Development of a suspension-mode seed train for adherent cells

Abstract

Advances in the use of recombinant viral vectors for gene therapy and DNA vaccination have created a need for the large-scale production of clinical-grade viral vectors, such as adeno-associated virus (rAAV) vectors, for the transport of genetic material. [Solution] This disclosure relates to a method for seed-train proliferation of adherent cells, the method comprising: culturing cells in serum-supplemented growth medium in an N-2 tank; removing cells from the serum-supplemented medium; inoculating cells from the process into serum-free growth medium in an N-1 tank; culturing cells in the N-1 tank under suspension conditions; and inoculating suspended cultured cells into growth medium in a bioreactor. In some embodiments, adherent cells are not suited to suspension. In some embodiments, adherent cells are suited to suspension.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Application No. 63 / 123,602, filed on December 10, 2020. The entire teachings of the above application are incorporated by reference. [Background technology]

[0002] Advances in the use of recombinant viral vectors for gene therapy and DNA vaccination have created a need for the large-scale production of clinical-grade viral vectors, such as adeno-associated virus (rAAV) vectors, for the transport of genetic material.

[0003] Mammalian adherent cells containing nucleic acids encoding recombinant proteins are often cultured in mass-production bioreactors to produce the therapeutic proteins of interest. Seed train processes are used to generate a sufficient number of such mammalian cells to inoculate mass-production bioreactors. Conventional seed train processes begin with thawing cryopreserved cell bank vials and are followed by multiple culture steps (e.g., five or more) in progressively larger culture vessels. Conventional seed train processes have several drawbacks, including the need for multiple manual operations during each step, which makes the entire process vulnerable to contamination and operator error. In particular, cell dissociation protocols for adherent cells during each passage often result in contamination, making this process unsuitable for scale-up production. These adherent seed train processes also fail to allow for the cultivation of the high cell numbers necessary to achieve the desired product yield in a sustainable and practical manner.

[0004] To overcome these problems, cell lines adapted to suspensions are being developed. However, developing suspension-adapted cell lines is time-consuming. Suspension-adapted cells also have a different transcriptome from their parent adherent cell lines. Therefore, a new suspension system is needed that enables the scale-up production of cells, for example, cells for virus production. [Overview of the project]

[0005] Several aspects of this disclosure relate to a cell proliferation method, the method comprising: (a) culturing cells in a first medium containing serum in an N-2 container; (b) removing cells from the first medium; (c) inoculating cells from step (b) into a second medium in an N-1 container, which is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing cells in the N-1 container under suspension conditions; and (e) inoculating cells from step (d) into a third medium in a bioreactor. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than that of the first medium. In some embodiments, the cells are adherent cells. In some embodiments, the N-1 container and the N-2 container are the same. In some embodiments, the N-1 container and the N-2 container are different. In some embodiments, the N-1 container includes a shaking flask or a wave bag. In some embodiments, the cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, or at least six times prior to step (e). In some embodiments, the third medium contains a serum concentration higher than that in the second medium.

[0006] In some embodiments, adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and their derivatives. In some embodiments, adherent cells are human. In some embodiments, adherent cells are animal cells, insect cells, or larvae. In some embodiments, adherent cells are HeLa cells or HEK-293 cells. In some embodiments, adherent cells are HEK-293.

[0007] In some embodiments, adherent cells are not adapted to suspension. In some embodiments, adherent cells are adapted to suspension. In some embodiments, culturing cells under suspension conditions does not alter the cell adhesion dependence. In some embodiments, the method does not alter cells to create new cell lines. In some embodiments, the method does not genetically alter cells.

[0008] In some embodiments, the method may further include culturing cells in a first medium in an N-3 container. In some embodiments, the method may further include culturing cells in a first medium in an N-4 container. In some embodiments, the method may further include culturing cells in a first medium in an N-5 container.

[0009] In some embodiments, the bioreactor is an adhesive bioreactor. In some embodiments, the bioreactor is selected from the group consisting of agitated tank bioreactors, bubble column bioreactors, airlift bioreactors, fluidized bed bioreactors, packed bed bioreactors, photobioreactor bioreactors, and stationary bed bioreactors. In some embodiments, the bioreactor is a stationary bed bioreactor.

[0010] In some embodiments, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor contains DMEM and 10% FBS.

[0011] In some embodiments, the cells are cultured under suspension conditions for approximately 24–120 hours. In some embodiments, the cells are cultured under suspension conditions for approximately 48–72 hours. In some embodiments, the N-1 container is a shaking flask. In some embodiments, the N-1 container is a wave bag.

[0012] In some embodiments, the method may further include contacting cells with a first polynucleotide sequence in a bioreactor. In some embodiments, the first polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

[0013] In some embodiments, the viral particle is AAV. In some embodiments, the method may further include contacting a cell with a second polynucleotide encoding a transgene. In some embodiments, the method includes containing a cell with a third polynucleotide encoding a helper gene. In some embodiments, the first polynucleotide comprises one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

[0014] In some embodiments, the method can further include culturing cells in a bioreactor.

[0015] In some embodiments, the culturing includes batch culturing. In some embodiments, the culturing includes fed-batch culturing. In some embodiments, the culturing includes perfusion culturing.

[0016] In some embodiments, the cells are cultured under conditions that produce recombinant virus particles.

[0017] Some embodiments of the present disclosure are directed to a method of cell growth, the method comprising: (a) culturing cells in a first medium containing serum in an N-3 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-2 container that does not contain serum or contains a lower concentration of serum than the first medium; (d) culturing the cells in the N-2 container under suspension conditions; (e) inoculating the cells from step (d) into the second medium in an N-1 container; (f) culturing the cells in the N-1 container under suspension conditions; and (g) inoculating the cells from step (d) into a third medium in a bioreactor.

[0018] In some embodiments, the cells are adherent cells. In some embodiments, the N-1, N-2, and N-3 containers are the same. In some embodiments, the N-1, N-2, and N-3 containers are different. In some embodiments, the cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, or at least six times before step (g). In some embodiments, the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and derivatives thereof. In some embodiments, the adherent cells are human. In some embodiments, the adherent cells are animal cells, insect cells, or larvae. In some embodiments, the adherent cells are HeLa cells or HEK-293 cells. In some embodiments, the adherent cells are HEK-293.

[0019] In some embodiments, adherent cells are not adapted to suspension. In some embodiments, adherent cells are adapted to suspension. In some embodiments, culturing cells under suspension conditions does not alter the cell adhesion dependence. In some embodiments, the method does not alter cells to create new cell lines. In some embodiments, the method does not genetically alter cells.

[0020] In some embodiments, the method may further include culturing cells in a first medium in an N-4 tank. In some embodiments, the method may further include culturing cells in a first medium in an N-5 container.

[0021] In some embodiments, the bioreactor is an adhesive bioreactor. In some embodiments, the bioreactor is selected from the group consisting of agitated tank bioreactors, bubble column bioreactors, airlift bioreactors, fluidized bed bioreactors, packed bed bioreactors, photobioreactor bioreactors, and stationary bed bioreactors. In some embodiments, the bioreactor is a stationary bed bioreactor.

[0022] In some embodiments, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor contains DMEM and 10% FBS.

[0023] In some embodiments, the cells are cultured under suspension conditions for approximately 24–120 hours. In some embodiments, the cells are cultured under suspension conditions for approximately 48–72 hours. In some embodiments, the N2 and N-1 containers are shaking flasks. In some embodiments, the N2 and N-1 containers are wave bags.

[0024] In some embodiments, the method may further include contacting cells with a first polynucleotide sequence in a bioreactor. In some embodiments, the first polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

[0025] In some embodiments, the viral particle is AAV. In some embodiments, the method may further include contacting a cell with a second polynucleotide sequence encoding a transgene. In some embodiments, the first polynucleotide sequence includes one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

[0026] In some embodiments, the method may further include culturing cells in a bioreactor.

[0027] In some embodiments, the culture includes batch culture. In some embodiments, the culture includes fed-batch culture. In some embodiments, the culture includes perfusion culture.

[0028] In some embodiments, cells are cultured under conditions that produce recombinant virus particles.

[0029] Some aspects of the present disclosure relate to a method for cell proliferation of adherent cells, the method comprising: (a) culturing adherent cells under adherent conditions in a first medium containing serum; (b) removing adherent cells from the first medium; (c) suspending adherent cells in a second medium that is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing the adherent cells from step (c) under suspension conditions; and (e) inoculating the adherent cells from step (d) into a third medium in a bioreactor.

[0030] In some embodiments, the method may further include subculturing the adherent cells of step (a) at least once under adherent conditions. In some embodiments, the method may further include subculturing the adherent cells of step (d) at least once under suspension conditions. In some embodiments, the method may further include subculturing the adherent cells of step (d) at least two, at least three, at least four, or at least five times under suspension conditions.

[0031] In some embodiments, the bioreactor is an adhesive bioreactor. In some embodiments, the bioreactor is selected from the group consisting of agitated tank bioreactors, bubble column bioreactors, airlift bioreactors, fluidized bed bioreactors, packed bed bioreactors, photobioreactor bioreactors, and stationary bed bioreactors. In some embodiments, the bioreactor is a stationary bed bioreactor.

[0032] In some embodiments, adherent cells are not adapted to suspension. In some embodiments, adherent cells are adapted to suspension. In some embodiments, culturing cells under suspension conditions does not alter the cell adhesion dependence. In some embodiments, the method does not alter cells to create new cell lines. In some embodiments, the method does not genetically alter cells.

[0033] In some embodiments, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor contains DMEM and 10% FBS.

[0034] In some embodiments, adherent cells are passaged up to two times under suspension conditions. In some embodiments, cells are passaged up to three, four, or five times under suspension conditions. In some embodiments, cells are cultured under suspension conditions for approximately 24 to 120 hours. In some embodiments, adherent cells are grown under suspension conditions for 48 to 72 hours.

[0035] In some embodiments, adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and their derivatives. In some embodiments, adherent cells are human. In some embodiments, adherent cells are animal cells, insect cells, or larvae. In some embodiments, adherent cells are HeLa cells or HEK-293 cells. In some embodiments, adherent cells are HEK-293 cells.

[0036] In some embodiments, the method may further include contacting adherent cells with a first polynucleotide sequence. In some embodiments, the first polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

[0037] In some embodiments, the viral particle is AAV. In some embodiments, the method may further include contacting adherent cells with a second polynucleotide sequence encoding a transgene. In some embodiments, the first polynucleotide sequence includes one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

[0038] In some embodiments, the method may further include culturing cells in a bioreactor.

[0039] In some embodiments, the culture includes batch culture. In some embodiments, the culture includes fed-batch culture. In some embodiments, the culture includes perfusion culture.

[0040] In some embodiments, cells are cultured under conditions that produce recombinant virus particles.

[0041] Some aspects of the present disclosure relate to a method for the proliferation of adherent cells, the method comprising: (a) culturing the cells under adherent conditions in a first medium containing serum; (b) removing the cells from the first medium; (c) suspending the cells in a second medium that is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing the cells under suspension conditions; (e) inoculating the cells from step (d) into a third medium in a bioreactor; (f) transfecting the cells with a polynucleotide encoding viral particles; and (g) culturing the cells in a medium under conditions that produce viral particles. In one embodiment, the second medium is a serum-free medium.

[0042] In some embodiments, the method may further include isolating the virus particles produced in step (g).

[0043] In some embodiments, the polynucleotide is a plasmid. In some embodiments, the viral particle is selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus. In some embodiments, the viral particle is an AAV.

[0044] In some embodiments, the bioreactor is an adhesive bioreactor. In some embodiments, the bioreactor is selected from the group consisting of agitated tank bioreactors, bubble column bioreactors, airlift bioreactors, fluidized bed bioreactors, packed bed bioreactors, photobioreactor bioreactors, and stationary bed bioreactors. In some embodiments, the bioreactor is a stationary bed bioreactor.

[0045] In some embodiments, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor contains DMEM and 10% FBS.

[0046] In some embodiments, the cells are adherent cells. In some embodiments, the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and their derivatives. In some embodiments, the adherent cells are human. In some embodiments, the adherent cells are animal cells, insect cells, or larvae.

[0047] In some embodiments, the adherent cells are HeLa cells or HEK-293 cells. In some embodiments, the adherent cells are HEK-293.

[0048] In some embodiments, adherent cells are not adapted to suspension. In some embodiments, adherent cells are adapted to suspension. In some embodiments, culturing cells under suspension conditions does not alter the cell adhesion dependence. In some embodiments, the method does not alter the cells to create new cell lines.

[0049] In some embodiments, the method may further include passage the cells of step (a) at least once under adhesive conditions. In some embodiments, the method may further include passage the cells of step (d) at least once under suspension conditions. In some embodiments, the cells are passaged two or fewer times under suspension conditions. In some embodiments, the cells are passaged two or fewer times, three or fewer times, four or five or fewer times under suspension conditions. In some embodiments, the cells are grown under suspension conditions for about 24 to 120 hours. In some embodiments, the cells are grown under suspension conditions for 48 to 72 hours.

[0050] In some embodiments, culturing cells in a bioreactor includes batch culture. In some embodiments, culturing cells in a bioreactor includes fed-batch culture. In some embodiments, culturing cells in a bioreactor includes perfusion culture. [Brief explanation of the drawing]

[0051] [Figure 1] This is a visual representation of the hybrid seed train propagation method described herein. [Figure 2] This is a visual representation of AAV particle production using the hybrid seed train propagation method disclosed herein. [Figure 3A] This graph shows the viability of HEK293 cells cultured according to the hybrid seed train growth method disclosed herein. [Figure 3B] This graph shows the survival rate of HEK293 cells cultured only under adhesion conditions. [Figure 4A]This graph shows the viable cell density of HEK293 cells cultured according to the hybrid seed train growth method disclosed herein. [Figure 4B] This graph shows the viable cell density of HEK293 cells cultured only under adhesion conditions. [Modes for carrying out the invention]

[0052] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, the definitions provided in this application shall prevail. Unless otherwise required by context, singular nouns shall include plural nouns, and plural nouns shall include singular nouns.

[0053] Throughout this disclosure, the terms “a” or “an” are understood to refer to one or more entities, for example, polynucleotide is understood to represent one or more polynucleotides. In such terms, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein.

[0054] Furthermore, where used herein, “and / or” is understood to mean each of two specified features or components, with or without the other, as specific disclosures. Thus, the term “and / or” as used in phrases, for example, “A and / or B” as used herein, is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases, for example, “A, B, and / or C” is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0055] The term "approximately" is used herein to mean roughly, broadly, before or after, or within that range. When the term "approximately" is used in conjunction with a numerical range, it thereby modifies the range by extending the upper and lower boundaries of the numerical value being expressed. Generally, the term "approximately" is used herein to change a numerical value above or below (higher or lower than) a 10 percent variation.

[0056] The term "at least" preceding a number or a series of numbers is understood, as is clear from the context, to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the characteristic indicated. When "at least" precedes a series of numbers or a range, it is understood that "at least" can modify each of the numbers in that series or range. "At least" is also not limited to integers, without considering the number of significant figures (for example, "at least 5%" includes 5.0%, 5.1%, and 5.18%).

[0057] Unless otherwise explicitly indicated, nucleotide sequences are presented herein in a single strand, from left to right, in the 5' to 3' direction only. Nucleotides and amino acids are represented herein in the format recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either a one-letter or three-letter code (both in accordance with 37 CFR §1.822 and established usage).

[0058] As used herein, “polynucleotide” or “nucleic acid” means a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in the 5' to 3' direction. The polynucleotides of this disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. The nucleotide bases are represented herein by the single-letter codes adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0059] As used herein, the term "polypeptide" encompasses both peptides and proteins unless otherwise indicated.

[0060] The terms “coding sequence” or “code” are used herein to mean a DNA or RNA region (transcription region) that “codes” a particular protein, such as insulin or glucokinase. A coding sequence, under the control of an appropriate regulatory region such as a promoter, is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo. The boundaries of a coding sequence are determined by a 5' (amino)-terminus start codon and a 3' (carboxy)-terminus translation termination codon. Examples of coding sequences include, but are not limited to, cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences. A transcription termination sequence may be located 3' relative to the coding sequence.

[0061] A gene may contain several operablely linked fragments, such as a promoter, a 5' leader sequence, an intron, a coding sequence, and a 3'-untranslated sequence, including, for example, a polyadenylation site or a signal sequence. As used herein, “gene expression” refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0062] The term “promoter” is used herein to mean a nucleic acid sequence or fragment that functions to control the transcription of one or more genes (or coding sequences) located upstream of the transcription start site of a gene, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence (including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequence of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from a promoter). A “constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions. A “tissue-specific” promoter is preferentially active in certain types of differentiated cells / tissues.

[0063] As used herein, the term “enhancer” refers to a cis-acting element that stimulates or inhibits the transcription of an adjacent gene. An enhancer that inhibits transcription is also called a “silencer.” Enhancers can function in any orientation over distances of up to several kilobase pairs (kb) from the coding sequence and from downstream locations of the transcription region (e.g., they may be associated with the coding sequence).

[0064] The terms “operatably linked,” “operatably inserted,” “operatably positioned,” “controlled,” or “transcriptionally regulated” mean that the promoter is in the correct position and orientation relative to the nucleic acid in order to control RNA polymerase initiation and gene expression. The term “operatably linked” means that the DNA sequence and regulatory sequence are linked in such a way that gene expression is enabled when the appropriate molecule (e.g., a transcription activator protein) is bound to the regulatory sequence. The term “operatably inserted” means that the target DNA introduced into a cell is positioned adjacent to a DNA sequence that induces transcription and translation of the introduced DNA (i.e., promotes the production of the polypeptide encoded by the target DNA).

[0065] The term “transgene” is used herein to mean a gene or nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid that encodes a therapeutic polypeptide. In some embodiments, a gene may be present, but in some cases, it is not normally expressed in the cell or is expressed at an insufficient level. In this context, “insufficient” means that the gene is normally expressed in the cell, but a condition and / or disease may still develop. In certain embodiments, a transgene enables increased or overexpression of a gene. A transgene may contain sequences native to the cell, sequences not native to the cell, or a combination of both. In certain embodiments, a transgene may contain sequences that can be operably linked to appropriate regulatory sequences for gene expression. In some embodiments, a transgene is not integrated into the host cell’s genome.

[0066] "Viral genome," "vector genome," or "viral vector" refers to a sequence containing one or more polynucleotide regions that encode or contain a molecule of interest, such as a protein, peptide, or polynucleotide, or a plurality thereof. Viral vectors are used to deliver genetic material into cells. Viral vectors may be modified for specific applications. In some embodiments, the delivery vector includes a viral vector selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, or retroviral vectors.

[0067] As used herein, the terms “adeno-associated virus vector” or “AAV vector” refer to any vector containing or derived from adeno-associated vector components and suitable for infecting mammalian cells, preferably human cells. The term AAV vector typically refers to an AAV-type virus particle or virion containing a payload. AAV vectors may be derived from a variety of serotypes, including combinations of serotypes (i.e., pseudotype AAV), or from a variety of genomes (e.g., single-stranded or self-complementary). In addition, AAV vectors may be replication-defective and / or targeted. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV1, AAV2, AAV3 (3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAVrh74, snake AAV, bird AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, their AAV serotypes and branchings disclosed by Gao et al. (J.Virol.78:6381(2004)) and Morris et al. (Virol.33:375(2004)), as well as any other AAV. See, for example, FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, “AAV vector” includes derivatives of known AAV vectors. In some embodiments, “AAV vector” includes modified or artificial AAV vectors. The terms “AAV genome” and “AAV vector” may be used interchangeably.

[0068] As used herein, “AAV particle” is an AAV virus comprising an AAV vector having at least one payload region (e.g., a polynucleotide encoding insulin and / or GcK) and at least one terminal inversion (ITR) region. In some embodiments, the term “AAV vector of the Disclosure” or “AAV vector disclosed herein” means, for example, an AAV vector comprising a polynucleotide or nucleic acid disclosed herein, which encodes insulin, GcK, or a combination thereof, encapsulated in an AAV particle.

[0069] "Genetic transfer" of cells by a virus means that there is a transfer of nucleic acids from a viral particle to a cell. In some embodiments, gene transfer refers to the delivery of nucleic acids encoding insulin and / or glucokinase to a recipient host cell by a viral vector. For example, gene transfer of a target cell by the rAAV vector of this disclosure results in the transfer of the rAAV genome (e.g., including the polynucleotides of this disclosure) contained in the vector to the gene transfer cell.

[0070] Cellular "transfection" refers to the introduction of genetic material into cells for the purpose of genetic modification. Transfection can be achieved by various means known in the art, such as gene transfer or electroporation.

[0071] As used herein, “vector” means a recombinant plasmid or virus containing polynucleotides that are delivered into a host cell, either in vitro or in vivo.

[0072] "Recombinant" generally means something that is different from what is commonly found in nature.

[0073] The "serotype" of a vector or viral capsid is defined by different immunological profiles based on the capsid protein sequence and capsid structure.

[0074] "AAV Cap" refers to the AAV Cap proteins VP1, VP2, and VP3, and their analogues.

[0075] "AAV Rep" refers to the AAV Rep protein and its analogues.

[0076] The term "adjacent" in relation to sequences where other elements are adjacent indicates the presence of one or more adjacent elements upstream and / or downstream of the sequence, i.e., at 5' and / or 3'. The term "adjacent" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the adjacent elements. A sequence (e.g., a transgene) where two other elements (e.g., ITRs) are "adjacent" indicates that one element is located at 5' relative to the sequence and the other at 3' relative to the sequence, but there may be intervening sequences between them.

[0077] As used herein, the term “gene therapy” means treating a disease or condition by inserting a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a therapeutic molecule as defined herein) into the cells and / or tissues of an individual. Gene therapy also includes the insertion of a transgene that is inherently inhibitory, i.e., a transgene that inhibits, reduces, or diminishes the expression, activity, or function of an endogenous gene or protein, such as an undesirable or abnormal (e.g., pathogenic) gene or protein. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that does not normally occur in the cells, tissues, and / or individual being treated. Both acquired and congenital diseases may be suitable for gene therapy.

[0078] As used herein, the terms “media,” “medium,” “cell culture medium,” “culture medium,” “tissue culture medium,” “tissue culture media,” and “growth medium” refer to a solution containing nutrients that nourish growing cultured eukaryotic cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. The solution may also contain components that enhance growth and / or survival beyond the minimum rate, including hormones and growth factors. The solution is formulated to the optimal pH and salt concentration for cell survival and proliferation. The medium may also be a “limited medium” or “known composition medium,” which is a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. A limited medium does not contain animal-derived components, and all components have known chemical structures. Those skilled in the art will understand that a limited medium may contain recombinant glycoproteins or proteins, for example, but not limited to hormones, cytokines, interleukins, and other signaling molecules.

[0079] As used herein, the terms “basic medium formulation” or “basic medium” refer to any cell culture medium used to culture cells that have not been modified by supplementation or selective removal of certain components.

[0080] As used herein, the terms “culture,” “cell culture,” and “eukaryotic cell culture” refer to a population of eukaryotic cells, whether surface-attached or in suspension, that is maintained or grown in a culture medium under conditions suitable for the survival and / or proliferation of the cell population. As will be apparent to those skilled in the art, as used herein, these terms may refer to a combination of mammalian cell populations and culture media in which the populations are suspended.

[0081] As used herein, the term “batch culture” refers to a method of culturing cells in which all components ultimately used in the cell culture, including the culture medium and the cells themselves, are provided at the start of the culture process. Batch culture is typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0082] As used herein, the term “fed-batch culture” refers to a method of culturing cells in which additional components are provided to the culture at some point after the start of the culture process. Fed-batch culture may be initiated using a basic medium. The culture medium in which additional components are provided to the culture at some point after the start of the culture process is a feed medium. The components provided typically include nutritional supplements for cells that have been depleted during the culture process. Fed-batch culture is typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0083] As used herein, the term “perfusion culture” refers to a method of culturing cells in which additional components are continuously or semi-continuously supplied to the culture after the start of the culture process. The supplied components typically include nutritional supplements for cells depleted during the culture process. The cells and / or components in the culture medium are typically harvested continuously or semi-continuously and optionally purified.

[0084] The “growth phase” in cell culture refers to the period of exponential cell growth (logarithmic phase) in which cells generally divide rapidly. During this phase, cells are cultured for a certain period, usually 1 to 4 days, and under conditions that maximize cell growth. The determination of the host cell growth cycle can be determined for a given host cell without excessive experimentation. “Period and conditions that maximize cell proliferation” refer to culture conditions that are judged to be optimal for cell growth and division for a particular cell line. In some embodiments, during the growth phase, cells are cultured in a nutrient medium containing necessary additives in a humidified controlled atmosphere, generally at about 25°C to 40°C, to achieve optimal growth for a particular cell line. In embodiments, cells are maintained in the growth phase for a period of about 1 to 7 days, for example, 2 to 6 days, for example, 6 days. The length of the growth phase for a particular cell can be determined without excessive experimentation. For example, the length of the growth phase is sufficient to allow a particular cell line to proliferate to a viable cell density within the range of approximately 20% to 80% of the maximum viable cell density possible, provided the culture is maintained under growth conditions. In some embodiments, "maximum growth rate" refers to the growth rate measured during the exponential growth phase of a particular cell line / clone while the cells are in fresh culture medium (e.g., measured during culture when nutrients are sufficient and there is no significant inhibition of growth from any component of the culture).

[0085] As used herein, the term “cell viability” refers to the ability of cells in a culture to survive under a given set of culture conditions or experimental variations. As used herein, this term also refers to the proportion of cells that survive at a given time, in relation to the total number of cells that are alive and dead in the culture at that time.

[0086] As used herein, the term “cell density” refers to the number of cells present in a given volume of culture medium.

[0087] The terms “cell derivative” or “derivative cell line” refer to a cell or cell line that is derived from, originates from, arises from, or arises from an original cell line, and that is in some respect different from the original cell line, for example, a derivative cell line may have one or more genetic modifications compared to the original cell line. The term “derivative cell line” does not imply any particular method or process for generating a cell line. In some embodiments, multiple derivative cell lines may arise from a single original cell line. A derivative cell line, for example, an HEK-293 derivative cell line derived from HEK-293 cells, is intended for use in the methods described herein.

[0088] As used herein, the terms “bioreactor” or “culture vessel” refer to any vessel used for growing mammalian cell cultures. Bioreactors may be of any size, as long as they are useful for culturing mammalian cells.

[0089] As used herein, the term “bioreactor run” may include one or more of the growth periods in a cell culture cycle, such as the lag phase, logarithmic phase, or plateau phase.

[0090] As used herein, the terms “Nl culture tank,” “Nl seed train culture tank,” “Nl tank,” “N1 culture,” or “N1 container” refer to a culture tank that is immediately preceding the N (production) culture tank and is used to grow cell cultures to a high viable cell density for subsequent inoculation into the N (production) culture tank. Cell cultures grown in the Nl culture tank may be obtained after culturing cells in several tanks, such as N-6, N-5, N-4, N-3, and N-2 tanks, prior to the Nl culture tank. As used herein, the terms “N culture tank,” “production culture tank,” “N tank,” “N bioreactor,” or “production bioreactor” refer to cell culture in a bioreactor after the Nl bioreactor. N cultures are used for AAV production.

[0091] As used herein, the terms “seeding” or “inoculating” refer to the process of providing a cell culture to a bioreactor or another tank. In one embodiment, the cells have been previously grown in another bioreactor or tank. In another embodiment, the cells are frozen and thawed immediately before being provided to the bioreactor or tank. The term refers to any number of cells, including a single cell.

[0092] Hybrid seed train proliferation of adherent cells Some aspects of the present disclosure relate to a method for cell proliferation, the method comprising: (a) culturing cells in a first medium containing serum in an N-2 container; (b) removing cells from the first medium; (c) inoculating cells from step (b) into a second medium in an N-1 container, which is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing cells in the N-1 container under suspension conditions; and (e) inoculating cells from step (d) into a third medium in a bioreactor. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than that of the first medium. In another embodiment, the third medium contains serum at a higher concentration than that of the second medium.

[0093] Some aspects of the present disclosure relate to a seed train propagation method, the method comprising: (a) culturing cells in a first medium containing serum in an N-3 container; (b) removing cells from the first medium; (c) inoculating cells from step (b) into a second medium in an N-2 container, which is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing cells in the N-2 container under suspension conditions; (e) inoculating cells from step (d) into a second medium in an N-1 tank; and (f) inoculating cells from step (d) into a third medium in a bioreactor. In one embodiment, the second medium is serum-free. In another embodiment, the second medium contains serum at a lower concentration than that of the first medium. In another embodiment, the third medium contains serum at a higher concentration than that of the second medium.

[0094] Some aspects of the present disclosure relate to a method for cell proliferation of adherent cells, the method comprising: (a) culturing adherent cells under adherent conditions in a first medium containing serum; (b) removing adherent cells from the first medium; (c) suspending adherent cells in a second medium that is serum-free or contains serum at a lower concentration than that of the first medium; (d) culturing adherent cells under suspension conditions; and (e) inoculating adherent cells from step (d) into a third medium in a bioreactor. In some aspects, the method may further comprise subculturing the adherent cells from step (a) at least once under adherent conditions. In some aspects, the method may further comprise subculturing the adherent cells from step (d) at least once under suspension conditions. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than that of the first medium in an N-1 container. In another embodiment, the third medium contains serum at a higher concentration than that of the second medium.

[0095] The first medium, the second medium, and the third medium may be any medium suitable for the specific cells to be cultured. In some embodiments, the medium may include, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., compositions containing albumin, growth factors, and growth inhibitors, e.g., fetal bovine serum, neonatal bovine serum, and equine serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins of plant or animal sources), and combinations thereof. The growth medium may be a commercially available medium such as 5× concentrated DMEM / F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA / GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma). In some embodiments, the first medium, the second medium, and the third medium may be restricted mediums as described herein. In some embodiments, the restricted medium may include DMEM and 10% FBS.

[0096] In some embodiments, a second serum-free growth medium, which contains no serum or serum at a lower concentration than that of the first serum, is substantially free (including trace levels or less) of extracellular matrix calcium ions, fetal bovine serum (FBS), fibronectin, collagen, laminin, or proteoglycans or non-proteoglycan polysaccharides that support cell fixation. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than that of the first medium.

[0097] In some embodiments, the growth medium is approximately 6.5-7.5, 6.5-7.4, 6.5-7.3, 6.5-7.2, 6.5-7.1, 6.5-7.0, 6.5-6.9, 6.5-6.8, 6.5-6.7, 6.6-7.5, 6.6-7.4, 6.6-7.3, 6.6-7.2, 6.6-7.1, 6.6-7.0, 6.6-6.9, 6.6-6.8, 6.7-7.5, 6.7-7.4, 6.7-7.3, 6.7-7.2, 6.7-7.1, 6.7- It can have a pH of approximately 7.0, approximately 6.7-6.9, approximately 6.8-7.5, approximately 6.8-7.4, approximately 6.8-7.3, approximately 6.8-7.2, approximately 6.8-7.1, approximately 6.8-7.0, approximately 6.9-7.5, approximately 6.9-7.4, approximately 6.9-7.3, approximately 6.9-7.2, approximately 6.9-7.1, approximately 7.0-7.5, approximately 7.0-7.4, approximately 7.0-7.3, approximately 7.0-7.2, approximately 7.1-7.5, approximately 7.1-7.4, approximately 7.1-7.3, approximately 7.2-7.5, approximately 7.2-7.4, or approximately 7.3-7.5.

[0098] In some embodiments, cells may be cultured at temperatures of approximately 32°C–39°C, approximately 32°C–37°C, approximately 32°C–37.5°C, approximately 34°C–37°C, approximately 35°C–37°C, approximately 35.5°C–37.5°C, approximately 36°C–37°C, or approximately 36.5°C. In some embodiments, cells may be cultured at a temperature of approximately 37°C from the start to the end of the culture period. In some embodiments, the temperature may be changed, for example, every hour or every day, or may fluctuate slightly during the culture period. In some embodiments, the temperature may be changed or shifted (e.g., increased or decreased) approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, or 15 days after the start of the culture period, or at any point during the culture period. In some embodiments, the temperature can be shifted upward by approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0°C. In some embodiments, the temperature can be shifted downward by approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10°C.

[0099] In some embodiments, cell culture may be carried out using an atmosphere containing about 1% to about 15% CO2. In some embodiments, cells may be cultured using an atmosphere containing about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5% CO2, 4% CO2, 3% CO2, 2% CO2, or about 1% CO2.

[0100] In some embodiments, cell culture is performed with approximately 3% to 55%, 3% to 50%, 3% to 45%, 3% to 40%, 3% to 35%, 3% to 30%, 3% to 25%, 3% to 20%, 3% to 15%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, and 5% to 25% in the cell culture. %, approximately 5% to 20%, approximately 5% to 15%, approximately 5% to 10%, approximately 10% to 55%, approximately 10% to 50%, approximately 10% to 45%, approximately 10% to 40%, approximately 10% to 35%, approximately 10% to 30%, approximately 10% to 25%, approximately 10% to 20%, approximately 15% to 55%, approximately 15% to 50%, approximately 15% to 45%, approximately 15% to 40%, approximately 15% to 35%, approximately 15% to 3 0%, approximately 15%~25%, approximately 15%~20%, approximately 20%~55%, approximately 20%~50%, approximately 20%~45%, approximately 20%~40%, approximately 20%~35%, approximately 20%~30%, approximately 20%~25%, approximately 25%~55%, approximately 25%~50%, approximately 25%~45%, approximately 25%~40%, approximately 25%~35%, approximately 25%~30%, approximately 30%~55%, approximately 30 This can be achieved by maintaining dissolved oxygen (dO2) at approximately 50%, 30%–45%, 30%–40%, 30%–35%, 35%–55%, 35%–50%, 35%–45%, 35%–40%, 40%–55%, 40%–50%, 40%–45%, 45%–55%, 45%–50%, or 50%–55%.

[0101] In some embodiments, the pH of a cell culture can be maintained at a specific pH value by adding a base solution, such as an alkaline base solution. The pH of cell culture is approximately 6.5-7.5, 6.5-7.4, 6.5-7.3, 6.5-7.2, 6.5-7.1, 6.5-7.0, 6.5-6.9, 6.5-6.8, 6.5-6.7, 6.6-7.5, 6.6-7.4, 6.6-7.3, 6.6-7.2, 6.6-7.1, 6.6-7.0, 6.6-6.9, 6.6-6.8, 6.7-7.5, 6.7-7.4, 6.7-7.3, 6.7-7.2, 6.7-7.1, 6.7-7. It can be maintained at a pH of 7.0, approximately 6.7-6.9, approximately 6.8-7.5, approximately 6.8-7.4, approximately 6.8-7.3, approximately 6.8-7.2, approximately 6.8-7.1, approximately 6.8-7.0, approximately 6.9-7.5, approximately 6.9-7.4, approximately 6.9-7.3, approximately 6.9-7.2, approximately 6.9-7.1, approximately 7.0-7.5, approximately 7.0-7.4, approximately 7.0-7.3, approximately 7.0-7.2, approximately 7.1-7.5, approximately 7.1-7.4, approximately 7.1-7.3, approximately 7.2-7.5, approximately 7.2-7.4, or approximately 7.3-7.5.

[0102] In some embodiments, cell culture under suspension conditions may be carried out in any type of cell culture flask suitable for stable or mixed / shaken suspension cell proliferation, for example, using a T-flask, roller bottle, spinner flask, or shaking flask, or a combination thereof. In some embodiments, the N-1 container is a shaking flask. In some embodiments, the N-1 container is a wave bag.

[0103] In some embodiments, the suspension conditions may include some form of stirring. In some embodiments, the stirring may be rotational stirring. In some embodiments, the stirring may be at speeds of approximately 25 RPM to 500 RPM, approximately 25 RPM to 480 RPM, approximately 25 RPM to 460 RPM, approximately 25 RPM to 440 RPM, approximately 25 RPM to 420 RPM, approximately 25 RPM to 400 RPM, approximately 25 RPM to 380 RPM, approximately 25 RPM to 360 RPM, approximately 25 RPM to 340 RPM, approximately 25 RPM to 320 RPM, approximately 25 RPM to 300 RPM, approximately 25 RPM to 280 RPM, approximately 25 RPM to 260 RPM, approximately 25 RPM to 240 RPM, and approximately 25 RPM to 22 0RPM, approximately 25RPM to approximately 200RPM, approximately 25RPM to approximately 180RPM, approximately 25RPM to approximately 160RPM, approximately 25RPM to approximately 140RPM, approximately 25RPM to approximately 120RPM, approximately 25RPM to approximately 100RPM, approximately 25RPM to approximately 80RPM, approximately 25RPM to approximately 60RPM, approximately 25RPM to approximately 40RPM, approximately 25RPM to approximately 35RPM, approximately 25RPM to approximately 30RPM, approximately 50RPM to approximately 500RPM, approximately 50RPM to approximately 480RPM, approximately 50RPM to approximately 460RPM, approximately 50RPM to approximately 440RPM, approximately 50RPM to approximately 4 20RPM, approximately 50RPM to approximately 400RPM, approximately 50RPM to approximately 380RPM, approximately 50RPM to approximately 360RPM, approximately 50RPM to approximately 340RPM, approximately 50RPM to approximately 320RPM, approximately 50RPM to approximately 300RPM, approximately 50RPM to approximately 280RPM, approximately 50RPM ~260RPM~240RPM, 50RPM~220RPM, 50RPM~200RPM, 50RPM~180RPM, 50RPM~160RPM, 50RPM~140RPM, 50RPM~120RPM, 50RPM~120RPM RPM ~ approx. 100 RPM, approx. 50 RPM ~ approx. 80 RPM, approx. 50 RPM ~ approx. 60 RPM, approx. 75 RPM ~ approx. 500 RPM, approx. 75 RPM ~ approx. 480 RPM, approx. 75 RPM ~ approx. 460 RPM, approx. 5RPM to about 400RPM, about 75RPM to about 380RPM, about 75RPM to about 360RPM, about 75RPM to about 340RPM, about 75RPM to about 320RPM, about 75RPM to about 300RPM, about 75RPM to about 280RPM, about 75RPM to about 260RPM,Approximately 75 RPM to 240 RPM, approximately 75 RPM to 220 RPM, approximately 75 RPM to 200 RPM, approximately 75 RPM to 180 RPM, approximately 75 RPM to 160 RPM, approximately 75 RPM to 140 RPM, approximately 75 RPM to 120 RPM, approximately 75 RPM to 100 RPM, approximately 75 RPM to 80 RPM, approximately 100 RPM to 500 RPM, approximately 100 RPM to 480 RPM, approximately 100 RPM to 460 RPM, approximately 100 RPM to 440 RPM, approximately 100 RPM to 420 RPM, approximately 100 RPM to 400 RPM, approximately 100 RPM to 380 RPM, approximately 10 0 RPM to approximately 360 RPM, approximately 100 RPM to approximately 340 RPM, approximately 100 RPM to approximately 320 RPM, approximately 100 RPM to approximately 300 RPM, approximately 100 RPM to approximately 280 RPM, approximately 100 RPM to approximately 260 RPM, approximately 100 RPM to approximately 240 RPM, approximately 100 RPM to approximately 220 RPM, approximately 100 RPM to approximately 200 RPM, approximately 100 RPM to approximately 180 RPM, approximately 100 RPM to approximately 160 RPM, approximately 100 RPM to approximately 140 RPM, approximately 100 RPM to approximately 120 RPM, approximately 150 RPM to approximately 500 RPM, approximately 150 RPM to approximately 480 RPM, approximately 150 RPM to approximately 460 RPM RPM, approximately 150 RPM to 440 RPM, approximately 150 RPM to 420 RPM, approximately 150 RPM to 400 RPM, approximately 150 RPM to 380 RPM, approximately 150 RPM to 360 RPM, approximately 150 RPM to 340 RPM, approximately 150 RPM to 320 RPM, approximately 150 RPM to 300 RPM, approximately 150 RPM to 280 RPM, approximately 150 RPM to 260 RPM, approximately 150 RPM to 240 RPM, approximately 150 RPM to 220 RPM, approximately 150 RPM to 200 RPM, approximately 150 RPM to 180 RPM, approximately 150 RPM to 160 RPM, approximately 200 RPM PM ~ approximately 500 RPM, approximately 200 RPM ~ 480 RPM, approximately 200 RPM ~ approximately 460 RPM, approximately 200 RPM ~ approximately 440 RPM, approximately 200 RPM ~ approximately 420 RPM, approximately 200 RPM ~ approximately 400 RPM, approximately 200 RPM ~ approximately 380 RPM, approximately 200 RPM ~ approximately 360 RPM, approximately 200 RPM ~ approximately 340 RPM, approximately 200 RPM ~ approximately 320 RPM, approximately 200 RPM ~ approximately 300 RPM, approximately 200 RPM ~ approximately 280 RPM, approximately 200 RPM ~ approximately 260 RPM, approximately 200 RPM ~ approximately 240 RPM, approximately 200 RPM ~ approximately 220 RPM, approximately 240 RPM ~ approximately 500 RPMApproximately 240RPM to approximately 480RPM, approximately 240RPM to approximately 460RPM, approximately 240RPM to approximately 440RPM, approximately 240RPM to approximately 420RPM, approximately 240RPM to approximately 400RPM, approximately 240RPM to approximately 380R PM, about 240RPM to about 360RPM, about 240RPM to about 340RPM, about 240RPM to about 320RPM, about 240RPM to about 300RPM, about 240RPM to about 280RPM, about 240RPM to about 26 0RPM, about 260RPM to about 500RPM, about 260RPM to about 480RPM, about 260RPM to about 460RPM, about 260RPM to about 440RPM, about 260RPM to about 420RPM, about 260RPM to about Approx. 400RPM, Approx. 260RPM ~ Approx. 380RPM, Approx. 260RPM ~ Approx. 360RPM, Approx. 260RPM ~ Approx. 340RPM, Approx. 260RPM ~ Approx. 320RPM, Approx. 260RPM ~ Approx. 300RPM, Approx. 260RPM M ~ about 280RPM, about 280RPM - about 500RPM, about 280RPM - about 480RPM, about 280RPM - about 460RPM, about 280RPM - about 440RPM, about 280RPM - about 420RPM, about 28 0RPM to approx. 400RPM, approx. 280RPM to approx. 380RPM, approx. 280RPM to approx. 360RPM, approx. 280RPM to approx. 340RPM, approx. 280RPM to approx. This can occur at rotational speeds of approximately 300 RPM to 500 RPM, 380 RPM to 480 RPM, 380 RPM to 460 RPM, 380 RPM to 440 RPM, 380 RPM to 420 RPM, 380 RPM to 400 RPM, 400 RPM to 500 RPM, 400 RPM to 480 RPM, 400 RPM to 460 RPM, 400 RPM to 440 RPM, or 400 RPM to 420 RPM. Stirring can be performed continuously or periodically.

[0104] In some embodiments, the cells are passaged two or fewer times under suspension conditions. In some embodiments, the cells are passaged three or fewer times under suspension conditions. In some embodiments, the cells are passaged four or fewer times under suspension conditions. In some embodiments, the cells are passaged five or fewer times under suspension conditions.

[0105] In some embodiments, cells are cultured in suspension culture for at least about 24 hours before subculturing to N-1 container. For example, if cells are subculturing five times under suspension conditions, the cells are cultured in N-5 container for at least about 24 hours before subculturing to N-4 container, the cells are cultured in N-4 container for at least about 24 hours before subculturing to N-3 container, the cells are cultured in N-3 container for at least about 24 hours before subculturing to N-2 container, and the cells are cultured in N-2 container for at least about 24 hours before subculturing to N-1 container.

[0106] In some embodiments, cells are cultured in suspension for approximately 24 to 120 hours. In some embodiments, cells are cultured in suspension for approximately 24 to 96 hours. In some embodiments, cells are cultured in suspension for approximately 36 to 84 hours. In some embodiments, cells are cultured in suspension for approximately 48 to 72 hours. In some embodiments, cells are cultured in suspension for approximately 54 to 66 hours. In some embodiments, cells are cultured in suspension for approximately 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours.

[0107] In some aspects of this disclosure, the cells are adherent cells. In some aspects, the adherent cells are HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and their derivatives. In some aspects, the adherent cells are human. In some aspects, the adherent cells are animal cells, insect cells, or larvae. In some aspects, the adherent cells are HeLa or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells.

[0108] In some embodiments, preferred adherent cells include, but are not limited to, CRL 1573 (ATCC), 293-F (GIBCO), HEK 293T (ATCC), 293 H, 293 MSR, Expi293, Flp-In(trademark)-293, 293 Met(-), and T-REx(trademark)293, encompassing a variety of commercially available HEK-293 cell line derivatives. The HEK-293 cell line is immortalized by the incorporation of a 4kbp adenovirus 5 (Ad5) genome fragment containing the E1A and E1B genes. Its expression enables continuous culture of HEK293 cells by inhibiting apoptosis and disrupting transcriptional and cell cycle regulatory pathways. (See Malm, M., Sci Rep 10, 18996 (2020)), which is incorporated herein in its entirety by reference.) Certain HEK-293 cell line derivatives were established through genetic modification. One non-limiting example is the HEK293T derivative. The HEK293T genome contains the SV40 large T antigen, which enables the production of recombinant proteins in plasmid vectors containing the SV40 promoter. Another non-limiting example is the HEK293MSR derivative. The HEK293MSR cell line is genetically modified to express the human macrophage scavenger receptor and adheres strongly to standard tissue culture dishes. In some aspects of this disclosure, preferred adherent cells are any of the commercially available genetically modified HEK-293 cell line derivatives.

[0109] In some embodiments, adherent cells are not adapted to suspension. In some embodiments, culturing cells under suspension conditions does not alter the cell adhesion dependency. In some embodiments, the method does not alter cells to create new cell lines. In some embodiments, the method does not genetically alter cells. The methods disclosed herein do not alter the genomic or transcriptome profile of cells. The methods disclosed herein do not alter the phenotype of cells.

[0110] In some embodiments, cells are passaged multiple times in serum-supplemented growth medium under adhesion conditions before inoculation into N-1 container. In some embodiments, cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, or at least six times in serum-supplemented growth medium under adhesion conditions before inoculation into N-1 container. In some embodiments, cells are cultured in N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9, or N-10 containers before inoculation into N-1 container. In some embodiments, cells are cultured in N-3 and N-2 containers. In some embodiments, cells are cultured in N-4, N-3, and N-2 containers. In some embodiments, cells are cultured in N-6, N-5, N-4, N-3, and N-2 containers. In some embodiments, cells are cultured in N-6, N-5, N-4, N-3, N-2, and N-1 containers. In some embodiments, the N-6, N-5, N-4, N-3, N-2, and N-1 containers are the same. In some embodiments, the N-6, N-5, N-4, N-3, N-2, and N-1 containers are different.

[0111] The N-1 container may include any type of N-1 container or cell culture vessel or tank known in the art for maintaining cell lines before inoculation into a bioreactor, such as a collapsible bag or flexible container, a non-collapsible or rigid container, and any other configuration with liquid storage. In some embodiments, the N-1 container may be a shaking flask. In some embodiments, the N-1 container may be a wave bag.

[0112] In some embodiments, the bioreactor is an adhesive bioreactor. In some embodiments, the bioreactor comprises at least one, more preferably, carriers to which proliferating cells are intended to adhere, which may be suspended or fixed within the bioreactor. Preferably, the carriers may be made using, for example, polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate, or acrylamide. In some embodiments, the bioreactor may contain bead-type microcarriers (e.g., Cytodex® brand beads, commercially available from GE Healthcare Inc. division of General Electric Corp.) or matrix-type carriers (e.g., Fibra-Cell® brand discs, commercially available from Eppendorf Corp.). In some embodiments, the bioreactor uses polyester fiber carriers, such as those used in iCELLis® nano or iCELLis® 500 bioreactors (commercially available from Advanced Technology Materials Inc. (Brussels, Belgium) and Pall Corporation (Fall River, Mass)).

[0113] In some embodiments, the bioreactor may be any commercially available bioreactor for bioprocesses. In some embodiments, the bioreactor may be a continuous agitated tank bioreactor. In some embodiments, the bioreactor may be a bubble column bioreactor. In some embodiments, the bioreactor may be an airlift bioreactor. In some embodiments, the bioreactor may be a fluidized bed bioreactor. In some embodiments, the bioreactor may be a packed bed bioreactor. In some embodiments, the bioreactor may be a photobioreactor. In some embodiments, the bioreactor may be a stationary bed bioreactor. In one embodiment, the bioreactor may be an ICELLIS stationary bed bioreactor (Pall Corporation, Port Washington, NY) for producing viral vectors.

[0114] In some embodiments, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the at least one factor that promotes cell adhesion may be added to the third medium immediately before, during, or immediately after inoculation of suspended cells into the bioreactor.

[0115] In some embodiments, the growth medium comprises DMEM and about 10 wt% FBS. In some embodiments, the growth medium comprises about 2 wt% to about 20 wt% FBS. In some embodiments, the growth medium comprises about 3 wt% to about 19 wt% FBS. In some embodiments, the growth medium comprises about 4 wt% to about 18 wt% FBS. In some embodiments, the growth medium comprises about 5 wt% to about 17 wt% FBS. In some embodiments, the growth medium comprises about 6 wt% to about 16 wt% FBS. In some embodiments, the growth medium comprises about 7 wt% to about 15 wt% FBS. In some embodiments, the growth medium comprises about 8 wt% to about 14 wt% FBS. In some embodiments, the growth medium comprises about 9 wt% to about 13 wt% FBS. In some embodiments, the growth medium comprises about 10 wt% to about 12 wt% FBS. In some embodiments, the growth medium comprises about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, or about 20 wt% FBS.

[0116] In some embodiments, the suspended proliferating cells from step (d) can be directly inoculated into the bioreactor. In some embodiments, the amount of cells inoculated into the bioreactor varies based on the size of the bioreactor. In some embodiments, a 4m 2 bioreactor (e.g., iCELLis® nanobioreactor) is used. In some embodiments, a 4m 2 bioreactor is inoculated with about 1×10 8 to 1×10 9 cells. In some embodiments, a 4m 2 bioreactor is inoculated with about 3×10 8 to 7×10 8 cells. A 4m 2 bioreactor is inoculated with about 4×10 8 to 6×10 8 cells. A 4m 2 bioreactor is inoculated with about 5×10 8Cells are inoculated. In some embodiments, equivalent cell densities are used in bioreactors of different sizes.

[0117] In some embodiments, the method of the present disclosure may further include culturing cells in a bioreactor. In some embodiments, the cell culture includes batch culture. In some embodiments, the cell culture includes fed-batch culture. In some embodiments, the cell culture includes perfusion culture.

[0118] Fed-batch culture involves the incremental (periodic) or continuous addition of feed culture medium to an initial cell culture without substantially or significantly removing growth medium from the cell culture. The cell culture in fed-batch culture may be placed in a bioreactor (e.g., a production bioreactor such as a 10,000 L production bioreactor). In some embodiments, the feed culture medium may be the same as the growth medium. The feed culture medium may be in liquid form or dry powder form. In some embodiments, the feed culture medium is a concentrated form of the growth medium and / or is added as a dry powder. In some embodiments, both a first liquid feed culture medium and a different second liquid feed culture medium may be added to the growth medium (e.g., continuously). In some embodiments, the addition of the first liquid feed culture medium and the second liquid feed culture medium to the culture may begin almost simultaneously. In some embodiments, the total volume of the first and second liquid feed culture media added to the culture over the entire culture period may be approximately the same.

[0119] When feed culture medium is added continuously, the rate of feed culture medium addition can be maintained constant or increased (e.g., steadily increasing) throughout the culture period. Continuous addition of feed culture medium may be used at specific points in time during the culture period (e.g., when cells reach a target viable cell density, e.g., approximately 1 × 10⁻⁶). 6 cells / mL, approximately 1.1×10 6 cells / mL, approximately 1.2×10 6 cells / mL, approximately 1.3×10 6 cells / mL, approximately 1.4×10 6cells / mL, approximately 1.5×10 6 cells / mL, approximately 1.6×10 6 cells / mL, approximately 1.7×10 6 cells / mL, approximately 1.8×10 6 cells / mL, approximately 1.9×10 6 Cells / mL, or approximately 2.0 × 10⁶ 6 It may be initiated when the viable cell density reaches cells / mL. In some embodiments, the continuous addition of feed culture medium may be initiated on day 2, day 3, day 4, or day 5 of the culture period.

[0120] In some embodiments, the gradual (periodic) addition of feed culture medium allows cells to reach a target viable cell density (e.g., approximately 1 × 10⁻⁶). 6 cells / mL, approximately 1.1×10 6 cells / mL, approximately 1.2×10 6 cells / mL, approximately 1.3×10 6 cells / mL, approximately 1.4×10 6 cells / mL, approximately 1.5×10 6 cells / mL, approximately 1.6×10 6 cells / mL, approximately 1.7×10 6 cells / mL, approximately 1.8×10 6 cells / mL, approximately 1.9×10 6 , or approximately 2.0 × 10 6 It can be initiated when the cell density reaches (cells / mL). In some embodiments, incremental feed medium addition may occur at regular intervals (e.g., daily, every other day, or every three days) or when the cells reach a specific target cell density (e.g., a target cell density that increases over the culture period). In some embodiments, the amount of feed medium added may be progressively increased between the first incremental addition of feed medium and subsequent additions of feed medium. In some embodiments, the volume of liquid culture feed medium added to the initial cell culture over any 24-hour period may be a percentage of the initial volume of the bioreactor containing the culture, or a percentage of the volume of the initial culture.

[0121] In some embodiments, the addition of liquid feed culture medium (continuous or periodic) occurs 6 to 7 days, approximately 6 to 6 days, approximately 6 to 5 days, approximately 6 to 4 days, approximately 6 to 3 days, approximately 6 to 2 days, approximately 6 to 1 day, approximately 12 to 7 days, approximately 12 to 6 days, approximately 12 to 5 days, approximately 12 to 4 days, approximately 12 to 3 days, approximately 12 to 2 days, approximately 1 day It can occur at approximately 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 3 to 4 days, 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 7 days, or 5 to 6 days.

[0122] In some embodiments, the volume of liquid feed culture medium added (continuously or periodically) to the initial cell culture over any given 24-hour period may be 0.01 × to approximately 0.3 × the capacity of the bioreactor.The proportions are approximately 0.01 × ~ 0.28 × of the bioreactor capacity, approximately 0.01 × ~ 0.26 ×, approximately 0.01 × ~ 0.24 ×, approximately 0.01 × ~ 0.22 ×, approximately 0.01 × ~ 0.20 ×, approximately 0.01 × ~ 0.18 ×, approximately 0.01 × ~ 0.16 ×, approximately 0.01 × ~ 0.14 ×, approximately 0.01 × ~ 0.12 ×, approximately 0.01 × ~ 0.10 ×, approximately 0.01 × ~ 0.08 ×, approximately 0.01 × ~ 0.06 ×, approximately 0.01 × ~ 0.04 ×, approximately 0.02 × ~ 0.3 ×, approximately 0.02 × ~ 0.28 ×, approximately 0.02 × ~ 0.26 ×, approximately 0.02×~approx. 0.24×, approx. 0.02×~approx. 0.22×, approx. 0.02×~approx. 0.20×, approx. 0.02×~approx. 0.18×, approx. 0.02×~approx. 0.16×, approx. 0.02×~approx. 0.14×, approx. 0.02×~approx. 0.12×, approx. 0.02×~approx. 0.10×, approx. 0.02×~approx. 0.08×, approx. 0.02×~approx. 0.06×, approx. 0.02×~approx. 0.05×, approx. 0.02×~approx. 0.04×, approx. 0.02×~approx. 0.03×, approx. 0.025×~approx. 0.3×, approx. 0.025×~approx. 0.28×, approx. 0.025×~approx. 0.26×, approx. 0.025×~approx. 0.24×, approx. 0.025×~approx. 0.22×, approx. 0.025×~approx. 0.20×, approx. 0.025×~approx. 0.18×, approx. 0.025×~approx. 0.16×, approx. 0.025×~approx. 0.14×, approx. 0.025×~approx. 0.12×, approx. 0.025×~approx. 0.10×, approx. 0.025×~approx. 0.08×, approx. 0.025×~approx. 0.06×, approx. 0.025×~approx. 0.04×, approx. 0.05×~approx. 0.3×, approx. 0.05×~approx. 0.28×, approx. 0.05×~approx. 0.26×, approx. 0.05×~approx. 0.24×, approx. 0.05×~approx. 0.22×, approx. 0.05×~approx. 0.20×, approx. 0.05×~approx. 0.18×, approximately 0.05× to approximately 0.16×, approximately 0.05× to approximately 0.14×, approximately 0.05× to approximately 0.12×, approximately 0.05× to approximately 0.10×, approximately 0.1× to approximately 0.3×, approximately 0.1× to approximately 0.28×, approximately 0.1× to approximately 0.26×, approximately 0.1× to approximately 0.24×, approximately 0.1× to approximately 0.22×, approximately 0.1× to approximately 0.20×, approximately 0.1× to approximately 0.18×, approximately 0.1× to approximately 0.16, approximately 0.1× to approximately 0.14×, approximately 0.1×, approximately 0.15× to approximately 0.3×, approximately 0.15× to approximately 0.2×, approximately 0.2× to approximately 0.3×, or approximately 0.25× to approximately 0.3× may.

[0123] In some embodiments, the volume of liquid feed culture medium added (continuously or periodically) to the initial cell culture over any 24-hour period during the culture period is approximately 0.02 × to approximately 1.0 ×, approximately 0.02 × to approximately 0.9 ×, approximately 0.02 × to approximately 0.8 ×, approximately 0.02 × to approximately 0.7 ×, approximately 0.02 × to approximately 0.6 ×, and approximately 0.02 × of the volume of the initial cell culture. × ~ approx. 0.5 ×, approx. 0.02 × ~ approx. 0.4 ×, approx. 0.02 × ~ approx. 0.3 ×, approx. 0.02 × ~ approx. 0.2 ×, approx. 0.02 × ~ approx. 0.1 ×, approx. 0.02 × ~ approx. 0.08 ×, approx. 0.02 × ~ approx. 0.06 ×, approx. 0.02 × ~ approx. 0.05 ×, approx. 0.02 × ~ approx. 0.04 ×, approx. 0.02 × ~ approx. 0.03 ×, approx. 0.05 × ~ approx. 1.0 ×, approx. 0.05 × ~approximately 0.8x, approximately 0.05x~approximately 0.7x, approximately 0.05x~approximately 0.6x, approximately 0.05x~approximately 0.5x, approximately 0.05x~approximately 0.4x, approximately 0.05x~approximately 0.3x, approximately 0.05x~approximately 0.2x, approximately 0.05x~approximately 0.1x, approximately 0.1x~approximately 1.0x, approximately 0.1x~approximately 0.9x, approximately 0.1x~approximately 0.8x, approximately 0.1x~approximately 0.7x, approximately 0 0.1 × ~ approximately 0.6 ×, approximately 0.1 × ~ approximately 0.5 ×, approximately 0.1 × ~ approximately 0.4 ×, approximately 0.1 × ~ approximately 0.3 ×, approximately 0.1 × ~ approximately 0.2 ×, approximately 0.2 × ~ approximately 1.0 ×, approximately 0.2 × ~ approximately 0.9 ×, approximately 0.2 × ~ approximately 0.8 ×, approximately 0.2 × ~ approximately 0.7 ×, approximately 0.2 × ~ approximately 0.6 ×, approximately 0.2 × ~ approximately 0.5 ×, or approximately 0.2 × ~ approximately 0.4 × may occur.

[0124] In some embodiments, the total amount of feed culture medium added (continuously or periodically) throughout the culture period is approximately 1% to 40% of the initial culture volume (e.g., approximately 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 4%, 2% to 40%, 2% to 35%). Approximately 2% to 30%, approximately 2% to 25%, approximately 2% to 20%, approximately 2% to 15%, approximately 2% to 10%, approximately 2% to 5%, approximately 3% to 40%, approximately 3% to 35%, approximately 3% to 30%, approximately 3% to 25%, approximately 3% to 20%, approximately 3% to 15%, approximately 3% to 10%, approximately 3% to 5%, approximately 4% to 40%, approximately 4% to 35%, approximately 4% to 30%, approximately 4% to 25% %, approximately 4% to 20%, approximately 4% to 15%, approximately 4% to 10%, approximately 4% to 8%, approximately 5% to 40%, approximately 5% to 35%, approximately 5% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, approximately 5% to 10%, approximately 10% to 40%, approximately 10% to 35%, approximately 10% to 30%, approximately 10% to 25%, approximately 10% to 20%, approximately 10% to 15% It could be approximately 15% to 40%, 15% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 40%, 30% to 35%, or 35% to 40%).

[0125] In some embodiments, two different feed culture media are added (continuously or incrementally) during fed-batch culture. In some embodiments, the amounts or volumes of the first and second feed culture media added may be substantially the same or different. In some embodiments, the first feed culture medium may be in liquid form, and the second feed culture medium may be in solid form. In some embodiments, the first and second feed culture media may be liquid feed culture media.

[0126] Perfusion culture involves removing a first volume of growth medium from a bioreactor and adding a second volume of second growth medium to a production bioreactor, wherein the first and second volumes are approximately equal. Cells are retained in the bioreactor by a cell retention device or by a technique such as cell sedimentation in a sedimentation cone. In some embodiments, the removal and addition of growth medium may be carried out simultaneously, sequentially, or in any combination thereof. In some embodiments, removal and addition can be carried out continuously at rates such as removing and replacing a volume of 0.1% to 800%, 1% to 700%, 1% to 600%, 1% to 500%, 1% to 400%, 1% to 350%, 1% to 300%, 1% to 250%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30% of the bioreactor's capacity.

[0127] In some embodiments, the first volume of the first growth medium removed and the second volume of the second growth medium added can be kept approximately the same over each 24-hour period. In some embodiments, the rate at which the first volume of the first growth medium is removed (volumes / hour) and the rate at which the second volume of the second growth medium is added (volumes / hour) can vary and depend on the conditions of the particular cell culture system. In some embodiments, the rate at which the first volume of the first growth medium is removed (volumes / hour) and the rate at which the second volume of the second growth medium is added (volumes / hour) can be approximately the same or different.

[0128] In some embodiments, the volumes removed and added can be varied by gradually increasing them over each 24-hour period. In some embodiments, the volume of the first growth medium removed and the volume of the second growth medium added over each 24-hour period can be increased over the culture period. In some embodiments, the volume can be increased over a 24-hour period to a volume of 0.5% to about 20% of the bioreactor's capacity. In some embodiments, the volume can be increased over the culture period to a volume of about 25% to about 150% of the bioreactor's capacity or the volume of the first liquid culture medium over a 24-hour period.

[0129] In some embodiments, after the first 48–96 hours of the culture period, over each 24-hour period, the first volume of the first growth medium removed and the second volume of the second growth medium added are approximately 10%–95%, 10%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–80%, 80%–90%, 85%–95%, 60%–80%, or 70% of the volume of the first growth medium.

[0130] In some embodiments, the first growth medium and the second growth medium may be of the same type. In some embodiments, the first growth medium and the second growth medium may be different. In some embodiments, the second liquid culture medium may be more concentrated with respect to one or more medium components.

[0131] In some embodiments, a first volume of the first growth medium can be removed by using any automated system. In some embodiments, alternating tangential flow filtration may be used. In some embodiments, a first volume of the first growth medium can be removed by leaching or gravity flow of the first volume of the first growth medium through a sterile membrane having a molecular weight cutoff that excludes cells. In some embodiments, a first volume of the first growth medium can be removed by stopping or significantly reducing the stirring speed for a period of at least 1 minute, at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 minutes, or 1 hour, and removing or aspirating the first volume of the growth medium from the top of the production bioreactor.

[0132] In some embodiments, a second volume of the second liquid culture medium can be added to the first liquid culture medium by a pump. In some embodiments, the second liquid culture medium can be added to the first liquid culture medium manually or in an automated manner, such as by directly dispensing or injecting the second volume of the second liquid culture medium onto the first liquid culture medium.

[0133] In some embodiments, the method further comprises contacting cells with a first polynucleotide sequence. In some embodiments, the method further comprises transfecting cells with the polynucleotide sequence. In some embodiments, the polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus. In some embodiments, the cells are transfected before inoculation into a bioreactor. In some embodiments, the cells are transfected after inoculation into a bioreactor. In some embodiments, the cells are contacted with or transfected with a second polynucleotide and contain a nucleic acid encoding a transgene. In some embodiments, the cells are contacted with or transfected with a third polynucleotide encoding a helper gene. In one embodiment, the helper gene is an adenovirus help gene. In one embodiment, the first polynucleotide comprises one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, or a nucleic acid encoding at least one AAV structural capsid protein.

[0134] In some embodiments, cells are cultured under conditions that produce a viral vector. In some embodiments, the method further includes isolating the produced viral vector.

[0135] In some embodiments, polynucleotides are viral vectors. In some embodiments, viral vectors are adenovirus and adeno-associated virus (AAV) vectors. These vectors infect numerous dividing and non-dividing cell types, including synovial cells and hepatocytes. The episomal properties of adenovirus and AAV vectors after cell entry make these vectors suitable for therapeutic use, as shown above (Russell, 2000, J.Gen.Virol.81:2573-2604, Goncalves, 2005, Virol J.2(1):43). AAV vectors can provide very stable, long-term expression of transgenes (up to 9 years in dogs (Niemeyer et al, Blood. 2009 Jan. 22;113(4):797-806) and up to 2 years in humans (Nathwani et al, N Engl J Med. 2011 Dec. 22;365(25):2357-65, Simonelli et al, Mol Ther. 2010 March;18(3):643-50. Epub 2009 Dec. 1.)). In some embodiments, adenovirus vectors are modified to reduce the host response, as reviewed by Russell (2000, see above). Methods for gene therapy using AAV vectors are described by Wang et al., 2005, J Gene Med. March 9 (pre-print electronic publication), Mandel et al., 2004, Curr Opin Mol Ther. 6(5):482-90, and Martin et al., 2004, Eye 18(11):1049-55, Nathwani et al, N Engl J Med. 2011 Dec. 22;365(25):2357-65, and Apparailly et al, Hum Gene Ther. 2005 April;16(4):426-34.

[0136] In some embodiments, the first polynucleotide sequence comprises one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof. In some embodiments, the second polynucleotide comprises a nucleic acid encoding a transgene. In some embodiments, the third polynucleotide encodes a helper gene.

[0137] In some embodiments, cells are cultured under conditions that produce recombinant virus particles. In some embodiments, the method further includes isolating the produced recombinant virus particles.

[0138] In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a lentivirus-based expression construct. Lentivirus vectors have the ability to infect and stably integrate into the genomes of dividing and non-dividing cells (Amado and Chen, 1999 Science 285:674-6). Methods for constructing and using lentivirus-based expression constructs are described in U.S. Patents No. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as by Federico (1999, Curr Opin Biotechnol 10:448-53) and Vigna et al. (2000, J Gene Med 2000;2:308-16).

[0139] In some embodiments, the viral vector is a herpesvirus vector, a polyomavirus vector, or a vacciniavirus vector.

[0140] In some embodiments, a viral vector comprises a transgene operably linked to a suitable regulatory sequence. The term “regulatory sequence” includes promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of a protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some embodiments, the regulatory sequence may include a promoter sequence. In some embodiments, the promoter sequence may be derived from a cytomegalovirus (CMV) intermediate early promoter, a viral long-terminal repeat promoter (LTR), e.g., mouse Moloney's leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, Simian virus 40 (SV40) early promoter, or a herpes simplex virus thymidine kinase promoter. In some embodiments, the promoter is a tissue-specific promoter, including but not limited to muscle, heart, CNS, and liver.

[0141] In some embodiments, the viral vector comprises a further nucleotide sequence encoding a further polypeptide. In some embodiments, the further polypeptide may be a (selectable) marker polypeptide that enables the identification, selection, and / or screening of cells containing the viral vector. In some embodiments, the marker polypeptide may be the fluorescent protein GFP, and selectable marker genes such as HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection in hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection in G418), and dihydrofolate reductase (DHFR) (for selection in methotrexate), CD20, and the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are found in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rd (edition), available from Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

[0142] Production of viral vectors Some aspects of this disclosure relate to methods for producing viral vectors, the methods comprising: growing cells according to any one of the seed-train growth methods disclosed herein; inoculating the cells into a growth medium in a bioreactor; transfecting the cells with a polynucleotide sequence encoding viral particles; and culturing the cells in the bioreactor under conditions that produce viral particles. In some aspects, the medium may be a limited medium or a conditioned medium. As used herein, the terms “defined medium” or “defined media” or their equivalents refer to a biochemically defined preparation consisting only of biochemically defined components. In some embodiments, the limited medium includes only components having a known chemical composition. In other embodiments, the limited medium includes components derived from a known source. For example, the limited medium may also include factors and other compositions secreted from known tissues or cells, but the limited medium does not include conditioned media from cultures of such cells. Thus, where indicated, “limited medium” may include certain compounds added to form a culture medium. Limited medium compositions are known in the art, for example, in PCT / US2007 / 062755 and are commercially available from Invitrogen, Carlsbad, California as StemPro~hESC SFM, which is incorporated herein in whole. Where used herein, the term “conditioned medium” refers to a medium that has been altered compared to a basal medium. For example, conditioned medium can be altered by adding molecules such as nutrients and / or growth factors to the original levels found in a basal medium or by depleting them therefrom. In some embodiments, a medium is conditioned by enabling a particular type of cell to grow or be maintained in the medium for a particular period of time under certain conditions.

[0143] Methods for introducing exogenous nucleic acids into host cells are well known in the art and vary depending on the host cell used. These techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Transfection may be transient or stable.

[0144] In some embodiments, the polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus. In some embodiments, the polynucleotide sequence is a viral vector. In some embodiments, the viral vector encodes a viral particle. In some embodiments, the viral particle is selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

[0145] In some embodiments, this disclosure provides recombinant viruses or viral vectors produced by methods described herein. In some embodiments, the recombinant viruses or viral vectors are AAV, lentiviruses, herpesviruses, polyomaviruses, or vacciniaviruses.

[0146] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector may include a recombinant AAV vector (rAAV). As used herein, “rAAV vector” refers to a recombinant vector containing a portion of the AAV genome capsidated in a protein shell of a capsid protein derived from an AAV serotype disclosed herein. In some embodiments, the AAV vector may include terminal inversion sequences (ITRs) derived from adeno-associated virus serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAVrh74, AAV11, AAV12, etc.

[0147] Typically, vector genomes require the use of adjacent 5' and 3' ITR sequences to enable efficient packaging of the vector genome into the rAAV capsid. In some embodiments, the rAAV genome present in the rAAV vector includes at least a nucleotide sequence of one terminal inversion region (ITR) of one of the AAV serotypes, or substantially identical nucleotide sequences, and a nucleic acid sequence encoding a transgene under the control of a suitable regulator (e.g., a promoter), the regulator and the modified nucleic acid sequence being inserted between the two ITRs.

[0148] Complete genomes of several AAV serotypes and their corresponding ITRs have been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, pp. 1309-1319). They can be cloned or synthesized by chemical synthesis known in the art, for example, by oligonucleotide synthesizers supplied by Applied Biosystems Inc. (Fosters, Calif., USA), or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from vectors containing AAV ITRs. ITR nucleotide sequences can be ligated at any end of nucleotide sequences encoding one or more therapeutic proteins using standard molecular biology techniques, or wild-type AAV sequences between ITRs can be replaced with desired nucleotide sequences.

[0149] In some embodiments, the viral capsid component of the packaged viral vector may be a parvovirus capsid, e.g., AAV Cap and / or a chimeric capsid. Examples of suitable parvovirus viral capsid components are capsid components derived from Parvoviridae, such as autonomous parvovirus or Dependvirus. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 capsid; those skilled in the art will know that there may be other variants that perform the same or similar functions but have not yet been identified) or may comprise components derived from two or more AAV capsids. The complete complement of the AAV Cap protein includes VP1, VP2, and VP3. ORFs containing nucleotide sequences encoding the AAV VP capsid protein may contain sub-complement of the AAV Cap protein, or they may provide the full complement of the AAV Cap protein.

[0150] In some embodiments, one or more AAV Cap proteins may be chimeric proteins comprising amino acid sequences AAV Cap derived from two or more viruses, preferably two or more AAVs. For example, a chimeric viral capsid may include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. In some embodiments, the rAAV genome present in the rAAV vector may not contain any nucleotide sequences encoding viral proteins, such as AAV rep (replication) or cap (capsid) genes. In some embodiments, the rAAV genome may further include marker or reporter genes, such as genes encoding antibiotic resistance genes, fluorescent proteins (e.g., GFP), or genes encoding products detectable and / or selectable by chemical, enzymatic, or other methods known in the art (e.g., lacZ, aph, etc.).

[0151] In some embodiments, the rAAV genome present in the rAAV vector may further include a promoter sequence operably ligated to the nucleotide sequence encoding the transgene.

[0152] In some embodiments, a suitable 3' untranslated sequence may also be operably linked to the modified nucleic acid sequence encoding the transgene. The suitable 3' untranslated region may be a region naturally associated with the nucleotide sequence, or it may be derived from a different gene, such as, for example, the bovine growth hormone 3' untranslated region (e.g., the bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0153] Unless otherwise indicated, methods known to those skilled in the art may be used to construct recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently and stably treated packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989) and AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0154] In some embodiments, the viral vector may be a lentiviral vector. Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Due to their greater complexity, lentiviruses can modulate their life cycle, such as being in the process of latent infection.

[0155] A typical lentivirus is the human immunodeficiency virus (HIV), the causative agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages. In vitro, HIV can infect primary cultures of monocyte-derived macrophages (MDM), as well as HeLa-Cd4 or T lymphocyte cells whose cell cycle has been arrested by treatment with affidicorin or gamma irradiation.

[0156] Cell infection depends on the active nuclear importation of the HIV pre-integration complex through the nuclear pores of target cells. This occurs through the interaction of several partially overlapping molecular determinants within the complex with the target cell's nuclear importation mechanism. Identified determinants include functional nuclear localization signals (NLS) in the gag matrix (MA) protein, nuclear affinity virion-associated proteins, vpr, and the C-terminal phosphotyrosine residue in the gag MA protein.

[0157] The lentiviral genome and proviral DNA contain three genes found in retroviruses: gag, pol, and env, each flanked by two long-terminal repeat (LTR) sequences. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), proteases, and integrases; and the env gene encodes the viral envelope glycoprotein. The 5' and 3' LTRs play a role in promoting virion RNA transcription and polyadenylation. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses also possess additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0158] Sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and efficient capsid formation of viral RNA into particles (Psi sites) are adjacent to the 5'LTR. If sequences necessary for capsid formation (or packaging of retroviral RNA into infectious virions) are lost from the viral genome, cis deletion prevents capsid formation of genomic RNA. However, the resulting mutants remain capable of inducing the synthesis of all virion proteins.

[0159] In some embodiments, recombinant lentiviruses can infect non-dividing cells by transfecting suitable host cells with two or more vectors carrying packaging functions, i.e., gag, pol, and env, as well as rev and tat. In some examples, vectors lacking the functional tat gene are desirable. For example, a first vector may provide nucleic acids encoding viral gag and viral pol, and another vector may provide nucleic acid encoding viral env to produce packaging cells. When a vector providing heterologous genes identified as transport vectors is introduced into packaging cells, producer cells are produced, which release infectious viral particles carrying the foreign genes of interest.

[0160] The gag, pol, and env genes of the target vector are also known in the art. Therefore, the relevant genes are cloned into the selected vector and then used to transform the target cells of interest.

[0161] According to the above-described configuration of the vector and foreign gene, the second vector can provide a nucleic acid encoding a viral envelope (env) gene. The env gene may be derived from any virus, including retroviruses. Preferably, the env is an amphiphilic envelope protein that enables gene transfer into human and other species cells.

[0162] To target receptors in specific cell types, it may be desirable to target recombinant viruses by linking their envelope proteins to antibodies or specific ligands. For example, by inserting the desired sequence (including regulatory regions) into a viral vector along with another gene encoding the ligand for the receptor on a specific target cell, the vector becomes target-specific at this point. Retroviral vectors can be made target-specific by inserting, for example, glycolipids or proteins. Targeting is often achieved by targeting the retroviral vector using the antigen-binding portion of an antibody or a recombinant antibody-type molecule such as a single-chain antibody. Those skilled in the art will know, or can easily verify, specific methods for achieving delivery of retroviral vectors to specific targets without excessive experimentation.

[0163] Examples of retrovirus-derived env genes include, but are not limited to, Moloney's mouse leukemia virus (MoMuLV or MMLV), Harvey's mouse sarcoma virus (HaMuSV or HSV), mouse mammary cancer virus (MuMTV or MMTV), gibbon leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV), and Rassarcoma virus (RSV). Other env genes, such as those of vesicular stomatitis virus (VSV) protein G (VSV G), hepatitis viruses, and influenza, may also be used.

[0164] A vector providing a viral env nucleic acid sequence is operably associated with a regulatory sequence, such as a promoter or enhancer. The regulatory sequence may be any eukaryotic cell promoter or enhancer, including, for example, a Moloney's mouse leukemia virus promoter-enhancer element, a human cytomegalovirus enhancer, or a vaccinia P7.5 promoter. In some cases, the promoter-enhancer element, such as the Moloney's mouse leukemia virus promoter-enhancer element, is located within or adjacent to the LTR sequence.

[0165] In some embodiments, the lentiviral genome present in the lentiviral vector further comprises a promoter sequence operably ligated to the nucleotide sequence encoding the transgene. In some embodiments, the promoter sequence is a promoter that confers expression in muscle cells and / or muscle tissue. Examples of such promoters include the CMV and RSV promoters disclosed herein.

[0166] In some embodiments, a suitable 3' untranslated sequence may also be operably linked to the nucleic acid sequence encoding the transgene. The suitable 3' untranslated region may be a region naturally associated with the nucleotide sequence, or it may be derived from a different gene, such as, for example, the bovine growth hormone 3' untranslated region (e.g., the bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0167] In some embodiments, additional nucleotide sequences can be operably linked to nucleic acid sequences encoding the transgene, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.

[0168] Unless otherwise indicated, methods known to those skilled in the art may be used to construct lentiviral constructs, vectors, and transiently and stably treated packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989) and AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0169] In some embodiments, the method further includes isolating the produced viral particles. Viral vectors replicate within cells, thereby being amplified and producing viral particles. Viral infection results in the lysis of transfected cells. Therefore, the lytic properties of viral vectors such as AAV allow for two different modes of production and isolation of viral particles. The first mode involves collecting viral particles before cell lysis and lysing the cells using an external factor. The second mode involves collecting viral particles from the supernatant after nearly complete cell lysis by the produced virus.

[0170] Methods that can be used for active cell lysis are known to those skilled in the art. In some embodiments, cells can be lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high-pressure extrusion, detergent lysis, or a combination of the above.

[0171] In some embodiments, cells may be lysed using at least one detergent. In some embodiments, the detergent may include anionic, cationic, zwitterionic, and nonionic detergents. In some embodiments, the concentration of the detergent may be about 0.1% to 5% (w / w). In some embodiments, the detergent may be Triton X-100.

[0172] In some embodiments, nucleases may be used to remove contaminating nucleic acids, i.e., native nucleic acids, from transfected cells. In some embodiments, the nuclease may be BENZONASE®, PULMOZYME®, or any other DNase and / or RNase commonly used in the art.

[0173] A method for obtaining or isolating a viral vector from transfected cells is broadly disclosed in WO 2005 / 080556, which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the time of collection or isolation of the viral vector is approximately 24–120 hours, 36–108 hours, 48–96 hours, or 60–84 hours after transfection. In some embodiments, the time of collection or isolation of the vector is approximately 72 hours after transfection.

[0175] In some embodiments, isolated virus particles may be further purified. In some embodiments, the purification of virus particles may be carried out in several steps, including purification, ultrafiltration, diafiltration, or separation by chromatography. Such methods are described in WO 2005 / 080556, which is incorporated herein by reference in whole. In some embodiments, purification may be carried out by a filtration step to remove cell debris and other impurities from the cell lysate. In some embodiments, the virus solution is concentrated using ultrafiltration. In some embodiments, salts, sugars, etc., can be removed and exchanged using diafiltration, buffer exchange, or ultrafiltration. Those skilled in the art know how to find the optimal conditions for each purification step.

[0176] In some embodiments, purification may be achieved by density gradient centrifugation. In some embodiments, purification may be performed using at least one chromatographic step. In some embodiments, the viral vector may be purified by anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0177] It should be understood that the section describing embodiments for carrying out the invention, rather than the section describing the summary and abstract of the invention, is intended to be used to interpret the claims. The section describing the summary and abstract of the invention may describe one or more but not all exemplary embodiments of the invention as contemplated by the inventors, and is therefore not intended to limit the invention and the appended claims in any way. [Examples]

[0178] HEK293 cells were passaged four times under adherent conditions. Before entering the second-to-last growth culture, cells were harvested, centrifuged to wash away serum (300g for 5 minutes), and resuspended in serum-free growth medium (EXPI293) in a suspension shaking flask at a seeding density of 0.5+E6 cells / mL. The cells were then grown for 48–72 hours to increase their number. Suspended cells were then collected according to the number of viable cells required for bioreactor inoculation and inoculated into shaking flasks or WAVE bags. At the end of 72 hours, the concentration of viable cells was determined using a cell counter. The required volume containing the desired total viable cells was then added to an adherent bioreactor containing DMEM and 10% FBS. Additional FBS was added appropriately to account for the addition of serum-free suspension culture volumes so that the final FBS concentration was maintained at 10%.

[0179] As shown in Figure 3, cell viability was similar in both the seed train system and the adhesion system. Regarding viable cell density (VCD), the hybrid seed train system showed a higher VCD after the 6th passage than after the 1st passage (Figure 4A), which is comparable to the adhesion system (Figure 4B). On the other hand, the seed train system with suspended cells or cells adapted to suspension allowed for more convenient passage than the conventional adhesion system. Furthermore, cell passage in the seed train system using suspension cell culture avoids the use of trypsin, minimizing contamination and thus enabling the scale-up production or manufacture of therapeutic viral vectors.

[0180] The present invention is described above using function building blocks that demonstrate the implementation of specified functions and their relationships. The boundaries of these function building blocks are arbitrarily defined herein for the sake of explanation. Alternative boundaries may be defined, provided that the specified functions and their relationships are adequately implemented.

[0181] The foregoing descriptions of specific embodiments reveal the general nature of the invention to such an extent that others, by applying knowledge within the skill of the art, can readily modify and / or adapt such specific embodiments for various uses without departing from the general concept of the invention or without excessive experimentation. Therefore, such adaptations and modifications are intended to be within the meaning and scope of equivalent embodiments of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that any words or phrases used herein are for illustrative purposes only, not limiting, so that they may be interpreted by those skilled in the art in light of the teachings and guidance.

[0182] The scope and breadth of the present invention should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims and their equivalents.

Claims

1. A method of cell proliferation, (a) Culturing cells in a first growth medium containing serum in container N-2, (b) Removing the cells from the first culture medium, (c) Inoculating the cells from step (b) into a second medium in container N-1, which is serum-free or contains serum at a lower concentration than that of the first medium, (d) Culturing the cells in the N-1 container under suspension conditions, (e) A method comprising inoculating the cells from step (d) with a third culture medium in a bioreactor.

2. The method according to claim 1, wherein the cells are adherent cells.

3. The method according to claim 1 or 2, wherein the N-1 container and the N-2 container are the same.

4. The method according to claim 1 or 2, wherein the N-1 container and the N-2 container are different.

5. The method according to any one of claims 1 to 4, wherein the N-1 container includes a shaking flask or a wave bag.

6. The method according to any one of claims 1 to 5, wherein the cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, or at least six times prior to step (e).

7. The method according to any one of claims 1 to 6, wherein the third culture medium contains a serum concentration higher than the serum concentration in the second culture medium.

8. The method according to any one of claims 2 to 7, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and derivatives thereof.

9. The method according to any one of claims 2 to 8, wherein the adherent cells are human cells, animal cells, insect cells, or larvae.

10. The method according to any one of claims 2 to 9, wherein the adherent cells are HeLa cells or HEK-293 cells.

11. The method according to any one of claims 2 to 10, wherein the adherent cells are HEK-293 cells.

12. The method according to any one of claims 2 to 11, wherein the adherent cells are not suitable for suspension.

13. The method according to any one of claims 2 to 11, wherein the adherent cells are suitable for suspension.

14. The method according to any one of claims 2 to 13, wherein culturing the cells under suspension conditions does not alter the adhesion dependence of the cells.

15. The method according to any one of claims 2 to 14, wherein the method does not genetically alter the cells.

16. The method according to any one of claims 1 to 15, further comprising culturing cells in the first culture medium in an N-3 container.

17. The method according to claim 16, further comprising culturing cells in the first culture medium in an N-4 container.

18. The method according to claim 17, further comprising culturing cells in the first culture medium in an N-5 container.

19. The method according to any one of claims 1 to 18, wherein the bioreactor is an adhesive bioreactor.

20. The method according to any one of claims 1 to 19, wherein the bioreactor is selected from the group consisting of a stirred tank bioreactor, a bubble column bioreactor, an airlift bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, a photobioreactor bioreactor, and a fixed bed bioreactor.

21. The method according to claim 20, wherein the bioreactor is a fixed-bed bioreactor.

22. The method according to any one of claims 1 to 21, wherein the third culture medium in the bioreactor contains at least one factor that promotes cell adhesion.

23. The method according to claim 22, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

24. The method according to claim 23, wherein the third culture medium in the bioreactor comprises DMEM and 10% FBS.

25. The method according to any one of claims 1 to 24, wherein the cells are cultured under suspension conditions for about 24 to 120 hours.

26. The method according to any one of claims 1 to 25, wherein the cells are cultured under suspension conditions for about 48 to 72 hours.

27. The method according to any one of claims 1 to 26, wherein the N-1 container is a shaking flask or a wave bag.

28. The method according to any one of claims 1 to 27, further comprising contacting the cells with a first polynucleotide sequence in the bioreactor.

29. The method according to claim 28, wherein the polynucleotide sequence is a plasmid.

30. The method according to claim 29, wherein the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentiviral vectors, herpesviruses, polyomaviruses, and vaccinia viruses.

31. The method according to claim 30, wherein the virus particle is AAV.

32. The method according to any one of claims 18 to 21, further comprising contacting the cells with a second polynucleotide encoding the introduced gene.

33. The method according to any one of claims 28 to 32, further comprising contacting the cells with a third polynucleotide encoding a helper gene.

34. The method according to any one of claims 1 to 33, further comprising culturing the cells in the bioreactor.

35. The method according to any one of claims 1 to 34, wherein the culture includes batch culture.

36. The method according to any one of claims 1 to 35, wherein the culture includes fed-batch culture.

37. The method according to any one of claims 1 to 36, wherein the culture includes perfusion culture.

38. The method according to any one of claims 28 to 37, wherein the cells are cultured under conditions that produce the recombinant virus particles.

39. A method of cell proliferation, (a) Culturing cells in the first culture medium in container N-3, (b) Removing the cells from the first culture medium, (c) Inoculating the cells from step (b) into a second medium in container N-2, which is serum-free or contains serum at a lower concentration than that of the first medium, (d) Culturing the cells in the N-2 container in the second culture medium, (e) Inoculating the cells from step (d) into the second culture medium in container N-1, (f) Culturing the cells in the N-1 container under suspension conditions, (g) A method comprising inoculating the cells from step (f) into a third culture medium in a bioreactor.

40. The method according to claim 39, wherein the cells are adherent cells.

41. The method according to claim 39 or 40, wherein the containers N-1, N-2, and N-3 are the same.

42. The method according to claim 39 or 40, wherein the containers N-1, N-2, and N-3 are different.

43. The method according to any one of claims 39 to 42, wherein the cells are passaged at least once, at least twice, at least three times, at least four times, at least five times, or at least six times in the second culture medium prior to step (g).

44. The method according to any one of claims 39 to 43, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and derivatives thereof.

45. The method according to any one of claims 40 to 44, wherein the adherent cells are human cells, animal cells, insect cells, or larvae.

46. The method according to any one of claims 40 to 45, wherein the adherent cells are HeLa cells or HEK-293 cells.

47. The method according to claim 46, wherein the adherent cells are HEK-293 cells.

48. The method according to any one of claims 40 to 47, wherein the adherent cells are not suitable for suspension.

49. The method according to any one of claims 40 to 47, wherein the adherent cells are suitable for suspension.

50. The method according to any one of claims 40 to 49, wherein culturing the cells under suspension conditions does not alter the adhesion dependence of the cells.

51. The method according to any one of claims 40 to 50, wherein the method does not genetically alter the cells.

52. The method according to any one of claims 39 to 51, further comprising culturing cells in the first culture medium in an N-4 container.

53. The method according to claim 52, further comprising culturing cells in the first culture medium in an N-5 container.

54. The method according to any one of claims 39 to 53, wherein the bioreactor is an adhesive bioreactor.

55. The method according to any one of claims 39 to 54, wherein the bioreactor is selected from the group consisting of a stirred tank bioreactor, a bubble column bioreactor, an airlift bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, a photobioreactor bioreactor, and a fixed bed bioreactor.

56. The method according to claim 55, wherein the bioreactor is a fixed-bed bioreactor.

57. The method according to any one of claims 39 to 56, wherein the third culture medium in the bioreactor contains at least one factor that promotes cell adhesion.

58. The method according to claim 57, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

59. The method according to claim 58, wherein the third culture medium in the bioreactor comprises DMEM and 10% FBS.

60. The method according to any one of claims 39 to 59, wherein the cells are cultured under suspension conditions for about 24 to 120 hours.

61. The method according to any one of claims 39 to 60, wherein the cells are cultured under suspension conditions for about 48 to 72 hours.

62. The method according to any one of claims 39 to 61, wherein the N-2 container and the N-1 container are shaking flasks or wave bags.

63. The method according to any one of claims 39 to 62, further comprising contacting the cells with a first polynucleotide sequence in the bioreactor.

64. The method according to claim 63, wherein the polynucleotide sequence is a plasmid.

65. The method according to claim 64, wherein the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentiviral vectors, herpesviruses, polyomaviruses, and vaccinia viruses.

66. The method according to claim 65, wherein the virus particle is AAV.

67. The method according to any one of claims 63 to 66, further comprising contacting the cells with a second polynucleotide encoding the introduced gene.

68. The method according to any one of claims 63 to 67, wherein the first polynucleotide comprises one or more of the following: a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, or a nucleic acid encoding at least one AAV structural capsid protein.

69. The method according to any one of claims 39 to 68, further comprising culturing the cells in the bioreactor.

70. The method according to any one of claims 39 to 69, wherein the culture includes batch culture.

71. The method according to any one of claims 39 to 69, wherein the culture includes fed-batch culture.

72. The method according to any one of claims 39 to 69, wherein the culture includes perfusion culture.

73. The method according to any one of claims 63 to 72, wherein the cells are cultured under conditions that produce recombinant virus particles.

74. A method for seed train proliferation of adherent cells, (a) Culturing the adherent cells in a first culture medium containing serum under adherent conditions, (b) Removing the adherent cells from the first culture medium, (c) The adherent cells are suspended in a second medium which is serum-free or contains serum at a lower concentration than that of the first medium, (d) Culturing the adherent cells from step (c) under suspension conditions, (e) A method comprising inoculating the adherent cells from step (d) into a third culture medium in a bioreactor.

75. The method according to claim 74, further comprising subculturing the adherent cells of step (a) at least once under adhesive conditions.

76. The method according to claim 74 or 75, further comprising subculturing the adherent cells of step (d) at least once under suspension conditions.

77. The method according to any one of claims 74 to 76, further comprising subculturing the adherent cells of step (d) at least two, at least three, at least four, or at least five times under suspension conditions.

78. The method according to any one of claims 74 to 77, wherein the bioreactor is an adhesive bioreactor.

79. The method according to any one of claims 74 to 78, wherein the bioreactor is selected from the group consisting of a stirred tank bioreactor, a bubble column bioreactor, an airlift bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, a photobioreactor bioreactor, and a fixed bed bioreactor.

80. The method according to claim 79, wherein the bioreactor is a fixed-bed bioreactor.

81. The method according to any one of claims 74 to 80, wherein the adherent cells are not suitable for suspension.

82. The method according to any one of claims 74 to 80, wherein the adherent cells are suitable for suspension.

83. The method according to any one of claims 74 to 82, wherein culturing the cells under suspension conditions does not alter the adhesion dependence of the cells.

84. The method according to any one of claims 74 to 83, wherein the method does not genetically alter the cells.

85. The method according to any one of claims 74 to 84, wherein the third culture medium in the bioreactor contains at least one factor that promotes cell adhesion.

86. The method according to claim 85, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

87. The method according to claim 86, wherein the third culture medium in the bioreactor comprises DMEM and 10% FBS.

88. The method according to any one of claims 74 to 87, wherein the cells are passaged two or fewer times under suspension conditions.

89. The method according to any one of claims 74 to 88, wherein the cells are passaged three or fewer times, four or fewer times, or five or fewer times under suspension conditions.

90. The method according to any one of claims 74 to 89, wherein the cells are cultured under suspension conditions for about 24 to 120 hours.

91. The method according to any one of claims 74 to 90, wherein the adherent cells are grown under suspension conditions for 48 to 72 hours.

92. The method according to any one of claims 74 to 91, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells, or derivatives thereof.

93. The method according to any one of claims 74 to 92, wherein the adherent cells are human cells, animal cells, insect cells, or larvae.

94. The method according to any one of claims 74 to 93, wherein the adherent cells are HeLa cells or HEK-293 cells.

95. The method according to claim 94, wherein the adherent cells are HEK-293 cells.

96. The method according to any one of claims 74 to 90, further comprising bringing the adherent cells into contact with a first polynucleotide sequence.

97. The method according to claim 96, wherein the polynucleotide sequence is a plasmid.

98. The method according to claim 97, wherein the plasmid encodes a capsid protein of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

99. The method according to claim 98, wherein the virus particle is AAV.

100. The method according to any one of claims 96 to 99, further comprising contacting the cells with a second polynucleotide encoding the introduced gene.

101. The method according to any one of claims 96 to 100, wherein the first polynucleotide comprises one or more of a terminal inversion sequence, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, or a nucleic acid encoding at least one AAV structural capsid protein.

102. The method according to any one of claims 74 to 101, further comprising culturing the cells in the bioreactor.

103. The method according to any one of claims 74 to 102, wherein the culture includes batch culture.

104. The method according to any one of claims 74 to 102, wherein the culture includes fed-batch culture.

105. The method according to any one of claims 74 to 102, wherein the culture includes perfusion culture.

106. The method according to any one of claims 96 to 105, wherein the cells are cultured under conditions that produce recombinant virus particles.

107. A method for producing a viral vector, (a) Culturing cells in the first culture medium under adhesion conditions, (b) Removing the cells from the first culture medium, (c) Suspending the cells in a second medium that does not contain serum or contains serum at a lower concentration than that of the first medium, (d) Culturing the cells from step c under suspension conditions, (e) Inoculating the cells from step (d) into a third culture medium in the bioreactor, (f) Transfecting the cells with a polynucleotide sequence encoding a viral particle, (g) A method comprising culturing the cells in the bioreactor under conditions in which the virus particles are produced.

108. The method according to claim 107, further comprising isolating the virus particles produced in step (g).

109. The method according to claim 107 or 108, wherein the polynucleotide is a plasmid.

110. The method according to any one of claims 107 to 109, wherein the virus particle is selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vacciniavirus.

111. The method according to claim 110, wherein the virus particle is an AAV vector.

112. The method according to any one of claims 107 to 111, wherein the bioreactor is an adhesive bioreactor.

113. The method according to any one of claims 107 to 112, wherein the bioreactor is selected from the group consisting of a stirred tank bioreactor, a bubble column bioreactor, an airlift bioreactor, a fluidized bed bioreactor, a packed bed bioreactor, a photobioreactor bioreactor, and a fixed bed bioreactor.

114. The method according to claim 113, wherein the bioreactor is a fixed-bed bioreactor.

115. The method according to any one of claims 107 to 114, wherein the third culture medium in the bioreactor contains at least one factor that promotes cell adhesion.

116. The method according to claim 115, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

117. The method according to claim 116, wherein the third culture medium in the bioreactor comprises DMEM and 10% FBS.

118. The method according to any one of claims 107 to 117e, wherein the cells are adherent cells.

119. The method according to claim 118, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, Sf9, Sf21, VERO cells, BHK cells, MDCK cells, MDBK cells, COS cells, and derivatives thereof.

120. The method according to claim 92 or 93, wherein the adherent cells are human cells, animal cells, insect cells, or larvae.

121. The method according to any one of claims 118 to 120, wherein the adherent cells are HeLa cells or HEK-293 cells.

122. The method according to claim 121, wherein the adherent cells are HEK-293 cells.

123. The method according to any one of claims 118 to 122, wherein the adherent cells are not suitable for suspension.

124. The method according to any one of claims 118 to 122, wherein the adherent cells are suitable for suspension.

125. The method according to any one of claims 107 to 124, wherein culturing the cells under suspension conditions does not alter the adhesion dependence of the cells.

126. The method according to any one of claims 107 to 125, wherein the method does not genetically alter the cells.

127. The method according to any one of claims 107 to 126, further comprising subculturing the cells of step (a) at least once under adhesive conditions.

128. The method according to any one of claims 107 to 127, further comprising subculturing the cells of step (d) at least once under suspension conditions.

129. The method according to any one of claims 107 to 128, wherein the cells are passaged two or fewer times under suspension conditions.

130. The method according to any one of claims 107 to 129, wherein the cells are passaged two or fewer times, three or fewer times, four or fewer times, or five or fewer times under suspension conditions.

131. The method according to any one of claims 107 to 130, wherein the cells grow under suspension conditions for about 24 to 120 hours.

132. The method according to any one of claims 107 to 131, wherein the cells grow under suspension conditions for about 48 to 72 hours.

133. The method according to any one of claims 107 to 132, wherein culturing the cells in the bioreactor includes batch culture.

134. The method according to any one of claims 107 to 132, wherein culturing the cells in the bioreactor includes fed-batch culture.

135. The method according to any one of claims 107 to 132, wherein culturing the cells in the bioreactor includes perfusion culture.

136. The method according to any one of claims 1, 39, 74, and 107, wherein the second culture medium is a serum-free medium.

137. The method according to any one of claims 1, 39, 74, and 107, wherein the second culture medium contains the serum at a concentration lower than the serum concentration in the first culture medium in the N-1 container.

138. The method according to any one of claims 1, 39, 74, and 107, wherein the third culture medium contains a serum concentration higher than the serum concentration in the second culture medium.

139. Recombinant virus produced by the method described in any one of claims 1 to 138.

140. The recombinant viruses are AAV, lentivirus, herpesvirus, polyomavirus, or vacciniavirus.

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