Vector Production Method

Abstract

The present disclosure provides a method for producing and purifying viral vectors.In particular, the present disclosure provides an improved method for producing lentiviral vectors from host cells grown in suspension.More specifically, the present disclosure provides an improved large-scale lentiviral production method, which includes growing cells to an appropriate number, transfecting with lentiviral packaging plasmid, transfer plasmid and transfection agent; treating with endonuclease; harvesting and clarifying suspension culture supernatant; capturing and concentrating lentiviral vectors using affinity chromatography or cation exchange chromatography; filtering concentrated lentiviral vectors; ultrafiltering and diafiltering lentiviral vectors; formulating lentiviral vectors; and sterile filtering formulated bulk lentiviral vectors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 223,249, filed July 19, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to improved methods for viral vector production and purification.The present invention also relates to improved methods for producing viral vectors. [Background technology]

[0003] Description of related fields Retroviral vectors, both of oncoretroviral and lentiviral origin, have a wealth of unmet potential as gene delivery vehicles. Large-scale manufacturing of clinical-grade viral gene therapy vectors faces many obstacles, including scalable unit operations for vector generation and purification that remove contaminants while maintaining vector stability, titer, and potency. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates generally, in part, to improved methods for producing lentiviral vectors. In particular, the present disclosure provides improved methods for producing lentiviral vectors from host cells grown in suspension.

[0005] In one aspect of the disclosure, a suspension method for producing lentiviral vectors (sLVV) is provided that includes inoculating a large-scale suspension culture with live host cells; transiently transfecting the host cells in the large-scale suspension culture with a mixture including a lentiviral packaging plasmid, a transfer plasmid, and a transfection agent; adding an endonuclease to the suspension culture supernatant about 36 hours to about 48 hours after transfection (after the start of transfection); harvesting and clarifying the suspension culture supernatant using tandem depth filters and bilayer filters; capturing and concentrating the lentiviral vector from the harvested and clarified suspension culture supernatant using chromatography; filtering the concentrated lentiviral vector; ultrafiltering and diafiltering the lentiviral vector using tangential flow filtration (TFF); and formulating the lentiviral vector to produce a formulated bulk lentiviral vector, and sterile filtering the formulated bulk lentiviral vector.

[0006] In various embodiments, the method includes inoculating a suspension culture of between 200 L and 2000 L. In some embodiments, the method includes inoculating a suspension culture of between 200 L and 1000 L. In some embodiments, the method includes inoculating a suspension culture of between 200 L and 500 L. In some embodiments, the method includes inoculating a suspension culture of 200 L.

[0007] In various embodiments, the large scale suspension culture comprises approximately 40.0×10 8 ~Approx. 120.0×10 8 In some embodiments, the host cells are selected from the group consisting of HEK293 cells, HEK293S cells, HEK293T cells adapted to suspension culture (HEK293T), HEK293F cells, HEK293FT cells, HEK293FTM cells, and HEK293E cells. In some embodiments, the host cells are HEK293T cells.

[0008] In various embodiments, the large-scale cell suspension culture comprises host cells cultured in a culture medium. In some embodiments, the large-scale cell suspension culture comprises host cells cultured in a culture medium for about 3 days, after which the culture medium is replaced with fresh culture medium. In some embodiments, the large-scale cell suspension culture comprises host cells cultured in a culture medium for about 3 days, after which the culture medium is replaced with fresh culture medium using alternating tangential flow filtration (ATF). In some embodiments, the culture medium is a serum-free, chemically defined cell culture medium.

[0009] In various embodiments, the host cells are transiently transfected with a mixture comprising a transfection agent selected from the group consisting of calcium phosphate, cationic lipid, and cationic polymer. In some embodiments, the host cells are transiently transfected with a mixture comprising a transfection agent that is a cationic polymer selected from the group consisting of DEAE-dextran, polybrene, dendrimer, and polyethyleneimine (PEI). In some embodiments, the host cells are transiently transfected with a mixture comprising a transfection agent that comprises PEI. In some embodiments, the transfection agent comprises PEI, and the mixture has a ratio of N (NH2 amine in PEI):P (phosphate group in DNA backbone) of about 5, about 5.5, about 6, about 6.4, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.

[0010] In various embodiments, the transfection agent is added to the suspension culture for about 14 to about 18 hours, and optionally, the suspension culture is subjected to a culture medium exchange with fresh culture medium using alternating tangential flow filtration (ATF).

[0011] In various embodiments, the lentiviral packaging plasmid encodes the lentiviral gag, pol, and rev as well as the heterologous envelope proteins.

[0012] In various embodiments, the lentiviral packaging plasmid encodes a heterologous envelope protein selected from the group consisting of a vesiculovirus envelope protein or variant thereof, a paramyxovirus envelope protein or variant thereof, an alphavirus envelope protein or variant thereof, a gammaretrovirus envelope protein or variant thereof, an orthohepadnavirus envelope protein or variant thereof, a hepacivirus envelope protein or variant thereof, and a lyssavirus envelope protein or variant thereof.

[0013] In certain embodiments, the lentiviral packaging plasmid encodes a heterologous envelope protein selected from the group consisting of Nipah virus envelope protein, Sendai virus (SeV) envelope protein, Morbillivirus envelope protein, Canine distemper (CDV) envelope protein, and Measles virus envelope protein.

[0014] In various embodiments, the lentiviral packaging plasmid encodes Sindbis virus (SINV) envelope proteins.

[0015] In various embodiments, the lentiviral packaging plasmid encodes a heterologous envelope protein selected from the group consisting of gibbon ape leukemia virus (GALV) envelope protein, feline leukemia virus (FeLV) envelope protein, feline endogenous retrovirus (RD114) envelope protein, and baboon endogenous retrovirus (BaEV) envelope protein.

[0016] In various embodiments, the lentiviral packaging plasmid encodes a Hepatitis B virus (HBV) envelope protein.

[0017] In various embodiments, the lentiviral packaging plasmid encodes a Hepatitis C virus (HCV) envelope protein.

[0018] In various embodiments, the lentiviral packaging plasmid encodes rabies virus (RABV).

[0019] In various embodiments, the lentiviral packaging plasmid is selected from the group consisting of a vesicular stomatitis virus (VSV) envelope protein or variant thereof (e.g., VSV-G), a coccus virus (COCV) envelope protein or variant thereof, a Maraba virus (MARAV) envelope protein or variant thereof, a Pili virus (PIRYV) envelope protein or variant thereof, a Nipah virus (NiV) envelope protein or variant thereof, a Sendai virus (SeV) envelope protein or variant thereof, a Morbillivirus envelope protein or variant thereof, a Canine Distemper (CDV) envelope protein or variant thereof, a Measles Virus (MRV) envelope protein or variant thereof, a Mycobacterium tuberculosis virus ... V) encoding an envelope protein or variant thereof, a Sindbis virus (SINV) envelope protein or variant thereof, a gibbon ape leukemia virus (GALV) envelope protein or variant thereof, a feline endogenous retrovirus (RD114) envelope protein or variant thereof, a feline leukemia virus (FeLV) envelope protein or variant thereof, a baboon endogenous retrovirus (BaEV) envelope protein or variant thereof, a hepatitis B (HBV) envelope protein or variant thereof, a hepatitis C (HCV) envelope protein or variant thereof, and a rabies virus (RABV) envelope protein or variant thereof.

[0020] In a preferred embodiment, the VSV envelope protein is VSV-G or a variant thereof.

[0021] In various embodiments, the transfer plasmid comprises a polynucleotide comprising a packageable lentiviral vector genome. In some embodiments, the transfer plasmid comprises a polynucleotide comprising a packageable lentiviral vector genome comprising a left chimeric (5') lentiviral LTR, and the promoter of 5'LTR is replaced with a heterologous promoter; a Psi (Ψ) packaging signal; a central polypurine tract / DNA flap (cPPT / FLAP); a retroviral transport element (RRE); a promoter operably linked to a polynucleotide of interest; and a right (3') self-inactivating (SIN) lentiviral LTR.

[0022] In various embodiments, the endonuclease is derived from Serratia marcescens, and optionally, the endonuclease is a recombinant NucA endonuclease. In some embodiments, the endonuclease has both DNA and RNA cleavage activity. In some embodiments, the endonuclease is Benzonase or Denarase. In some embodiments, the endonuclease is added at a concentration of about 60 U / ml or about 30 U / ml.

[0023] In various embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours to about 72 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours to about 48 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 48 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 44 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 40 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours after transfection.

[0024] In various embodiments, the endonuclease is added to the culture for about 1 to about 2 hours.

[0025] In various embodiments, the harvesting and clarification steps include filtering the suspension culture supernatant through a tandem depth filter that retains contaminants of at least about 40 μm or at least about 60 μm and a bilayer filter with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.22 μm to about 0.45 μm.

[0026] In various embodiments, the clarified suspension culture is optionally adjusted to about pH 7.0 or about pH 7.2 with 1 M HEPES.

[0027] In various embodiments, the lentiviral vector is captured and concentrated from the harvested and clarified suspension culture supernatant using affinity chromatography or cation exchange chromatography. In some embodiments, the supernatant is passed through an affinity chromatography column or a cation exchange chromatography column. In some embodiments, the affinity chromatography is heparin affinity chromatography. In some embodiments, the cation exchange chromatography is sulfate cation exchange chromatography. In some embodiments, the sulfate cation exchange chromatography comprises a column with a bead size of about 45 μm and / or an average pore size of about 100 nm.

[0028] In various embodiments, a wash buffer comprising about 50 mM HEPES, about 100 mM NaCl, pH 7 is pumped onto the chromatography column.

[0029] In various embodiments, an elution buffer comprising about 50 mM HEPES, about 400 mM NaCl, pH 8 is pumped onto the chromatography column.

[0030] In various embodiments, a wash buffer comprising about 50 mM HEPES, about 300 mM NaCl, pH 7.2 is passed through the chromatography column. In various embodiments, an elution buffer comprising about 50 mM HEPES, about 1 M NaCl, pH 7.5 is passed through the chromatography column.

[0031] In various embodiments, filtering the concentrated vector comprises filtering the concentrated lentiviral vector through a bilayer filter with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.2 μm to about 0.45 μm.

[0032] In various embodiments, the lentiviral vector is ultrafiltered and diafiltered using a hollow fiber tangential flow filtration (TFF) filter with a pore size or molecular weight cutoff of about 100 kDa to about 500 kDa. In some embodiments, the hollow fiber TFF filter has a pore size or molecular weight cutoff of about 100 kDa. In some embodiments, the hollow fiber TFF filter has a pore size or molecular weight cutoff of about 300 kDa. In some embodiments, the hollow fiber TFF filter has a pore size or molecular weight cutoff of about 500 kDa.

[0033] In various embodiments, the lentiviral vector is diafiltered into a diafiltration buffer, optionally, the diafiltration buffer is about 50 mM HEPES, about 100 mM NaCl, pH 7.50.

[0034] In various embodiments, the lentiviral vector is diafiltered into a diafiltration buffer, optionally, the diafiltration buffer is about 50 mM HEPES, pH 7.0.

[0035] In various embodiments, the lentiviral vector is diafiltered into a diafiltration buffer, optionally, the diafiltration buffer is about 50 mM L-histidine, pH 7.0.

[0036] In various embodiments, the lentiviral vector is formulated 1:1 in 2× stem cell growth medium (SCGM) to generate a formulated bulk lentiviral vector.

[0037] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer comprising HEPES and sucrose, optionally, the buffer further comprises L-proline, poloxamer 188, or NaCl.

[0038] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, and about 100 mM L-proline.

[0039] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 0.2 to about 2.0 mg / ml poloxamer 188.

[0040] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 150 mM NaCl.

[0041] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, about 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188.

[0042] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188.

[0043] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer containing L-histidine, sucrose, and L-proline.

[0044] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer comprising about 5 mM L-histidine, about 146 mM sucrose, and about 100 mM L-proline, and optionally, the formulation further comprises about 0.2 to about 2.0 mg / mL poloxamer 188.

[0045] In various embodiments, the formulated bulk lentiviral vector is optionally sterile filtered through a 0.22 μm filter with a 0.45 μm prefilter.

[0046] In various embodiments, the method further comprises fill-finishing the formulated bulk lentiviral vector to produce a final lentiviral vector, and freezing the final lentiviral vector.

[0047] In various embodiments, the method further comprises freezing the formulated bulk lentiviral vector.

[0048] In various embodiments, the method further comprises thawing the formulated bulk lentiviral vector, sterile filtering the formulated bulk lentiviral vector, filling finish the formulated bulk lentiviral vector to produce a final lentiviral vector, and freezing the final lentiviral vector. In some embodiments, the final lentiviral vector is frozen at ≦−65° C.

[0049] In another embodiment, about 10.0 x 10 cells / mL of culture medium in a P0 suspension culture. 6 ~Approx. 15.0×10 6 Inoculate approximately 30.0 x 10 live HEK293T cells obtained from the P0 suspension culture into a P1 suspension culture containing approximately 100 mL of culture medium. 6 ~Approx. 70.0×10 6Inoculate approximately 11.0 x 10 live HEK293T cells obtained from the P1 suspension culture into three P2 suspension cultures, each containing approximately 200 mL of culture medium. 7 ~Approx. 19.0×10 7 Inoculate approximately 55.0 x 10 viable HEK293T cells obtained from the pooled P2 suspension cultures into three P3 suspension cultures, each containing approximately 1.0 L of culture medium. 7 ~Approx. 95.0×10 7 Inoculate approximately 40.0 x 10 viable HEK293T cells obtained from the pooled P3 suspension culture into a P4 suspension culture containing approximately 20.0 L of culture medium. 8 ~Approx. 120.0×10 8 Inoculate approximately 40.0 x 10 viable HEK293T cells obtained from the P4 suspension culture into a P5 suspension culture containing approximately 200.0 L of culture medium. 8 ~Approx. 120.0×10 8inoculating the P5 suspension culture with viable HEK293T cells; culturing the P5 suspension culture for about 3 days and replacing the culture medium of the P5 suspension culture with about 190.0 L of fresh culture medium using alternating tangential flow filtration (ATF); transfecting the P5 suspension culture after the culture medium replacement to produce a lentiviral vector, the transfecting step including adding about 10.0 L of culture medium containing a transfer plasmid and plasmid DNA encoding gag, pol, rev, and VSV-g complexed with polyethylenimine (PEI); replacing the culture medium of the P5 suspension culture with about 200.0 L of fresh culture medium after transfection using ATF; about 36 hours to about 48 hours after transfection, treating the P5 suspension culture with an endonuclease for about 1 hour to about 2 hours; a tandem depth filter that retains particles of 60 μm or larger, and harvesting and clarifying the P5 suspension culture supernatant using a bilayer filter with pore sizes of 0.8 μm and 0.45 μm; capturing and concentrating the lentiviral vector from the harvested and clarified P5 suspension culture supernatant comprising heparin chromatography or sulfate cation exchange chromatography; filtering the concentrated lentiviral vector using a bilayer filter with pore sizes of 0.8 μm and 0.45 μm; ultrafiltering the lentiviral vector using tangential flow filtration (TFF) to further concentrate the lentiviral vector and diafiltering the lentiviral vector into a diafiltration buffer, thereby generating a bulk lentiviral vector; and formulating the lentiviral vector to generate a formulated bulk lentiviral vector.

[0050] In various embodiments, the culture medium is a serum-free, chemically defined cell culture medium.

[0051] In various embodiments, the ratio of N (NH2 amines in PEI):P (phosphate groups in the DNA backbone) for the PEI / DNA mixture is about 5, about 5.5, about 6, about 6.4, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.

[0052] In various embodiments, PEI is added to the suspension culture for about 14 to about 18 hours, and optionally, the suspension culture is subjected to a culture medium exchange with fresh culture medium using alternating tangential flow filtration (ATF).

[0053] In various embodiments, the transfer plasmid comprises a polynucleotide comprising a packageable lentiviral vector genome. In some embodiments, the transfer plasmid comprises a polynucleotide comprising a packageable lentiviral vector genome comprising a left chimeric (5') lentiviral LTR, and the promoter of 5'LTR is replaced with a heterologous promoter; a Psi (Ψ) packaging signal; a central polypurine tract / DNA flap (cPPT / FLAP); a retroviral transport element (RRE); a promoter operably linked to a polynucleotide of interest; and a right (3') self-inactivating (SIN) lentiviral LTR.

[0054] In various embodiments, the endonuclease is derived from Serratia marcescens, and optionally, the endonuclease is a recombinant NucA endonuclease. In some embodiments, the endonuclease has both DNA and RNA cleavage activity. In some embodiments, the endonuclease is Benzonase or Denarase. In some embodiments, the endonuclease is added at a concentration of about 60 U / ml or about 30 U / ml.

[0055] In various embodiments, the clarified suspension culture is optionally adjusted to about pH 7.0 or about pH 7.2 with 1 M HEPES.

[0056] In various embodiments, the sulfate cation exchange chromatography comprises a column having a bead size of about 45 μm and / or an average pore size of about 100 nm.

[0057] In various embodiments, a wash buffer comprising about 50 mM HEPES, about 300 mM NaCl, pH 7.2 is pumped onto the affinity chromatography column or the cation exchange chromatography column.

[0058] In various embodiments, an elution buffer comprising about 50 mM HEPES, about 1 M NaCl, pH 7.5 is pumped onto an affinity chromatography column or a cation exchange chromatography column.

[0059] In various embodiments, the lentiviral vector is ultrafiltered using hollow fiber TFF filters with a pore size or molecular weight cutoff of about 100 kDa, about 300 kDa, or about 500 kDa, and diafiltered into a diafiltration buffer.

[0060] In various embodiments, the diafiltration buffer is about 50 mM HEPES, about 100 mM NaCl, pH 7.50.

[0061] In various embodiments, the diafiltration buffer is about 50 mM HEPES, pH 7.0.

[0062] In various embodiments, the lentiviral vector is formulated 1:1 in 2× stem cell growth medium (SCGM) to generate a formulated bulk lentiviral vector.

[0063] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer comprising HEPES and sucrose, optionally, the buffer further comprises L-proline, poloxamer 188, or NaCl.

[0064] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, and about 100 mM L-proline.

[0065] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 0.2 mg / ml poloxamer 188.

[0066] In various embodiments, the lentiviral vector is formulated 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 150 mM NaCl.

[0067] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, about 150 mM NaCl, and about 0.2 mg / ml poloxamer 188.

[0068] In various embodiments, the lentiviral vector is formulated 1:1 with a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 150 mM NaCl, and about 0.2 mg / ml poloxamer 188.

[0069] In various embodiments, the method further comprises fill-finishing the formulated bulk lentiviral vector to produce a final lentiviral vector, and freezing the final lentiviral vector.

[0070] In various embodiments, the method further comprises freezing the formulated bulk lentiviral vector.

[0071] In various embodiments, the method further comprises thawing the formulated bulk lentiviral vector, sterile filtering the formulated bulk lentiviral vector, filling finish the formulated bulk lentiviral vector to produce a final lentiviral vector, and freezing the final lentiviral vector. In some embodiments, the final lentiviral vector is frozen at ≦−65° C.

[0072] In another aspect, a method for reducing host cell protein (HCP) from the suspension method for producing viral vector is provided.In various embodiments, the method for reducing HCP includes: (a) preparing the harvested and clarified suspension culture supernatant containing viral vector (for example, the viral vector produced as contemplated herein); (b) using cation exchange chromatography to capture and concentrate viral vector from the harvested and clarified suspension culture supernatant; (c) filtering the concentrated viral vector; (d) using tangential flow filtration (TFF) to ultrafilter and diafilter viral vector; and (e) formulating the viral vector to produce formulated bulk viral vector, and sterile filtering the formulated bulk viral vector.

[0073] In various embodiments, the viral vector is a lentiviral vector.

[0074] In various embodiments, the viral vector comprises a vesicular stomatitis virus (VSV) envelope protein or variant thereof (e.g., VSV-G), a coccococcal virus (COCV) envelope protein or variant thereof, a Maraba virus (MARAV) envelope protein or variant thereof, a Pili virus (PIRYV) envelope protein or variant thereof, a Nipah virus (NiV) envelope protein or variant thereof, a Sendai virus (SeV) envelope protein or variant thereof, a Morbillivirus envelope protein or variant thereof, a Canine Distemper (CDV) envelope protein or variant thereof, a Measles Virus (MV) envelope protein or barrier protein thereof, In some embodiments, the viral vector is pseudotyped with a heterologous envelope protein selected from the group consisting of a vesicular stomatitis virus (VSV) envelope protein or variant thereof, a sindbis virus (SINV) envelope protein or variant thereof, a gibbon ape leukemia virus (GALV) envelope protein or variant thereof, a feline endogenous retrovirus (RD114) envelope protein or variant thereof, a feline leukemia virus (FeLV) envelope protein or variant thereof, a baboon endogenous retrovirus (BaEV) envelope protein or variant thereof, a hepatitis B (HBV) envelope protein or variant thereof, a hepatitis C (HCV) envelope protein or variant thereof, and a rabies virus (RABV) envelope protein or variant thereof. In some embodiments, the viral vector is pseudotyped with a heterologous envelope protein consisting of a vesicular stomatitis virus (VSV) envelope protein or variant thereof (e.g., VSV-G).

[0075] In various embodiments, the harvesting and clarification steps include filtering the suspension culture supernatant through a tandem depth filter that retains contaminants of at least about 40 μm or at least about 60 μm and a bilayer filter with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.22 μm to about 0.45 μm.

[0076] In various embodiments, the clarified suspension culture is optionally adjusted to about pH 7.0 or about pH 7.2 with 1 M HEPES.

[0077] In various embodiments, the supernatant is passed through a cation exchange chromatography column.

[0078] In various embodiments, the cation exchange chromatography is sulfate cation exchange chromatography. In some embodiments, the sulfate cation exchange chromatography comprises a column having a bead size of about 45 μm and / or an average pore size of about 100 nm.

[0079] In various embodiments, a wash buffer comprising about 50 mM HEPES, about 300 mM NaCl, pH 7.2 is passed through the chromatography column.

[0080] In various embodiments, an elution buffer comprising about 50 mM HEPES, about 1 M NaCl, pH 7.5 is passed through the chromatography column.

[0081] In various embodiments, the filtering step (c) comprises filtering the concentrated viral vector through a bilayer filter with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.2 μm to about 0.45 μm.

[0082] In various embodiments, the viral vector is ultrafiltered and diafiltered using hollow fiber tangential flow filtration (TFF) filters with a pore size or molecular weight cutoff of about 100 kDa to about 500 kDa. In some embodiments, the hollow fiber TFF filters have a pore size or molecular weight cutoff of about 100 kDa. In some embodiments, the hollow fiber TFF filters have a pore size or molecular weight cutoff of about 300 kDa. In some embodiments, the hollow fiber TFF filters have a pore size or molecular weight cutoff of about 500 kDa.

[0083] In various embodiments, the viral vector is diafiltered into a diafiltration buffer, optionally, the diafiltration buffer is about 50 mM HEPES, pH 7.0.

[0084] In various embodiments, the viral vector is formulated 1:1 in a buffer comprising HEPES and sucrose, optionally further comprising L-proline, poloxamer 188, or NaCl. In some embodiments, the viral vector is formulated 1:1 in a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, and about 100 mM L-proline. In some embodiments, the viral vector is formulated 1:1 in a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 0.2 to about 2.0 poloxamer 188. In some embodiments, the viral vector is formulated 1:1 in a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 150 mM NaCl. In some embodiments, the viral vector is formulated 1:1 with a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, about 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188. In some embodiments, the viral vector is formulated 1:1 with a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188.

[0085] In various embodiments, the formulated bulk viral vector is optionally sterile filtered through a 0.22 μm filter with a 0.45 μm prefilter.

[0086] In various embodiments, the method further includes fill-finishing the formulated bulk viral vector to produce a final viral vector, and freezing the final viral vector.

[0087] In various embodiments, the method further comprises freezing the formulated bulk viral vector. In some embodiments, the method further comprises thawing the formulated bulk viral vector, sterile filtering the formulated bulk viral vector, fill finishing the formulated bulk viral vector to produce a final viral vector, and freezing the final viral vector.

[0088] In various embodiments, the final viral vector is frozen at ≦-65°C. [Brief description of the drawings]

[0089] [Figure 1] FIG. 1 shows an example of the upstream process flow, downstream process flow and fill-to-fill for the production of lentiviral vectors from suspension cultures. [Diagram 2] FIG. 2 shows an example of the upstream process flow, downstream process flow and fill-to-fill for the production of lentiviral vectors from suspension cultures. [Figure 3A] 3A-3C show plasmid maps of the gag, pol, rev, and vsv-g packaging plasmids. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4] FIG. 4 shows an example of the upstream process flow, downstream process flow and fill-to-fill for the production of lentiviral vectors from suspension cultures. [Diagram 5] FIG. 5 shows a comparison of total infectious titer yields between different LVV production methods. [Figure 6] FIG. 6 shows a comparison of infectious titers between different LVV production methods. [Figure 7] FIG. 7 shows a comparison of the ratio of particle to infectivity between different LVV production methods. [Figure 8] FIG. 8 shows a comparison of normalized host cell proteins (HCPs) between different LVV production methods. [Figure 9]FIG. 9 shows a comparison of cumulative host cell protein (HCP) log reduction between different LVV production methods. [Figure 10] FIG. 10 shows a comparison of process host cell protein (HCP) log reduction between different LVV production methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] A. Overview The present disclosure generally relates in part to an improved large-scale process for producing retroviral vectors for clinical use. Without wishing to be bound by any particular theory, the present disclosure is the first to provide a manufacturing process for producing clinical grade retroviral and lentiviral vectors. The vectors produced using the manufacturing process contemplated herein are produced at a clinical scale with infectivity and purity that cannot be matched by methods existing in the art. Furthermore, the present disclosure provides a method for reducing host cell proteins (HCPs) from the supernatant of produced viral vectors.

[0091] The manufacturing processes existing in the art usually err on the side of increasing viral vector quantity at the expense of vector quality. The manufacturing process contemplated herein solves the problem in the art of trading volume for quality, allowing the production of high-quality clinical grade vectors on a commercial scale. In fact, the process contemplated herein shows higher infectious titers (TU / ml) and, in some embodiments, comparable or lower host cell protein (HCP) levels.

[0092] The contemplated manufacturing process includes an upstream process of producing viral vectors and a downstream process of purifying viral vectors.The contemplated manufacturing process includes: establishing a large-scale host cell culture in a bioreactor; transiently transfecting host cells with a mixture containing packaging plasmids and transfer plasmids encoding viral accessory genes; culturing the transfected host cells to produce virus; and collecting and processing the culture supernatant containing crude lentiviral vectors to remove impurities, concentrate, and formulate viral vectors for clinical use.

[0093] In certain embodiments, the manufacturing process contemplated herein includes an upstream process that includes thawing, culturing, and expanding host cells in progressively larger quantities until there is enough host cells to seed a large-scale, e.g., at least 200 L working volume, bioreactor. The seeded host cells are cultured to a desired density, the medium is exchanged, and the cells are transfected with a mixture that includes a transfection agent, a packaging plasmid encoding a viral accessory gene, and a transfer plasmid encoding a packageable viral vector genome that includes a therapeutic transgene. After a sufficient time for transfection, another medium exchange is performed, and the transfected host cells are cultured to produce the viral vector for about one to about three days. In a preferred embodiment, the host cells are cultured in a serum-free, chemically defined cell culture medium.

[0094] In certain embodiments, the manufacturing process contemplated herein comprises downstream processes including: treating the contents of bioreactor with DNA endonuclease; harvesting and clarifying suspension culture supernatant by filtration; capturing and concentrating viral vector in the resulting filtrate using affinity chromatography or cation exchange chromatography; filtering the eluate containing viral vector; ultrafiltering and diafiltering viral vector using tangential flow filtration (TFF); formulating viral vector in culture medium to produce formulated bulk viral vector.In one embodiment, formulated bulk lentiviral vector is sterile filtered, filled, and frozen; then thawed, sterile filtered, and subjected to final filling finish.In another embodiment, bulk lentiviral vector is sterile filtered, subjected to final filling finish, and frozen.

[0095] In a preferred embodiment, the viral vector is a retroviral vector, and in an even more preferred embodiment, the viral vector is a lentiviral vector.

[0096] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, which are cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I&II (IRL Press, Oxford Univ. Press USA, 1985), Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach,Warren Strober 2001 John Wiley&Sons,NY,NY)、Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK、Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)、Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991)、Oligonucleotide Synthesis(N.Gait,Ed.,1984)、Nucleic Acid The Hybridization(B.Hames&S.Higgins,Eds.,1985)、Transcription and Translation(B.Hames&S.Higgins,Eds.,1984)、Animal Cell Culture(R.Freshney,Ed.,1986)、Perbal,A Practical Guide to Molecular Cloning(1984)、Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH)、PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press)、Immobilized Cells And Enzymes(IRL Press,1986)、the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.)、Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology are examples of such research papers.

[0097] B. Definition Before describing the present disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test specific embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below.

[0099] The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e. to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0100] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0101] The term "and / or" should be understood to mean either or both of the alternatives.

[0102] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0103] In one embodiment, ranges such as, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refer to each value subsumed within the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0104] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces about the same effect, e.g., a physiological effect, as the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0105] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to everything preceding the word "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to any elements recited thereafter, and other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the recited elements.

[0106] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. As such, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Additionally, it should be understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0107] The term "vector" is used herein to refer to a nucleic acid molecule, microorganism, or virus capable of transferring or transporting another nucleic acid molecule into a cell or genome. Illustrative examples of vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, bacteria, and viral vectors.

[0108] The term "viral vector" is used in certain embodiments to refer to a nucleic acid molecule that contains a virus-derived nucleic acid element that typically facilitates the transfer or integration of the nucleic acid molecule into a cell and / or genome. The term "viral vector" also refers, in particularly preferred embodiments, to any modified virus or virus particle that is capable of transporting nucleic acid into a cell and / or genome. A viral vector may contain structural and / or functional genetic elements that are primarily derived from a virus. Viral vectors suitable for use in preferred embodiments include, but are not limited to, retroviral vectors and lentiviral vectors.

[0109] Retroviral vectors are common tools for gene delivery (Miller, 2000, Nature. 357:455-460). As used herein, the term "retrovirus" or "retroviral vector" refers to a viral vector that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, which then covalently integrates the genomic DNA into the host genome. Exemplary retroviral vectors suitable for use in certain embodiments include, but are not limited to, those derived from Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV), and lentiviruses.

[0110] As used herein, the term "lentivirus" refers to a group (or species) of complex retroviruses. Examples of lentiviral vectors suitable for use in certain embodiments contemplated herein include, but are not limited to, those derived from HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2); Visnamaedivirus (VMV); Caprine arthritis encephalitis virus (CAEV), Equine infectious anemia virus (EIAV), Feline immunodeficiency virus (FIV), Bovine immunodeficiency virus (BIV), and Simian immunodeficiency virus (SIV).

[0111] The term "provector" or "provirus" refers to a viral vector that has integrated into a host genome. Provectors are similar to viral vectors but contain two copies of the 3'LTR generated during reverse transcription, see e.g., Pluta and Kacprzak, 2009.

[0112] In certain embodiments, the viral vector comprises a 5'LTR, a packaging signal, a cPPT / FLAP element, an RNA transport element, a transgene, and a 3'LTR. The viral vector may optionally comprise post-transcriptional control elements and a polyadenylation signal / sequence.

[0113] The term "long terminal repeat (LTR)" refers to the domains of base pairs located at the ends of retroviral genomes; in the original sequence, LTRs are direct repeats and contain U3, R, and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain a number of control signals, including transcriptional regulatory elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, called U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region, and contains polyadenylation sequences. The R (repeat) region is flanked by the U3 and U5 regions. LTRs are composed of the U3, R, and U5 regions and are present at both the 5' and 3' ends of the viral genome. Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site).

[0114] As used herein, the term "modified LTR" refers to one or more nucleotide additions, deletions or substitutions in the native virus 5'LTR and / or 3'LTR. Those skilled in the art will be able to determine whether an LTR is modified compared to a reference LTR. One or both of the LTRs may contain one or more modifications. Modification of the 3'LTR is often performed to improve the safety of the viral vector system, including but not limited to making the viral vector replication-defective.

[0115] As used herein, the term "replication defective" refers to a viral vector that is incapable of replicating completely and efficiently, such that infectious viral particles are not produced (e.g., replication defective viral progeny).

[0116] A "self-inactivating" (SIN) viral vector refers to a vector that lacks replication competence, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to inhibit viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and thus viral transcripts cannot be made without the U3 enhancer-promoter. Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to generate the vector RNA. This deletion is then transferred to the 5' LTR of the proviral DNA during reverse transcription. In the case of lentiviral vectors, it has been shown that such vectors can tolerate large U3 deletions, including removal of the LTR TATA box (e.g., deletion of -418 to -18), without significant loss of vector titer.

[0117] Additional safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter (i.e., chimeric 5'LTR) to drive transcription of the viral vector genome during viral vector production. The chimeric 5'LTR promoter can drive high levels of transcription in a Tat-independent manner. Examples of heterologous promoters suitable for use in certain embodiments contemplated herein include, but are not limited to, simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0118] "R region" refers to the region within the LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just before the start of the polyA tract. The R region is also defined as being adjacent to the U3 and U5 regions. The R region plays a role in allowing the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0119] In certain embodiments, the viral vector contemplated comprises a TAR element. The term "TAR" refers to the "transactivation response" genetic element located in the R region of LTR. This element interacts with the transactivator (tat) genetic element to enhance viral replication. However, this element is not required in viral vectors in which the U3 region of 5'LTR is replaced by a heterologous promoter.

[0120] As used herein, the term "packaging signal" or "packaging sequence" refers to a sequence located in the viral genome and is required for inserting viral RNA into the viral capsid or viral particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4, pp. 2101-2109. Some viral vectors use a minimal packaging signal (also referred to as psi [Ψ] or [Ψ+] sequence) required for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence", "packaging signal", "psi" and the symbol "Ψ" are used in reference to the non-coding sequence required for encapsidation of the viral RNA strand during viral particle formation.

[0121] As used herein, the term "FLAP element" or "cPPT / FLAP" refers to a nucleic acid whose sequence comprises the central polypurine tract and central termination sequences (cPPT and CTS) of a viral vector. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During viral reverse transcription, the central initiation of positive-stranded DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) result in the formation of a triple-stranded DNA structure called central DNA FLAP. Without wishing to be bound by any theory, DNA FLAP may act as a cis-acting determinant of viral vector genome nuclear import and / or increase viral titer.

[0122] The term "export factor" refers to a cis-acting post-transcriptional regulator that controls the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423).

[0123] As used herein, the term "post-transcriptional control element" or "PRE" refers to a cis-acting element that controls expression at the mRNA level, for example, by controlling capping, splicing, poly(A) tailing, and mRNA stability. Illustrative examples of PTEs include, but are not limited to, the woodchuck hepatitis virus post-transcriptional control element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional control element present in Hepatitis B virus (HPRE) (Huang and Yen, 1995, Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

[0124] As used herein, the term "poly(A) site" or "poly(A) sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of a mammalian pre-mRNA has two recognition elements adjacent to the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA).

[0125] As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-stranded RNA (RNA(+)), negative-stranded RNA (RNA(-)), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Illustrative examples of polynucleotides contemplated in certain embodiments include, but are not limited to, transfer plasmids, plasmids encoding viral structural and / or accessory proteins, e.g., gag, poly, tat, rev, and / or env, and polynucleotides of interest.

[0126] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the reference sequences described herein, and typically, the variant maintains at least one biological activity of the reference sequence. Polynucleotide variants include polynucleotides in which one or more nucleotides are added or deleted or replaced with different nucleotides compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to the reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0127] As used herein, the term "polynucleotide of interest" refers to one or more polynucleotides, including a therapeutic polypeptide, inserted into a vector that is desired to be expressed, e.g., a polynucleotide encoding a polypeptide (i.e., a polypeptide of interest). In certain embodiments, the vector and / or plasmid comprises one or more therapeutic RNAs, e.g., shRNA, miRNA, or shmiR, and / or one or more polynucleotides of interest that encode a therapeutic polypeptide.

[0128] Polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., as disclosed elsewhere herein or known in the art, so that their overall length may vary greatly. It is therefore contemplated that polynucleotide fragments of almost any length may be employed, with the overall length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0129] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

[0130] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and its variants and synthetic analogs. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of corresponding natural amino acids, as well as naturally occurring amino acid polymers. Illustrative examples of polypeptides include, but are not limited to, globin polypeptides suitable for use in the compositions and methods of certain embodiments. See also, for example, U.S. Patent Nos. 6,051,402, 7,901,671, and 9,068,199, the entire disclosures and claims of which are specifically incorporated herein by reference in their entirety.

[0131] Certain embodiments contemplated herein also include polypeptide "variants." A recited polypeptide "variant" refers to a polypeptide that is distinguished from a reference polypeptide by the addition, deletion, truncation, modification, and / or substitution of at least one amino acid residue and retains biological activity. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions that may be conservative or non-conservative as known in the art. In certain embodiments, a variant polypeptide comprises an amino acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity with the corresponding sequence of the reference polypeptide. In certain embodiments, the amino acid addition or deletion occurs at the C-terminus and / or N-terminus of the reference polypeptide.

[0132] Additional definitions are set forth throughout this disclosure.

[0133] C. Upstream viral vector manufacturing process The upstream manufacturing process contemplated herein includes culturing a population of host cells in a large-scale bioreactor, transfecting the host cells with a mixture including a transfer plasmid, a packaging plasmid, and a transfection agent; and culturing the transfected host cells to produce the viral vector. In certain embodiments, the upstream manufacturing process contemplated herein can be used in combination with various downstream manufacturing processes.

[0134] The upstream manufacturing process contemplated in certain embodiments herein includes thawing a working cell bank of host cells; culturing and expanding the host cells in gradually larger quantities until there is a sufficient amount of host cells to seed a large-scale bioreactor; seeding the large-scale bioreactor with the host cells and culturing the host cells until a sufficient density is reached for transfection; exchanging the culture medium in the bioreactor; transfecting the host cells with a mixture comprising a transfection agent, a packaging plasmid encoding a viral accessory gene, and a transfer plasmid encoding a packageable viral vector genome comprising a therapeutic transgene; culturing the transfected cells for a period sufficient for complete transfection; exchanging the culture medium in the bioreactor; and culturing the cells for about one to about three days to produce the viral vector.

[0135] Upstream manufacturing processes contemplated in certain embodiments herein include thawing a working cell bank of host cells; culturing and expanding the host cells in gradually larger quantities until there is a sufficient amount of host cells to seed a large-scale bioreactor; seeding the large-scale bioreactor with the host cells and culturing the host cells until they reach a density sufficient for transfection; exchanging the culture medium in the bioreactor; transfecting the host cells with a mixture comprising a transfection agent, a packaging plasmid encoding a viral accessory gene, and a transfer plasmid encoding a packageable viral vector genome comprising a therapeutic transgene; culturing the transfected cells for a period sufficient for complete transfection; exchanging the culture medium in the bioreactor; and culturing the cells for about one to about three days to produce the viral vector, e.g., a retroviral or lentiviral vector.

[0136] In various embodiments, the upstream manufacturing process of retroviral vectors, e.g., lentiviral vectors, includes thawing a working cell bank of host cells, expanding the host cells in gradually larger quantities in serum-free, chemically defined cell culture medium over a period of about 18 days; inoculating a quantity of live host cells into a large-scale bioreactor, e.g., of at least 200 liters working volume, and culturing the host cells in serum-free, chemically defined cell culture medium for about three days; replacing the cell culture medium in the bioreactor with fresh serum-free, chemically defined cell culture medium; transfecting the cells with a mixture comprising a transfection agent, a packaging plasmid encoding a viral accessory gene, and a transfer plasmid encoding a packageable viral vector genome comprising a therapeutic transgene; replacing the culture medium in the bioreactor with fresh serum-free, chemically defined cell culture medium for about 12 hours to about 20 hours of transfection or about 14 hours to about 18 hours of transfection; and culturing the transfected host cells for about one day or about three days to produce the retroviral vector. In a preferred embodiment, the culture supernatant is collected once during about one or about three days of retroviral production. In a specific embodiment, the culture supernatant is collected once, twice, or three times during about one to about three days of retroviral vector production.

[0137] 1.Host cells The large-scale viral vector production process contemplated herein involves introducing transfer vectors and plasmids encoding viral structural and / or accessory genes, such as gag, pol, env, rev, tat, vif, vpr, vpu, vpx, and / or nef genes, into a population of host cells.

[0138] "Host cells" refer to cells that are modified to produce viral vectors. In certain embodiments, host cells include packaging cells and producer cells. "Packaging cells" are host cells that are modified to express viral structures and / or accessory genes that allow packaging of a viral vector genome into a viral vector. Packaging cells do not contain a packaging signal for packaging the viral vector genome into a viral vector. "Producer cells" are packaging cells that contain a viral vector genome that includes a packaging signal for packaging the viral vector genome into a viral vector.

[0139] In a preferred embodiment, the host cells are mammalian cells that can be cultured in suspension culture or that are capable of being adapted to suspension culture.

[0140] In certain embodiments, host cells suitable for use in certain embodiments include, but are not limited to, CHO cells, A549 cells, and HEK293 cells and their derivatives.In preferred embodiments, the host cell is selected from the group consisting of HEK293 cells, HEK293S cells, HEK293T cells adapted to suspension culture (HEK392T), HEK293F cells, HEK293FT cells, HEK293FTM cells, and HEK293E cells.In more preferred embodiments, the host cell is HEK293T cells.

[0141] 2. Expansion Culture Host cells are often stored in aliquots as part of a working cell bank. A working cell bank is a convenient way to store substantially similar aliquots of host cells to maximize reproducibility in host cell expansion culture. The upstream manufacturing process contemplated in certain embodiments contemplates thawing the host cells and culturing the host cells in gradually larger quantities to generate a sufficient number of live host cells to inoculate a large-scale bioreactor. Large-scale bioreactors suitable for use in certain embodiments include, but are not limited to, bioreactors having a working volume of at least 100L, at least 200L, at least 250L, at least 500L, at least 1000L, at least 1500L, or at least 2000L.

[0142] In certain embodiments, the working cell bank is thawed and the host cells are expanded (or passaged) through 1, 2, 3, 4, 5, 6 or more culture rounds (e.g., P0, P1, P2, P3, P4, P5) in gradually larger quantities over a total period of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days.

[0143] The host cells are cultured for expansion in a chemically defined cell culture medium. In a preferred embodiment, the host cells are cultured in a serum-free chemically defined cell culture medium. Illustrative examples of serum-free chemically defined cell culture media suitable for use in certain embodiments include, but are not limited to, Freestyle 293 Expression Medium, Ex-Cell 293 Serum-Free Medium, Expi293 Expression Medium, and Opti-MEM Reduced Serum Medium. The host cells are cultured at about 37° C. and about pH 7.

[0144] In certain embodiments, the working cell bank is thawed and the host cell culture is cultured to a concentration of at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% viable cells at least about 0.5×10 6 Cells / mL, at least approximately 1 x 10 6Cells / mL, at least approximately 1.5 x 10 6 Cells / mL, at least approximately 2 x 10 6 cells / mL, or at least about 2.5 x 10 6 The host cells are cultured in a volume of about 50 mL of medium (e.g., in a culture vessel of at least 250 mL) until a viable cell density of 1000 cells / mL is reached. In certain embodiments, the 50 mL culture is referred to as the P0 (first passage) culture.

[0145] In certain embodiments, after the viable host cells from the P0 culture reach sufficient density, the host cell culture is characterized in that at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the viable cells are at least about 2.5×10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 5.5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 6.5 x 10 6 Cells / mL, at least approximately 7 x 10 6 Cells / mL, at least approximately 7.5 x 10 6 Cells / mL, at least approximately 8 x 10 6 cells / mL, 8.5×10 6 Cells / mL, at least approximately 9 x 10 6 Cells / mL, at least approximately 9.5 x 10 6 Cells / mL, at least approximately 10 x 10 6 Cells / mL, at least approximately 10.5 x 10 6 Cells / mL, at least about 11 x 10 6 Cells / mL, at least about 115 x 10 6 Cells / mL, at least approximately 12 x 10 6 Cells / mL, at least approximately 12.5 x 10 6 Cells / mL, at least about 13 x 10 6Cells / mL, at least approximately 13.5 x 10 6 Cells / mL, at least approximately 14 x 10 6 Cells / mL, at least approximately 14.5 x 10 6 cells / mL, or at least about 15 x 10 6 The cells are expanded to a culture volume of about 100 mL of medium (e.g., at least a 500 mL culture vessel) until a viable cell density of 100 cells / mL is reached. In certain embodiments, the 100 mL culture is referred to as the P1 (second passage) culture.

[0146] In certain embodiments, after the viable host cells from the P1 culture reach sufficient density, the host cell culture is characterized in that at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the viable cells are at least about 2.5×10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 5.5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 6.5 x 10 6 Cells / mL, at least approximately 7 x 10 6 Cells / mL, at least approximately 7.5 x 10 6 Cells / mL, at least approximately 8 x 10 6 cells / mL, 8.5×10 6 Cells / mL, at least approximately 9 x 10 6 Cells / mL, at least approximately 9.5 x 10 6 Cells / mL, at least approximately 10 x 10 6 Cells / mL, at least approximately 10.5 x 10 6 Cells / mL, at least about 11 x 10 6 Cells / mL, at least about 115 x 10 6 Cells / mL, at least approximately 12 x 10 6 Cells / mL, at least approximately 12.5 x 10 6Cells / mL, at least about 13 x 10 6 Cells / mL, at least approximately 13.5 x 10 6 Cells / mL, at least approximately 14 x 10 6 Cells / mL, at least approximately 14.5 x 10 6 cells / mL, or at least about 15 x 10 6 The cells are expanded into single, double or triple cultures, each with a culture volume of about 200 mL of medium (e.g., at least 1 L culture vessel), until a viable cell density of 10000000 cells / mL is reached. In certain embodiments, the 200 mL cultures are referred to as P2 (passage 3) cultures.

[0147] In certain embodiments, after the viable host cells from the P2 culture reach sufficient density, the host cell culture is characterized in that at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the viable cells are at least about 2.5×10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 5.5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 6.5 x 10 6 Cells / mL, at least approximately 7 x 10 6 Cells / mL, at least approximately 7.5 x 10 6 Cells / mL, at least approximately 8 x 10 6 cells / mL, 8.5×10 6 Cells / mL, at least approximately 9 x 10 6 Cells / mL, at least approximately 9.5 x 10 6 Cells / mL, at least approximately 10 x 10 6 Cells / mL, at least approximately 10.5 x 10 6 Cells / mL, at least about 11 x 10 6 Cells / mL, at least about 115 x 10 6 Cells / mL, at least approximately 12 x 106 Cells / mL, at least approximately 12.5 x 10 6 Cells / mL, at least about 13 x 10 6 Cells / mL, at least approximately 13.5 x 10 6 Cells / mL, at least approximately 14 x 10 6 Cells / mL, at least approximately 14.5 x 10 6 cells / mL, or at least about 15 x 10 6 The cells are expanded into single, double or triple cultures, each with a culture volume of about 1 L of medium (e.g., at least a 3 L culture vessel), until a viable cell density of 100000000 cells / mL is reached. In certain embodiments, the 1 L culture is referred to as a P3 (fourth passage) culture.

[0148] In certain embodiments, after the viable host cells from the P3 culture reach sufficient density, the host cell culture is at least about 85%, or at least about 90%, of the viable cells are at least about 2.5×10 6 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 5.5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 6.5 x 10 6 Cells / mL, at least approximately 7 x 10 6 Cells / mL, at least approximately 7.5 x 10 6 Cells / mL, at least approximately 8 x 10 6 cells / mL, 8.5×10 6 Cells / mL, at least approximately 9 x 10 6 Cells / mL, at least approximately 9.5 x 10 6 Cells / mL, at least approximately 10 x 10 6 Cells / mL, at least approximately 10.5 x 10 6 Cells / mL, at least about 11 x 10 6 Cells / mL, at least about 115 x 10 6Cells / mL, at least approximately 12 x 10 6 Cells / mL, at least approximately 12.5 x 10 6 Cells / mL, at least about 13 x 10 6 Cells / mL, at least approximately 13.5 x 10 6 Cells / mL, at least approximately 14 x 10 6 Cells / mL, at least approximately 14.5 x 10 6 cells / mL, or at least about 15 x 10 6 The cells are expanded into culture in a bioreactor with a volume of about 20 L of medium (e.g., a bioreactor with a volume of at least 50 L) until a viable cell density of 1000000 cells / mL is reached. In certain embodiments, the 20 mL culture is referred to as a P4 (fifth passage) culture.

[0149] In certain embodiments, after the viable host cells from the P4 culture reach a sufficient density, a large-scale bioreactor (P5, sixth passage culture) having a working volume of at least about 200 L is cultured at a density of at least about 0.1×10 6 At least approximately 0.15 x 10 viable cells / mL 6 At least approximately 0.2 x 10 cells / mL 6 At least approximately 0.25 x 10 cells / mL 6 At least approximately 0.3 x 10 cells / mL 6 Cells / mL, at least approximately 0.35 x 10 6 At least approximately 0.4 x 10 cells / mL 6 At least approximately 0.45 x 10 cells / mL 6 cells / mL, or at least about 0.5 x 10 6 Cells / mL are seeded.

[0150] In various embodiments, the large-scale suspension bioreactor comprises approximately 40.0×10 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 50.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 60.0×10 live host cells.8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 70.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 80.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 90.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 100.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 110.0×10 live host cells. 8 ~Approx. 120.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 110.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 100.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 90.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 80.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 70.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 60.0×10 8In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 ~Approx. 50.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 50.0×10 live host cells. 8 ~Approx. 110.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 60.0×10 live host cells. 8 ~Approx. 100.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 70.0×10 live host cells. 8 ~Approx. 90.0×10 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 40.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 50.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 60.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 70.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 80.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with approximately 90.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with 100.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with 110.0×10 live host cells. 8 In some embodiments, the large-scale suspension bioreactor is seeded / inoculated with 120.0×10 live host cells. 8 are seeded / inoculated into live host cells.

[0151] Cells were cultured at a viable cell density of at least approximately 2.5×106 Cells / mL, at least approximately 3 x 10 6 Cells / mL, at least approximately 3.5 x 10 6 Cells / mL, at least approximately 4 x 10 6 Cells / mL, at least approximately 4.5 x 10 6 Cells / mL, at least approximately 5 x 10 6 Cells / mL, at least approximately 5.5 x 10 6 Cells / mL, at least approximately 6 x 10 6 Cells / mL, at least approximately 6.5 x 10 6 Cells / mL, at least approximately 7 x 10 6 cells / mL, or at least about 7.5 x 10 6 The cells are cultured for about two, about three, or about four days until the cells reach a density of about 100 cells / mL.

[0152] In certain embodiments, the host cells cultured in the large-scale bioreactor reach a desired pre-transduction viable cell density, and the cell culture medium in the bioreactor is replaced with fresh chemically defined cell culture medium. In certain embodiments, an alternating tangential flow filtration (ATF) filter unit, a TFF filter unit, or an acoustic filter unit is used to perform the medium exchange. In one embodiment, an ATF filter unit is used to perform the medium exchange. In one embodiment, a TFF filter unit is used to perform the medium exchange. In one embodiment, an acoustic filter unit is used to perform the medium exchange. In a preferred embodiment, after the bioreactor medium is exchanged, the host cells are ready for transfection.

[0153] 3. Transfection Transfection is the process of introducing one or more polynucleotides into a host cell by physical or chemical methods (non-viral). "Transfection" refers to the process of introducing naked DNA into a cell by non-viral methods. Transfection can be stable or transient. In certain embodiments, a host cell is transiently transfected with a mixture comprising one or more plasmids encoding one or more viral structures and / or accessory genes for packaging the viral vector genome, a transfer plasmid containing a packaging signal and the viral vector genome containing a transgene (e.g., a therapeutic gene, a gene of interest, or a polynucleotide of interest), and a transfection agent.

[0154] A "transfection agent" is a molecule that increases the transfection of DNA into a host cell. Illustrative examples of transfection agents suitable for use in certain embodiments contemplated herein include, but are not limited to, calcium phosphate, cationic lipids, and cationic polymers.

[0155] Illustrative examples of cationic lipids suitable for use in certain embodiments contemplated herein include, but are not limited to, N-[1-(2,3-dioleoyloxy)proper]-N,N,N-trimethylammonium (DOTMA); 2,3-dioleyloxy-N-[2-sperminecarboxamido]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA, Lipofectamine); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); N-[1-(2,3-dimyristoyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 3-β-[N-(N,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol); dioctadecylamidoglyceryl spermine (DOGS, Transfectam); and dimethyldioctadecylammonium bromide (DDAB). In certain embodiments, the cationic lipid is lipofectamine.

[0156] Illustrative examples of cationic polymers suitable for use in certain embodiments contemplated herein include, but are not limited to, DEAE-dextran, polybrene, dendrimers, and polyethyleneimine (PEI).

[0157] In certain embodiments, host cells in large-scale bioreactors are transiently transfected with a mixture comprising a transfection agent, one or more plasmids encoding viral structural and / or accessory genes, and a transfer plasmid comprising a packageable viral vector genome encoding a human therapeutic transgene. In preferred embodiments, the transfection agent comprises a cationic polymer, in more preferred embodiments the cationic polymer comprises PEI, and in even more preferred embodiments the cationic polymer comprises linear PEI.

[0158] In certain embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 5 to about 10, or about 5, about 5.5, about 6, about 6.4, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 5 to about 10. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 5. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 5.5. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 6. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 6.4. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 6.5. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 7. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 7.5. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 8. In some embodiments, the ratio of N(NH2 amine in PEI):P(phosphate group in DNA backbone) for the PEI / DNA mixture is about 9.5. In some embodiments, the ratio of N (NH 2 amines in PEI):P (phosphate groups in the DNA backbone) for the PEI / DNA mixture is about 10.

[0159] In a preferred embodiment, the one or more plasmids comprise polynucleotides encoding retroviral gag, pol, rev, heterologous envelope proteins, and optionally, tat; more preferably, the one or more plasmids comprise polynucleotides encoding lentiviral gag, pol, rev, and heterologous envelope proteins.

[0160] In particularly preferred embodiments, the one or more plasmids include a plasmid comprising a polynucleotide encoding lentiviral gag and pol, a plasmid encoding lentiviral rev, and a plasmid encoding a heterologous envelope glycoprotein, including, but not limited to, an envelope glycoprotein derived from a vesiculovirus envelope protein or variant thereof, a paramyxovirus envelope protein or variant thereof, an alphavirus envelope protein or variant thereof, a gammaretrovirus envelope protein or variant thereof, an orthohepadnavirus envelope protein or variant thereof, a hepacivirus envelope protein or variant thereof, and a lyssavirus envelope protein or variant thereof.

[0161] In particularly preferred embodiments, the one or more plasmids include a plasmid comprising a polynucleotide encoding lentiviral gag and pol, a plasmid encoding lentiviral rev, and a plasmid encoding a vesicular stomatitis virus (VSV) envelope protein or variant thereof (e.g., VSV-G), a coccus virus (COCV) envelope protein or variant thereof, a Maraba virus (MARAV) envelope protein or variant thereof, a Pili virus (PIRYV) envelope protein or variant thereof, a Nipah virus (NiV) envelope protein or variant thereof, a Sendai virus (SeV) envelope protein or variant thereof, a Morbillivirus envelope protein or variant thereof, a Canine Distemper (CDV) envelope protein or variant thereof, a Measles virus (MV) envelope protein or variant thereof, a Mycobacterium tuberculosis virus (MRV ... The present invention also includes plasmids encoding heterologous envelope glycoproteins, including, but not limited to, envelope glycoproteins from human rabies virus (MV) envelope protein or variant thereof, sindbis virus (SINV) envelope protein or variant thereof, gibbon ape leukemia virus (GALV) envelope protein or variant thereof, feline endogenous retrovirus (RD114) envelope protein or variant thereof, feline leukemia virus (FeLV) envelope protein or variant thereof, baboon endogenous retrovirus (BaEV) envelope protein or variant thereof, hepatitis B (HBV) envelope protein or variant thereof, hepatitis C (HCV) envelope protein or variant thereof, and rabies virus (RABV) envelope protein or variant thereof.

[0162] In a preferred embodiment, the transfer vector comprises an HIV lentiviral vector backbone containing packaging sequences and encoding a human therapeutic transgene, preferably the transfer vector comprises an HIV-1 lentiviral vector backbone containing packaging sequences and encoding a human therapeutic transgene for the treatment of a severe genetic disease or cancer. In a specific embodiment, the transfer vector comprises an HIV-1 lentiviral vector backbone containing packaging sequences and encoding a human therapeutic globin for the treatment of hemoglobinopathies, the ABCD1 gene for the treatment of CALD, or a chimeric receptor, e.g., a chimeric antigen receptor, a T cell receptor, or DARIC for the treatment of cancer.

[0163] In certain embodiments, host cells in a large-scale bioreactor are transiently transfected with a mixture comprising linear PEI, a plasmid encoding lentiviral gag and pol, a plasmid encoding rev, a plasmid encoding VSV-G, and a transfer plasmid comprising a packageable HIV-1-based lentiviral vector genome encoding a human therapeutic transgene. In certain embodiments, the lentiviral gag and pol, rev, and / or VSV-G are codon optimized for expression and / or stability in human cells. In certain embodiments, the transfer plasmid and the packaging plasmid comprise a selection cassette or gene. In a preferred embodiment, the transfer plasmid and the packaging plasmid comprise an RNAout selection cassette (Nature Technology). In certain embodiments, the host cells are transfected for about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, or about 20 hours. In particular embodiments, the host cells are transfected for about 14 hours, about 15 hours, about 16 hours, about 17 hours, or about 18 hours. In more particular embodiments, the host cells are transfected for about 14 hours to about 18 hours.

[0164] After host cell transfection is complete, for example, about 14 hours to about 18 hours after transfection, medium exchange is performed using an alternating tangential flow filtration (ATF) filter unit, a TFF filter unit, or an acoustic filter unit. In one embodiment, medium exchange is performed using an ATF filter unit. In one embodiment, medium exchange is performed using a TFF filter unit. In one embodiment, medium exchange is performed using an acoustic filter unit. In certain embodiments, after the bioreactor medium is exchanged, the host cells are cultured for viral vector production.

[0165] In certain embodiments, viral vector production occurs in serum-free, chemically defined cell culture medium about 36 hours to about 48 hours post-transfection, about 36 hours to about 48 hours post-transfection, about 36 hours to about 46 hours post-transfection, about 36 hours to about 44 hours post-transfection, about 38 hours to about 48 hours post-transfection, about 38 hours to about 46 hours post-transfection, about 38 hours to about 44 hours post-transfection, or about 38 hours to about 42 hours post-transfection (e.g., 36 hours post-transfection is 36 hours after addition of the transfection mixture to the host cells). In certain embodiments, viral vector production occurs in serum-free, chemically defined cell culture medium up to about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 48 hours after transfection. In certain embodiments, viral vector production occurs in serum-free, chemically defined cell culture medium up to about 38 hours, about 39 hours, about 40 hours, about 41 hours, or about 42 hours after transfection.

[0166] In certain embodiments, viral vector production occurs in serum-free, chemically defined cell culture medium for about 12 hours to about 48 hours, about 18 hours to about 48 hours, about 18 hours to about 36 hours, about 18 hours to about 30 hours, about 20 hours to about 28 hours, or about 22 hours to about 26 hours. In certain embodiments, viral vector production occurs for about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours in serum-free, chemically defined cell culture medium.

[0167] In certain embodiments, suspension culture supernatants are sampled and analyzed one or more times to ensure sufficient viral vector production.

[0168] In various embodiments, the suspension culture supernatant is collected once, twice, three or more times during viral vector production. In certain embodiments, the suspension culture supernatant is collected once during viral vector production. In preferred embodiments, after sufficient viral vector production, the suspension culture supernatant is not collected prior to the start of downstream manufacturing processes.

[0169] D. Downstream Viral Vector Manufacturing Process Viral vector production marks the end of the upstream manufacturing process. The downstream manufacturing process starts at the end of the viral vector production period. The downstream manufacturing process contemplated herein includes adding nuclease to suspension culture supernatant at the end of the viral vector production period; harvesting and clarifying suspension culture supernatant using filtration; capturing and concentrating viral vector from the harvested and clarified suspension culture supernatant using chromatography; filtering viral vector; ultrafiltering and diafiltering viral vector; and formulating viral vector. In certain embodiments, bulk formulated viral vector is sterile filtered and frozen; then thawed and sterile filtered; subjected to final fill finish; and then frozen. In other specific embodiments, bulk formulated viral vector is sterile filtered and subjected to final fill finish; and then frozen.

[0170] The downstream manufacturing process contemplated in certain embodiments herein includes adding endonuclease to suspension culture supernatant at the end of the viral vector production period; harvesting and clarifying suspension culture supernatant using tandem depth filtration; capturing and concentrating viral vector from the harvested and clarified suspension culture supernatant using affinity chromatography or cation exchange chromatography; filtering the viral vector; ultrafiltering and diafiltering the viral vector using tangential flow filtration; and formulating the viral vector in cell culture medium. In certain embodiments, the bulk formulated viral vector is sterile filtered and frozen at ≦-65°C; then thawed and sterile filtered; subjected to final fill finish; and then frozen at ≦-65°C. In other specific embodiments, the bulk formulated viral vector is sterile filtered and subjected to final fill finish; and then frozen at ≦-65°C.

[0171] The downstream manufacturing process contemplated in certain embodiments herein includes adding endonuclease to suspension culture supernatant at the end of the retroviral vector, e.g., lentiviral vector, production period; harvesting and clarifying suspension culture supernatant using tandem depth filtration and a second filter; capturing and concentrating retroviral vector from the harvested and clarified suspension culture supernatant using heparin affinity chromatography or cation exchange chromatography; filtering the concentrated retroviral vector; ultrafiltering and diafiltering retroviral vector using hollow fiber tangential flow filtration; and formulating the retroviral vector. In certain embodiments, the bulk formulated retroviral vector is sterile filtered and frozen at ≦-65°C; then thawed and sterile filtered; subjected to final fill finish; and then frozen at ≦-65°C. In other specific embodiments, the bulk formulated retroviral vector is sterile filtered and subjected to final fill finish; and then frozen at ≦-65°C.

[0172] In various embodiments, the downstream manufacturing process for retroviral vector production and purification includes adding a DNA endonuclease, e.g., Benzonase or Denarase, to the suspension culture supernatant at the end of the retroviral vector, e.g., lentiviral vector, production period; harvesting and clarifying the suspension culture supernatant using tandem depth filtration and a second filter, the second filter comprising a prefilter membrane and a filtration membrane; capturing and concentrating the retroviral vector from the harvested and clarified suspension culture supernatant using heparin affinity chromatography or cation exchange chromatography; filtering the concentrated retroviral vector using a filter comprising a prefilter membrane and a filtration membrane; ultrafiltering and diafiltering the retroviral vector using hollow fiber tangential flow filtration, the TFF having a molecular weight cutoff of about 100 kDa to about 500 kDa; and formulating the eluate containing the retroviral vector in a formulation buffer (e.g., 2× stem cell growth medium (SCGM)), optionally, and in some embodiments, preferably, in a 1:1 ratio. In certain embodiments, the bulk formulated retroviral vector is sterile filtered, frozen at ≦-65° C., then thawed, sterile filtered, subjected to final fill finish, and then frozen at ≦-65° C. In other certain embodiments, the bulk formulated retroviral vector is sterile filtered, subjected to final fill finish, and then frozen at ≦-65° C.

[0173] In certain embodiments, the downstream manufacturing processes contemplated herein result in at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold or more of viral vector concentration.

[0174] 1. Nuclease Digestion The upstream viral vector manufacturing process is terminated after sufficient viral vector is produced. The viral vector supernatant may contain residual nucleic acid, including but not limited to RNA, plasmid DNA from host cell transfection and genomic DNA from host cell lysate during viral vector production. Such residual nucleic acid is potentially toxic and reduces the efficacy of any viral vector produced from the manufacturing process contemplated herein. The purpose of the nuclease digestion step is to reduce the amount of these residual nucleic acids in the viral vector production supernatant.

[0175] In various embodiments, the nuclease is added to the viral vector production supernatant at the end of the viral vector production process. In certain embodiments, the nuclease is an endonuclease, and in preferred embodiments, the nuclease is a DNA / RNA endonuclease (an endonuclease that cleaves both DNA and RNA). Illustrative examples of endonucleases suitable for use in certain embodiments of the downstream manufacturing processes contemplated herein include, but are not limited to, Benzonase® endonuclease (EMD Millipore), Denarase® endonuclease (c-LEcta GmbH), Decontaminase™ endonuclease (AG Scientific), and recombinant NucA protein from Serratia marcescens.

[0176] In a preferred embodiment, Benzonase® endonuclease from Serratia marcescens or recombinant NucA protein is added to the viral vector production supernatant at the end of the viral vector production process.

[0177] In certain embodiments, MgCl 2is added to the viral vector production supernatant along with the endonuclease to ensure that the endonuclease is catalytically active. In various embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours to about 72 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours to about 48 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 48 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 44 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 40 hours after transfection. In some embodiments, the endonuclease is added to the suspension culture supernatant about 36 hours after transfection.

[0178] In certain embodiments, the nuclease digestion step of the downstream viral vector manufacturing process is carried out at an appropriate temperature and for a time sufficient to digest contaminating nucleic acids present in the viral vector production supernatant. In certain embodiments, the nuclease digestion is carried out overnight at about 2°C to about 8°C. In certain embodiments, the nuclease digestion is carried out at about 36°C to about 38°C for about one, about two, or about three hours. In preferred embodiments, the endonuclease digestion is carried out at about 36°C, about 37°C, or about 38°C for about one, about two, or about three hours. In more preferred embodiments, the Benzonase® endonuclease digestion is carried out at about 37°C for about one to two hours.

[0179] In various embodiments, the endonuclease digestion is carried out at a concentration of about 20 U / ml to about 70 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 50 U / ml to about 70 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 55 U / ml to about 65 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 20 U / ml to about 40 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 25 U / ml to about 35 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 50 U / ml to about 70 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 20 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 25 U / ml. In some embodiments, the endonuclease digestion is carried out at a concentration of about 30 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 35 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 40 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 45 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 50 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 55 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 60 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 65 U / ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 70 U / ml.

[0180] After the nuclease has sufficiently digested the extracellular nucleic acids present in the viral vector production supernatant, the supernatant is clarified and filtered.

[0181] 2. Clarification In certain embodiments, the downstream viral vector manufacturing process further includes a clarification step. The ultimate purpose of clarification is to prepare the viral vector production supernatant for downstream chromatography and purification of the viral vector. The clarification step removes contaminants, such as host cells and cell debris, prior to viral vector capture, chromatography, and purification.

[0182] In certain embodiments, the clarification step comprises a primary clarification step and a secondary clarification step, hi certain embodiments, the primary clarification step comprises depth filtration and the secondary clarification step comprises membrane filtration.

[0183] Depth filters do not have a defined pore size or structure. Depth filters comprise a gradient density structure specifically designed to retain particles of a defined size. Particles are retained throughout the depth of the filter media. Depth filter media may include cellulose, diatomaceous earth, or other materials suitable for retaining contaminants of a specific size. In contrast, membrane filters retain particles of a specific size at the membrane surface that are excluded by the membrane's pore size. In certain embodiments, membrane filters have pre-filtration and filtration membranes. In preferred embodiments, the pre-filtration membrane has a larger pore size than the filtration membrane and functions to reduce clogging or fouling of the filtration membrane. Several types of depth filters and membrane filters are commercially available.

[0184] In various embodiments, harvesting and clarifying the suspension culture supernatant comprises the steps of tandem depth filtration (primary clarification) and a membrane filtration step (secondary clarification).

[0185] In certain embodiments, the tandem depth filter retains contaminants of at least about 40 μm to about 60 μm. In certain embodiments, the tandem depth filter retains contaminants of at least about 40 μm, about 50 μm, or about 60 μm. In preferred embodiments, the tandem depth filter retains contaminants of at least about 60 μm. In preferred embodiments, the tandem depth filter retains contaminants of about 60 μm or greater.

[0186] In certain embodiments, the tandem depth filter retains contaminants having a size greater than about 40 μm to about 60 μm. In certain embodiments, the tandem depth filter retains contaminants having a size greater than about 40 μm, about 50 μm, or about 60 μm. In preferred embodiments, the tandem depth filter retains contaminants having a size greater than about 60 μm.

[0187] In certain embodiments, the membrane filtration is bilayer filtration. In certain embodiments, the bilayer filter comprises a prefilter membrane and a filtration membrane. In certain embodiments, the bilayer filter comprises a prefilter membrane with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filtration membrane with a final filter pore size of about 0.22 μm to about 0.45 μm. In certain embodiments, the bilayer filter comprises a prefilter membrane with a prefilter pore size of about 0.45 μm to about and a final filtration membrane with a final filter pore size of about 0.22 μm. In certain embodiments, the bilayer filter comprises a prefilter membrane with a prefilter pore size of about 0.8 and a final filtration membrane with a final filter pore size of about 0.45 μm.

[0188] In various embodiments, harvesting and clarifying the suspension culture supernatant includes a step of tandem depth filtration (primary clarification) using a tandem depth filter that retains contaminants having a size greater than about 40 μm, about 50 μm, or about 60 μm, and a membrane filtration step (secondary clarification) using a bilayer filter with a prefilter membrane having a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filtration membrane having a final filter pore size of about 0.22 μm to about 0.45 μm.

[0189] In certain embodiments, harvesting and clarifying the suspension culture supernatant comprises a step of tandem depth filtration (primary clarification) using a tandem depth filter that retains contaminants having a size of about 60 μm or larger or a membrane filtration step (secondary clarification) using a bilayer filter with a prefilter membrane having a prefilter pore size of about 0.45 μm and a final filtration membrane having a final filter pore size of about 0.22 μm.

[0190] In certain embodiments, harvesting and clarifying the suspension culture supernatant includes a step of tandem depth filtration (primary clarification) using a tandem depth filter that retains contaminants having a size of about 60 μm or more, or a membrane filtration step (secondary clarification) using a bilayer filter with a prefilter membrane having a prefilter pore size of about 0.8 μm and a final filtration membrane having a final filter pore size of about 0.45 μm. In various embodiments, the clarified suspension culture is adjusted to about pH 7.0 or pH 7.2 with 1 M HEPES. In some embodiments, the clarified suspension culture is adjusted to about pH 7.0. In some embodiments, the clarified suspension culture is adjusted to about pH 7.2.

[0191] After the viral vector production supernatant is harvested and clarified, it may optionally be stored for about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours at a suitable temperature, for example, about 4° C., about 20° C., about 30° C., or about 37° C. After harvesting and clarification, and optionally storage, the viral vectors are captured and concentrated using chromatography.

[0192] 3. Chromatography The downstream viral vector manufacturing process contemplated in certain embodiments further comprises a chromatography step. Chromatography is typically performed in a column packed with resins or beads designed to capture the viral vector from the harvested and clarified viral vector production supernatant and allow undesired impurities in the harvested and clarified viral vector production supernatant to pass through the column. The captured viral vector is then displaced or eluted from the column using a desorption agent.

[0193] In certain embodiments, the chromatography is ion exchange chromatography, size exclusion chromatography, affinity chromatography, or multi-mode chromatography.

[0194] Ion exchange chromatography (IEX) involves the separation of ionizable molecules based on their total charge. IEX includes both anion exchange chromatography and cation exchange chromatography. Anion exchange chromatography (AEX) utilizes the negatively charged surface of viral vector particles for purification purposes. AEX has been used to prepare inactivated HIV-1 vaccines and for the purification of retroviruses, such as lentiviral vectors. In certain embodiments, viral vectors from harvested and clarified viral vector production supernatants are captured and concentrated using anion exchange chromatography. Cation exchange chromatography is another form of ion exchange chromatography (IEX). Cation exchange chromatography uses a negatively charged ion exchange resin that has an affinity for molecules with a net positive surface charge. Here, the pH of the lentiviral supernatant is adjusted below the isoelectric point of the LVV to give the LVV an overall positive net surface charge and can bind to the negatively charged resin beads.

[0195] Thus, in various embodiments, the LVV supernatant is pumped onto an ion exchange chromatography column. In some embodiments, the LVV supernatant is pumped onto a cation exchange chromatography column. In certain embodiments, the LVV supernatant is pumped onto a sulfate cation exchange chromatography column (e.g., Toyopearl™ Sulfate-650F). In some embodiments, the sulfate cation exchange chromatography comprises a column having a bead size of about 45 μm and / or an average pore size of about 100 nm.

[0196] Size exclusion chromatography (SEC) separates molecules based on their size using a resin containing beads of defined pore size. Molecules elute from the SEC resin in size order: larger molecules that are not trapped in the bead pores travel shorter distances, and the first and smaller molecules that are eluted by the bead pores elute last. Beads of different pore sizes can be purchased to obtain the desired resolution. SEC has been used to purify wild-type retroviruses and retroviral vectors. Retroviral vectors are excluded from the bead pores due to their large size and elute in the void volume of the column, while smaller molecular weight contaminants are retarded by the column and elute in later fractions. In certain embodiments, viral vectors from harvested and clarified viral vector production supernatants are captured and concentrated using size exclusion chromatography.

[0197] Affinity chromatography (AC) separates molecules based on their highly selective affinity for a particular chromatographic adsorbent. Unfortunately, little is known about the composition of viral membranes complicating the selection of an appropriate adsorbent. Viral vectors have been engineered to express affinity tags on their surface to facilitate purification, for example, MoMLV has been modified to express a hexahistidine affinity tag that was purified by immobilized metal affinity chromatography (IMAC). MoMLV viral vectors have also been purified by exploiting the interaction between streptavidin and biotin. Heparin affinity chromatography has been used to purify viral vectors that use heparan sulfate as a cell surface receptor, including pseudotyped retroviral vectors, for example, VSV-G pseudotyped lentiviral vectors. In certain embodiments, viral vectors from harvested and clarified viral vector production supernatants are captured and concentrated using affinity chromatography.

[0198] Multimode or mixed-mode chromatography (MMC) incorporates multiple chromatographic modes on a single resin. MMC enhances the selectivity of the resin since molecules can be separated based on several of their characteristics rather than a single one.

[0199] In a preferred embodiment, the viral vectors from the harvested and clarified viral vector production supernatant are captured and concentrated using heparin affinity chromatography. In a specific embodiment, the harvested and clarified viral vector production supernatant is adjusted to a pH of about 7.0 as necessary. The harvested and clarified viral vector production supernatant is passed through a heparin affinity chromatography column, and the column is washed one or more times with a washing buffer (e.g., 50 mM HEPES, 100 mM NaCl, pH 7.0) and eluted (e.g., 50 mM HEPES, 400 mM NaCl, pH 8.0).

[0200] In a preferred embodiment, the viral vectors from the harvested and clarified viral vector production supernatant are captured and concentrated using cation exchange chromatography. In various embodiments, the chromatography is sulfate cation exchange chromatography. In a particular embodiment, the harvested and clarified viral vector production supernatant is adjusted to a pH of about 7.2, if necessary. The harvested and clarified viral vector production supernatant is pumped through / up a sulfate cation exchange chromatography column, and the column is washed one or more times with a wash buffer (e.g., about 50 mM HEPES, about 300 mM NaCl, pH 7.2) and eluted (e.g., about 50 mM HEPES, about 1 M NaCl, pH 7.5).

[0201] The resulting eluate, containing the concentrated viral vector, is filtered to further remove impurities.

[0202] 4. Post-chromatographic filtration A downstream viral vector manufacturing process contemplated in certain embodiments includes a filtration step after chromatographic purification of the viral vector. Post-chromatographic filtration protects unit operations downstream of the chromatography step by further removing impurities that may interfere with the purification of the viral vector.

[0203] In certain embodiments, the filtering step comprises filtering the concentrated viral vector through a bilayer filter with a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.22 μm to about 0.45 μm.

[0204] In certain embodiments, the filtering step comprises filtering the concentrated viral vector through a bilayer filter with a prefilter pore size of about 0.45 μm and a final filter pore size of about 0.22 μm.

[0205] In certain embodiments, the filtering step comprises filtering the concentrated viral vector through a bilayer filter with a prefilter pore size of about 0.8 μm and a final filter pore size of about 0.45 μm.

[0206] In certain embodiments, after the concentrated viral vectors have passed through the filter, the filter is chased with diafiltration buffer to maximize recovery of the viral vectors.

[0207] The filtered viral vector solution is then further purified and concentrated using ultrafiltration and then diafiltered.

[0208] 5.Ultrafiltration / Diafiltration The downstream viral vector manufacturing process contemplated in certain embodiments further comprises an ultrafiltration step to further purify and concentrate the viral vector in preparation for formulation, and a diafiltration step to buffer exchange the concentrated, filtered viral vector buffer with a diafiltration buffer (e.g., about 50 mM HEPES, about 100 mM NaCl, pH 7.5; or about 50 mM HEPES, pH 7.0; or about 50 mM L-histidine, pH 7.0).

[0209] In certain embodiments, the viral vector is ultrafiltered to further remove impurities and concentrate the viral vector, and then subsequently diafiltered into a buffer appropriate for bulk viral vector formulation.

[0210] In certain embodiments, viral vectors are filtered, concentrated, and then diafiltered using tangential flow filtration. In a preferred embodiment, viral vectors are filtered, concentrated, and then diafiltered using tangential flow filtration. Hollow fiber TFF modules or filters are used to simultaneously concentrate and remove impurities to obtain highly active retroviral vectors. Hollow fiber TFF modules or filters have also been used as a convenient tool to diafilter viral vectors into buffers suitable for bulk viral vector formulation.

[0211] In certain embodiments, the TFF system includes pumping a feed solution containing the viral vector into a hollow fiber TFF module, the pore size of which is selected such that the viral vector does not pass through the pores and is concentrated in a recovery solution, which is a solution retained in the TFF module, while a permeate containing impurities passes through the pores.

[0212] In certain embodiments, the TFF system is used to perform diafiltration or buffer exchange of the viral vector-containing solution. The TFF system is an effective method for removing, modifying, and / or exchanging altered ion concentrations, pH, salts, sugars, non-aqueous solvents, separating unbound molecules, and removing low molecular weight contaminants.

[0213] In certain embodiments, hollow fiber TFF modules or filters are used to perform diafiltration and / or ultrafiltration, further purification, concentration, and buffer exchange. Suitable TFF systems for use in certain embodiments contemplated herein are commercially available from, for example, EMD Millipore, Sigma, GE Healthcare, Sartorius, and Repligen.

[0214] In certain embodiments, the hollow fiber TFF module or filter has a pore size of about 100 kDa to about 500 kDa and a pore size of about 0.5 mm. 2 , about 1.0m 2 , about 2.5m 2 , about 5.0m 2 , about 10m 2 , or about 20m 2 In certain embodiments, the hollow fiber TFF module or filter has a pore size of about 100 kDa to about 500 kDa and a surface area of ​​about 1.00 m 2 , about 1.05m 2 , about 1.10m 2 , about 1.15m 2 , about 1.20m 2 , about 1.25m 2 , about 1.30m 2 , about 1.35m2 , about 1.40m 2 , about 1.45m 2 , about 1.50m 2 , about 1.55m 2 , about 1.60m 2 , about 1.65m 2 , about 1.70m 2 , about 1.75m 2 , about 1.80m 2 , about 1.85m 2 , about 1.90m 2 , about 1.95m 2 , about 2.00m 2 , 2.00m 2 , about 2.05m 2 , about 2.10m 2 , about 2.15m 2 , about 2.20m 2 , about 2.25m 2 , about 2.30m 2 , about 2.35m 2 , about 2.40m 2 , about 2.45m 2 , or approximately 2.50 m 2 It has a surface area of.

[0215] In certain embodiments, the hollow fiber TFF module or filter has a pore size of about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, or about 500 kDa and a pore size of about 1.00 m 2 , about 1.05m 2 , about 1.10m 2 , about 1.15m 2 , about 1.20m 2 , about 1.25m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , about 1.50m 2 , about 1.55m 2 , about 1.60m 2 , about 1.65m 2 , about 1.70m 2 , about 1.75m 2 , about 1.80m 2 , about 1.85m 2 , about 1.90m 2, about 1.95m 2 , about 2.00m 2 , 2.00m 2 , about 2.05m 2 , about 2.10m 2 , about 2.15m 2 , about 2.20m 2 , about 2.25m 2 , about 2.30m 2 , about 2.35m 2 , about 2.40m 2 , about 2.45m 2 , or approximately 2.50 m 2 It has a surface area of.

[0216] In certain embodiments, the hollow fiber TFF module or filter has a pore size of about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, or about 500 kDa and a pore size of about 1.00 m 2 , about 1.05m 2 , about 1.10m 2 , about 1.15m 2 , about 1.20m 2 , about 1.25m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , about 1.50m 2 , about 1.55m 2 , about 1.60m 2 , about 1.65m 2 , about 1.70m 2 , about 1.75m 2 , about 1.80m 2 , about 1.85m 2 , about 1.90m 2 , about 1.95m 2 , about 2.00m 2 , 2.00m 2 , about 2.05m 2 , about 2.10m 2 , about 2.15m 2 , about 2.20m 2 , about 2.25m 2 , about 2.30m 2 , about 2.35m 2 , about 2.40m 2 , about 2.45m2 , or approximately 2.50 m 2 It has a surface area of.

[0217] In a particular embodiment, the hollow fiber TFF module or filter has a pore size of about 100 kDa and a pore size of about 1.25 m. 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , or about 1.50 m 2 In a particular embodiment, the hollow fiber TFF module or filter has a pore size of about 200 kDa and a surface area of ​​about 1.25 m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , or about 1.50 m 2 In a particular embodiment, the hollow fiber TFF module or filter has a pore size of about 300 kDa and a surface area of ​​about 1.25 m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , or about 1.50 m 2 In a particular embodiment, the hollow fiber TFF module or filter has a pore size of about 400 kDa and a surface area of ​​about 1.25 m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , or about 1.50 m 2 In a particular embodiment, the hollow fiber TFF module or filter has a pore size of about 500 kDa and a surface area of ​​about 1.25 m 2 , about 1.30m 2 , about 1.35m 2 , about 1.40m 2 , about 1.45m 2 , or about 1.50 m 2 It has a surface area of.

[0218] In certain embodiments, the downstream viral vector manufacturing process includes an ultrafiltration step performed using a hollow fiber TFF module having a pore size of about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, or about 500 kDa, and further includes a diafiltration step performed using a hollow fiber TFF module to exchange the buffer containing the viral vector into a diafiltration buffer (e.g., 50 mM HEPES, 100 mM NaCl, pH 7.5; or 50 mM HEPES, pH 7.0) in preparation for the formulation.

[0219] 6. Formulation In certain embodiments, the downstream viral vector manufacturing process further comprises formulating the viral vector in an appropriate buffer and / or pharma- ceutically acceptable medium, the viral vector being formulated to stabilize the vector and retain vector activity through freeze / thaw cycles.

[0220] The ultrafiltration and diafiltration steps contemplated in certain embodiments result in the purification, concentration, and diafiltration of the viral vector into a diafiltration buffer (e.g., about 50 mM HEPES, about 100 mM NaCl, pH 7.5; or about 50 mM HEPES, pH 7.0; or about 50 mM L-histidine, pH 7.0).

[0221] In certain embodiments, to formulate the viral vector, the volume of the diafiltered viral vector is diluted with an equal volume of 2x concentrated suitable formulation buffer or pharmaceutical cell culture medium. In certain embodiments, the viral vector is formulated by diluting an equal volume of the diafiltered viral vector into 2x concentrated serum-free chemically defined cell culture medium. Illustrative examples of suitable formulation media include, but are not limited to, 2x Freestyle 293 expression medium, 2x Ex-Cell 293 serum-free medium, 2x Expi293 expression medium, 2x Opti-MEM reduced serum medium, and 2x Stem Cell Growth Medium (SCGM, CellGenix).

[0222] In certain embodiments, the viral vector is formulated by diluting an equal volume of the diafiltered viral vector into 2x concentrated SCGM. In some embodiments, the viral vector diafiltered into 50 mM HEPES, 100 mM NaCl, pH 7.50 is formulated by diluting 1:1 in 2x SCGM. In some embodiments, the viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer containing HEPES and sucrose, optionally the buffer further containing L-proline, poloxamer 188, or NaCl. In some embodiments, the viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, and about 100 mM L-proline. In some embodiments, a viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 0.2 to about 2.0 mg / mL poloxamer 188. In some embodiments, a viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer comprising about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 150 mM NaCl. In some embodiments, a viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, about 150 mM NaCl, and about 0.2 to about 2.0 mg / mL poloxamer 188. In some embodiments, a viral vector diafiltered into about 50 mM HEPES, pH 7.0 is formulated by diluting 1:1 in a buffer containing about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 150 mM NaCl, and about 0.2 to about 2.0 mg / mL poloxamer 188.

[0223] In various embodiments, the viral vector diafiltered into about 50 mM L-histidine, pH 7.0 is formulated by diluting 1:1 in a buffer comprising L-histidine, sucrose, and L-proline. In various embodiments, the viral vector diafiltered into about 50 mM L-histidine, pH 7.0 is formulated by diluting 1:1 in a buffer comprising about 5 mM L-histidine, about 146 mM sucrose, and about 100 mM L-proline, optionally, the formulation further comprising about 0.2 to about 2.0 mg / mL poloxamer 188.

[0224] In various embodiments, the formulation buffer for the 1:1 dilution comprises about 0.2 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.3 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.4 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.5 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.6 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.7 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.8 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 0.9 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.0 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.1 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.2 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.3 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.4 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.5 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.6 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.7 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.8 mg / mL poloxamer 188. In some embodiments, the formulation buffer for the 1:1 dilution comprises about 1.9 mg / mL poloxamer 188. In some embodiments, the formulation buffer for a 1:1 dilution contains about 2.0 mg / mL poloxamer 188.

[0225] Viral vectors are formulated in bulk, often and, in certain embodiments, preferably referred to as formulated bulk viral vectors.

[0226] 7. Post-formulation filtration In certain embodiments, the downstream viral vector manufacturing process further comprises a post-formulation filtration step of the viral vector, which further removes particles and impurities in the formulated bulk viral vector.

[0227] In certain embodiments, the filtration step comprises filtering the formulated bulk viral vector through a bilayer filter with a prefilter pore size of about 0.5 μm and a final filter pore size of about 0.2 μm.

[0228] In certain embodiments, after the formulated bulk viral vector has passed through the filter and been collected, the filtered formulated bulk viral vector is either stored frozen or subjected to a holding step (without cryopreservation) until final fill finish can be performed.

[0229] In certain embodiments, after the formulated bulk viral vector has passed through the filter and been harvested, a final fill polish is performed on the filtered formulated bulk viral vector, which is then stored frozen.

[0230] 8. Cryopreservation In certain embodiments, the downstream viral vector manufacturing process further includes cryopreserving the filtered formulated bulk viral vector until such time as final fill finish can be performed on the bulk viral vector. The cryopreservation of the formulated bulk viral vector is performed such that the stability and biological activity of the vector is substantially maintained and / or loss of the stability and biological activity of the viral vector is minimized.

[0231] As used herein, "cryopreserving" or "cryopreservation" refers to preserving a viral vector by cooling to a temperature below zero. In certain embodiments, the filtered formulated bulk viral vector further comprises a cryoprotectant. In certain embodiments, the viral vector is formulated to provide cryoprotection.

[0232] In various embodiments, the filtered formulated bulk viral vector is at a temperature below about -20°C, below about -21°C, below about -22°C, below about -23°C, below about -24°C, below about -25°C, below about -26°C, below about -27°C, below about -28°C, below about -29°C, below about -30°C, below about -31°C, below about -32°C, below about -33°C, below about -34°C, below about -35°C, below about -36°C, below about -37°C, below about -38°C, below about -39°C, below about -40°C, below about -41°C, below about -42°C, below about -43°C, below about -44°C, below about -45°C, below about -46°C, below about -47°C, below about -48°C, below about -49°C , or frozen at less than about -50°C, less than about -51°C, less than about -52°C, less than about -53°C, less than about -54°C, less than about -55°C, less than about -56°C, less than about -57°C, less than about -58°C, less than about -59°C, less than about -60°C, less than about -61°C, less than about -62°C, less than about -63°C, less than about -64°C, less than about -65°C, less than about -66°C, less than about -67°C, less than about -68°C, less than about -69°C, less than about -70°C, less than about -71°C, less than about -72°C, less than about -73°C, less than about -74°C, less than about -75°C, less than about -76°C, less than about -77°C, less than about -78°C, less than about -79°C, or less than about -80°C. One of ordinary skill in the art would understand that a temperature of -80°C is less than a temperature of -20°C.

[0233] In various embodiments, the filtered formulated bulk viral vector is stored frozen or frozen at below about -65°C, below about -70°C, below about -75°C, or below about -80°C.

[0234] In a particular embodiment, the cooling rate is between 1° C. / min and 3° C. / min.

[0235] 9. Filling and finishing Downstream manufacturing processes contemplated herein further include a fill-finish step. Viral vectors are typically aliquoted into single-use volumes and stored frozen to protect the stability and biological activity of the vector and to minimize heat inactivation of the viral vector.

[0236] "Fill finish" or "fill and finish" refers to the part of the downstream manufacturing process that involves filling containers, e.g., vials, ampoules, etc., with a formulated viral vector and completing the process of packaging the viral vector for distribution.

[0237] In certain embodiments, fill-finish is carried out on the filtered formulated viral vector without any intervening cryopreservation step.After fill-finish, the viral vector is cryopreserved according to the method contemplated herein.In certain embodiments, the viral vector is cryopreserved or frozen at less than about -65°C, less than about -70°C, less than about -75°C, or less than about -80°C.

[0238] In certain embodiments, fill-finishing is carried out on the filtered formulated viral vector with an intervening holding step but without an intervening cryopreservation step.After fill-finishing, the viral vector is cryopreserved according to the method contemplated herein.In certain embodiments, the viral vector is cryopreserved or frozen at less than about -65°C, less than about -70°C, less than about -75°C, or less than about -80°C.

[0239] In certain embodiments, the frozen and filtered formulated bulk viral vector is thawed and sterile filtered before filling finish. In certain embodiments, the filtration step comprises filtering the frozen formulated bulk viral vector through a double-layer filter with a prefilter pore size of about 0.5 μm and a final filter pore size of about 0.2 μm. After filtration, filling finish is performed with the formulated viral vector. After filling finish, the viral vector is frozen and stored according to the method contemplated herein. In certain embodiments, the viral vector is frozen and stored or frozen at less than about -65°C, less than about -70°C, less than about -75°C, or less than about -80°C.

[0240] E. Composition The compositions contemplated herein may include viral vectors, such as retroviral or lentiviral vectors. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition that is formulated for administration to a cell or animal in a pharma- ceutical or physiologically acceptable solution, alone or in combination with one or more other therapeutic modalities. There is virtually no limit to the other components that may be included in the composition, except that any added agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0241] As used herein, the phrase "pharmacologically acceptable" is employed to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0242] As used herein, a "pharmaceutical acceptable carrier" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, phosphate buffers, and any other compatible substance employed in pharmaceutical formulations.

[0243] In certain embodiments, a composition comprises a viral vector and a physiologically acceptable buffer or medium.

[0244] In certain embodiments, the composition comprises a viral vector and a diafiltration buffer (eg, about 50 mM HEPES, about 100 mM NaCl, pH 7.5; or about 50 mM HEPES, pH 7.0; or about 50 mM L-histidine, pH 7.0).

[0245] In certain embodiments, the composition comprises a viral vector and a pharmaceutical cell culture medium. Illustrative examples of suitable medium include, but are not limited to, 2x Freestyle 293 expression medium, 2x Ex-Cell 293 serum-free medium, 2x Expi293 expression medium, 2x Opti-MEM reduced serum medium, and 2x Stem Cell Growth Medium (SCGM, CellGenix).

[0246] In certain embodiments, the composition comprises a viral vector, diafiltration buffer, and 1×SCGM.

[0247] In certain embodiments, the composition comprises a viral vector in a HEPES-based formulation.

[0248] In certain embodiments, the composition comprises a viral vector in an L-histidine-based formulation.

[0249] In some embodiments, the composition comprises a viral vector, HEPES, and sucrose, and optionally, the buffer further comprises L-proline, poloxamer 188, or NaCl.

[0250] In some embodiments, the composition comprises a viral vector, about 27.5 mM HEPES (pH 7.0), about 73 mM sucrose, and about 50 mM L-proline.

[0251] In some embodiments, the composition comprises a viral vector, about 27.5 mM HEPES (pH 7.0), about 73 mM sucrose, about 50 mM L-proline, and about 0.1 to about 1.0 mg / ml poloxamer 188.

[0252] In some embodiments, the composition comprises a viral vector, about 27.5 mM HEPES (pH 7.0), about 73 mM sucrose, about 50 mM L-proline, and about 75 mM NaCl.

[0253] In some embodiments, the composition comprises a viral vector, about 27.5 mM HEPES (pH 7.0), about 73 mM sucrose, about 50 mM L-proline, about 75 mM NaCl, and about 0.1 to about 1.0 mg / ml poloxamer 188.

[0254] In some embodiments, the composition comprises a viral vector, about 27.5 mM HEPES (pH 7.0), about 73 mM sucrose, about 75 mM NaCl, and about 0.1 to about 1.0 mg / ml poloxamer 188.

[0255] In some embodiments, the composition comprises a viral vector, L-histidine, sucrose, and L-proline.

[0256] In some embodiments, the composition comprises a viral vector, about 27.5 mM L-histidine, about 73 mM sucrose, and about 50 mM L-proline, and optionally, the formulation further comprises about 0.1 to about 1.0 mg / mL poloxamer 188.

[0257] In some embodiments, the composition comprises about 0.1 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.2 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.3 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.4 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.5 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.6 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.7 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.8 mg / mL poloxamer 188. In some embodiments, the composition comprises about 0.9 mg / mL poloxamer 188. In some embodiments, the composition comprises about 1.0 mg / mL poloxamer 188.

[0258] Those of skill in the art will appreciate that certain embodiments of the compositions contemplated herein are well known in the pharmaceutical arts and may be found, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II.22, which are incorporated herein by reference in their entirety. nd It will be understood that the composition may include other components such as those described in the above-mentioned US Pat. No. 6,312,312, Published Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012.

[0259] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0260] The foregoing embodiments have been described in detail in the figures and examples for purposes of clarity and understanding, but in light of the teachings contemplated herein, it will be readily apparent to one skilled in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0261] Example 1 Lentiviral Vector Suspension Manufacturing Process 2.0 A HEK293T working cell bank was cultured in a 250 mL culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P0 culture, the culture was passaged into a 500 mL culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P1 culture, the culture was passaged into a 1 L culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P2 culture, the culture was passaged into a 3 L culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P3 culture, the culture was passaged into a 50 L culture vessel and cultivated in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P4 culture, the culture was passaged into a 200 L bioreactor (P5 culture) and cultivated in serum-free, chemically defined cell culture medium at 37.0° C. and pH 7.0. After confirming the appropriate viable cell density of the P5 culture, the culture medium was replaced with 190 L of fresh serum-free, chemically defined cell culture medium using alternating tangential flow filtration (ATF).

[0262] Prior to transfection, the packageable lentiviral vector genome and the transfer plasmid containing the gag / pol, rev, and VSV-G packaging plasmids and PEI components were mixed together in serum-free, chemically defined cell culture medium in a volume of 10 L. The DNA / PEI mixture was added to the P5 suspension cultures for approximately 14 to 18 hours, after which they were subjected to a culture medium exchange with fresh serum-free, chemically defined cell culture medium using ATF.

[0263] Approximately 36–48 h after transfection, cells were cultured with Benzonase endonuclease (50–75 U / mL final concentration) and MgCl2 diluted in serum-free, chemically defined cell culture medium. 2was added to the 200 L bioreactor culture and incubated at 37°C for approximately 1-2 hours.

[0264] The benonzase-treated culture was pumped through a tandem depth filter that retained contaminants greater than about 60 μm and then through a bilayer filter with a prefilter pore size of about 0.8 μm and a final filter of about 0.45 μm.

[0265] The clarified vector production supernatant was captured and concentrated using pseudoaffinity heparin chromatography. The supernatant was pumped onto a chromatography column, washed, and eluted in elution buffer (e.g., 50 mM HEPES, 400 mM NaCl, pH 8). The concentrated vector solution was pumped through a bilayer filter with a prefilter pore size of about 0.8 μm and a final filter of about 0.45 μm.

[0266] The filtered concentrated vector was pumped through a hollow fiber TFF column with a molecular weight cutoff or pore size of about 300 kDa to about 500 kDa. The retentate was then diafiltered against diafiltration buffer (e.g., 50 mM HEPES, 100 mM NaCl, pH 7.5) and formulated with a 1:1 dilution of 2x / 1x SCGM formulation stock solution to form formulated bulk lentiviral vector (LVV). The formulated bulk LVV was filtered through a sterilizing grade filter, filled into bulk storage containers, and stored at ≦-65°C until fill / finish.

[0267] Frozen formulated bulk LVV was thawed and aliquots were pooled together and mixed before final filtration and then fill / finish using ready-to-use containers / closures and an automated filling line from West Pharmaceuticals.

[0268] Example 2 Lentiviral Vector Suspension Manufacturing Process 2.5 A HEK293T working cell bank was cultured in a 250 mL culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P0 culture, the culture was passaged into a 500 mL culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P1 culture, the culture was passaged into a 1 L culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P2 culture, the culture was passaged into a 3 L culture vessel and cultured in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P3 culture, the culture was passaged into a 50 L culture vessel and cultivated in serum-free, chemically defined cell culture medium at 37.0° C. and 8.0% CO2. After confirming the appropriate viable cell density of the P4 culture, the culture was passaged into a 200 L bioreactor (P5 culture) and cultivated in serum-free, chemically defined cell culture medium at 37.0° C. and pH 7.0. After confirming the appropriate viable cell density of the P5 culture, the culture medium was replaced with 190 L of fresh serum-free, chemically defined cell culture medium using alternating tangential flow filtration (ATF).

[0269] Prior to transfection, the packageable lentiviral vector genome and the transfer plasmid containing the gag / pol, rev, and VSV-G packaging plasmids and PEI components were mixed together in serum-free, chemically defined cell culture medium in a volume of 10 L. The DNA / PEI mixture was added to the P5 suspension cultures for approximately 14 to 18 hours, after which they were subjected to a culture medium exchange with fresh serum-free, chemically defined cell culture medium using ATF.

[0270] Approximately 36-48 hours after transfection, cells were cultured with Denarase endonuclease (~30 U / mL) and MgCl2 diluted in serum-free, chemically defined cell culture medium. 2was added to the 200 L bioreactor culture at 37°C for approximately 1-2 hours.

[0271] The Denarase treated culture was pumped through a tandem depth filter that retained contaminants greater than about 60 μm and then through a bilayer filter with a prefilter pore size of about 0.8 μm and a final filter of about 0.45 μm.

[0272] The clarified vector production supernatant was captured and concentrated using cation exchange sulfate chromatography. The supernatant was pumped onto a chromatography column, washed in a wash buffer (e.g., 50 mM HEPES, 300 mM NaCl, pH 7.2), and eluted in an elution buffer (e.g., 50 mM HEPES, 1 M NaCl, pH 7.5). The concentrated vector solution was pumped through a bilayer filter with a prefilter pore size of about 0.8 μm and a final filter of about 0.45 μm.

[0273] The filtered concentrated vector was pumped through a hollow fiber TFF column with a molecular weight cutoff or pore size of approximately 300 kDa to approximately 500 kDa. The retentate was then diafiltered against diafiltration buffer (e.g., 50 mM HEPES, pH 7.0) and formulated in a 1:1 dilution of concentrated formulation medium (e.g., 5 mM HEPES, 146 mM sucrose, 100 mM L-proline, pH 7.0) to form formulated bulk lentiviral vector (LVV). The formulated bulk LVV was filtered through a sterilizing grade filter and stored at 2-8 °C until fill / finish.

[0274] The cooled intermediate bulk was sterile filtered and filled / finished using ready-to-use containers / closures (West Pharmaceuticals) and an automated filling line.

[0275] Example 3 Comparison of lentiviral vector manufacturing processes The suspension manufacturing processes described in Examples 1 and 2 (i.e., processes sLVV 2.0 and sLVV 2.5) were compared to an adherent manufacturing process (aLVV). aLVV was produced as described in Gorman et al., Molecular Therapy, Volume 23, Supplement 1, May 2015. sLVV 2.0 was produced as described in Example 1 (see also FIG. 1), while sLVV 2.5 was produced at a 2 L final culture scale (scaled volumes accordingly) and purified to the intermediate bulk unit operation described in Example 2 (see also FIG. 4). The intermediate bulk step represents the final LVV product in terms of remaining impurities and is therefore compared to the final LVV product produced by the 2.0 sLVV process (FIG. 1) and the aLVV process. The process yield is determined by the infectious titer yield, which is measured by viral transduction of HOS cells. The purity of the LVV products is compared based on residual host cell protein (HCP) concentration, measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the relative residual p24 capsid protein concentration versus infectious titer (particle-to-infectivity ratio) is compared between platforms as a measure of LVV purity. Residual p24 is also measured by ELISA.

[0276] As shown in Figure 5, the suspension 2.0 sLVV process exhibits a roughly 10-fold increase in total infectious titer (TU) compared to the aLVV generation process, due in part to the increased production cell culture scale (200 L for 2.0 sLVV versus 40 L for aLVV), as well as increased infectious titer concentration, as shown in Figure 6. The 2.5sLVV process produces similar intermediate bulk titer concentrations as the 2.0sLVV (Figure 6) and maintains a higher infectious titer than the original aLVV process. Additionally, the 2.0 and 2.5sLVV processes demonstrate a more consistent particle-to-infectivity (VP / TU) ratio compared to the aLVV process (see Figure 7).

[0277] Additionally, the sLVV 2.5 process results in a significant improvement in host cell protein (HCP) impurity reduction compared to the sLVV 2.0 process. The sLVV 2.0 process resulted in a 10-fold increase in infectious titer compared to the aLVV process, but also a >10-fold increase in HCP levels throughout the process when normalized to infectious titer. However, the sLVV 2.5 process was able to achieve substantially higher host cell protein reduction from intermediate bulk LVV while resulting in normalized HCP levels comparable to the LVV production process (Figure 8). HCP log reduction values ​​were similar between the aLVV and sLVV 2.5 processes, indicating similar impurity removal performance (Figure 9). Without wishing to be bound by any particular theory, one potential reason for the improved HCP removal in the 2.5 sLVV process is due to the introduction of a sulfate cation capture chromatography column. As shown in FIG. 10, the heparin affinity binding column achieves an average HCP log reduction of <1.0 log over the chromatography unit operation steps, while the sulfate column exhibits approximately 1.5 log reduction during chromatography, contributing to an overall increase in HCP reduction over the sLVV 2.5 process of 1.0 log HCP (>10-fold reduction in HCP concentration at intermediate bulk).

[0278] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

**Claim 1** A suspension method for generating a lentiviral vector (sLVV), comprising: (a) inoculating a production host cell into a large-scale suspension culture; (b) transiently transfecting the host cells in the large-scale suspension culture with a mixture comprising a lentiviral packaging plasmid, a transfer plasmid, and a transfection agent; (c) adding an endonuclease to the suspension culture supernatant about 36 to about 48 hours after transfection (after the start of transfection); (d) collecting and clarifying the suspension culture supernatant using a tandem depth filter and a bilayer filter; (e) capturing and concentrating the lentiviral vector from the collected and clarified suspension culture supernatant using chromatography; (f) filtering the concentrated lentiviral vector; (g) using tangential flow filtration (TFF) to ultrafilter and diafilter the lentiviral vector; and (h) formulating the lentiviral vector to produce a formulated bulk lentiviral vector and sterile filtering the formulated bulk lentiviral vector. **Claim 2** The method according to claim 1, comprising inoculating a suspension culture of 200 L to 2000 L, 200 L to 1000 L, 200 L to 500 L, or 200 L. **Claim 3** The large-scale suspension culture is inoculated with about 40.0×10 ８ to about 120.0×10 ８ live host cells, the method according to any one of claims 1 to 2. **Claim 4** The method according to any one of claims 1 to 2, wherein the host cell is selected from the group consisting of HEK293 cells, HEK293S cells, HEK293T cells adapted for suspension culture (HEK293T), HEK293F cells, HEK293FT cells, HEK293FTM cells, and HEK293E cells. **Claim 5** The method according to any one of claims 1 to 2, wherein the large-scale cell suspension culture comprises host cells cultured in a culture medium. **Claim 6** The method according to any one of claims 1 to 2, wherein the large-scale cell suspension culture comprises host cells cultured in a culture medium for about 3 days, and after the 3 days, the culture medium is replaced with fresh culture medium. **Claim 7** The method according to any one of claims 1 to 2, wherein the large-scale cell suspension culture comprises host cells cultured in a culture medium for about 3 days, and after the 3 days, the culture medium is replaced with fresh culture medium using alternating tangential flow filtration (ATF).

8. The method according to claim 5, wherein the culture medium is a serum-free chemically defined cell culture medium.

9. The method according to any one of claims 1 to 2, wherein the host cell is transiently transfected with the mixture comprising a transfection agent selected from the group consisting of calcium phosphate, cationic lipids, and cationic polymers.

10. The method according to any one of claims 1 to 2, wherein the endonuclease is derived from Serratia marcescens, and optionally, the endonuclease is a recombinant NucA endonuclease.

11. The method according to any one of claims 1 to 2, wherein the endonuclease has both DNA and RNA cleavage activities.

12. The method according to any one of claims 1 to 2, wherein the endonuclease is Benzonase or Denarase.

13. The method according to any one of claims 1 to 2, wherein the endonuclease is added at a concentration of about 60 U / ml or about 30 U / ml.

14. The method according to any one of claims 1 to 2, wherein the endonuclease is added to the suspension culture supernatant about 36 hours to about 72 hours after transfection, about 36 hours to about 48 hours after transfection, about 48 hours after transfection, about 44 hours after transfection, about 40 hours after transfection, or about 36 hours after transfection.

15. The method according to any one of claims 1 to 2, wherein the harvesting and clarification step comprises filtering the suspension culture supernatant through a tandem depth filter that retains contaminants of at least about 40 μm or at least about 60 μm and a double layer filter having a pre-filter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.22 μm to about 0.45 μm.

16. The method according to any one of claims 1 to 2, wherein the lentiviral vector is captured and concentrated from the harvested and clarified suspension culture supernatant using affinity chromatography or cation exchange chromatography.

17. The method according to claim 16, wherein the cation exchange chromatography is sulfated cation exchange chromatography.

18. The method according to claim 17, wherein the sulfuric acid cation exchange chromatography comprises a column having a bead size of about 45 μm and / or an average pore size of about 100 nm.

19. (i) about 50 mM HEPES, about 100 mM NaCl, pH 7, (ii) about 50 mM HEPES, about 400 mM NaCl, pH 8, (iii) about 50 mM HEPES, about 300 mM NaCl, pH 7.2, or (iv) about 50 mM HEPES, about 1 M NaCl, pH 7.5, wherein the washing buffer is pumped onto the chromatography column. The method according to any one of claims 1 to 2.

20. The method according to any one of claims 1 to 2, wherein the filtration step (f) comprises filtering the concentrated lentiviral vector through a bilayer filter having a prefilter pore size of about 0.45 μm to about 0.8 μm and a final filter pore size of about 0.2 μm to about 0.45 μm.

21. The method according to any one of claims 1 to 2, wherein the lentiviral vector is ultrafiltered and diafiltered using a hollow fiber tangential flow filtration (TFF) filter having a pore size or molecular weight cut-off of about 100 kDa to about 500 kDa.

22. The method according to claim 21, wherein the lentiviral vector is diafiltered into a diafiltration buffer, and optionally, the diafiltration buffer is (i) about 50 mM HEPES, about 100 mM NaCl, pH 7.50, (ii) about 50 mM HEPES, pH 7.0, or (iii) about 50 mM L-His, pH 7.

0.

23. The method according to any one of claims 1 to 2, wherein the lentiviral vector is formulated 1:1 in 2× stem cell growth medium (SCGM) to produce the formulated bulk lentiviral vector.

24. The lentiviral vector is formulated 1:1 in a buffer comprising (i) HEPES and sucrose, (ii) about 5 mM HEPES (pH 7.0), about 146 mM sucrose, and about 100 mM L-proline, (iii) about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and about 0.2 to about 2.0 poloxamer 188, (iv) about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, and 150 mM NaCl, (v) about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 100 mM L-proline, 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188, (vi) about 5 mM HEPES (pH 7.0), about 146 mM sucrose, about 150 mM NaCl, and about 0.2 to about 2.0 mg / ml poloxamer 188, (vii) L-histidine, sucrose, and L-proline, or (vii) about 5 mM L-histidine, about 146 mM sucrose, and about 100 mM L-proline, optionally, the formulation further comprises about 0.2 to about 2.0 mg / mL poloxamer 188, the method according to any one of claims 1 to 2.

25. The method according to any one of claims 1 to 2, wherein the formulated bulk lentiviral vector is sterile filtered through a 0.22 μm filter.

26. The method according to any one of claims 1 to 2, further comprising filling and finishing the formulated bulk lentiviral vector to produce a final lentiviral vector, and freezing the final lentiviral vector.

27. A method for generating a suspension lentiviral vector, comprising: (a) Inoculating a P0 suspension culture containing approximately 50 mL of culture medium with approximately 10.0×10 ６ to approximately 15.0×10 ６ viable HEK293T cells; (b) Inoculating about 30.0×10 ６ to about 70.0×10 ６ live HEK293T cells obtained from the P0 suspension culture into a P1 suspension culture containing about 100 mL of culture medium; (c) Inoculating each of three P2 suspension cultures containing about 200 mL of culture medium with about 11.0×10 ７ to about 19.0×10 ７ live HEK293T cells obtained from the P1 suspension culture; (d) Inoculating each of three P3 suspension cultures containing approximately 1.0 L of culture medium with approximately 55.0×10 ７ to approximately 95.0×10 ７ viable HEK293T cells obtained from the pooled P2 suspension culture; (e) Inoculating about 40.0×10 ８ to about 120.0×10 ８ viable HEK293T cells obtained from the pooled P3 suspension culture into a P4 suspension culture containing about 20.0 L of culture medium; (f) Inoculating about 40.0 × 10 ８ to about 120.0 × 10 ８ live HEK293T cells obtained from the P4 suspension culture into a P5 suspension culture containing about 200.0 L of culture medium; (g) culturing the P5 suspension culture for about 3 days and using alternating tangential flow filtration (ATF) to replace the culture medium of the P5 suspension culture with about 190.0 L of fresh culture medium; (h) After the culture medium exchange, transfecting the P5 suspension culture to generate a lentiviral vector, wherein the transfection step comprises adding about 10.0 L of a culture medium containing a transfer plasmid complexed with polyethyleneimine (PEI) and plasmid DNA encoding gag, pol, rev, and VSV-g; (i) After transfection, using ATF to exchange the culture medium of the P5 suspension culture with about 200.0 L of fresh culture medium; (j) About 36 to about 48 hours after transfection, treating the P5 suspension culture with an endonuclease for about 1 to about 2 hours; (k) Collecting and clarifying the supernatant of the P5 suspension culture using a tandem depth filter that retains particles of 60 μm or larger and a bilayer filter having pore sizes of 0.8 μm and 0.45 μm; (l) Capturing and concentrating the lentiviral vector from the collected and clarified supernatant of the P5 suspension culture, including heparin chromatography or cationic sulfate exchange chromatography; (m) Filtering the concentrated lentiviral vector using a bilayer filter having pore sizes of 0.8 μm and 0.45 μm; (n) Using tangential flow filtration (TFF) to ultrafilter the lentiviral vector to further concentrate the lentiviral vector and diafilter the lentiviral vector into a diafiltration buffer, thereby generating a bulk lentiviral vector; and (o) A method comprising formulating the lentiviral vector to generate a formulated bulk lentiviral vector.

28. A method for reducing host cell protein (HCP) from a suspension method for generating a viral vector, comprising: (a) Preparing a collected and clarified suspension culture supernatant containing a viral vector; (b) Capturing and concentrating the viral vector from the collected and clarified suspension culture supernatant using cation exchange chromatography; (c) Filtering the concentrated viral vector; (d) Using tangential flow filtration (TFF) to ultrafilter and diafilter the viral vector; and A method comprising formulating the viral vector to produce a formulated bulk viral vector and sterile filtering the formulated bulk viral vector. **Claim 29** The method according to claim 28, wherein the viral vector is pseudotyped with a heterologous envelope protein consisting of a vesicular stomatitis virus (VSV) envelope protein or a variant thereof.

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