses (SIV), or from non-primate mammal lentiviruses such as the equine infectious anemia virus (EIAV), from the feline immunodeficiency virus (FIV), the caprine arthritis-encephalitis virus (CAEV), or the ovine visna-maedi virus (VMV).

[0149] In some examples, the enveloped virus, e.g., the retrovirus, is pseudotyped, i.e., it comprises an envelope glycoprotein derived from a virus different from the virus from which it is derived, a modified envelope glycoprotein or a chimeric envelope glycoprotein.

[0150] In some examples, the enveloped virus comprises 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 a transgene coding for a therapeutic RNA (e.g. encoding an antisense complementary RNA of a target RNA or DNA sequence), a transgene encoding for a protein that is deficient or absent in a subject affected with a pathology, or a transgene used for vaccination with DNA, i.e. a transgene coding for a protein, the expression of which will induce vaccination of the recipient body against said protein. In some examples, the transgene encodes a protein or nucleic acid useful for treating a hemoglobinopathy, e.g., sickle cell disease or a thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating a primary immunodeficiency. In some examples, the transgene encodes a protein or nucleic acid useful for treating Wiskott-Aldrich Syndrome. In some examples, the transgene encodes a protein or nucleic acid useful for treating X linked agammaglobulinemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating Sickle Cell Disease (SCD). In some examples, the transgene encodes a protein or nucleic acid useful for treating Deficiency of Interleukin-1 Receptor Antagonist (DIRA). In some examples, the transgene encodes a protein or nucleic acid useful for treating Adenosine deaminase 2 deficiency (DADA2).

[0151] In some examples, an enveloped virus is produced by introducing the four following elements into a host cell: an expression cassette comprising a lentiviral gene gagpol, an expression cassette comprising a lentiviral gene rev, a transgene, all positioned between a lentiviral LTR-5’ and a lentiviral LTR-3’, and an expression cassette encoding envelope glycoprotein(s).

[0152] In some examples, the enveloped virus is produced from a stable line expressing one or several elements required for producing an enveloped virus (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra). In one example, the enveloped virus is produced from a mammal host cell transfected transiently with one or several plasmids coding for the elements required for producing the virus. According to an alternative example, the elements are introduced into the cell by means of multiple plasmids: one plasmid bearing an expression cassette comprising a lentiviral gagpol gene, one plasmid bearing an expression cassette comprising a lentiviral rev gene, one plasmid bearing an expression cassette encoding the envelope glycoprotein(s), one plasmid bearing an expression cassette comprising a tetracycline transactivator (ITA) gene, and / or one plasmid bearing an expression cassette comprising a lentiviral tat gene. A transfer plasmid comprising an expression cassette with the transgene, comprised between a lentiviral LTR-5’ and LTR-3’, can be introduced as a concatemer along with a helper plasmid with an antibiotic resistance cassette to confer resistance to the producer cells.

[0153] The host cell may be selected from any cell allowing production of an enveloped virus. According to one example, the cell is selected from a human cell (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), a musteli cell (NIH-3T3), a mustelidae cell (Mpf), a canid cell (DI 7). According to one example, the cell is 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, NH43T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells. According to one example, the cell is selected from the GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. In another example, the cell is selected from a cell line derived from any of the above cell lines.

[0154] In one example, the enveloped virus is produced from stable producer cells. Stable producer cells can be derived from packaging cell lines, including as any of the cell lines disclosed herein. In some embodiments the packaging cell lines are GPRG or GPRTG cell lines (Throm et al. (2009) Blood 113(21): 5104-5110; and Bonner et al. (2015) Molecular Therapy, Vol. 23, Suppl. 1, S35). In one example, stable producer cell line cells are generated by synthesizing a vector by cloning one or more genes into a recombinant plasmid; forming a concatemeric array from an expression cassette excised from the synthesized vector, and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfecting packaging cell line cells with the formed concatemeric array; and isolating the stable producer cell line cells. Virus is produced by inducing the inducible promoters of the stable producer cell line cells.

[0155] The cells are cultivated in a medium suitable for cultivation of mammal cells and for producing an enveloped virus. The cells can be cultivated in an adherent environment, e.g., while attached to a surface, or in a suspension environment, e.g., suspended in the medium. The medium may moreover be supplemented with additives known in the field such as antibiotics, serum (notably fetal calf serum, etc.) added in suitable concentrations. The medium may be supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell Boost™ 5, Gibco™ Cholestrol Lipid Concentrate (250x); FastGro Synthetic, chemically defined FBS replacement; LS250 (Cytiva); Synthecol (500x) (Sigma) and / or an antidumping agent. The medium used may notably comprise serum or be serum-free. Culture media for mammal cells are known and include, for example, DMEM (Dulbecco’s Modified Eagle’s medium) medium, RPMI1640 or a mixture of various culture media, including for example DMEM / F12, or a serum-free medium like optiMEM®, optiPRO®, optiPRO-SFM®, CD293® (ThermoFisher), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies), HyClone™ peak expression (Cytiva); HyClone™ CDM4HEK293 (Cytiva); or Ex-Cell® 293 (Sigma-Aldrich).

[0156] In a process using transiently transfected cells, any agent allowing transfection of plasmids may be used. Exemplary agents include calcium phosphate or polyethyleneimine. The conditions (e.g., amount of plasmid(s), ratio between the plasmids, ratio between the plasmid(s) and the transfection agent, the type of medium, etc.) and the transfection time may be adapted by one skilled in the art according to the characteristics of the produced virus and / or of the transgene introduced into the transfer plasmid.

[0157] According to some examples, the culture medium used has a neutral pH (e.g. comprised between 7 and 7.4, notably 7, 7.1, 7.2, 7.3 or 7.4) conventionally used in the state of the art for cultivating cells and producing viruses. In other examples, the production process used comprises the cultivation of producing cells in a moderately acid medium. The expression “moderately acid condition” designates the pH of an 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 selected pH will also depend on the buffering power of the culture medium used, which one skilled in the art may easily determine taking into account his / her general knowledge. One skilled in the art is able to modify the pH of a solution.

[0158] In one example, the production of the enveloped virus comprises: transient transfection of HEK293T cells or derivatives thereof by means of one or several plasmids coding for the elements required for production of said enveloped vector, or by the use of stable producing cells, e.g., GPRG or GPRTG, producing the vectors constitutively or after induction; culturing the cells in a suitable medium, for which the pH is of about 6 or of about 7; harvesting cell culture fluid containing the enveloped virus.

[0159] Methods of the disclosure are applicable to purifying enveloped viruses from both small- and large-scale productions. The methods are particularly useful for their ability to be scaled up for manufacturing pharmaceutical products at commercial scale.

[0160] In one example, cells are grown in an adherent or fixed-bed environment. In one example, cells are grown in a cell culture chamber, such as a CellSTACK® (Corning). In one example, cells are grown in an adherent bioreactor, such as iCELLis® (Pall), scale-X™ orNevoLine™ (Univercells Technologies). An adherent cell culture chamber or bioreactor may have an available growth surface of greater than about 0.1 m2, greater than about 1 m2, greater than about 10 m2, greater than about 30 m2, greater than about 100 m2, greater than about 200 m2, greater than about 500 m2, or greater than about 600 m 2.

[0161] In one example, cells are grown in a suspension environment. In one example, cells are grown in a stirred tank bioreactor. In examples, the cells are grown in a Biostat® or Univessel® bioreactor (Sartorius).

[0162] In embodiments, the volume of a harvest of cell culture fluid can be 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 a harvest of cell culture fluid is about 5 L. In other embodiments, the volume of a harvest of cell culture fluid can be 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 harvest is between about 35 and 150 L. In one example, the volume of the harvest is about 35-150 L. For example, the volume of the harvest is about 20 L. In one example, the volume of the harvest is about 50-70 L. For example, the volume of the harvest is about 50 L.

[0163] In one example, the cell culture is operated at a temperature that permits cell growth and viral production. For example, the cell culture has a temperature conventionally used in the state of the art for cultivating cells and producing viruses. In one example, the cell culture is at a temperature of between 35-39 °C. For example, at a temperature of 37 ± 0.5 °C or at a temperature of 38 ± 0.5 °C.

[0164] In one example, the cell culture is operated for a period of at least 1 day. For example, the cell culture is operated for a period of at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 8 days, or at least 9 days. In one example, the cell culture is operated for a period of at least 10 days. For example, the cell culture is operated for a period of between about 10 and 50 days. In one example, the cell culture is operated for a period of between 10 and 35 days, for example, about 10 days or about 12 days, or about 15 days, or about 18 days, or about 20 days, or about 22 days, or about 25 days, or about 28 days, or about 30 days or about 32 days or about 35 days. In one example, the cell culture is operated for at least 15 days. For example, the cell culture is operated for about 20 days. In another example, the cell culture is operated for at least 20 days. In a further example, the cell culture is operated for at least 25 days. For example, the cell culture is operated for about 28 days. In one example, the cell culture is operated for at least 30 days. In one example, the cell culture is operated for at least 32 days. For example, the cell culture is operated for a period of 35 days. In one example, the cell culture is operated for at least 35 days.

[0165] Endonuclease treatment

[0166] In one example, the cell culture fluid is contacted with the endonuclease prior to harvest filtration, i.e., filtration to remove cells and cellular debris.

[0167] Suitable endonucleases will be apparent to the skilled artisan based on the disclosure herein. In one example, the endonuclease cleaves in a sequence non-specific manner. For example, the endonuclease cleaves DNA (and, optionally RNA) into short oligonucleotides, e.g., 2-10bp long, such as 3-7bp long, e.g., 3-5bp long.

[0168] In one example, the endonuclease is from Serratia marcescens. Anabaena sp., Saccharomyces cerevisiae. Bos Taurus, Syncephalostrum racemosum and / or Borrelia burgdorferi. In one example, the endonuclease is a Serratia nuclease, NucA, Nucl and / or endonuclease G. In one example, the endonuclease is from Serratia marcescens. Such an endonuclease is also referred to as Golden nuclease. This nuclease is sold under the tradenames Benzonase® or Denarase®.

[0169] The endonuclease may be isolated or purified from the recited source. Alternatively, the endonuclease can be produced recombinantly.

[0170] The endonuclease can also be obtained from a suitable commercial source, as will be apparent to the skilled artisan and / or described herein. For example, endonucleases are available from New England Biolabs, Inc or c-LEcta GmbH.

[0171] In one example, the method involves adjusting the concentration of Mg2+in the cell culture fluid to achieve a concentration of up to 10 mM Mg2+. In one example, the concentration of Mg2+is adjusted to about 1-3 mM. In one example, the concentration of Mg2+is adjusted to 2 mM. In one example, the concentration of Mg2+in the cell culture fluid is about 0.8 mM and is not adjusted further.

[0172] In one example, the method involves adjusting the pH of the cell culture fluid to 6.0 to 10.0. In one example, the method involves adjusting the pH of the cell culture fluid to 8.0 to 9.2.

[0173] In one example, the cell culture fluid is at a temperature of between 0 °C and 42 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of between 2 °C and 8 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of 4 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of between 35 °C and 40 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of about 37 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of between 18 °C and 22 °C during contact with the endonuclease. In one example, the cell culture fluid is at a temperature of 20 °C during contact with the endonuclease.

[0174] In one example, the endonuclease is added to the culture medium at a concentration of 0.001 U / mL cell culture fluid to 100 U / mL cell culture fluid. For example, the endonuclease is added to the cell culture fluid at a concentration of 10 U / mL cell culture fluid to 50 U / mL cell culture fluid. For example, the endonuclease is added to the cell culture fluid at a concentration of 30 U / mL of cell culture fluid. In one example, the endonuclease is added to the cell culture fluid at a concentration of 0.01 U / mL cell culture fluid to 10 U / mL cell culture fluid. For example, the endonuclease is added to the cell culture fluid at a concentration of 0.1 U / mL cell culture fluid to 1 U / mL cell culture fluid. In one example, the endonuclease is added to the cell culture fluid at a concentration of 1 U / mL cell culture fluid. Purifying Enveloped Viruses

[0175] The present disclosure provides methods for improving the purity and / or recovery of enveloped viruses from cell culture fluid or filtered cell culture fluid.

[0176] Harvest filtration

[0177] The downstream process for purifying and concentrating viral vector from a cell culture fluid includes a harvest filtration step (also known as “clarification filtration” or “harvest clarification filtration” or “bioburden reduction”) to remove cellular debris and components from the harvest, a purification step (e.g., a charged depth filtration step) to reduce overall volume and to separate viral vector from host cell DNA, proteins, and media components, and an ultrafiltration / diafiltration step to concentrate the viral vector into a final formulation buffer. In some examples, the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.

[0178] As used herein, “harvesting” refers to removal of the cell culture fluid containing virus particles from the producer cells for downstream processing, and “harvest” refers to the cell culture fluid containing virus particles that has been removed for the purpose of downstream processing. A harvesting process may include collecting one or more harvests. “Harvest filtration” refers to either a harvest that has been filtered or cell culture fluid containing virus particles that has been filtered to remove the producer cells for downstream processing.

[0179] In one example, a harvested cell culture fluid is filtered following production of the enveloped virus.

[0180] In one example, the harvest filtration is performed using membrane filtration. For example, the harvest filtration is performed using a 0.8pm filter and a 0.45pm filter, which may be included within a single unit.

[0181] As used herein, the term “filtered cell culture fluid” will be understood to encompass the cell culture fluid after it has been subjected to harvest filtration.

[0182] Charged depth filtration

[0183] Following harvest filtration, the enveloped virus is purified using a charged depth filtration step. For example, the enveloped virus is purified using an anionic charged depth filtration step.

[0184] As discussed herein, the inventors determined that the purification step accounted for the largest losses in virus yield. In addition, existing purification methods include capture chromatography columns which require high salt content, and increasing the salt concentration is complicated by several factors, including the salt is harmful to virus and the high-salt elution must then be diluted. This results in larger volumes, creating an additional burden on downstream processing.

[0185] The inventors’ solution to these problems is to purify the cell culture fluid with a charged depth filter (i.e., an anionic charged depth filter) that is typically used in flowthrough-mode as a bioburden reduction step for protein and antibody purification. The inventors determined performing this purification step in bind-and-elute mode achieves improved purification of anionic viruses, such as lentiviruses.

[0186] In one example, the filtration step is performed in bind-elute mode. In this regard, the enveloped virus binds to the filtration medium while contaminants flow through. The virus is subsequently eluted from the filtration medium. Performing the filtration step in this manner reduces the volume of liquid in which the virus is suspended and removes contaminants, such as host cell DNA, host cell proteins and medium components like fetal bovine serum.

[0187] In one example, the filtration medium comprises a non-woven substrate and a plurality of quaternary ammonium (Q) monomers. An exemplary filtration medium useful in the method of the present disclosure includes EMPHAZE® AEX Hybrid Purifier.

[0188] As exemplified herein, the filtration medium comprises a plurality of Q ion exchange groups. The exemplified filtration medium further comprises a non-woven susbtrate.

[0189] In one example, the non-woven substrate comprises a thermoplastic polymeric material. For example, the thermoplastic polymeric material is selected from the group consisting of polyolefins, poly (isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates), polyesters such as poly(lactic acid), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers), poly(vinyl alcohols), poly (carbonates) and combinations thereof.

[0190] In one example, the thermoplastic polymeric material is a hydrophobic thermoplastic polyolefin. For example, the polyolefin is selected from the group consisting of poly (ethylene), poly(propylene), poly(l-butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1 -butene, 1 -hexene, 1 -octene, and 1 -decene), poly (ethylene -co- 1 -butene), poly(ethylene-co- 1 -butene-co- 1 -hexene) and combinations thereof. As exemplified herein, the filtration medium comprises a plurality of Q ion exchange groups grafted onto the surface of a poly(propylene) non-woven substrate. For example, the filtration medium is an EMPHAZE® AEX Hybrid Purifier.

[0191] In one example, during loading the filtration medium is contacted with an enveloped virus from a cell culture fluid or a filtered cell culture fluid. For example, during loading the filtration medium is contacted with a cell culture fluid or a filtered cell culture fluid.

[0192] In one example, during loading the filtration medium is contacted with multiple membrane volumes (MVs) of a cell culture fluid or a filtered cell culture fluid. In this regard, a volume of solution approximately equal to the volume of the filtration medium is equal to one MV of cell culture fluid or a filtered cell culture fluid. In some examples, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 20 or 30 or 40 or 50 MVs of a cell culture fluid or a filtered cell culture fluid are flowed through the filtration medium. In some examples, 50 or 60 or 70 or 80 or 90 or 100 MVs of a cell culture fluid or a filtered cell culture fluid are flowed through the filtration medium. For example, during loading the filtration medium is contacted with 90 to 100 MVs of a cell culture fluid or a filtered cell culture fluid. In one example, 110 or 120 or 130 or 140 or 150 or 160 or 170 or 180 or 190 or 200 MVs of a cell culture fluid or a filtered cell culture fluid are flowed through the filtration medium. For example, during loading the filtration medium is contacted with 150 to 200 MVs of a cell culture fluid or a filtered cell culture fluid. In one example, during loading the filtration medium is contacted with 170 to 190 MVs of a cell culture fluid or a filtered cell culture fluid.

[0193] In one example, during loading the filtration medium is contacted with cell culture fluid or a filtered cell culture fluid containing about 1 x 108TU / mL of membrane volume to about 1 x 1010TU / mL of membrane volume. For example, the filtration medium is contacted with cell culture fluid or a filtered cell culture fluid containing about 1 x 108TU / mL of membrane volume, or about 2 x 108TU / mL of membrane volume, or about 3 x 108TU / mL of membrane volume, or about 4 x 108TU / mL of membrane volume, or about 5 x 108TU / mL of membrane volume, or about 6 x 108TU / mL of membrane volume, or about 7 x 108TU / mL of membrane volume, or about 8 x 108TU / mL of membrane volume, or about 9 x 108TU / mL of membrane volume, or about 1 x 109TU / mL of membrane volume, or about 2 x 109TU / mL of membrane volume, or about 3 x 109TU / mL of membrane volume, or about 4 x 109TU / mL of membrane volume, or about 5 x 109TU / mL of membrane volume, or about 6 x 109TU / mL of membrane volume, or about 7 x 109TU / mL of membrane volume, or about 8 x 109TU / mL of membrane volume, or about 9 x 109TU / mL of membrane volume, or about 1 x IO10TU / mL of membrane volume.

[0194] In one example the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of at least 0.5 MV / min. In one example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of between 0.5 and 10 MV / min. For example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of 0.5, 0.75, 1, 1.25, 1.5, 1.75 or 2 MV / min. In another example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 1 MV / min, 2 MV / min, 3 MV / min, 4 MV / min or 5 MV / min. For example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 1 MV / min. In one example, example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 3.5 MV / min. In one example, example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 5 MV / min. In a further example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 6 MV / min, 7 MV / min, 8 MV / min, 9 MV / min or 10 MV / min. In one example, example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 10 MV / min.

[0195] In one example the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of at least 0.5 mL / min. In one example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of between 0.5 and 10 mL / min. For example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of 0.5, 0.75, 1, 1.25, 1.5, 1.75 or 2 mL / min. In another example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min or 5 mL / min. For example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 1 mL / min. In one example, example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 5 mL / min. In a further example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min or 10 mL / min. In another example, the cell culture fluid or filtered cell culture fluid is loaded at a flow rate of 8 mL / min. In another example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of between 10 mL / min and 100 mL / min. For example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 mL / min. In another example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of between 100 mL / min and 1000 mL / min. For example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 mL / min. In one example, the cell culture fluid or a filtered cell culture fluid is loaded at a flow rate of about 450 mL / min. In one example, the filtration medium is washed with one or more wash solutions.

[0196] In one example, during washing the filtration medium is contacted with multiple MVs of one or more wash solutions. In this regard, a volume of solution approximately equal to the volume of the filtration medium is equal to one MV of wash solution. In some examples, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 20 or 30 or 40 or 50 MVs of a wash solution are flowed through the filtration medium. For example, during washing the filtration medium is contacted with 10 MVs of a wash solution. For example, during washing the filtration medium is contacted with 20 to 40 MVs of a wash solution. In one example, during washing the filtration medium is contacted with 20 to 25 MVs of a wash solution. In another example, during washing the filtration medium is contacted with 35 to 40 MV of a wash solution.

[0197] In one example, the bound enveloped virus is eluted from the filtration medium with an elution solution.

[0198] In one example, during elution of the bound enveloped virus the filtration medium is contacted with multiple MVs of an elution solution. In this regard, a volume of solution approximately equal to the volume of the filtration medium is equal to one MV of elution solution. In some examples, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 20 or 30 or 40 or 50 MVs of an elution solution are flowed through the filtration medium. For example, during elution the filtration medium is contacted with 20 to 40 MVs of an elution solution. In one example, during elution the filtration medium is contacted with 20 to 25 MVs of an elution solution. In another example, during elution the filtration medium is contacted with 35 to 40 MV of an elution solution. In another example, during elution the filtration medium is contacted with 1 to 20 MVs of an elution solution. For example, during elution the filtration medium is contacted with 5 to 10 MVs of an elution solution.

[0199] In one example elution is performed at a flow rate of at least 0.5 MV / min. In one example, elution is performed at a flow rate of between 0.5 and 10 MV / min. For example, elution is performed at a flow rate of 0.5, 0.75, 1, 1.25, 1.5, 1.75 or 2 MV / min. In another example, elution is performed at a flow rate of 1 MV / min, 2 MV / min, 3 MV / min, 4 MV / min or 5 MV / min. For example, elution is performed at a flow rate of 1 MV / min. For example, elution is performed at a flow rate of 2 MV / min. In one example, elution is performed at a flow rate of 3 MV / min, 4 MV / min, 5 MV / min, 6 MV / min, 7 MV / min, 8 MV / min, 9 MV / min or 10 MV / min. In one example, elution is performed at a flow rate of 10 MV / min.

[0200] The inventors also recognized that during protein and antibody purification, charged depth filters are typically only single-use and replacement of filters is time- consuming and costly at commercial scale. However, the inventors surprisingly found that the charged depth filters could be cleaned in place and re-equilibrated permitting reuse. This method permitted multiple rounds of purification, without manual handling or adding any process time, and is performed in-line in a continuous or semi-continuous manner, thereby streamlining the downstream process.

[0201] For example, the method disclosed herein further comprises optionally cleaning in place the filtration medium. For example, the method further comprises cleaning in place the filtration medium following elution of the enveloped virus from the filtration medium.

[0202] In one example, the filtration medium is cleaned in place with a cleaning in place solution. For example, the filtration medium is cleaned in place with one or more MVs of a cleaning in place solution.

[0203] In one example, the cleaning in place solution comprises sodium hydroxide and / or sodium chloride. For example, the cleaning in place solution comprises sodium hydroxide and sodium chloride.

[0204] In one example, the cleaning in place solution comprises sodium hydroxide at a concentration of 0.5 M to 1.5 M. For example, the cleaning in place solution comprises 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, IM, 1.1 M, 1.2 M, 1.3 M, 1.4 M or 1.5 M sodium hydroxide. In one example, the cleaning in place solution comprises 1 M sodium hydroxide.

[0205] In one example, the cleaning in place solution comprises sodium chloride at a concentration of 1.5 M to 2.5 M. For example, the cleaning in place solution comprises 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M or 2.5 M sodium chloride. In one example, the cleaning in place solution comprises 2M sodium chloride.

[0206] In one example, the cleaning in place solution comprises 1 M sodium hydroxide and 2 M sodium chloride.

[0207] In one example, the filtration medium is cleaned in place with one or more MVs of a cleaning in place solution comprising sodium hydroxide and sodium chloride. For example, the filtration medium is cleaning in place with one or more MVs of a cleaning in place solution comprising 1 M sodium hydroxide and 2 M sodium chloride.

[0208] In one example, the filtration medium is contacted with multiple MVs of a cleaning solution to clean in place the filtration medium. In this regard, a volume of solution approximately equal to the volume of the filtration medium is equal to one MV. In some examples, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 20 or 30 or 40 or 50 MVs of a solution are flowed through the filtration medium. In one example, the filtration medium is contacted with between 40 and 50 MVs of a cleaning solution to clean in place the filtration medium. For example, the filtration medium is contacted with about 45 MVs of a cleaning solution to clean in place the filtration medium.

[0209] In one example, the method disclosed herein further comprises re-equilibrating the filtration medium. For example, the method further comprises re-equilibrating the filtration medium following cleaning in place of the filtration medium.

[0210] In one example, the filtration medium is re-equilibrated with a re-equilibration solution. For example, the filtration medium is cleaned in place with one or more MVs of a re-equilibration solution.

[0211] In one example, the re-equilibration solution comprises sodium chloride.

[0212] In one example, the re-equilibration solution comprises sodium chloride at a concentration of 0.1 M to 1.5 M. For example, the cleaning in place solution comprises 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M or 1.5 M sodium chloride.

[0213] In one example, the filtration medium is re-equilibrated with one or more MVs of a re-equilibration solution comprising sodium chloride.

[0214] In one example, the filtration medium is re-equilibrated with one or more MVs of a first re-equilibration solution comprising sodium chloride and one or more MVs of a second re-equilibration solution comprising sodium chloride. In one example, the first re-equilibration solution comprises a higher concentration of sodium chloride than the second re-equilibration solution. For example, the concentration of sodium chloride in the first re-equilibration solution is at least twice the concentration of sodium chloride in the second re-equilibration solution. In one example, the concentration of sodium chloride in the first re-equilibration solution is three times the concentration of sodium chloride in the second re-equilibration solution. For example, the filtration medium is reequilibrated with one or more MVs of a first re-equilibration solution comprising 1.2 M sodium chloride and one or more MVs of a second re-equilibration solution comprising 0.4 M sodium chloride.

[0215] In one example, the filtration medium is contacted with multiple MVs of a reequilibration solution to re-equilibrate the filtration medium. In this regard, a volume of solution approximately equal to the volume of the filtration medium is equal to one MV. In some examples, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 20 or 30 or 40 or 50 one or more MVs of of a solution are flowed through the filtration medium.

[0216] In one example, the filtration medium is contacted with between 10 and 50 MVs of a re-equilibration solution to re-equilibrate the filtration medium. For example, the filtration medium is contacted with between 10 and 20 MVs of a first re-equilibration solution and between 20 and 30 MVs of a second re-equilibration solution to re- equilibrate the filtration medium. In one example, the filtration medium is contacted with 18 MVs of a first re-equilibration solution and 25 MVs of a second re-equilibration solution to re-equilibrate the filtration medium.

[0217] The present disclosure additionally provides a method of cleaning in place and reequilibrating a filtration medium (i.e., charged depth filter). For example, the method comprises cleaning in place the filtration medium followed by re-equilibrating the filtration medium.

[0218] It will be apparent to the skilled person from the disclosure herein that performing the above cleaning in place and re-equilibration method of the disclosure on the filtration medium between each purification (i.e., batch of cell culture fluid) extends the lifetime of the medium. For example, the method extends the lifetime of the medium or enables reuse of the medium for up to a total of 13 cycles or more. In one example, the method extends the lifetime of the medium or enables reuse of the medium for multiple batches of cell culture fluid.

[0219] The filtration medium may undergo multiple cycles (e.g. 13 cycles) of medium equilibration, loading, binding, washing and / or elution of the enveloped virus, along with cleaning in place of the filtration medium. Multiple batch runs may be performed using the filtration medium. The total lifetime of the medium can be in the range of 1 to 13 cycles (if not more) before the medium is unusable.

[0220] In one example, the filtration medium is operated in continuous mode. For example, the continuous mode is selected from the group consisting of rapid cycling and periodic counter current mode. In one example, the filtration medium is operated in rapid cycling mode. In another example, the filtration medium is operated in periodic counter current mode.

[0221] In another example, the cleaning in place and re-equilibration method is not performed, and the medium is used as a single-use medium.

[0222] Additional steps

[0223] In one example, an enveloped virus eluted from the filtration medium is further purified on the basis of its size. In one example, the buffer in which virus was eluted from the filtration medium, is exchanged more or less at the same time. In the process of the disclosure, tangential flow filtration is preferred. This method permits impurity removal and buffer exchange at almost the same time. The eluted virus can be frozen for subsequent manipulation.

[0224] Tangential flow ultrafiltration / diafiltration is a method which may be used to remove residual protein and nucleic acids as well as for exchanging working buffer into a final formulation buffer. The final formulation buffer may be a cell culture medium, such as X-VIVO 10 medium (available from Lonza), HSC Brew (with or without HSC Brew Supplement) (available from Miltenyi Biotec), StemPro-34 (with or without StemPro-34 Supplement), StemPro HSC (with or without StemPro-HSC Expansion Supplement) (available from ThermoFisher), or stem cell growth medium (SCGM) (available from CellGenix). Ultrafiltration using tangential flow is preferred and different devices can be used (e.g. Proflux and LAB SCALE (ultrafiltration system) TFF System, both Millipore or the KR2i system from Repligen). The particular ultrafiltration membrane selected will be of a filter pore size sufficient small to retain enveloped virus but large enough to allow penetration of impurities. Depending on the manufacturer and membrane type, nominal molecular weight cut-offs between 100 and 1000 kDa may be appropriate (e.g. UFP-750-E-5A, GE Healthcare; BIOMAX. (ultrafiltration device) NMWC 1000, Millipore). In one example, the molecular weight cut-off is 500kDa. The membrane composition may be, but it is not limited to, regenerate cellulose, (modified) polyethersulfone, poly sulfone. Membranes can be of flat sheet or hollow fibre type. The main parameters that must be optimized are flux rate and trans-membrane pressure. In combination with nominal molecular weight cut-off these two parameters will enable efficient purification and buffer exchange and high virus yield. The diluted eluate or final retentate can be frozen for subsequent manipulation.

[0225] As an additional step sterile filtration may be performed to eliminate bioburden. Therefore, diluted eluate or final retentate from the ultrafiltration step may be filtered through a filter, for example a 0.22 pm filter. The filter may be constructed from various materials, which may include but are not limited to polypropylene, hydrophilic PVDF, cellulose, hydrophilic regenerated cellulose, cellulose esters, wetting agent-free cellulose acetate, cellulose acetate, nylon, hydrophilic nylon membrane, polyethersulfone, hydrophilic polyethersulfone, hydrophilic asymmetric PES, or any other material which is consistent with low unspecific influenza virus binding. The filter may have a single membrane layer or more than one layer or may incorporate a prefilter of the same or different material, for example a 0.45 pm prefilter. The sterile filtrated virus can be held frozen for subsequent manipulation.

[0226] In one example, the sterile filter has a filtration area of at least 15 cm2. For example, the sterile filter has a filtration area of about 17.8 cm2or about 20 cm2. In one example, the sterile filter has a filtration area of at least 200 cm2. For example, the sterile filter has a filtration area of about 210 cm2or about 220 cm2.

[0227] In one example, the sterile filter has a filtration capacity of at least 2.5 mL / cm2. For example, the sterile filter has a filtration capacity of at least 4.0 mL / cm2. The present disclosure is further defined in the following numbered paragraphs. Unless this would result in a contradiction, the embodiments of the following paragraphs can be combined with any of the above examples and provide further details on certain aspects of the disclosure.

[0228] 1. A method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a charged depth filtration medium.

[0229] 2. The method of paragraph 1, wherein the method comprises eluting the enveloped virus from the charged depth filtration medium.

[0230] 3. The method of paragraph 1, wherein the filtration medium is an anionic charged depth filtration medium.

[0231] 4. The method of any one of paragraphs 1 to 3, wherein the filtration medium comprises a non-woven substrate and a plurality of quaternary ammonium monomers.

[0232] 5. The method of paragraph 4, wherein the filtration medium comprises a hydrogel.

[0233] 6. A method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a filtration medium comprising a non-woven substrate and a plurality of quaternary ammonium monomers.

[0234] 7. The method of any one of paragraphs 4 to 6, wherein the plurality of quaternary ammonium monomers is grafted to the surface of the non-woven substrate as a copolymer comprising interpolymerized monomer units of the quaternary ammonium monomers, thereby producing a co-polymer grafted non-woven substrate.

[0235] 8. The method of paragraph 7, wherein the grafted co-polymer further comprises an amide monomer and / or an oxy monomer.

[0236] 9. The method of paragraph 8, wherein the grafted copolymer comprises interpolymerized monomer units of: a) 10 to 50 parts by weight of the quaternary ammonium monomer; b) 10 to 80 parts by weight of the amide monomer; and c) 10 to 40 parts by weight of the oxy monomer; wherein the sum of a) to c) is 100 parts by weight.

[0237] 10. The method of any one of paragraphs 4 to 9, wherein the quaternary ammonium monomers comprise methacrylamidopropyltrimethylammonium chloride (MAPTAC).

[0238] 11. The method of paragraph 9 or paragraph 10, wherein the amide monomer is N- vinyl pyrrolidone (NVP) and / or wherein the oxy monomer is glycidyl methacrylate (GMA).

[0239] 12. The method of any one of paragraphs 4 to 11, wherein the non-woven substrate comprises a thermoplastic polymeric material.

[0240] 13. The method of paragraph 12, wherein the thermoplastic polymeric material is a hydrophobic thermoplastic polyolefin.

[0241] 14. The method of paragraph 13, wherein the polyolefin is poly(propylene).

[0242] 15. The method of any one of paragraphs 7 to 13, wherein the non-woven substrate has one or more of the following properties: a) a tensile strength of at least 4.0 newtons prior to grafting of the co-polymer; b) a surface area of 15 to 50 m2per square meter of nonwoven substrate; c) a mean pore size of 1-40 microns; d) a solidity of less than 20%; and e) an effective fiber diameter of about 3 to about 10 mm.

[0243] 16. The method of any one of paragraphs 7 to 15, wherein the grafted co-polymer is 0.5 to 3 times the weight of the non-woven substrate.

[0244] 17. The method of any one of paragraphs 7 to 16, wherein the filtration medium comprises at least four layers of the co-polymer grafted non-woven substrate.

[0245] 18. The method of paragraph 17, wherein each downstream layer of the co-polymer grafted non-woven substrate has a smaller effective fiber diameter. 19. The method of any one of paragraphs 4 to 18, wherein the filtration media further comprises a non-functionalized substrate downstream of the non-woven substrate.

[0246] 20. The method of paragraph 19, wherein the non-functionalized substrate comprises a microporous, non-functionalized size-exclusion membrane.

[0247] 21. The method of paragraph 20, wherein the non-functionalized size-exclusion membrane comprises an asymmetric pore structure.

[0248] 22. The method of paragraph 20 or paragraph 21 , wherein the non-functionalized size exclusion membrane comprises a gradient or a multizone pore morphology, wherein the pore size decreases from the upstream surface toward the downstream surface.

[0249] 23. The method of any one of paragraphs 20 to 22, wherein the non-functionalized size-exclusion membrane comprises a 0.2-micron rated, asymmetric, non- functionalized polyamide size-exclusion membrane.

[0250] 24. The method of any one of paragraphs 1 to 23, wherein the filtration medium is configured in a filter unit as a planar or lenticular disk.

[0251] 25. The method of any one of paragraphs 1 to 24, wherein the filtration medium is pleated.

[0252] 26. The method of any one of paragraphs 1 to 25, wherein the filtration medium has a volume of at least about 1 mL per L of the cell culture fluid and / or the filtered cell culture fluid.

[0253] 27. The method of any one of paragraphs 1 to 26, wherein the cell culture fluid is harvested from stable producer cells.

[0254] 28. The method of any one of paragraphs 1 to 27, wherein the cell culture fluid or the filtered cell culture fluid is contacted with an endonuclease prior to purifying the virus.

[0255] 29. The method of paragraph 28, wherein the endonuclease is a non-specific endonuclease and degrades both DNA and RNA without sequence specificity. 30. The method of paragraph 28 or paragraph 29, wherein the endonuclease is from Serratia marcescens, Anabaena sp., Saccharomyces cerevisiae, Bos Taurus, Syncephalostrum racemosum and / or Borrelia burgdorferi .

[0256] 31. The method of any one of paragraphs 28 to 30, wherein the endonuclease is a Serratia nuclease, NucA, Nucl, endonuclease G, DNase I, or micrococcal nuclease.

[0257] 32. The method of any one of paragraphs 28 to 31, wherein the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of 0.001 to 100 units / mL of cell culture fluid or filtered cell culture fluid.

[0258] 33. The method of paragraph 32, wherein the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of about 1 unit / mL of cell culture fluid or filtered cell culture fluid.

[0259] 34. The method of paragraph 32, wherein the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of about 5 unit / mL of cell culture fluid or filtered cell culture fluid.

[0260] 35. The method of paragraph 32, wherein the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of about 30 unit / mL of cell culture fluid or filtered cell culture fluid.

[0261] 36. The method of any one of paragraphs 1 to 35, wherein the method further comprises adjusting a concentration of Mg2+in the cell culture fluid or the filtered cell culture fluid to about 1-3 mM prior to purifying the virus.

[0262] 37. The method of paragraph 36, wherein the concentration of Mg2+in the cell culture fluid or the filtered cell culture fluid is adjusted to about 2 mM.

[0263] 38. The method of any one of paragraphs 27 to 37, wherein the cell culture fluid is harvested from cells cultivated in an adherent environment or from cells cultivated in a suspension environment. 39. The method of any one of paragraphs 1 to 38, wherein the method comprises performing a harvest filtration on the cell culture fluid to produce the filtered cell culture fluid before subjecting the filtered cell culture fluid to the filtration medium.

[0264] 40. The method of any one of paragraphs 1 to 39, wherein the filtered cell culture fluid is contacted with a high concentration salt solution to form a salt-spiked cell culture fluid prior to or during loading on to the filtration medium.

[0265] 41. The method of paragraph 40, wherein the high concentration salt solution and the filtered cell culture fluid are mixed in-line.

[0266] 42. The method of paragraph 40 or paragraph 41, wherein the high concentration salt solution comprises a monovalent and / or a divalent salt.

[0267] 43. The method of paragraph 42, wherein the monovalent salt is sodium chloride.

[0268] 44. The method of any one of paragraphs 42 to 43, wherein the high concentration salt solution is at a concentration of 5M.

[0269] 45. The method of any one of paragraphs 42 to 44, wherein the salt-spiked cell culture fluid has a target conductivity of 38 to 44 mS / cm at 25°C.

[0270] 46. The method of any one of paragraphs 1 to 45, wherein the method further comprises washing the filtration medium with one or more wash solutions.

[0271] 47. The method of paragraph 46, wherein the one or more wash solutions comprises a salt solution comprising a monovalent and / or a divalent salt.

[0272] 48. The method of paragraph 47, wherein the monovalent salt is sodium chloride.

[0273] 49. The method of paragraph 47 or paragraph 48, wherein the salt solution is at a concentration of between 0.1M and IM.

[0274] 50. The method of paragraph 49, wherein the salt solution is at a concentration of 0.6M. 51. The method of any one of paragraphs 1 to 50, wherein the method further comprises eluting bound enveloped virus from the filtration medium with an elution solution.

[0275] 52. The method of paragraph 51, wherein the elution solution comprises a buffer selected from the group consisting of Tris, histidine and HEPES.

[0276] 53. The method of paragraph 51 or paragraph 52, wherein the elution solution comprises a buffer at a concentration of 10 mM to 50 mM.

[0277] 54. The method of any one of paragraphs 51 to 53, wherein the elution solution has a pH of between 5 and 10.

[0278] 55. The method of any one of paragraphs 51 to 54, wherein the elution solution comprises: a) 50 mM Tris, pH8.0; b) 10 mM histidine, pH7.0; or c) 10 mM HEPES, pH7.5.

[0279] 56. The method of any one of paragraphs 51 to 55, wherein the elution solution comprises 10 mM HEPES at pH 7.5.

[0280] 57. The method of any one of paragraphs 51 to 56, wherein the elution solution further comprises a monovalent and / or a divalent salt.

[0281] 58. The method of paragraph 57, wherein the monovalent salt is sodium chloride.

[0282] 59. The method of paragraph 57 or paragraph 58, wherein the salt is at a concentration of between 0.5M and 2M.

[0283] 60. The method of paragraph 59, wherein the salt is at a concentration of 1 ,2M.

[0284] 61. The method of any one of paragraphs 1 to 60, wherein the method increases the virus infectious titer yield by at least 10%. 62. The method of any one of paragraphs 51 to 61, wherein the method results in a viral infectious titer yield of at least 1 x 108transducing units (TU) / mL of eluted virus.

[0285] 63. The method of any one of paragraphs 51 to 62, wherein the method results in a residual dsDNA content of less than 100 pg / mL of eluted virus.

[0286] 64. The method of any one of paragraphs 51 to 63, wherein the method further comprises diluting the eluted virus with histidine, Tris or HEPES.

[0287] 65. The method of paragraph 64, wherein the eluted virus was diluted to achieve a salt concentration of 150 mM.

[0288] 66. The method of any one of paragraphs 51 to 65, wherein the method further comprises concentrating and / or diafiltering the eluted virus or the diluted eluted virus.

[0289] 67. The method of any one of paragraphs 1 to 66, wherein the enveloped virus is a retrovirus.

[0290] 68. The method of paragraph 67, wherein the retrovirus is a lentivirus.

[0291] 69. The method of any one of claims 1 to 68, wherein the cell culture fluid and / or the filtered cell culture fluid 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.

[0292] 70. The method of any one of paragraphs 1 to 69, additionally comprising formulating the enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

[0293] 71. A purified enveloped virus produced by the method according to any one of paragraphs 1 to 69.

[0294] 72. A method of purifying an enveloped virus from a cell culture fluid, comprising:

[0295] (i) providing a cell culture fluid comprising viral vector produced from a stable producer cell line;

[0296] (ii) contacting the cell culture fluid with an endonuclease; (iii) contacting the endonuclease treated cell culture fluid to a filter to produce a filtered cell culture fluid;

[0297] (iv) loading the filtered cell culture fluid and a high concentration salt solution to a charged depth filtration medium; (v) optionally, washing the filtration medium with one or more wash solutions;

[0298] (vi) eluting the bound enveloped virus from the filtration medium with an elution solution;

[0299] (vii) diluting the eluted virus with a buffer; and

[0300] (viii) concentrating and diafiltering the eluted virus.

[0301] The present disclosure is described further in the following non-limiting examples.

[0302] EXAMPLES

[0303] Example 1: Alternative purification methods of an enveloped virus

[0304] The purification step was identified as the step with the largest viral yield losses. Accordingly, alternative purification methods were compared to the existing Mustang® Q membrane adsorber, strong anion exchange purification step, including:

[0305] • alternative membrane adsorber, strong anion exchangers (Sartobind® Q)

[0306] • membrane adsorber, weak anion exchanger (Sartobind® D)

[0307] • monolithic column, strong anion exchanger (CIM® QA)

[0308] • monolithic column, weak anion exchanger (CIM® DEAE)

[0309] • hybrid woven filter matrix with anion exchange function (Emphaze®)

[0310] All devices were loaded with IL of filtered cell culture harvest comprising enveloped virus. The differing process parameters are set out in Table 1.

[0311] Table 1: Process parameters

[0312] Analytical analyses included p24, infectious titer and total DNA. As the load volume and material was the same for all experiments, the absolute numbers can be compared.

[0313] As shown in Figure 1, infectious titer results were higher for Sartobind® Q, CIM® QA and Emphaze® than for Mustang® Q baseline. Sartobind® D weak anion exchanger showed the worst performance. CIM® DEAE was similar to Mustang Q. p24 results were similar for all membranes tested, except Sartobind® D which was about 30 - 40 % lower. DNA results varied widely and were best for CIM® QA and Emphaze®. Mustang® Q. Both weak anion exchangers were similar, with Sartobind® Q 50 % worse than Mustang® Q.

[0314] A notable difference was observed for the Emphaze® device. The UV traces of the chromatography run were significantly smaller compared to the Mustang® Q run (Figure 2), despite that the virus concentration was similar according to p24 ELISA, which suggests that less UV-absorbing species (impurities of media components) were co-purified.

[0315] Furthermore, the observed pressure across the Emphaze® was comparably low.

[0316] In a second experiment (data not shown), the parameters for the Emphaze® were optimized to assess the device’s potential. In particular, a higher load flow rate of 5 MV / min was used successfully without pressure issues. Infectious titre yield was 58 % and p24 yield was 75 % for elution at 1.0 M NaCl. No further virus was found in the 1.5 M NaCl fraction. The material was successfully processed on a TFF (62 % TU yield).

[0317] Example 2: Purification of enveloped virus from suspension and adherent cell cultures using charged depth filtration

[0318] Methods

[0319] Cell culture and lentivirus production

[0320] Cells were grown in adherent or suspension bioreactors and virus production induced.

[0321] For adherent cells, cell growth and lentiviral production was performed in one or two 10-layer cell stacks and up to 8 harvests were collected. In the 2 first production campaigns no nuclease treatment was performed. In the subsequent productions, each harvest was treated with 0.3 U / mL of benzonase after addition of MgCL for a final concentration of 2 mM. Clarification was performed with 300 cm2Sartopore 2, 0.8 + 0.45 pm capsule filters at a flow rate of 50 mL / min.

[0322] For suspension cells, cell growth and lentiviral production was performed in a bioreactor with appropriate cell culture fluid. Benzonase and MgCh were added to a final concentration of 0.3 U / ml and 2 mM, respectively, before filtration.

[0323] Purification

[0324] Chromatography was performed in batch mode, rapid cycling (RC) mode, or periodic counter current (PCC) mode. In batch mode, the harvest material is all loaded onto a chromatography column large enough to purify the material in a single run. RC purifies a batch over a large number of very short cycles instead of running a single long purification cycle, using a smaller chromatography column. In RC mode, the column is loaded, eluted, washed, and re-equilibrated multiple times until all of the harvest material has gone through the column. In PCC mode, multiple columns operate in parallel cycling through the chromatography program such that there is always one column in the loading phase at any given time, and feedstock can be continuously loaded.

[0325] Chromatography was performed on Akta Avant 150, or Avant 25 systems controlled by Unicorn software (Cytiva) except for continuous periodic counter current (PCC) runs that were performed on an in-house modified Akta Explorer 100. On Akta Avant 150 and Avant 25, flow restrictor was set off-line and, on Akta Explorer 100, no restrictor was used.

[0326] Three anion exchange chromatography (AEX) membranes were used: 0.86 mL Mustang® Q, 3 and 1 mL Sartobind® Q and 1.4 and 8 mL Emphaze® AEX Hybrid Purifier. Prior to chromatography, membranes were prepared according to the manufacturers’ instructions and equilibrated with buffer until pH and conductivity traces were stable.

[0327] For adherent cells, chromatography was run at 10 membrane volumes (MV) / min for Mustang® Q, 5 MV / min for Sartobind® Q, 3 mL, 15 MV / min for Sartobind® Q, 1 mL, and 5 MV / min for 8 mL Emphaze®. Buffer systems include 50 mM Tris, pH 8.0, 10 mM Histidine, pH 7.0 and 10 mM Hepes, pH 7.5.

[0328] Clarified stocks were NaCl spiked with a 5 M solution (63 mL / kg clarified) to increase conductivity and match NaCl concentration of AEX equilibration buffers (0.4 M NaCl). Virus elution was performed by stepwise increase of NaCl concentration. Eluates were immediately diluted with cold (4 - 8 °C) buffer to reach a final NaCl concentration of 150 mM and frozen at - 80 °C. In two runs, eluates were not diluted or diluted to final NaCl concentrations of 500, 250 and 150 mM and a fraction of these solutions was immediately frozen while the other fraction was frozen 90 min after dilution to estimate virus stability.

[0329] For suspension cells, chromatography was performed in 10 mM Hepes, pH 7.5 buffer using 0.86 mL Mustang® Q and 1.4 mL Emphaze® membranes run at 10 and 3.4 MV / min (8.6 and 5 mL / min), respectively. Spiking was performed off-line (as in adherent cultures) in the case some of rapid cycling experiments whereas, in others and in PCC runs, in-line spiking was used. Elution from Mustang® Q was performed with 1.5 M NaCl after a washing step with 0.75 M NaCl and, from Emphaze, elution was accomplished with a 1.2 M NaCl step. As in adherent cell runs, eluates were immediately diluted with cold (4 - 8 °C) buffer to reach a final NaCl concentration of 150 mM and frozen at - 80 °C.

[0330] The methods used for continuous purification of suspension cells with Mustang® Q and Emphaze®, operated in PCC or RC modes included several steps: membrane loading, washing of unbound material with equilibration 0.4 M NaCl buffer, elution steps at ~0.6 and 1.5 M NaCl for Mustang® Q or at 1.2 M NaCl for Emphaze®, cleaning in place (CIP) with 1 M NaOH, 2 M NaCl solution and re-equilibration of the membrane (see Table 2). To decrease pH more rapidly after CIP, re-equilibration was performed initially with high NaCl concentration buffer and then with 0.4 M NaCl equilibration buffer.

[0331] Table 2 - Process parameters

[0332] Tangential flow filtration

[0333] Following elution, tangential flow filtration (TFF) was performed. TFF was performed using a peristaltic Sartoflow 200 pump equipped with a Tandem 1081 head. Hollow fibers from Repligen (MicroKros, Polysulfone, 500 kDa MWCO, 0.5 mm id, 28 cm2area) and from Cytiva (Polysulfone, 750 kDa MWCO, 0.5 mm id, 26 cm2) and also cassettes from Millipore (Polyethersulfone, 300 kDa MWCO, 50 cm2) were evaluated.

[0334] Each device was sanitized according to the manufacturers’ instructions and water flushed until a volume of 2 mL / cm2permeated the membrane. For Cytiva hollow fibers, clean water permeability was determined from the slope of permeate flow rate vs transmembrane pressures plot.

[0335] Before concentration and diafiltration of diluted eluate, TFF devices were equilibrated with the same buffer used for purification but with a NaCl concentration of 150 mM except in two runs in which hollow fibers were equilibrated with X-Vivo 10 growth medium, the final formulation solution. After concentration to the minimum volume possible, and diafiltered with 7 volumes of X-Vivo 10. For the hollow fibers, feed flow rate was set to achieve shear rates of 1200, 4000 or 8000 s-1 and, for cassettes, the feed flow rate was 40 mL / min as indicated by the manufacturer. Transmembrane pressures (TMP) between 0.2 and 0.6 bar were set by partially closing a pinch valve in the retentate line. In one experiment, permeate flow rate was controlled to be 10 % of the feed flow rate using a peristaltic pump in the permeate line.

[0336] Sterilizing filtration

[0337] Two filters were evaluated for the final sterilizing filtration step: Mini Kleenpak, 0.2 pm polyethersulfone EKV Supor syringe filter and Millex-GP, 0.22 pm poly ethersulfone syringe filter. The filters were equilibrated with 2 mL / cm2of formulation solution (X-Vivo 10 without gentamycin or phenol red growth medium) before virus filtration.

[0338] Titration (infectious and total particle concentration)

[0339] Infectious and total concentration of lentiviral vectors from adherent and suspension cultures was performed.

[0340] Both vectors harbor a GFP encoding gene and infectious particles were determined by FACS and are represented in transducing units (TU) / mL.

[0341] For lentiviral from adherent cultures, total particle content was determined by quantifying the virus-associated p24 protein using a commercial ELISA kit.

[0342] For lentiviral from suspension cultures, total particles concentration was determined by quantification of RNA content by ddPCR after a reverse transcriptase step.

[0343] Total protein was assessed with PierceTM BCA assay kit, (ThermoFisher) and total DNA was quantified with Quant-iT PicoGreen dsDNA assay kit, (ThermoFisher).

[0344] Results

[0345] Infectious viral particles from adherent cultures

[0346] Different AEX membranes and buffer systems were evaluated for the purification of lentiviral vector from adherent cultures (Table 3).

[0347] (1) Membranes and volumes: MSTG - Mustang® Q, 0.86 mL; EMPH - Emphaze®, 8 mL; SART - Sartobind® Q, 3 mL; (2) Sartobind® Q membrane, 1 mL

[0348] The baseline process (Run 1) was performed with Mustang® Q and 50 mM Tris, pH 8.0 and elution with 1.5 M NaCl after a washing step with 0.75 M NaCl. The process was successfully implemented and a TU yield of 38 ± 12 % was obtained (Figure 3). With the same buffer, Emphaze® and Sartobind® Q membranes were performed (Runs 2 and 3, respectively). Elution steps of Sartobind® Q were equal to those of Mustang® Q while, for Emphaze®, no washing step was performed, and bound virus eluted with 1.0 M NaCl. TU yield with Emphaze® was similar to that of Mustang® Q (47 ± 21 %) but a higher recovery was observed with Sartobind® Q (66 ± 26 %) (Figure 3).

[0349] After AEX, eluates were diluted to achieve a final NaCl concentration of 150 mM and loaded onto the TFF feed vessel. In the baseline process (run 1), concentration and formulation was performed with a Repligen hollow fiber with 500 kDa cut-off, an area of 28 cm2and an inner diameter of 0.5 mm (Table 4). The feed flow rate was set to achieve a shear rate of 1200 s-1 and the transmembrane pressure (TMP) was adjusted to 0.3 bar by partially closing a pinch valve in the retentate line. Eluate from Mustang® Q run with 50 mM Tris, pH 8.0 was concentrated 20-fold, diafiltered with 7 volumes of X- Vivo 10 and a recovery yield of 48 ± 13 % was obtained (Figure 4).

[0350] Experiments with a shear rate of 4000 s-1 were also performed (runs 11, 12, 13, 15) both with Repligen hollow fiber as well as with a Cytiva hollow fiber with 750 kDa cut-off, an area of 26 cm2and an inner diameter of 0.5 mm. Using Histidine buffer (run 11) and Repligen hollow fiber at 0.3 bar TMP, a VCF of ~ 30 was achieved and recoveries of Cal-H of 72 ± 12 were obtained. An experiment was performed with the permeate flow rate controlled at 3 mL / min which corresponds to 10 % of the feed flow rate (run 12). TMP increased from 0.2 and 0.6 bar and, after that, the permeate pump was removed (pressure in the permeate line reached 0 bar). TMP continued to increase up to 0.8 bar during concentration and, during diafiltration, a value of 0.7 bar was obtained. This experiment was performed in Hepes buffer and 76 ± 17 % of virus were recovered. Two experiments at 4000 s-1 shear rate were performed in which hollow fibers were equilibrated with X-Vivo 10 before feeding diluted eluates from Sartobind Q run in Histidine or Hepes buffer (runs 13 and 15, respectively). A Cytiva 750 kDa hollow fiber and a Repligen 500 kDa hollw fiber were used being the TMP set at 0.8 bar and recovery yields of 57 ± 6 and 106 ± 20 % were obtained.

[0351] Shear rates of 8000 s-1 were also assayed to concentrate and formulate virus eluted from Emphaze membranes. Hollow fibers from Cytiva using Histidine or Hepes buffers and with a Repligen hollow fiber using Hepes buffer setting the TMP at 0.5 bar (runs 14, 19 and 16, respectively). Yields of infectious particles from Cytiva fibers with Histidine or Hepes buffer were 99 ± 16 and 62 ± 15 %, respectively. Recovery from Repligen fiber was 73 ± 12 %.

[0352] After formulation, the solution was sterile filtered using EKV or Millex-GP syringe filters. In general, higher than 80 % TU yield was obtained (Figure 5). Excluding run 16 in which yields were lower than 60 %, an average recovery of 88 ± 7 % can be calculated for the sterilizing filtration step.

[0353] In summary, lentivirus production from adherent cells and purification processes as well as the required analytical methods were successfully implemented. The performance of three anion exchange membranes, Mustang Q, Sartobind Q and Emphaze, was evaluated using different buffers at pH values 8.0, 7.5 and 7.0. In the baseline process, using Mustang Q with Tris buffer at pH 8.0, a TU yield of - 40 % was obtained. With the same experimental conditions, Emphaze and Sartobind Q showed elution yields around 50 and 65 %, respectively. However, by decreasing the pH to 7.0 (Histidine buffer), recoveries improved to - 60 % for Mustang Q and - 70 % for Emphaze. Results from Sartobind Q at pH 7.0 were not much improved with yields ranging from 50 to 60 %. Similar results were also obtained with Emphaze and Sartobind Q at pH 7.5 (Hepes buffer). In summary, consistent results were obtained, particularly for Mustang and Emphaze membranes, showing that lowering the pH to 7.0 or 7.5 results in a greater than 20% improvement in recovery yield (Table 4). Table 4 - Summary of infectious titre yields

[0354] For formulation, Repligen, 500 kDa hollow fiber, Cytiva, 750 kDa hollow fiber and a Millipore, 300 kDa cassette were evaluated. Cassettes’ performance was much worse than that of hollow fibers. TFF results were less reproducible but, experiments performed at different shear rates and buffers seem to indicate that shear rates between 4000 and 8000 s-1 in Histidine or Hepes buffer led to more than 70 % infectious recoveries.

[0355] Two filters were used for the final sterilizing step and around 90 % recovery of infectious particles was observed.

[0356] Infectious viral particles from suspension cultures

[0357] AEX using Emphaze or Mustang Q membranes operated in rapid cycling (RC) or periodic counter current (PCC) modes was performed as shown in Table 5.

[0358] Table 5 - Process parameters

[0359] (1) Membranes and volumes: MSTG - Mustang Q, 0.86 mL run at 8.6 mL / min; EMPH - Emphaze, 1.4 mL run at 5 mL / min; (2) RC - Rapid cycling; PCC - Periodic counter current with 3 membranes Performance of Emphaze in RC mode to purify lentivirus was evaluated three times (runs 34, 39 and 43). In the first experiment (run 34), 3 cycles were completed, and no pressure increase was observed over the cycles. In respect to the spiked load, TU recoveries of 92 ± 20, 77 ± 17 and 71 ± 16 % were determined for the elution peak from cycles 1 to 3, respectively. Regarding total viral particles, an average yield of 62 ± 12 % was obtained.

[0360] Emphaze RC operation for higher number of cycles was assessed (run 39), and different results were obtained depending on membrane orientation: when membrane inlet was facing down, TU and RNA yields (relative to clarified harvest) of 42 ± 10 and 59 ± 5 % were obtained, respectively; in the case membrane inlet was facing up, the yields were 24 ± 6 and 40 ± 4 %. In this experiment, clarified harvest and AEX load (i.e., NaCl spiked clarified) were titrated and no losses were observed for the RNA content but TU yields of 42 ± 10 and 24 ± 8 % were determined for the first and sixth loads, respectively.

[0361] An Emphaze RC run was performed with in-line NaCl spiking (run 43). In opposition to the previous run (in which chromatograms of different cycles were very reproducible), in run 43, elution peaks were similar up to cycle 6 but decreased and showed an increasing shoulder in the following cycles (Figure 6). A slight increase in the differential pressure was observed (up to 0.1 MPa) during the 13 run cycles but below the 0.24 MPa limit of the membrane. RNA recovery yield in the elution peaks was reproducible for the different cycles (only 10 first cycles were analyzed) with an average value of 50 ± 5 %. Regarding TU yield, there was an apparent decrease after the 5th cycle from ~25 % for elutions 1, 3 and 4 to values around 10 % for elutions 6 to 10.

[0362] The performance of Emphaze running in PCC mode was also evaluated (run 38) and it was possible to run 2 cycles with 3 membranes. There was an increase in the system pressure (0.4 MPa) but without compromising the experiment accomplishment. Reproducible TU and RNA yield were determined for the elution of the different switch intervals with average values of 22 ± 5 and 49 ± 9 %, respectively.

[0363] By evaluating RNA concentration in different flowthrough fractions of Emphaze, the DBC io% was determined to be 1.3 x 1013RNA copies / mL of Emphaze was calculated for the (run 41). Correcting for the amount of virus in the membrane’s flowthrough, a recovery of 52 ± 5 % RNA copies was determined.

[0364] Regarding impurities, almost all protein elutes in membrane’s flowthrough and elution fractions of Emphaze contain approximately 24 ± 5 % of residual dsDNA, both when operated in RC or PCC mode. Two purification runs were also performed using Mustang Q operated in RC mode (runs 36 and 42). Three cycles were performed with off-line NaCl spiking in the first experiment (run 36) and no pressure increase was observed. The 1.5 M NaCl elution peaks were superimposable and TU and RNA recoveries, in respect to spiked load, were 41 ± 3 and 23 ± 2, respectively. In the second experiment (run 42), performed with inline NaCl spiking of the clarified thawed harvest, pressure increased gradually, especially for the 5th cycle and exceeded the maximum value during the 8th load, thus, seven complete cycles were run. Elution peaks of the 1.5 M NaCl step were also decreasing in cycles 5, 6 and 7 in comparison to peaks from the initial cycles. Average recovery TU yield was 27 ± 1 % in cycles 1 to 4 and decreased to 20 ± 1 % in 5 to 7 cycles. As regards RNA yield, an average value of 31 ± 3 % was obtained.

[0365] The use of Mustang Q in PCC mode was evaluated (run 35) and 2 cycles with 3 membranes were performed and an increase in system pressure of 0.1 MPa was observed (Figure 7). Chromatograms were not reproducible, but recoveries were similar for the several elution fractions with average values of 38 ± 9 and 27 ± 5 % for TU and RNA copies, respectively. DBCio% of Mustang Q was determined as 2.9 x 10° RNA copies / mL of Mustang Q (run 40). A recovery of 51 ± 6 % RNA copies was determined, taking into account the virus in the membrane’s flowthrough.

[0366] As observed with Emphaze, no residual protein is found in elution peaks of Mustang Q operated either in RC or PCC mode, however, lower dsDNA removal was obtained with residual amounts varying between 30 and 60 %.

[0367] In summary, purification by continuous chromatography was evaluated using Mustang Q and Emphaze membranes. It was observed that the lentivirus purified is sensitive to NaCl concentration, as spiking the clarified harvest with NaCl to a final concentration of 0.4 M resulted in the loss of at least 60% of infectious particles. Similar results were obtained for both membranes in PCC or RC modes.

[0368] Pressure issues were more evident with Mustang Q with which no more than 7 RC cycles were possible to be performed while with Emphaze, after 13 RC cycles, pressure had not reached the maximum operation limit. Although consistent results were obtained for the elution of the different cycles in one of the two RC -Emphaze experiments, the other run, which used in-line spiking, showed a decrease in TU recovery from ~ 25 to 10 % after the 5th cycle. For Mustang Q, results obtained indicate a slightly better performance as ~ 27 % TU recovery in the initial 4 cycles and ~ 20 % for the remaining cycles were found. Regarding RNA copies yield, ~ 50 and 30 % were obtained for Emphaze and Mustang Q, respectively. Considering impurity clearance, higher dsDNA removal was obtained with Emphaze. Example 3: Large scale purification of enveloped virus using charged depth filtration

[0369] Suspension cell culture and lentivirus production was performed as described in Example 2.

[0370] Purification was performed as previously described in Example 2 without a wash step using a 60 mL Emphaze® membrane, compared to a 60 mL Mustang® Q membrane adsorber.

[0371] The buffers used were:

[0372] • Equilibration: 10 mM HEPES, 150 mM NaCl, 7.5 pH;

[0373] • Elution: 10 mM HEPES, 1200 mM NaCl, 7.5 pH; and

[0374] • Dilution: 10 mM HEPES, 7.5 pH.

[0375] Eluates were analyzed for total yield (by ddPCR), volume-adjusted yield (by ddPCR), and RNA content, as described in Example 2.

[0376] As shown in Figure 8, the process was successfully implemented and a higher TU yield of 61% was achieved using the Emphaze® membrance compared to 43% with the Mustang® Q membrane, with similar volume adjusted yields. Regarding RNA copies yield, ~ 38 and 49 % were obtained for Emphaze® and Mustang® Q, respectively.

[0377] Purification was also performed as previously described in Example 2 without a wash step using a 120 mL Emphaze® membrane, compared to a 60 mL Mustang® Q membrane adsorber.

Claims

CLAIMS1. A method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a charged depth filtration medium.

2. The method of claim 1, wherein the method comprises eluting the enveloped virus from the charged depth filtration medium.

3. The method of claim 1, wherein the filtration medium:(i) is an anionic charged depth filtration medium;(ii) comprises a non-woven substrate and a plurality of quaternary ammonium monomers; and / or(iii)comprises a hydrogel.

4. A method of purifying an enveloped virus from a cell culture fluid or a filtered cell culture fluid, comprising subjecting the cell culture fluid or the filtered cell culture fluid to a filtration medium comprising a non-woven substrate and a plurality of quaternary ammonium monomers.

5. The method of claim 3, wherein the plurality of quaternary ammonium monomers is grafted to the surface of the non-woven substrate as a co-polymer comprising interpolymerized monomer units of the quaternary ammonium monomers, thereby producing a co-polymer grafted non-woven substrate.

6. The method of claim 5, wherein the grafted co-polymer further comprises an amide monomer and / or an oxy monomer; and / or the grafted copolymer comprises interpolymerized monomer units of: a) 10 to 50 parts by weight of the quaternary ammonium monomer; b) 10 to 80 parts by weight of the amide monomer; and c) 10 to 40 parts by weight of the oxy monomer; wherein the sum of a) to c) is 100 parts by weight.

7. The method of claim 3, wherein the quaternary ammonium monomers comprise methacrylamidopropyltrimethylammonium chloride (MAPTAC).

8. The method of claim 6, wherein the amide monomer is N-vinyl pyrrolidone (NVP) and / or wherein the oxy monomer is glycidyl methacrylate (GMA).

9. The method of claim 5, wherein the non-woven substrate:(i) comprises a thermoplastic polymeric material; and / or(ii)has one or more of the following properties: a) a tensile strength of at least 4.0 newtons prior to grafting of the co-polymer; b) a surface area of 15 to 50 m2per square meter of nonwoven substrate; c) a mean pore size of 1-40 microns; d) a solidity of less than 20%; and e) an effective fiber diameter of about 3 to about 10 mm.

10. The method of claim 9, wherein the thermoplastic polymeric material is a hydrophobic thermoplastic polyolefin.

11. The method of claim 10, wherein the polyolefin is poly(propylene).

12. The method of claim 5, wherein:(i) the grafted co-polymer is 0.5 to 3 times the weight of the non-woven substrate; and / or(ii)the filtration medium comprises at least four layers of the co-polymer grafted nonwoven substrate.

13. The method of claim 12, wherein each downstream layer of the co-polymer grafted non-woven substrate has a smaller effective fiber diameter.

14. The method of claim 3, wherein the filtration media further comprises a nonfunctionalized substrate downstream of the non-woven substrate.

15. The method of claim 14, wherein the non-functionalized substrate comprises a microporous, non-functionalized size-exclusion membrane.

16. The method of claim 15, wherein the non-functionalized size-exclusion membrane comprises:(i) an asymmetric pore structure; and / or(ii) a gradient or a multizone pore morphology, wherein the pore size decreases from the upstream surface toward the downstream surface; and / or(iii)a 0.2-micron rated, asymmetric, non- functionalized polyamide size-exclusion membrane.

17. The method of claim 1, wherein the filtration medium is:(i) configured in a filter unit as a planar or lenticular disk; and / or(ii)is pleated.

18. The method of claim 1, wherein the filtration medium has a volume of at least about 1 mL per L of the cell culture fluid and / or the filtered cell culture fluid.

19. The method of claim 1, wherein:(i) the cell culture fluid is harvested from stable producer cells; and / or(ii)the cell culture fluid or the filtered cell culture fluid is contacted with an endonuclease prior to purifying the virus.

20. The method of claim 19, wherein the endonuclease is:(i) a non-specific endonuclease and degrades both DNA and RNA without sequence specificity; and / or(ii)from Serratia marcescens, Anabaena sp., Saccharomyces cerevisiae, Bos Taurus, Syncephalostrum racemosum and / or Borrelia burgdorferi,' and / or(iii)a Serratia nuclease, NucA, Nucl, endonuclease G, DNase I, or micrococcal nuclease.

21. The method of claim 19, wherein the endonuclease is contacted to the cell culture fluid or the filtered cell culture fluid at a concentration of:(i) 0.001 to 100 units / mL of cell culture fluid or filtered cell culture fluid; or(ii) about 1 unit / mL of cell culture fluid or filtered cell culture fluid; or(iii)about 5 unit / mL of cell culture fluid or filtered cell culture fluid; or(iv) about 30 unit / mL of cell culture fluid or filtered cell culture fluid.

22. The method of claim 1, wherein the method further comprises adjusting a concentration of Mg2+in the cell culture fluid or the filtered cell culture fluid to about 1- 3 mM prior to purifying the virus and / or to about 2 mM.

23. The method of claim 19, wherein the cell culture fluid is harvested from cells cultivated in an adherent environment or from cells cultivated in a suspension environment.

24. The method of claim 1, wherein the method comprises performing a harvest filtration on the cell culture fluid to produce the filtered cell culture fluid before subjecting the filtered cell culture fluid to the filtration medium.

25. The method of claim 1, wherein the filtered cell culture fluid is contacted with a high concentration salt solution to form a salt-spiked cell culture fluid prior to or during loading on to the filtration medium.

26. The method of claim 25, wherein:(i) the high concentration salt solution and the filtered cell culture fluid are mixed inline; and / or(ii)the high concentration salt solution comprises a monovalent and / or a divalent salt.

27. The method of claim 26, wherein:(i) the monovalent salt is sodium chloride; and / or(ii)the high concentration salt solution is at a concentration of 5M.

28. The method of claims 25, wherein the salt-spiked cell culture fluid has a target conductivity of 38 to 44 mS / cm at 25°C.

29. The method of claim 1, wherein the method further comprises washing the filtration medium with one or more wash solutions.

30. The method of claim 29, wherein the one or more wash solutions comprises a salt solution comprising a monovalent and / or a divalent salt.

31. The method of claim 30, wherein:(i) the monovalent salt is sodium chloride; and / or(ii)the salt solution is at a concentration of between 0.1M and IM; and / or(iii)the salt solution is at a concentration of 0.6M.

32. The method of claim 1, wherein the method further comprises eluting bound enveloped virus from the filtration medium with an elution solution.

33. The method of claim 32, wherein the elution solution:(i) comprises a buffer selected from the group consisting of Tris, histidine and HEPES;(ii) comprises a buffer at a concentration of 10 mM to 50 mM;(iii)has a pH of between 5 and 10;(iv) comprises: a) 50 mM Tris, pH8.0; b) 10 mM histidine, pH7.0; or c) 10 mM HEPES, pH7.5; and / or(v)comprises 10 mM HEPES at pH 7.5.

34. The method of claim 32, wherein the elution solution further comprises a monovalent and / or a divalent salt.

35. The method of claim 34, wherein:(i) the monovalent salt is sodium chloride;(ii)the salt is at a concentration of between 0.5M and 2M; and / or(iii)the salt is at a concentration of 1.2M.

36. The method of claim 1, wherein the method increases the virus infectious titer yield by at least 10%.

37. The method of claim 32, wherein the method results in:(i) a viral infectious titer yield of at least 1 x 108transducing units (TU) / mL of eluted virus; and / or(ii) a residual dsDNA content of less than 100 pg / mL of eluted virus.

38. The method of claim 32, wherein the method further comprises diluting the eluted virus with histidine, Tris or HEPES.

39. The method of claim 38, wherein the eluted virus is diluted to achieve a salt concentration of 150 mM.

40. The method of claim 32, wherein the method further comprises concentrating and / or diafiltering the eluted virus or the diluted eluted virus.

41. The method of claim 1, wherein the enveloped virus is a retrovirus.

42. The method of claim 41, wherein the retrovirus is a lentivirus.

43. The method of claim 1, wherein the cell culture fluid and / or the filtered cell culture fluid 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.

44. The method of claim 1, additionally comprising formulating the enveloped virus into a pharmaceutical formulation or into a solution suitable for infecting a cell.

45. A purified enveloped virus produced by the method according to claim 1.

46. A method of purifying an enveloped virus from a cell culture fluid, comprising:(i) providing a cell culture fluid comprising viral vector produced from a stable producer cell line;(ii) contacting the cell culture fluid with an endonuclease;(iii) contacting the endonuclease treated cell culture fluid to a filter to produce a filtered cell culture fluid;(iv) loading the filtered cell culture fluid and a high concentration salt solution to a charged depth filtration medium;(v) optionally, washing the filtration medium with one or more wash solutions;(vi) eluting the bound enveloped virus from the filtration medium with an elution solution;(vii) diluting the eluted virus with a buffer; and(viii) concentrating and diafiltering the eluted virus.

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