Lentivirus manufacturing platform

Abstract

In accordance with embodiments of the present disclosure, apparatuses, systems, and methods (e.g., end-to-end manufacturing platforms) for viral vectors are provided that include upstream production and downstream purification of stages of drug substances that are unaffected by therapeutic vector payloads.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 319,690, filed on March 14, 2022, which is hereby incorporated by reference in its entirety for all purposes. Background Art

[0003] The viral vector gene therapy industry lacks a Good Manufacturing Practice (GMP)-friendly, scalable, transient transfection suspension manufacturing platform that performs consistently across multiple therapeutic target viral vector payloads and is also able to achieve the yield and quality standards required by biopharmaceutical companies and regulatory agencies. Summary of the invention

[0004] The present disclosure provides compositions, methods, systems, and platforms (eg, end-to-end manufacturing platforms) for producing and purifying lentiviral vectors.

[0005] In certain aspects, provided herein is a composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl. In some embodiments, the lentiviral vector is a VSV-G lentiviral vector or any pseudotype or recombinant derivative thereof. In some embodiments, the lentiviral vector is a GaLV-TR lentiviral vector or any pseudotype or recombinant derivative thereof.

[0006] In some embodiments, the composition comprises at least 20 mM proline (e.g., at least about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline). In some embodiments, the composition comprises no more than 110 mM proline (e.g., no more than about 110 mM, 105 mM, 100 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, or 25 mM proline). In certain embodiments, the composition comprises about 25 mM-100 mM proline (e.g., about 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline).

[0007] In some embodiments, the composition comprises at least 1 mM MgCl2 (e.g., at least about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2). In some embodiments, the composition comprises no more than 11 mM MgCl2 (e.g., no more than about 1 1 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, or 2 mM MgCl2). In certain embodiments, the composition comprises about 2 mM-10 mM MgCl2 (e.g., about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2).

[0008] In some embodiments, the composition comprises at least 90 mM NaCl (e.g., at least about 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl). In some embodiments, the composition comprises no more than 160 mM NaCl (e.g., no more than about 160 mM, 155 mM, 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, 110 mM, 105 mM, or 100 mM NaCl). In certain embodiments, the composition comprises about 100 mM-150 mM NaCl (e.g., about 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl).

[0009] In some embodiments, the composition comprises at least 10 mM sodium phosphate buffer at pH 6-7 (e.g., at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7). In some embodiments, the composition comprises no more than 60 mM sodium phosphate buffer at pH 6-7 (e.g., no more than about 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, or 20 mM sodium phosphate buffer at pH 6-7). In certain embodiments, the composition comprises about 20 mM-50 mM sodium phosphate buffer at pH 6-7 (e.g., about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7).

[0010] In certain embodiments, the composition further comprises lactose. In some embodiments, the composition comprises at least 1% lactose by weight (e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight). In some embodiments, the composition comprises no more than 11% lactose by weight (e.g., no more than about 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% lactose by weight). In certain embodiments, the composition comprises about 2%-10% lactose by weight (e.g., about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight).

[0011] In certain aspects, provided herein is a method of producing a purified lentiviral vector composition, the method comprising subjecting a composition provided herein (eg, a composition set forth above) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition.

[0012] In some aspects, provided herein is a method for producing a purified lentiviral vector composition, the method comprising: (i) subjecting a composition provided herein (e.g., a composition described above) to depth filtration to produce a depth-filtered lentiviral vector composition; and (ii) subjecting the depth-filtered lentiviral vector composition produced in step (i) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition.

[0013] In certain embodiments of the method provided herein, the deep filtration step is carried out using a deep filter having a pore size gradient starting from about 8 microns-60 micron pores to about 0.45 micron pores. In some embodiments, the maximum pore size in the deep filter is at least 20 microns. In some embodiments, the pore size gradient is a continuous gradient. In certain embodiments, the gradient is a discontinuous gradient. In some embodiments, the deep filtration step is carried out using a deep filter made of cellulose or modified polyethylsulfone (mPES) material.

[0014] In some embodiments of the methods provided herein, the AEX chromatography step is performed using a bind and elute mode. In certain embodiments, the AEX step is performed using a balanced, positively charged fiber membrane column.

[0015] In some embodiments, the AEX chromatography step is performed with a buffer containing sodium phosphate, lactose, proline, and sodium chloride. In some embodiments, the buffer comprises about 20mM-50mM sodium phosphate, about 2%-10% lactose, and / or about 25mM-100mM proline. In certain embodiments, the buffer further comprises magnesium chloride. In some embodiments, the concentration of magnesium chloride is about 10mM-12mM. In some embodiments, the pH of the buffer is about 6.0-6.5. In certain embodiments, the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 150mM-250mM. In some embodiments, the buffer is an elution buffer, and the NaCl concentration is about 800mM-1000mM. In some embodiments, the buffer is a stripping buffer, and the NaCl concentration is about 1200mM-2000mM. In certain embodiments, the AEX chromatography step comprises the use of a column equilibration and / or post-load wash buffer provided herein, an elution buffer provided herein, and / or a stripping buffer provided herein.

[0016] In certain embodiments of the methods provided herein, the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF). In some embodiments, the purified lentiviral vector composition is purified using tangential flow filtration (TFF). In some embodiments, TFF is performed using a modified polyethersulfone (mPES) hollow fiber membrane. In certain embodiments, the mPES hollow fiber membrane has a fiber with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa. In some embodiments, TFF is performed at about 20 L / m 2 -40L / m 2 In some embodiments, TFF is performed at a flow rate controlled by a permeate flux of about 10 liters / square meter / hour to 50 liters / square meter / hour (LMMH). In some embodiments, TFF is performed at an inlet pressure of no more than 8 psi and a transmembrane pressure of no more than 5 psi.

[0017] In certain aspects, provided herein is a method for producing a purified lentiviral vector (e.g., a VSV-G lentiviral vector or any pseudotype or recombinant derivative thereof or a GaLV-TR lentiviral vector or any pseudotype or recombinant derivative thereof) composition, the method comprising: (A) harvesting a lentiviral vector from a culture of a lentiviral vector production cell to produce a lentiviral vector composition; (B) adding a DNase to the lentiviral vector composition; (C) adding an additive to the lentiviral vector composition to produce a stable lentiviral vector composition, the stable lentiviral vector composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl; and (D) subjecting the stable lentiviral vector composition produced in step (C) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition. In some embodiments, step (C) is performed before step (B).

[0018] In certain embodiments, the DNase is a universal nuclease.In some embodiments, about 40U / ml-80U / ml DNase is added to the lentiviral vector composition during step (B).

[0019] In some embodiments, the stable lentiviral vector composition comprises at least 20mM proline (e.g., at least about 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM proline). In some embodiments, the stable lentiviral vector composition comprises no more than 110mM proline (e.g., no more than about 110mM, 105mM, 100mM, 95mM, 90mM, 85mM, 80mM, 75mM, 70mM, 65mM, 60mM, 55mM, 50mM, 45mM, 40mM, 35mM, 30mM, or 25mM proline). In certain embodiments, the stable lentiviral vector composition comprises about 25 mM-100 mM proline (e.g., about 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline).

[0020] In some embodiments, the stable lentiviral vector composition comprises at least 1 mM MgCl2 (e.g., at least about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2). In some embodiments, the stable lentiviral vector composition comprises no more than 11 mM MgCl2 (e.g., no more than about 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, or 2 mM MgCl2). In certain embodiments, the stable lentiviral vector composition comprises about 2 mM-10 mM MgCl2 (e.g., about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2).

[0021] In some embodiments, the stable lentiviral vector composition comprises at least 90mM NaCl (e.g., at least about 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, or 150mM NaCl). In some embodiments, the stable lentiviral vector composition comprises no more than 160mM NaCl (e.g., no more than about 160mM, 155mM, 150mM, 145mM, 140mM, 135mM, 130mM, 125mM, 120mM, 115mM, 110mM, 105mM, or 100mM NaCl). In certain embodiments, the stable lentiviral vector composition comprises about 100 mM-150 mM NaCl (e.g., about 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl).

[0022] In some embodiments, the stable lentiviral vector composition comprises at least 10 mM sodium phosphate buffer at pH 6-7 (e.g., at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7). In some embodiments, the stable lentiviral vector composition comprises no more than 60 mM sodium phosphate buffer at pH 6-7 (e.g., no more than about 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, or 20 mM sodium phosphate buffer at pH 6-7). In certain embodiments, the stable lentiviral vector composition comprises about 20 mM-50 mM sodium phosphate buffer at pH 6-7 (e.g., about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7).

[0023] In certain embodiments, the stable lentiviral vector composition further comprises lactose. In some embodiments, the stable lentiviral vector composition comprises at least 1% lactose by weight (e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight). In some embodiments, the stable lentiviral vector composition comprises no more than 11% lactose by weight (e.g., no more than about 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% lactose by weight). In certain embodiments, the stable lentiviral vector composition comprises about 2%-10% lactose by weight (e.g., about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight).

[0024] In some embodiments, the AEX chromatography step is performed with a buffer containing one or more of sodium phosphate, lactose, proline, sodium chloride, or any combination thereof. In some embodiments, the buffer comprises about 20mM-50mM sodium phosphate, about 2%-10% lactose, and / or about 25mM-100mM proline. In certain embodiments, the buffer further comprises magnesium chloride. In some embodiments, the concentration of magnesium chloride is about 10mM-12mM. In some embodiments, the pH of the buffer is about 6.0-6.5. In certain embodiments, the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 250mM. In some embodiments, the buffer is an elution buffer, and the NaCl concentration is about 800mM-1000mM. In some embodiments, the buffer is a stripping buffer, and the NaCl concentration is about 1200mM-2000mM. In certain embodiments, the AEX chromatography step comprises using a column equilibration and / or post-loading wash buffer provided herein, an elution buffer provided herein, and / or a stripping buffer provided herein. In certain embodiments, the AEX step is performed using a balanced, positively charged fiber membrane column.

[0025] In certain embodiments of the methods provided herein, the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF). In some embodiments, the purified lentiviral vector composition is purified using tangential flow filtration (TFF). In some embodiments, TFF is performed using a modified polyethersulfone (mPES) hollow fiber membrane. In certain embodiments, the mPES hollow fiber membrane has a fiber with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa. In some embodiments, TFF is performed at about 20 L / m 2 -40L / m 2In some embodiments, TFF is performed at a flow rate controlled by a permeate flux of about 10 liters / square meter / hour to 50 liters / square meter / hour (LMH). In some embodiments, TFF is performed at an inlet pressure of no more than 10 psi and a transmembrane pressure of no more than 6 psi.

[0026] In certain embodiments, the stable lentiviral vector composition is deep filtered before step (D). In some embodiments, the deep filtration step is performed using a deep filter having a pore size gradient starting from about 8 microns-60 microns pores to about 0.45 microns pores. In some embodiments, the maximum pore size in the deep filter is at least 20 microns. In some embodiments, the pore size gradient is a continuous gradient. In certain embodiments, the gradient is a discontinuous gradient. In some embodiments, the deep filtration step is performed using a deep filter made of cellulose (with or without diatomaceous earth) or modified polyethyl sulfone (mPE S) material.

[0027] In some embodiments, the method provided herein also includes the step of transfecting cells from cell culture with one or more lentiviral encoding plasmids to produce a culture of lentiviral vector production cells. In some embodiments, the cell is HEK293 cell and / or HEK293T cell. In certain embodiments, transfection is carried out using a cationic lipid-based transfection reagent (e.g., Thermo Fisher Scientific LV-MAX transfection reagent, lipofectamine 2000). In some embodiments, cells are transfected for 3 generations to 8 generations after cell thawing. In some embodiments, the method also includes amplifying cells to produce a cell culture for use in a transfection step. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a process flow diagram of an exemplary manufacturing platform for producing and purifying viral vectors.

[0029] Figure 2 The elution and stripping chromatograms for Experiment 2 (using only the 0.9 M NaCl elution step) are depicted. DETAILED DESCRIPTION

[0030] In certain aspects, provided herein are methods and compositions that can be used as part of an end-to-end manufacturing platform (e.g., production and purification) for lentiviral vectors. (See, e.g., exemplary embodiments of Figure 1 ). In some embodiments, the lentiviral vector is a VSV-G pseudotyped lentiviral vector. In some such embodiments, upstream production and downstream purification to the drug substance stage is not affected by the therapeutic vector payload. For example, virus production is performed using Thermo Fisher Transient transfection of suspension HEK cells consists of proprietary cells, media, plasmids and transfection reagents. Virus purification may also include clarification by depth microfiltration, anion exchange (AEX) chromatography in bind and elute mode, followed by tangential flow filtration (TFF) to concentrate and formulate the virus.

[0031] In some aspects, provided herein are lentiviral vector compositions, such as lentiviral compositions prepared by the methods disclosed herein. In addition to lentiviral vectors, such lentiviral compositions may also include lactose, proline, MgCl2, NaCl, or any combination thereof. In some embodiments, the composition comprises each of lentiviral vectors, lactose, proline, MgCl2, and NaCl. In some embodiments, the lentiviral composition comprises an aqueous sodium phosphate buffer.

[0032] In certain aspects, provided herein is a method for producing a purified lentiviral vector composition, the method comprising subjecting a lentiviral vector composition disclosed herein to anion exchange chromatography. For example, but not limited to, such a method may comprise subjecting a lentiviral vector composition disclosed herein to depth filtration to produce a depth-filtered lentiviral vector composition; and subjecting the depth-filtered lentiviral vector composition to anion exchange chromatography to produce a purified lentiviral vector composition.

[0033] In some aspects, provided herein is a method for producing a purified lentiviral vector composition, the method comprising harvesting a lentiviral vector from a culture of a lentiviral vector production cell to produce a lentiviral vector composition; adding a DNA enzyme to the lentiviral vector composition; adding an additive to the lentiviral vector composition to produce a stable lentiviral vector composition, the stable lentiviral vector composition comprising: a lentiviral vector, lactose, proline, MgCl2 and NaCl; and performing anion exchange chromatography on the stable lentiviral vector composition. In some embodiments, the additive is added to the lentiviral vector composition before adding the DNA enzyme.

[0034] In certain aspects, provided herein is a composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl. In some embodiments, the lentiviral vector is a VSV-G lentiviral vector or any pseudotype or recombinant derivative thereof. In some embodiments, the lentiviral vector is a GaLV-TR lentiviral vector or any pseudotype or recombinant derivative thereof.

[0035] In some embodiments, the composition comprises at least 20 mM proline (e.g., at least about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline). In some embodiments, the composition comprises no more than 110 mM proline (e.g., no more than about 110 mM, 105 mM, 100 mM, 95 mM, 90 mM, 85 mM, 80 mM, 75 mM, 70 mM, 65 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, or 25 mM proline). In certain embodiments, the composition comprises about 25 mM-100 mM proline (e.g., about 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline).

[0036] In some embodiments, the composition comprises at least 1 mM MgCl2 (e.g., at least about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2). In some embodiments, the composition comprises no more than 11 mM MgCl2 (e.g., no more than about 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, or 2 mM MgCl2). In certain embodiments, the composition comprises about 2 mM-10 mM MgCl2 (e.g., about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2).

[0037] In some embodiments, the composition comprises at least 90 mM NaCl (e.g., at least about 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl). In some embodiments, the composition comprises no more than 160 mM NaCl (e.g., no more than about 160 mM, 155 mM, 150 mM, 145 mM, 140 mM, 135 mM, 130 mM, 125 mM, 120 mM, 115 mM, 110 mM, 105 mM, or 100 mM NaCl). In certain embodiments, the composition comprises about 100 mM-150 mM NaCl (e.g., about 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl).

[0038] In some embodiments, the composition comprises at least 10 mM sodium phosphate buffer at pH 6-7 (e.g., at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7). In some embodiments, the composition comprises no more than 60 mM sodium phosphate buffer at pH 6-7 (e.g., no more than about 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, or 20 mM sodium phosphate buffer at pH 6-7). In certain embodiments, the composition comprises about 20 mM-50 mM sodium phosphate buffer at pH 6-7 (e.g., about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7).

[0039] In certain embodiments, the composition further comprises lactose. In some embodiments, the composition comprises at least 1% lactose by weight (e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight). In some embodiments, the composition comprises no more than 11% lactose by weight (e.g., no more than about 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% lactose by weight). In certain embodiments, the composition comprises about 2%-10% lactose by weight (e.g., about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight).

[0040] definition

[0041] As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, a value of "about 50 mM" means a concentration of 45 mM to 55 mM.

[0042] As used herein, the term "weight / volume percentage" or "% w / v" means the weight percentage (in grams) of a single component relative to the total volume of the mixture containing the component. For example, 500 mg of a component in a total volume of 8 ml is 6.25% w / v, and 500 mg of a component in a total volume of 5 ml is 10% w / v.

[0043] As used herein, the term "viral titer" refers to the number of infectious vector particles, such as "transduction units" (TU) that result in the production of viral and / or transgene products in target cells, or "infectious units" (IU) that produce LV particles that enter cells, reverse transcribe their RNA genome into DNA, and integrate into the host cell genome. TU = LV particles that enter cells. Viral titer can be measured by functional assays known in the art.

[0044] As used herein, the term "viral vector" refers to a viral particle with the ability to introduce a nucleic acid molecule into a host. "Lentiviral vector" includes a viral vector containing a sequence derived from HIV-1. Lentiviral vectors carrying exogenous genes can be packaged into infectious viral particles via viral packaging using a specific cell line with the aid of a packaging plasmid. Infectious viral particles infect cells to achieve the expression of exogenous genes. "Recombinant" viral vectors refer to viral vectors constructed by genetic recombination technology. Recombinant viral vectors can be constructed using methods known in the art, such as by transducing a packaging cell line with a nucleic acid encoding a viral genome, followed by isolation of newly packaged viral particles. The term "virus" also refers to pseudoviral particles, i.e., viral particles that do not have any envelope glycoproteins or a genome on their surface and are obtained by spontaneous assembly of the structure and / or enzyme proteins of the virus.

[0045] Cell culture materials, methods and techniques are well known to those skilled in the art. For example, recombinant virus seed stock (e.g., slow virus seed stock) is used to infect a confluent host cell group or a host cell group of a specific density (e.g., 293 cell culture) at a given multiplicity of infection in a bioreactor; the virus grows in a cell culture at a given time and temperature; and newborn virus offspring are harvested in a cell culture fluid. As defined below, the terms "culture fluid", "cell culture fluid", "cell culture medium", "culture medium" and / or "bioreactor fluid" are used interchangeably and refer to a culture medium or solution in which a cell culture grows.

[0046] As used herein, the term "host cell" or "host cell line" refers to any mammalian cell line that supports the replication of a corresponding virus, such as a wild-type, modified or recombinant lentivirus. It will be appreciated that any such host cell line, grown in culture to a growth period known in the art, followed by infection with a seed stock of the corresponding virus, followed by additional culturing under physiologically acceptable conditions, results in the production of additional viral populations that can be harvested by the methods disclosed herein.

[0047] Virus composition containing additives

[0048] Disclosed herein are virus preparations (e.g., lentiviral preparations) that exhibit improved retention properties, which prevent the loss of viral vectors in subsequent purification steps. Without being bound by any particular theory, and purely for exemplary purposes, the virus preparations disclosed herein may include stabilizing additives such as carbohydrates and sugars, amino acids, salts, reducing agents, chelating agents, and / or cationic peptides. Exemplary carbohydrates and sugars include sucrose, trehalose, lactose, mannose, mannitol, glucose, sorbitol, dextran, polysorbate, polyethylene glycol; such as lactose. Exemplary amino acids include proline, lysine, leucine, histidine, arginine, alanine, isoleucine, methionine, aspartic acid, glutamic acid, and glycine; such as proline. Exemplary salts that can be used for stable virus preparations disclosed herein include MgCl2, NaCl, Na2SO4, CaCl2, sodium phosphate, sodium citrate, sodium succinate; for example, MgCl2 and / or NaCl. The virus vector preparation may be an aqueous mixture, such as an aqueous solution or a suspension.

[0049] In some such embodiments, the amino acid, salt, reducing agent, chelating agent, and / or cationic peptide can be present in the viral formulation, for example, each alone or in any combination, at a concentration of about 1 mM to about 1 M (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 100 mM, 12 mM, 800mM, 825mM, 850mM, 875mM, 900mM, 925mM, 950mM, 957mM or 1 M). In some embodiments, the concentration is from about 1 mM to about 250 mM, from about 2 mM to about 200 mM, or from about 10 mM to about 150 mM (e.g., 2 mM-10 mM, 25 mM-100 mM, or 100 mM-150 mM). In some such embodiments, the carbohydrate / sugar can be present, for example, at a concentration of 1% w / v-10% w / v, such as 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v, 5% w / v, 5.5% w / v, 6% w / v, 6.5% w / v, 7% w / v, 7.5% w / v, 8% w / v, 8.5% w / v, 9% w / v, 9.5% w / v, or 10% w / v.

[0050] In some embodiments, the viral composition (e.g., a lentiviral composition) comprises 2%-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2, 100mM-150mM NaCl, or any combination thereof. Other buffers used in conjunction with the lentiviral preparations disclosed herein include phosphate buffer, sodium citrate buffer, MES buffer, and MOPS buffer. In some such embodiments, the viral composition (e.g., a lentiviral composition) comprises 2% w / v-10% w / v lactose, 25mM-100mM proline, 2mM-10mM MgCl2, 100mM-150mM NaCl in a 20mM-50mM sodium phosphate buffer at a pH of about 6.0-7.0.

[0051] The lentiviral vectors contemplated herein for use with the compositions and methods disclosed herein may include one or more transgenes, e.g., protein encoding genes designed for delivery into a host cell and / or integration into its chromosomal DNA. Such lentiviral preparations as described herein may be used in conjunction with purification techniques such as filtration and chromatography procedures to purify the lentiviral vector with improved recovery.

[0052] The lentiviral vector can be present in the lentiviral preparations disclosed herein within a range of concentrations. For example, the lentiviral vector can be present at, for example, about 2×10 8 Infectious units / mL (IU / mL) is about 1×10 9 IU / mL (e.g., 2×10 8 IU / mL, 2.5×10 8 IU / mL, 3×10 8 IU / mL, 3.5×10 8 IU / mL, 4×10 8 IU / mL, 4.5×10 8 IU / mL, 5×10 8 IU / mL, 5.5×10 8 IU / mL, 6×10 8 IU / mL, 6.5×10 8 IU / mL, 7×10 8 IU / mL, 7.5×10 8 IU / mL, 8×10 8 IU / mL, 8.5×10 8 IU / mL, 9×10 8 IU / mL, 9.5×10 8 IU / mL or 1×10 9 IU / mL) is present in the lentiviral preparation. When desired, the lentiviral preparation may contain a concentration of about 3×10 8 IU / mL to about 5×10 8 IU / mL (e.g., 3×10 8 IU / mL, 3.5×10 8 IU / mL, 4×10 8 IU / mL, 4.5×10 8 IU / mL or 5×10 8 IU / mL) of lentiviral vector.

[0053] In certain aspects, provided herein is a method for producing a purified lentiviral vector (e.g., a VSV-G lentiviral vector or any pseudotype or recombinant derivative thereof or a GaLV-TR lentiviral vector or any pseudotype or recombinant derivative thereof) composition, the method comprising: (A) harvesting a lentiviral vector from a culture of a lentiviral vector production cell to produce a lentiviral vector composition; (B) adding a DNase to the lentiviral vector composition; (C) adding an additive to the lentiviral vector composition to produce a stable lentiviral vector composition, the stable lentiviral vector composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl; and (D) subjecting the stable lentiviral vector composition produced in step (C) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition. In some embodiments, step (C) is performed before step (B).

[0054] In certain embodiments, the DNase is a universal nuclease.In some embodiments, about 40U / ml-80U / ml DNase is added to the lentiviral vector composition during step (B).

[0055] In some embodiments, the stable lentiviral vector composition comprises at least 20mM proline (e.g., at least about 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM proline). In some embodiments, the stable lentiviral vector composition comprises no more than 110mM proline (e.g., no more than about 110mM, 105mM, 100mM, 95mM, 90mM, 85mM, 80mM, 75mM, 70mM, 65mM, 60mM, 55mM, 50mM, 45mM, 40mM, 35mM, 30mM, or 25mM proline). In certain embodiments, the stable lentiviral vector composition comprises about 25 mM-100 mM proline (e.g., about 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM proline).

[0056] In some embodiments, the stable lentiviral vector composition comprises at least 1 mM MgCl2 (e.g., at least about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2). In some embodiments, the stable lentiviral vector composition comprises no more than 11 mM MgCl2 (e.g., no more than about 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, or 2 mM MgCl2). In certain embodiments, the stable lentiviral vector composition comprises about 2 mM-10 mM MgCl2 (e.g., about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM MgCl2).

[0057] In some embodiments, the stable lentiviral vector composition comprises at least 90mM NaCl (e.g., at least about 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, or 150mM NaCl). In some embodiments, the stable lentiviral vector composition comprises no more than 160mM NaCl (e.g., no more than about 160mM, 155mM, 150mM, 145mM, 140mM, 135mM, 130mM, 125mM, 120mM, 115mM, 110mM, 105mM, or 100mM NaCl). In certain embodiments, the stable lentiviral vector composition comprises about 100 mM-150 mM NaCl (e.g., about 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM NaCl).

[0058] In some embodiments, the stable lentiviral vector composition comprises at least 10 mM sodium phosphate buffer at pH 6-7 (e.g., at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7). In some embodiments, the stable lentiviral vector composition comprises no more than 60 mM sodium phosphate buffer at pH 6-7 (e.g., no more than about 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, or 20 mM sodium phosphate buffer at pH 6-7). In certain embodiments, the stable lentiviral vector composition comprises about 20 mM-50 mM sodium phosphate buffer at pH 6-7 (e.g., about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM sodium phosphate buffer at pH 6-7).

[0059] In certain embodiments, the stable lentiviral vector composition further comprises lactose. In some embodiments, the stable lentiviral vector composition comprises at least 1% lactose by weight (e.g., at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight). In some embodiments, the stable lentiviral vector composition comprises no more than 11% lactose by weight (e.g., no more than about 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% lactose by weight). In certain embodiments, the stable lentiviral vector composition comprises about 2%-10% lactose by weight (e.g., about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% lactose by weight).

[0060] In some embodiments, the AEX chromatography step is performed with a buffer containing sodium phosphate, lactose, proline, and sodium chloride. In some embodiments, the buffer comprises about 20mM-50mM sodium phosphate, about 2%-10% lactose, and / or about 25mM-100mM proline. In certain embodiments, the buffer further comprises magnesium chloride. In some embodiments, the concentration of magnesium chloride is about 10mM-12mM. In some embodiments, the pH of the buffer is about 6.0-6.5. In certain embodiments, the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 150mM-250mM. In some embodiments, the buffer is an elution buffer, and the NaCl concentration is about 800mM-1000mM. In some embodiments, the buffer is a stripping buffer, and the NaCl concentration is about 1200mM-2000mM. In certain embodiments, the AEX chromatography step comprises the use of a column equilibration and / or post-load wash buffer provided herein, an elution buffer provided herein, and / or a stripping buffer provided herein.

[0061] In certain embodiments of the methods provided herein, the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF). In some embodiments, the purified lentiviral vector composition is purified using tangential flow filtration (TFF). In some embodiments, TFF is performed using a modified polyethersulfone (mPES) hollow fiber membrane. In certain embodiments, the mPES hollow fiber membrane has a fiber with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa. In some embodiments, TFF is performed at about 20 L / m 2 -40L / m 2In some embodiments, TFF is performed at a flow rate controlled by a permeate flux of about 10 liters / square meter / hour to 50 liters / square meter / hour (LMH). In some embodiments, TFF is performed at an inlet pressure of no more than 10 psi and a transmembrane pressure of no more than 6 psi.

[0062] In certain embodiments, the stable lentiviral vector composition is deep filtered before step (D). In some embodiments, the deep filtration step is performed using a deep filter having a pore size gradient starting from about 8 microns-60 microns and ending at about 0.45 microns. In some embodiments, the maximum pore size in the deep filter is at least 20 microns. In some embodiments, the pore size gradient is a continuous gradient. In certain embodiments, the gradient is a discontinuous gradient. In some embodiments, the deep filtration step is performed using a deep filter made of cellulose or modified polyethylsulfone (mPES) material. In certain embodiments, the deep filtration step is performed using a balanced, positively charged fiber membrane column.

[0063] In some embodiments, the method provided herein also includes the step of transfecting cells from cell culture with one or more lentiviral encoding plasmids to produce a culture of lentiviral vector production cells. In some embodiments, the cell is HEK293 cell and / or HEK293T cell. In certain embodiments, transfection is carried out using a cationic lipid-based transfection reagent (e.g., Thermo Fisher Scientific LV-MAX transfection reagent, lipofectamine 2000). In some embodiments, cells are transfected for 3 generations to 8 generations after cell thawing. In some embodiments, the method also includes amplifying cells to produce a cell culture for use in a transfection step.

[0064] Viral vectors

[0065] A variety of viral vectors can be used in the methods disclosed herein. For example, but not limited to, the enveloped virus purified according to the methods disclosed herein is an enveloped recombinant virus. In some embodiments, the enveloped virus is a pseudotyped recombinant retrovirus, such as a lentivirus. In some embodiments, the plasmids transfected into the cells in culture contain four plasmids, including a plasmid encoding an envelope protein (Env plasmid), which can be derived from a virus of interest (e.g., a lentivirus), but can also be derived from other enveloped viruses, such as plasmids encoding lentiviral Gag and Pol proteins (Gag-Pol plasmids), plasmids encoding lentiviral Rev proteins (Rev plasmids), and plasmids containing transgenes of interest (TOI). In some such embodiments, the TOI is encoded by an expression cassette (TOI plasmid) between the lentiviral 3′-LTR and the lentiviral 5′LTR. In some embodiments, the envelope protein is derived from a GaLV virus (particularly a modified GaLVTR glycoprotein for a lentiviral vector), a VSV virus (particularly a VSV-G envelope), or a measles virus (MV). In certain embodiments, the enveloped virus purified according to the methods disclosed herein is a pseudotyped recombinant retrovirus, more particularly a lentivirus, wherein the envelope protein is derived from a GaLV virus (particularly a modified GaLVTR glycoprotein of a lentiviral vector).

[0066] As non-limiting examples, the lenti expression (transfer) plasmid pLenti6.3 / V5-GW / EmGFP and the lenti packaging plasmid ViraPower TM One or more of the lentiviral packaging mixture, such as packaging plasmids pLP1, pLP2, and pLP / VSVG, can be used with a transgenic plasmid of interest in a predetermined appropriate molar ratio. Other lentiviral vectors can be used, including #277.pCCLsin.cPT.hPGK.eGFP, Wpre(277-eGFP). Other non-restrictive lentiviral plasmids can also be used. Without being bound by theory, both integration-competent and integration-deficient lentiviral vectors (ICLV and IDLV, respectively) can be used. In some such embodiments, a serum-free suspension lentiviral production protocol can be used, wherein such a protocol is easy to use, scalable, and efficient in delivering lentiviral expression and packaging vectors to serum-free suspension growing cells at high cell density.

[0067] Cell culture

[0068] As used herein, the term "cell" refers to all types of eukaryotic and prokaryotic cells. In some embodiments, the term refers to eukaryotic cells, especially mammalian cells. In certain exemplary but non-limiting embodiments, the term "cell" means human embryonic kidney (HEK) or human 293 cells or variants thereof, such as 293 variants that can grow in suspension. In some such embodiments, variants of 293 cells that can grow, proliferate, and transfect in suspension culture, particularly variants that can grow at high density (e.g., ≥ about 2×10 6 cells / ml, ≥ about 3×10 6 cells / ml, ≥ about 4×10 6 cells / ml, ≥ about 6×10 6 cells / ml or up to about 20×10 6 cells / ml) culture.

[0069] In some embodiments, when used in the context of culturing cells and performing transfection workflows, the term "high density" generally refers to a known cell line or variant of a known cell line that can be grown or cultured in an appropriate cell culture medium to a density of ≥ about 1×10 6 cells / ml, ≥ about 2×10 6 cells / ml, ≥ about 3×10 6 cells / ml or even optionally ≥ about 6×10 6 cells / ml, or ≥ about 20×10 6 cells / ml while still maintaining the ability to efficiently transfect and express the target protein.

[0070] In some embodiments, the cell is suitable for high-density cell culture. This refers to a cell lineage or (non-clone) cell group derived from the same parent cell lineage, which has been suitable for high-density growth in high-density culture medium, while maintaining cell viability at or above about 80%. By maintaining the cell at high density ≥ about 40 times, 50 times, 60 times, 70 times or 80 times of continuous passage, and gradually replacing the ratio of growth medium with the required high-density culture medium, it is possible to separate or select such cells from the parent cell group. Optionally, during the process, different cell pools can be bred individually and undergo a selection procedure, while synchronously assessing transfection efficiency and or lentiviral vector production efficiency, so that a non-clone cell group can be selected, which can be maintained and grown at high density, efficiently transfected, and express a high level of desired recombinant protein. Although it will be readily apparent to skilled practitioners that a variety of cell types and lineages can undergo this selection procedure, it has been determined that the cell lineage derived from 293 fibroblasts is particularly suitable for the selection process adapted to high-density growth conditions. In some cases, cells suitable for high density growth culture and suitable for use herein are also capable of being transfected with high efficiency and / or capable of expressing recombinant protein at a yield of more than at least about 200 μg / mL cell culture to about 2 mg / mL cell culture, more typically about 500 μg / ml cell culture to about 1 mg / mL cell culture. In some cases, cells suitable for high density culture are capable of expressing recombinant protein at a yield of about 1×10 6 cells / ml to about 20×10 6 cells / ml, about 2×10 6 cells / ml to about 2×10 6 cells / ml or about 2.5×10 6 cells / ml to about 6×10 6 In some embodiments, cells can be cultured at high densities and maintained and transfected at approximately 1×10 cells / ml. 6 About 20×10 6 , about 1×10 6 About 4×10 6 , about 1×10 6 About 3×10 6 , about 1×10 6 About 2×10 6 Transfection density within a range.

[0071] In some embodiments, the cells are grown in suspension culture. This includes cell cultures in which most or all of the cells in the culture vessel are in suspension, and a few or no cells in the culture vessel are attached to the surface of the vessel or another surface within the vessel. In some embodiments, the suspension culture has ≥ about 75% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel. In some embodiments, the suspension culture has ≥ about 85% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel. In some embodiments, the suspension culture has about 95% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel.

[0072] Embodiments disclosed herein include culturing the cells disclosed herein in a number of culture devices, such as bioreactors suitable for culturing cells in suspension. The bioreactor can be a single-use (disposable) or reusable bioreactor. The bioreactor can be, for example, selected from a culture vessel or a bag and tank reactor. Non-limiting representative bioreactors include glass bioreactors (e.g., 2L-10L, Sartorius), single-use bioreactors that utilize rocking motion agitation such as wave bioreactors (e.g., Cultibag 10L-25L, Sartorius), single-use stirred tank bioreactors (Cultibag 50 L, Sartorius) or stainless steel tank bioreactors. Growth is performed under controlled conditions (e.g., pH = 7.2, pO2 = 50%, 37°C and specific agitation according to the system, such as for the cultivation of HEK 293 cells as disclosed herein).

[0073] As is known in the art, the methods disclosed herein may also include a transfection reagent or composition that promotes the entry of macromolecules into cells. In some such embodiments, the transfection reagent may include a cationic lipid. In other embodiments, the transfection reagent may include a cationic lipid and one or more neutral / helper lipids as a "lipid aggregate". The lipid aggregate may include at least a first cationic lipid and optionally at least a first neutral lipid, wherein the lipid aggregate is suitable for forming a cationic complex with nucleic acid under aqueous conditions. In some embodiments, a transfection mixture applied to cultured cells is prepared in parts. For example, but not limited to, a transfection mixture is prepared in two parts. The production plasmid can be diluted into a complex culture medium. The plasmid can be a mixture of packaging plasmids pLP1, pLP2 and pLP-VSVG with a transgenic plasmid in an appropriate molar ratio, which will be recognized by those skilled in the art. The transfection reagent is then diluted in an appropriate complex culture medium and added to the plasmid mixture before being added to the cell culture. In some embodiments, the incubation duration of the transfection-plasmid complex is defined as the beginning of adding the diluted transfection reagent to the diluted plasmid to the end of adding the transfection-plasmid mixture to the cell culture. In some embodiments, the mixing process of the transfection-plasmid complex can be performed by methods known in the art, such as simply shaking or swirling the container in a stirred tank mixer before adding to the culture. The transfection-plasmid mixture can then be transferred to the cell culture using gravity or a peristaltic pump, but the duration of the transfer process should be ≤ 5 minutes.

[0074] In some embodiments, an enhancer balanced to ambient temperature can be added to the transfected cell culture. Such enhancers may include sodium propionate, sodium butyrate, caffeine, or any combination thereof. In some embodiments, the enhancer optionally includes valproic acid. In some embodiments, the enhancer does not include valproic acid. In some embodiments, the enhancer may include about 0.5mM to 1mM valproic acid. In some embodiments, the enhancer is added at one or more than one time point, such as at the time of transfection (about 0 hours) until about 48 hours after transfection. A lentiviral production enhancer can be added about 4 hours to 16 hours after transfection to enhance the cell packaging of the lentiviral vector. In some embodiments, a lentiviral production enhancer can be added at the time of transfection. In some embodiments, a lentiviral production enhancer can be added at the time of transfection and about 16 hours after transfection. In some embodiments, a lentiviral production enhancer can be added about 4 hours to 16 hours after transfection. The enhancer can be added to the transfected VPC culture by gravity or a peristaltic pump, but the duration of the transfer process should be ≤ 10 minutes. In some embodiments, the transfected culture is allowed to incubate until the viral particles, eg, lentiviral particles, are harvested.

[0075] Harvesting of viral particles

[0076] Virus particles, for example, lentiviral particles, are then harvested from the supernatant of the culture (i.e., culture medium) according to methods well known in the art. For example, the clarification and purification methods disclosed herein may also include one or more steps of treating the sample with one or more nucleases. In some embodiments, one or more nucleases include at least one DNA enzyme. In some such embodiments, the DNA enzyme is Benzonase. In some embodiments, after the plasmid transfection step, in some embodiments, before the clarification and / or purification step, nucleases are used in the culture medium of the production cell (e.g., in a bioreactor). In some embodiments, additives disclosed herein are added to the supernatant of the culture medium / harvest. For exemplary purposes, at harvest, additives comprising at least one sugar / carbohydrate, at least one amino acid, at least one salt, or any combination thereof are added to the culture medium (e.g., in a bioreactor). In some embodiments, the additive comprises 2%-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2 and 100mM-150mM NaCl. In certain embodiments, the additive consists of 2%-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2, and 100mM-150mM NaCl in 20mM-50mM sodium phosphate buffer at pH 6.0-7.0. The additive can be added before, after, or simultaneously with the nuclease.

[0077] Clarification and purification

[0078] After harvesting the cell culture medium, the subsequent purification process includes clarification of the cell culture medium and anion exchange chromatography steps.

[0079] Clarification can be performed by a filtration step to remove cell debris and other impurities from the harvested cell culture medium. Suitable filters can utilize cellulose filters, regenerated cellulose fibers, cellulose fibers combined with inorganic filter aids (e.g., diatomaceous earth, perlite, fumed silica), cellulose filters combined with inorganic filter aids and organic resins, or any combination thereof, and polymer filters (examples include, but are not limited to, nylon, polypropylene, polyethersulfone) to achieve effective removal and acceptable recovery. In some embodiments, a multi-stage process can be used, such as a two-stage or three-stage process, which includes a coarse filter to remove large precipitates and cell debris, followed by polishing a second-stage filter with a nominal pore size greater than 0.2 microns but less than 1 micron. A single-stage process using, for example, a relatively small pore size filter or centrifugation can also be used for clarification. More generally, any clarification method, including but not limited to dead-end filtration, microfiltration, centrifugation or a combination of a main feed of a filter aid (e.g., diatomaceous earth) and dead-end filtration or deep filtration, which provides a filtrate with a suitable clarity so as not to contaminate the membrane and / or resin in a subsequent purification step, may be acceptable for use in the clarification step disclosed herein. In some such embodiments, deep filtration using a physical porous filter medium can be used, which can retain the material through the entire depth of the filter. Deep filtration materials and methods are well known to those skilled in the art. For example, the filter material is typically composed of a thick and fibrous cellulose structure, and may also contain an inorganic filter aid, such as diatomaceous earth particles embedded in the fiber openings. Such filter materials have a large internal surface area, which is key to particle capture and filtration capabilities. Such deep filters have a pore size gradient (continuous or discontinuous) starting from 8 microns-60 microns and ending at 0.45 microns, and are made of cellulose or modified polyethylsulfone (mPES) materials. In some embodiments, the filter is made of cellulose. In some such embodiments, the maximum pore size is ≥20 microns. Those skilled in the relevant art will recognize that the filter size and flow rate required to process the material depends on the combination of pore sizes used.

[0080] In some embodiments, the filter is rinsed with water, culture medium or buffer in advance. In some embodiments, the filter is rinsed with anion exchange column (AEX) equilibration buffer in advance. In some embodiments, the filter is rinsed after use to clarify the filter. In some such embodiments, the clarified harvest can be collected with the rinsed volume to increase the recovery of the virus (e.g., slow virus) in this step. In some embodiments, accurate viral titer can be assessed in this step. In certain embodiments, the AEX step is carried out using a balanced, positively charged fiber membrane column.

[0081] Once the viral preparation has been recovered by clarification as described above, the viral preparation is further purified on a balanced, positively charged membrane (AEX; anion exchange column) such as a positively charged fiber membrane. Membrane materials are well known to those skilled in the art and are commercially available from suppliers such as Sartorius Corp. (Edgewood, NY), Pall Corp. (East Hills, NY), and Sigma-Aldrich Corp. (St. Louis, MO). Exemplary anion exchange membrane adsorbers include, but are not limited to, Sartobind. TM Q membrane adsorber (Sartorius Corp.) and Mustang TM Q membrane adsorber (Pall Corp.). Generally, methods and buffers known from conventional ion exchange chromatography can be directly applied to membrane adsorption chromatography, which is known to those skilled in the art. In certain embodiments, anion exchange membrane adsorption chromatography is performed at room temperature. Therefore, in certain embodiments, the virus preparation is purified via an AEX column, wherein the clarified preparation is loaded onto an AEX column balanced with a first pH buffered saline solution (also referred to as "column equilibration buffer", which can also be used as a post-loading wash). The virus preparation is eluted from the AEX column with a second pH buffered saline solution ("elution buffer"), and the eluted virus fraction is recovered.

[0082] In certain embodiments, the first pH buffered saline solution or equilibrium buffer is a NaCl or KCL saline solution. NaCl or KCl is present in the solution with an ionic strength of about at least 0.1M to about 0.4M. Therefore, the ionic strength of the salt includes at least 0.1M, 0.2M, 0.3M and 0.4M, including the fractional ionic strength therebetween. In a specific embodiment, the salt is NaCl and the ionic strength of the NaCl solution is 150mM to 250mM. The buffer solution can be a phosphate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer or tris (hydroxymethyl) aminomethane (TRIS) buffer. In certain embodiments, these buffers have a pH between about 6.0 and about 8.0, that is, at least 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0 pH or pH number therebetween. In a specific embodiment, the first pH buffered saline solution has a pH of 6.0-6.5. In other embodiments, the first buffer of the anion exchange membrane adsorption step has a pKa between 6.0 and 8.5, i.e., at least 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4 and 8.5 or a pKa number therebetween.

[0083] In some embodiments, the equilibration buffer further comprises about 20 mM-50 mM sodium phosphate, or a molar concentration number therebetween. In some such embodiments, the equilibration buffer comprises about 2% lactose to about 10% lactose, or a percentage therebetween. In some embodiments, the equilibration buffer further comprises about 25 mM-100 mM proline, or a molar concentration number therebetween. In some embodiments, the equilibration buffer further comprises about 4 mM-12 mM MgCl2, or a molar concentration number therebetween.

[0084] The second pH buffered saline solution ("elution buffer") may also include the same buffer components as the first (equilibrium) buffer. In certain embodiments, the second pH buffered saline solution or equilibrium buffer is a NaCl or KCL saline solution. In some embodiments, the salt in the second pH buffered saline solution is NaCl. NaCl or KCl is present in the solution with an ionic strength between about at least 0.5M and about 1M. Therefore, the ionic strength of the salt includes at least 0.5M, 0.6M, 0.7M, 0.8M, 0.9M and 1M, including fractional ionic strengths therebetween. In some such embodiments, the salt is NaCl and the ionic strength of the NaCl solution is 800mM to 1000mM (0.8M to 1.0M). The buffer solution can be a phosphate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer or tris(hydroxymethyl)aminomethane (TRIS) buffer. In some embodiments, these buffers have a pH between about 6.0 and about 8.0, i.e., at least 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0 pH or pH numbers therebetween. In some embodiments, the second pH buffered saline solution has a pH of 6.0-6.5. In other embodiments, the second buffer of the anion exchange membrane adsorption step has a pKa between 6.0 and 8.5, i.e., at least 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, and 8.5 pKa or pKa numbers therebetween.

[0085] In some embodiments, the elution buffer further comprises about 20 mM-50 mM sodium phosphate, or a molar concentration number therebetween. In some such embodiments, the equilibration buffer comprises about 2% lactose to about 10% lactose, or a percentage therebetween. In some embodiments, the equilibration buffer further comprises about 25 mM-100 mM proline, or a molar concentration number therebetween. In some embodiments, the equilibration buffer further comprises about 4 mM-12 mM MgCl2, or a molar concentration number therebetween.

[0086] In some embodiments, the performance of each chromatography step is measured by conductivity and UV absorbance at a wavelength of 280 nm.

[0087] After the clarified harvest is passed through the column, the column is washed with an equilibrium buffer until the UV absorbance is stable near the baseline. In order to elute the viral particles from the AEX column, the salt concentration (ionic strength) of the elution buffer can be increased by a linear gradient or in a single-step elution process. In some embodiments, the viral particles bound to the AEX column are eluted into a separate container with an elution buffer.

[0088] After eluting the viral particles from the AEX column, the eluted viral particles are diluted in a final buffer composition comprising 20mM-50mM sodium phosphate, 2%-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2 pH 6.0-6.5, 100mM-200mM NaCl. The purified intermediate can be further processed by ultrafiltration and diafiltration (UFDF) using tangential flow filtration (TFF) immediately. In some embodiments, the purified can be stored at 2°C-8°C for up to 24 hours before further processing. In some such embodiments, the viral particles are purified via TFF (and / or further). Typically, TFF is a pressure-driven process that uses a membrane to separate components in a liquid solution (or suspension), wherein a fluid (feed stream) is pumped tangentially along the surface of the membrane, and the applied pressure is used to force a "part" of the fluid to pass through the membrane to the filtrate side (of the membrane). In some embodiments, TFF is performed at room temperature. In such a process, the buffer is exchanged and the viral particles are concentrated. In some embodiments, TFF comprises concentrating the viral particles recovered from the AEX column at least 5 times, followed by at least one buffer exchange. In other embodiments, TFF comprises concentrating the viral particles recovered from the AEX column at least five to twenty times, followed by at least five or at least six buffer exchanges. Other embodiments involve performing at least two, at least three, at least four, at least five, or at least six buffer exchanges after concentrating the viral particles recovered from the AEX column.

[0089] TFF materials (e.g., hollow fibers, spirally wound flat sheets) and methods (e.g., ultrafiltration (UF), diafiltration (DF), microfiltration) are well known to those skilled in the art. In some embodiments, the TFF membrane has a molecular weight cutoff of 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, or 700 kDa. In some embodiments, the TFF membrane has a pore size of 100 kD-500 kD. In some embodiments, the TFF membrane is a mPES hollow fiber membrane.

[0090] In some embodiments, the buffer used in the buffer exchange of TFF is a phosphate buffer, HEPES buffer or TRIS buffer as described above. However, in certain embodiments, the buffer can have a concentration of 5mM to 15mM, including at least 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM and 15mM, and also includes the mM concentration therebetween. In certain embodiments, the buffer has a pH between about 6.0 and 7.5. Therefore, in one embodiment, the buffer has a pH of 6.0, 6.5, 7.0 or 7.5 or a fractional pH value therebetween. In some embodiments, the buffer exchange buffer also includes lactose, proline and MgCl .

[0091] In some embodiments, the viral particle fractions from the AEX column are combined, and the combined solution is concentrated, and the buffer is exchanged by TFF using a hollow fiber TFF membrane filter as described herein. The flow rate through the filter can be controlled by the permeate flux, but is maintained within an acceptable inlet pressure (≤10psi) and transmembrane pressure (≤6psi). The viral particles can be concentrated up to 20 times, and 5-10 diafiltration volumes are used to exchange the buffer into the desired buffer. In some embodiments, after concentration and buffer exchange, the retentate is collected.

[0092] In certain aspects, provided herein is a method of producing a purified lentiviral vector composition, the method comprising subjecting a composition provided herein (eg, a composition set forth above) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition.

[0093] In some aspects, provided herein is a method for producing a purified lentiviral vector composition, the method comprising: (i) subjecting a composition provided herein (e.g., a composition described above) to depth filtration to produce a depth-filtered lentiviral vector composition; and (ii) subjecting the depth-filtered lentiviral vector composition produced in step (i) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition.

[0094] In certain embodiments of the method provided herein, the deep filtration step is carried out using a deep filter having a pore size gradient starting from about 8 microns-60 micron pores to about 0.45 micron pores. In some embodiments, the maximum pore size in the deep filter is at least 20 microns. In some embodiments, the pore size gradient is a continuous gradient. In certain embodiments, the gradient is a discontinuous gradient. In some embodiments, the deep filtration step is carried out using a deep filter made of cellulose or modified polyethyl sulfone (mPES) material. In certain embodiments, the deep filtration step is carried out using a balanced, positively charged fiber membrane column.

[0095] In some embodiments of the methods provided herein, the AEX chromatography step is performed using a bind and elute mode.

[0096] In some embodiments, the AEX chromatography step is performed with a buffer containing sodium phosphate, lactose, proline, and sodium chloride. In some embodiments, the buffer comprises about 20mM-50mM sodium phosphate, about 2%-10% lactose, and / or about 25mM-100mM proline. In certain embodiments, the buffer further comprises magnesium chloride. In some embodiments, the concentration of magnesium chloride is about 10mM-12mM. In some embodiments, the pH of the buffer is about 6.0-6.5. In certain embodiments, the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 150mM-250mM. In some embodiments, the buffer is an elution buffer, and the NaCl concentration is about 800mM-1000mM. In some embodiments, the buffer is a stripping buffer, and the NaCl concentration is about 1200mM-2000mM. In certain embodiments, the AEX chromatography step comprises the use of a column equilibration and / or post-load wash buffer provided herein, an elution buffer provided herein, and / or a stripping buffer provided herein.

[0097] In certain embodiments of the methods provided herein, the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF).

[0098] In some embodiments of the methods provided herein, the purified lentiviral vector composition (processed by UFDF or not processed by UFDF) is further purified using tangential flow filtration (TFF). In some embodiments, TFF is carried out using a modified polyethersulfone (mPES) hollow fiber membrane. In certain embodiments, the mPES hollow fiber membrane has a fiber with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa. In some embodiments, TFF is carried out with an inlet pressure of no more than 8 psi and a transmembrane pressure of no more than 5 psi.

[0099] In some embodiments, kits are also provided herein, which include an aqueous composition as described herein (e.g., an aqueous composition such as one comprising an additive as described herein) and optional package instructions (e.g., a package instruction that instructs the user of the kit to express the plasmid in cells in culture according to the methods described herein). The kit may optionally also include one or more reagents that can be used to prepare and use (e.g., harvest and / or purify) a viral composition and / or formulation as described herein.

[0100] Kits contemplated herein include the use of aqueous compositions as described herein in methods for delivering plasmids encoding viral particles (which may include transgenes) into host cells and further harvesting and collecting viral compositions disclosed herein. The contemplated viral compositions (e.g., lentiviral compositions) can be used in methods for preventing or treating diseases or disorders and / or delivering transgenes (e.g., genes encoding chimeric antigen receptors, therapeutic peptides or nucleic acids, etc.), and can involve administering compositions described herein.

[0101] Example

[0102] The methods and compositions provided herein will be more readily understood by reference to the following examples, which are included to illustrate certain aspects and embodiments provided herein, but are not limited thereto. The following examples describe an end-to-end manufacturing (production and purification) platform for transiently transfected lentiviral vectors using HEK, which can be produced using the platform system disclosed herein (e.g., according to Figure 1 The exemplary embodiment depicted in FIG.

[0103] Example 1: Cell expansion

[0104] The production seed line was grown in production medium supplemented with Glutamax using a vial from one of the VPC pools. Upon thawing and at each subsequent passage, the culture vessels were inoculated with VPC at a cell density of 2.5E+05 to 5.5E+05 vc / mL VPC and grown to a cell density of 2E+06 to 6E+06 vc / mL before scaling up the culture in the next passage.

[0105] Example 2: Transfection

[0106] Transfection of VPCs occurred between passages 3 and 8 after thawing. Prior to transfection, cultures were incubated with LV at a final concentration of 5% v / v. Supplementation. The transfection mixture is prepared in two parts. First, the production plasmid is diluted to 2.5% to 5.0% v / v complex medium of the final VPC culture volume, or a similar medium can be used as a complex medium. The total amount of plasmid used is 2.0 mg / L to 2.5 mg / L final VPC culture volume. The plasmid is a mixture of pLP 1, pLP2, pLP-VSVG and transgenic plasmids in appropriate molar ratios. As needed, the diluted DNA is incubated at ambient temperature (15°C to 25°C) for 2 minutes to at least 60 minutes (min) (e.g., 10 minutes to 60 minutes). Secondly, the LV Max transfection reagent is diluted and incubated at ambient temperature for ≤15 minutes. The diluted transfection reagent is added to the diluted plasmid mixture, mixed briefly, and then incubated at ambient temperature. The mixing process of the transfection-plasmid complex can be performed by simple shaking or rotation of the container, or on a platform shaker. The transfection-plasmid mixture can be transferred to the cell culture using gravity or a peristaltic pump, but the duration of the transfer process should be ≤5 minutes. Once all of the transfection-plasmid mixture has been added to the cell culture, it is defined as post-transfection time = 0. At 4 to 16 hours post-transfection (hpt) (e.g., 4 to 6 hpt), an enhancer equilibrated to ambient temperature is added to the transfected cell culture to a final concentration of 40 mL / L final VPC culture volume. The enhancer can be added to the transfected VPC culture by gravity or a peristaltic pump. The transfected VPC culture is then allowed to incubate until LV harvest.

[0107] Example 3: Harvesting of vectors

[0108] In the bioreactor at the time of harvest, the culture is treated with 40U / mL to 80U / mL DNA enzyme (e.g., Benzonase), and additives are added to the harvest to prevent the loss of LV in the subsequent purification step. The additive can be added after the DNA enzyme treatment or simultaneously. The additive consists of 0-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2 and 100mM-150mM NaCl in 20mM-50mM sodium phosphate buffer at pH 6.0-7.0. The treated harvest is then clarified by deep filtration. The deep filter has a pore size gradient (continuous or discontinuous) starting from 8 microns-60 microns to 0.45 microns and is made of cellulose or modified polyethyl sulfone (mPES) material. In some cases, cellulose is a material, and the maximum pore size is ≥20 microns. The filter size and flow rate required for the treatment material depend on the combination of the pore size used. The filter is rinsed with water, culture medium or buffer in advance. In some cases, the buffer is an AEX equilibration buffer (described below). This clarified harvest is the first process intermediate for assessing accurate viral titer.

[0109] Example 4: Filtration / Purification Steps

[0110] The clarified harvest was further purified using a positively charged membrane (AEX column) using a bind and elute mode. The AEX column was operated using a buffer containing sodium phosphate (20mM-50mM), lactose (2%-10%), proline (25mM-100mM) and sodium chloride, the concentrations of which were step-dependent (see Table 1), and a pH of 6.0-6.5. The following Table 1 provides the appropriate salt concentrations for each chromatography step. The performance of each chromatography step was measured by conductivity and UV absorbance at a wavelength of 280nm. After passing the clarified harvest through the column, the column was washed with an equilibrium buffer until the UV absorbance was stable near the baseline. The LV particles bound to the AEX column were eluted into a separate container with an elution buffer.

[0111] Table 1

[0112]

[0113] The final buffer composition of the LV particles is 20mM-50mM sodium phosphate, 2%-10% lactose, 25mM-100mM proline, 2mM-10mM MgCl2 pH 6.0-6.5, 100mM-200mM NaCl. At this point, the purified LV intermediate can be further processed immediately by ultrafiltration and diafiltration (UFDF) using tangential flow filtration (TFF).

[0114] Finally, LV was further purified and formulated via TFF using mPES hollow fiber membranes with pore sizes of 100 kDa-500 kDa with fibers of 1.0 mm inner diameter, with size based on harvest volume. The flow rate through the filter was controlled by permeate flux and pressure. LV was concentrated up to 20 times and buffer exchanged into the desired buffer using 5-10 diafiltration volumes. After concentration and buffer exchange, the retentate was collected.

[0115] Table 2

[0116]

[0117] AEX membrane testing: Capture chromatography was developed as part of a manufacturing platform for lentivirus (LV) using triple transfection of suspended HEK293 cells. Historically, due to the large and fragile nature of LV and the lack of affinity chromatography specific for LV, anion exchange (AEX) columns in membrane or monolithic form have been used to purify LV. Past experience with AEX membrane columns (Mustang Q) resulted in low yields (<20%) or high column inlet pressures when AEX monolithic columns (CIM-Q, 0.5 nm channels) were used. Of all the resins tested, AEX membrane columns conjugated to amines had the highest probability of success. Therefore, LV purification was optimized on AEX membrane columns as described herein.

[0118] Example 5: Optimization of LV Chromatographic Purification

[0119] The variables varied for each experiment are disclosed herein in their corresponding Experimental and Results sections. The procedures described herein are common to all small-scale experiments.

[0120] The frozen clarified harvest was balanced to ambient temperature and then centrifuged to remove the precipitate produced by freezing / thawing. Proline, lactose, sodium chloride and / or magnesium chloride were added to the clarified harvest at different concentrations, which are listed in each experiment. The AEX column was operated at a flow rate of 5MV / min in a downward flow mode, except during the column stripping step, which was operated with an upward flow for half of the buffer volume and with a downward flow for the latter half of the buffer volume. The harvest material flowing through the column was collected in equal fractions (4 or 8). These fractions were sampled separately, then merged together and sampled again. The merged fractions were tested to reduce the test burden. After the harvest material was loaded, the column was washed with an equilibrium buffer and then eluted with various salt concentrations. The eluate was collected in a container containing enough neutralization buffer to reduce the NaCl concentration to about 200mM. After elution, the column was stripped with 1.2 M or 1.5 M NaCl, and the stripping effluent was also diluted with neutralization buffer to reduce the NaCl concentration to approximately 200 mM-400 mM. The samples were initially tested for infectious titer and physical titer, but the infectious titer assay did not perform as expected, so we only performed physical titer testing using the P24 assay to determine LV titer and recovery. All samples were frozen at ≤-65 °C prior to testing.

[0121] Results: I (Experiment 1). The potential of using a fiber AEX membrane capture column was evaluated. During this evaluation, it was realized that purification was improved when proline was used instead of arginine and lactose was used instead of sorbitol. Therefore, a fiber AEX membrane evaluation was performed using proline and / or lactose.

[0122] Table 3: Harvested materials for Experiment 1

[0123]

[0124] Table 4: Buffers used in Experiment 1

[0125]

[0126] Table 5: Volumes and results of Experiment 1

[0127]

[0128] Results: II (Experiment 2). It was determined whether more LV particles could be eluted if the 0.4 M NaCl elution step was omitted and only the 0.9 M NaCl elution step was used. Twice as many flow-through fractions were also collected to determine whether the LV in the flow-through was a result of LV not binding to the column or due to column breakthrough when 200 mL of harvest (approximately 4.58E+1 1 vp per 1 mL of membrane) was loaded. The harvest was first treated with loading conditioning buffer to a final proline concentration of 10 mM by adding an equal volume of conditioning buffer. The flow-through was collected in 8x approximately 13 mL fractions. (See Figure 2 . )

[0129] Table 6: Harvested materials for Experiment 2

[0130]

[0131] Table 7: Buffers used in Experiment 2

[0132] Buffer name preparation Loading Conditioning Buffer 100mM NaPO4, 5% lactose, 20mM MgCl2, 20mM proline, pH 6.5, EQ / Wash Buffer 50mM NaPO4, 120mM NaCl, pH 7.0; DOM 24Sep2020 Elution buffer 20 mM NaPO4, 0.9 M NaCl, 5% lactose, 1 00 mM proline, pH 6.5; Stripping buffer 20 mM NaPO4, 1.2 M NaCl, 5% lactose, 1 00 mM proline, pH 6.5; Neutralization buffer 100mM NaPO4, 5% lactose, 20mM MgCl2, 20mM proline, pH 6.0,

[0133] Table 8: Volumes and results of Experiment 2

[0134]

[0135] Results: III (Experiment 3). The same conditions as Experiment 2 were used, but less virus was loaded (3.66E+11 vp per 1 mL membrane) to see if this would reduce breakthrough and / or improve recovery of LV in the eluate. Four equal-volume fractions were collected instead of 8 to keep the fraction volumes the same between II (Experiment 2) and III (STT-Experiment 3).

[0136] Table 9: Harvested materials for Experiment 3

[0137] Sample adjustment Add an equal volume of adjustment buffer

[0138] Table 10: Buffers used in Experiment 3

[0139] Buffer name preparation Loading Conditioning Buffer 100mM NaPO4, 5% lactose, 20mM MgCl2, 20mM proline, pH 6.5, EQ / Wash Buffer 50mM NaPO4, 120mM NaCl, pH 7.0; Elution buffer 20 mM NaPO4, 0.9 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Stripping buffer 20 mM NaPO4, 1.2 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Neutralization buffer 100mM NaPO4, 5% lactose, 20mM MgCl2, 20mM proline, pH 6.5,

[0140] Table 11: Volumes and results of Experiment 3

[0141]

[0142] Results: IV (Experiment 4). To improve LV recovery and overall mass balance in the purification step, the concentration of proline, known to reduce protein aggregation, was increased to a final concentration of about 100 mM loading (previously 10 mM-20 mM), and the step elution was repeated with 0.4 M, 0.6 M, 0.8 M, and 1.0 M NaCl. The salt in the stripping buffer was also increased from 1.2 M to 1.5 M. The elution buffer was prepared by mixing the stripping buffer and the equilibration buffer online on the chromatography slide.

[0143] Table 12: Harvested materials of Experiment 4

[0144] Sample adjustment Add an equal volume of adjustment buffer

[0145] Table 13: Buffers used in Experiment 4

[0146]

[0147]

[0148] Table 14: Volumes and results of Experiment 4

[0149]

[0150] Results: V (Experiment 5). IV (Experiment 4) was repeated except that only two elutions were performed: 0.4 M and 0.9 M NaCl. In addition, a new neutralization buffer with lower MgCl2 and higher proline was used to ensure that the virus did not aggregate and that MgCl2 did not interfere with the assay.

[0151] Table 15: Harvest materials of V (Experiment 5)

[0152] Sample adjustment Add an equal volume of adjustment buffer

[0153] Table 16: Buffer for V (Experiment 5)

[0154]

[0155] Table 17: Volume of V and results (Experiment 5)

[0156]

[0157] Results: VI (Experiment 6). V (Experiment 5) was repeated except that the lactose concentration in the harvest was increased from 2.5% to 5%.

[0158] Table 18: Harvested materials of Experiment 5

[0159] Sample adjustment Add an equal volume of adjustment buffer

[0160] Table 19: Buffers used in Experiment 6

[0161] Buffer name preparation Loading Conditioning Buffer 40mM NaPO4, 8mM MgCl2, 0.2M proline, 10% lactose, pH 6.5 EQ / Wash Buffer 50 mM NaPO4, 120 mM NaCl, 5% lactose, pH 6.5; Elution Buffer #1 20 mM NaPO4, 0.4 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Elution Buffer #2 20 mM NaPO4, 0.9 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Stripping buffer 20 mM NaPO4, 1.2 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Neutralization buffer 10mM NaPO4, 5% lactose, 12mM MgCl2, 120mM proline, pH 6.5

[0162] Table 20: Volume and LV titer results for Experiment 6

[0163]

[0164] Results: VII (Experiment 7). In order to increase the recovery of all LV particles in the 0.9M elution, complete removal of lactose from the harvest load was tested. By removing lactose, a more concentrated stock solution of the loading adjustment buffer could be prepared. The sodium phosphate concentration in all buffers was increased to increase the pH buffering capacity. At this point, a larger format of the column was tested; 15 mL.

[0165] Table 21: Harvested materials of Experiment 7

[0166]

[0167] Table 22: Buffers used in Experiment 7

[0168] Buffer name preparation Loading Conditioning Buffer 125 mM PBS, 0.6 M proline, 60 mM MgCl2, pH 6.0 EQ / Wash Buffer 50 mM NaPO4, 200 mM NaCl, 5% lactose, pH 6.0; Elution Buffer #1 50 mM NaPO4, 0.9 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Stripping buffer 50 mM NaPO4, 1.5 M NaCl, 5% lactose, 100 mM proline, pH 6.5; Neutralization buffer 20mM NaPO4, 5% lactose, 12mM MgCl2, 50mM proline, pH 6.5

[0169] Table 23: Volume and LV titer results for Experiment 7

[0170]

[0171]

[0172] Table 24: Purity results of Experiment 7

[0173]

[0174] Example 6: Harvest Processing and Clarification Optimization

[0175] Evaluate harvest treatment and clarification to reduce culture contaminants prior to chromatography. The purpose of these treatments is to remove cells and large debris that could clog chromatography columns and to reduce free-floating DNA that would compete with the LV on anion exchange chromatography and result in DNA impurities in the purified LV product. Typical treatment and clarification for large-scale LV manufacturing processes involves DNase treatment, typically utilizing DNase Benzonase at concentrations of 40 U / mL-80 U / mL, and clarification by microfiltration using a set of microfilters working in series to first remove large fragments (>5 μm) and then smaller fragments (5 μm to 0.45 μm). A number of manufacturing platforms also include a 3rd clarification step with ultrafiltration in tangential flow filtration mode following the microfiltration step to improve LV purification on the subsequent column chromatography step. Evaluate and / or optimize the following features / steps:

[0176] The efficacy of different DNA enzymes in removing DNA impurities,

[0177] Clarification through several different microfiltration filter sets

[0178] • Chemical conditioning of the clarified harvest rather than adding ultrafiltration tangential flow filtration as an additional unit operation to improve subsequent chromatography.

[0179] Harvest was generated using the following procedure: Using LV-Max TM Kit components Transfection of virus production cells 1.0, the kit components include 3 LV helper plasmids, lipid-based LV-Max TM Transfection reagents and LV-Max TM enhancer and use a transfer vector containing the LV self-inactivating long terminal repeat (Sin LTR), Rev response element (RRE), LV psi packaging signal, and GFP as a replacement therapeutic gene driven by the CMV promoter. TM The kit explains that the cell passage number between 3-8 generations after the cell-vial is thawed is transfected, and the virus is harvested 48 hours after transfection. Unless otherwise stated, the crude harvest is treated with DNA enzyme. The harvest treated with DNA enzyme is clarified by centrifugation or deep filtration / 0.45 μm filtration. Any other specific experimental conditions that change between experiments are described in each subsection below. In most cases, the infection titer and / or physical titer (P24ELISA) of the samples from these experiments are tested, and residual extracellular DNA is tested in some experiments. All samples are frozen at ≤-65°C before testing.

[0180] Results: DNase treatment. The experiment was designed to compare potential alternative DNases with Benzonase under normal harvest conditions (i.e., no pretreatment of the harvest). All nucleases tested were selected based on their activity levels in the normal harvest matrix, with the exception of SAN-HQ, which was optimal at high salt concentrations (>0.4M). SAN-HQ DNase served as a reference point for suboptimal nuclease activity. Cells in the DNase-treated harvest were then removed by centrifugation as described in the methods section. DNase efficiency was measured by qPCR assays of residual plasmid and residual host cell DNA as a function of time and / or enzyme concentration. The LLOQ for the host cell DNA assay was 1.00E+06ng / mL.

[0181] Table 25: Related LV production and harvest records

[0182] Experimental scale 50mL shake bottle 60mL shake bottle Clarification method Centrifugation Centrifugation

[0183] Table 26: DNase Experiment #1 Conditions

[0184]

[0185]

[0186] Table 27: DNase Experiment #1 Results

[0187]

[0188] Table 28: DNase Experiment #2 Conditions

[0189]

[0190] Table 29: DNase Experiment #2 Results

[0191]

[0192] Most commercially available DNases appear to be relatively interchangeable, as long as the harvesting composition allows the enzyme to have most of its activity.

[0193] Clarification. Determine the optimal filter selection, size, flux, and filter wash parameters for removing cells and other biological contaminants from DNase-treated harvests while retaining infectious LV titers. The general consensus across the industry is that cellulose filters give better LV recoveries than polypropylene filters. To test this, a cellulose 8μm-60μm filter was placed in series with either a cellulose 1μm-4μm or a polypropylene 5μm filter as a secondary filter (Conditions 1 and 3 below). The terminal filter in both studies was cellulose 1.2 / 0.45. Alternative filters were tested in Conditions 4-6 below.

[0194] Table 30: Related LV production and harvesting

[0195]

[0196] Table 31: Evaluation of Clarifying Filters

[0197]

[0198] Table 32: Experimental conditions

[0199]

[0200] Table 33: In-process measurements for conditions #1 and #2

[0201]

[0202]

[0203] Table 34: In-process measurements for condition #3

[0204]

[0205] Table 35: In-process measurements for condition #3

[0206]

[0207]

[0208] Table 36: Results of clarification experiments conditions #4 and #5 (no titer for condition #6)

[0209]

[0210] Table 37: Condition #4 / 5 end filter and #6 in-process measurements

[0211]

[0212]

[0213] Confirmation Run. Validate harvest processing and clarification parameters at 50 L upstream harvest scale. Use the following conditions.

[0214] Table 38: Harvest materials used for confirmation runs

[0215]

[0216] Table 39: Buffers for confirmation runs

[0217] Buffer name preparation EQ / Wash Buffer 50mM NaPO4, 200mM NaCl, 5% lactose, 100mM proline, pH 6.0 10x Additive 25mM NaPO4, 60mM MgCl2, 0.6M proline, pH 6.0

[0218] Table 40: Confirmation Run Filter Load and Flux

[0219]

[0220]

[0221] Table 41: LV titers in clarified harvest results for confirmation runs

[0222]

[0223] Example 7: Harvest Processing and Clarification Optimization

[0224] The manufacturing platform of triple transfected lentivirus (LV) using suspended HEK293 cells involves using tangential flow filtration (TFF) to concentrate and buffer exchange LV into the final formulation. There are two filter formats that can be used for TFF, i.e. flat sheet membrane or hollow fiber (HF) membrane. It is known that the HF format requires a smaller operating pressure and flow (shear) rate than the flat sheet membrane to achieve the same amount of concentration and buffer exchange. Since LV is a relatively fragile virus, HF filters are commonly used for LV TFF. Flow rate, pressure, operating pressure and operating time are all interdependent, so a proper balance of parameters is required to maximize LV recovery. There are also several filters that can be rated for pore sizes of 100kDa, 300kDa, 500kDa or 750kD. LV should not be able to pass through any of these pore sizes, which is a requirement for successful TFF concentration and buffer exchange. Generally, process design principles indicate that the filter pore size should be selected to be about 30% smaller than the biological product of interest, so as to maximize the number of potential contaminants that can be filtered. However, published studies evaluating lentivirus using TFF filters of varying pore sizes have shown that recovery improves as the pore size becomes smaller. Therefore, a balance needs to be struck between maximizing LV recovery and maximizing impurity removal. The buffer composition of the final formulation buffer can have a significant impact on the performance of TFF, so TFF parameters were tested in both PBS (without additives) and formulation buffers containing cryoprotectants and other stabilizing elements (buffers listed in each experiment below).

[0225] For the following experiments, the starting material was LV generated by triple transfection of suspension HEK 293 cells, clarified by depth filtration and purified using an AEX column. The AEX eluate loaded on the column was concentrated 10-fold to 20-fold and the buffer was exchanged using 1 DV to 10 DV. After the buffer exchange, the TFF retentate containing the LV was collected. The HF filter was then backwashed with 1.2x hold-up volume of the final formulation buffer. The rinse and retentate were combined and tested for LV titer. Any other specific experimental conditions that varied between experiments are described in each subsection below. All samples were frozen at ≤-65 °C prior to testing.

[0226] Result: Final TFF.

[0227] Table 42: TFF conditions using a hollow fiber filter with a pore size of 500 kDa and a fiber with an inner diameter of 1.0 mm.

[0228]

[0229] Table 43: TFF process measurements

[0230]

[0231] Table 44: LV titers of retentate + flushing fluid

[0232]

[0233] Table 45: Residue levels

[0234]

Claims

1. A composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl.

2. The composition according to claim 1, comprising 25 mM-100 mM proline.

3. A composition according to claim 1 or claim 2, comprising 2 mM-10 mM MgCl2.

4. The composition according to any one of claims 1 to 3, comprising 100 mM - 150 mM NaCl.

5. The composition according to any one of claims 1 to 4, wherein the composition further comprises 20 mM-50 mM sodium phosphate buffer at pH 6-7.

6. The composition according to any one of claims 1 to 5, wherein the lentiviral vector is a VSV-G lentiviral vector or any pseudotype or recombinant derivative thereof.

7. The composition according to any one of claims 1 to 5, wherein the lentiviral vector is a GaLV-TR lentiviral vector or any pseudotype or recombinant derivative thereof.

8. A method for producing a purified lentiviral vector composition, comprising subjecting the lentiviral vector composition according to any one of claims 1 to 6 to anion exchange chromatography to produce a purified lentiviral vector composition.

9. A method for purifying a lentiviral vector, comprising: (i) performing depth filtration on the lentiviral vector composition according to any one of claims 1 to 6 to produce a depth-filtered lentiviral vector composition; as well as (ii) subjecting the depth-filtered lentiviral vector composition produced in step (i) to anion exchange (AEX) chromatography to produce a purified lentiviral vector composition.

10. The method of claim 9, wherein step (i) is performed using a depth filter having a pore size gradient starting from about 8 microns to 60 microns pores and ending at about 0.45 microns pores.

11. The method of claim 10, wherein the maximum pore size in the depth filter is at least 20 microns.

12. The method according to claim 10 or 11, wherein the pore size gradient is a continuous gradient.

13. The method according to claim 10 or 11, wherein the pore size gradient is a discontinuous gradient.

14. The method according to any one of claims 9 to 13, wherein step (i) is performed using a depth filter made of cellulose or modified polyethylsulfone (mPES) material.

15. The method according to any one of claims 9 to 14, wherein step (ii) is performed using a balanced, positively charged fiber membrane column.

16. The method according to any one of claims 9 to 15, wherein step (ii) is performed using a bind and elute mode.

17. The method according to any one of claims 9 to 18, wherein step (ii) is performed with a buffer containing sodium phosphate, lactose, proline and sodium chloride.

18. The method of claim 17, wherein the buffer comprises about 20 mM-50 mM sodium phosphate, about 2%-10% lactose and / or about 25 mM-100 mM proline.

19. The method of claim 17 or 18, wherein the buffer further comprises magnesium chloride.

20. The method of claim 19, wherein the concentration of magnesium chloride is about 10 mM-12 mM.

21. The method of any one of claims 17 to 20, wherein the pH of the buffer is about 6.0-6.

5.

22. The method according to any one of claims 17 to 21, wherein the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 150 mM-250 mM.

23. The method of any one of claims 17 to 21, wherein the buffer is an elution buffer and the NaCl concentration is about 800 mM-1000 mM.

24. The method of any one of claims 17 to 21, wherein the buffer is a stripping buffer and the NaCl concentration is about 1200 mM-2000 mM.

25. The method of any one of claims 9 to 24, wherein the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF).

26. The method of any one of claims 9 to 25, wherein the purified lentiviral vector composition is further purified using tangential flow filtration (TFF).

27. The method of claim 26, wherein the TFF is performed using a modified polyethersulfone (mPES) hollow fiber membrane.

28. The method of claim 27, wherein the mPES hollow fiber membrane has fibers with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa.

29. The method of claim 28, wherein the TFF is carried out at about 20 L / m 2 -40L / m 2 The harvest volume was performed.

30. The method of any one of claims 26 to 29, wherein the TFF is performed at a flow rate controlled by a permeate flux of about 10-50 L / m2 / hr (LMMH).

31. The method of claim 30, wherein the TFF is performed at an inlet pressure of no more than 8 psi and a transmembrane pressure of no more than 5 psi.

32. A method for producing a purified lentiviral vector composition, comprising: (A) harvesting lentiviral vectors from a culture of lentiviral vector producer cells to produce a lentiviral vector composition; (B) adding DNase to the lentiviral vector composition; (C) adding an additive to the lentiviral vector composition to produce a stable lentiviral vector composition, the stable lentiviral vector composition comprising: (a) a lentiviral vector; (b) proline; (c) MgCl2; and (d) NaCl; and (D) subjecting the stable lentiviral vector composition produced in step (C) to anion exchange chromatography to produce a purified lentiviral vector composition.

33. The composition of claim 32, wherein the lentiviral vector is a VSV-G lentiviral vector or any pseudotyped or recombinant derivative thereof.

34. The composition of claim 32, wherein the lentiviral vector is a GaLV-TR lentiviral vector or any pseudotyped or recombinant derivative thereof.

35. The method according to any one of claims 32 to 34, wherein step (C) is performed before step (B).

36. according to the method described in any one in claim 32 to 35, wherein said DNA enzyme is a universal nuclease.

37. The method of any one of claims 32 to 36, wherein about 40 U / ml-80 U / ml DNase is added to the lentiviral vector composition during step (B).

38. The method of any one of claims 32 to 37, wherein the stable lentiviral vector composition comprises 25 mM-100 mM proline.

39. The method of any one of claims 32 to 38, wherein the stable lentiviral vector composition comprises 2 mM-10 mM MgCl2.

40. The method of any one of claims 32 to 39, wherein the stable lentiviral vector composition comprises 100 mM-150 mM NaCl.

41. The method of any one of claims 32 to 40, wherein the stable lentiviral vector composition further comprises 20 mM-50 mM sodium phosphate buffer at pH 6-7.

42. The method of any one of claims 32 to 41, wherein step (D) is performed using a balanced, positively charged fiber membrane column.

43. The method according to any one of claims 32 to 42, wherein step (D) is performed using a bind and elute mode.

44. The method of any one of claims 32 to 43, wherein step (D) is performed with a buffer containing sodium phosphate, lactose, proline and sodium chloride.

45. The method of claim 44, wherein the buffer comprises about 20 mM-50 mM sodium phosphate, about 2%-10% lactose, and / or about 25 mM-100 mM proline.

46. ​​The method of claim 44 or 45, wherein the buffer further comprises magnesium chloride.

47. The method of claim 46, wherein the concentration of magnesium chloride is about 10 mM-12 mM.

48. The method of any one of claims 44 to 47, wherein the pH of the buffer is about 6.0-6.

5.

49. The method according to any one of claims 44 to 48, wherein the buffer is a column equilibration and / or post-loading wash buffer, and the NaCl concentration of the buffer is about 150 mM-250 mM.

50. The method of any one of claims 44 to 48, wherein the buffer is an elution buffer and the NaCl concentration is about 800 mM-1000 mM.

51. The method of any one of claims 44 to 48, wherein the buffer is a stripping buffer and the NaCl concentration is about 1200 mM-2000 mM.

52. The method of any one of claims 32 to 51, wherein the purified lentiviral vector composition is further processed by ultrafiltration and diafiltration (UFDF).

53. The method of any one of claims 32 to 52, wherein the purified lentiviral vector composition is further purified using tangential flow filtration (TFF).

54. The method of claim 53, wherein the TFF is performed using a modified polyethersulfone (mPES) hollow fiber membrane.

55. The method of claim 54, wherein the mPES hollow fiber membrane has fibers with an inner diameter of about 1.0 mm and a pore size of about 100 kDa-500 kDa.

56. The method of claim 55, wherein the TFF is carried out at about 20 L / m 2 -40L / m 2 The harvest volume was performed.

57. The method of any one of claims 53 to 56, wherein the TFF is performed at a flow rate controlled by a permeate flux of about 10-50 L / m2 / hr (LMMH).

58. The method of claim 57, wherein the TFF is performed at an inlet pressure of no more than 8 psi and a transmembrane pressure of no more than 5 psi.

59. The method of any one of claims 32 to 58, wherein the stable lentiviral vector composition is depth filtered prior to step (D).

60. The method of claim 59, wherein the depth filtration is performed using a depth filter having a pore size gradient starting from about 8-60 micron pores and ending at about 0.45 micron pores.

61. The method of claim 60, wherein the maximum pore size in the depth filter is at least 20 microns.

62. The method of claim 60 or 61, wherein the pore size gradient is a continuous gradient.

63. The method of claim 60 or 61, wherein the pore size gradient is a discontinuous gradient.

64. The method of any one of claims 59 to 63, wherein the depth filtration is performed using a depth filter made of cellulose or modified polyethylsulfone (mPES) material.

65. The method of any one of claims 32 to 64, further comprising the step of transfecting cells from the cell culture with one or more lentiviral encoding plasmids to produce a culture of lentiviral vector producer cells prior to step (A).

66. The method of claim 65, wherein the cells are HEK293 cells and / or HEK293T cells.

67. The method of claim 65 or 66, wherein transfection is performed using a cationic lipid-based transfection reagent.

68. The method of any one of claims 65 to 67, further comprising expanding cells to produce the cell culture for use in the transfection step.

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