# US 11,111,470 B2 · Packed-bed bioreactor systems and methods of using the same

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- Number: US 11,111,470 B2
- Document ID: US11111470B2
- Status: Granted
- Kind: B2
- Application number: 17/039,218
- Filed: 2020-09-30
- Published: 2021-01-28
- Granted: 2021-09-07
- Earliest claimed: 2019-02-05
- Assignees: Corning Incorporated
- Applicants: Corning Incorporated
- Inventors: Ann MeeJin Ferrie; Vasiliy Nikolaevich Goral
- Web page: https://patentgrep.com/patent/US11111470B2

## CPC

- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12M 23/02: {Form or structure of the vessel (large containers B65D88/00)}
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12M 23/44: {Multiple separable units; Modules}
- C12M 25/04: {in combination with well or multiwell plates, i.e. culture inserts}
- C12M 29/10: {Perfusion}
- C12M 29/26: {Conditioning fluids entering or exiting the reaction vessel}
- C12M 41/26: {of pH}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0602: {Vertebrate cells}
- C12N 2513/00: 3D culture
- C12N 2533/30: Synthetic polymers
- C12N 2533/40: Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2535/00: Supports or coatings for cell culture characterised by topography
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A packed-bed bioreactor system for culturing cells is provided, the system including a cell culture vessel having at least one interior reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and a cell culture matrix disposed in the reservoir. The cell culture matrix includes a structurally defined multi-layered substrate for adhering cells thereto, and each layer of the multi-layered substrate has a physical structure and a porosity that are substantially regular and uniform.

## Claims

### Claim 1

A packed-bed bioreactor system for culturing cells, the system comprising:
a cell culture vessel comprising at least one interior reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and
a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a structurally defined multi-layered substrate configured for adhering cells thereto,
wherein a plurality of layers in the structurally defined multi-layered substrate are not separated by a spacer material or a physical barrier, and
wherein each layer of the multi-layered substrate comprises a physical structure and a porosity that are in an ordered array that is substantially regular and uniform.

### Claim 2 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the cell culture matrix comprises a substantially uniform porosity.

### Claim 3 (depends on 2)

The packed-bed bioreactor system of claim 2, wherein the cell culture matrix is configured for uniform fluid flow therethrough.

### Claim 4 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the reservoir is defined by a length and a width, the length extending from a first end of the reservoir adjacent to the inlet to a second end of the reservoir adjacent to the outlet, the substrate layers each having a width extending substantially across a width of the reservoir.

### Claim 5 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the structurally defined multi-layered substrate comprises a plurality of substrate disks stacked in the reservoir.

### Claim 6 (depends on 5)

The packed-bed bioreactor system of claim 5, wherein the stack of the plurality of substrate disks is configured to exhibit substantially uniform fluid flow across a width of each of the plurality of substrate disks.

### Claim 7 (depends on 5)

The packed-bed bioreactor system of claim 5, wherein structurally defined multi-layered substrate comprises a plurality of openings defining the porosity, the plurality of openings being arrayed in a regular or uniform pattern in each disk of the plurality of substrate disks.

### Claim 8 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the structurally defined multi-layered substrate comprises a woven substrate.

### Claim 9 (depends on 8)

The packed-bed bioreactor system of claim 8, wherein the structurally defined multi-layered substrate comprises the woven substrate having a plurality of interwoven fibers with surfaces configured for adhering cells thereto, the plurality of interwoven fibers defining a plurality of openings.

### Claim 10 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the structurally defined multi-layered substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide.

### Claim 11 (depends on 9)

The packed-bed bioreactor system of claim 9, wherein the cell culture matrix comprises a plurality of woven substrates, each woven substrate having a first side and a second side opposite the first side and separated from the first side by a thickness of the woven substrate.

### Claim 12 (depends on 11)

The packed-bed bioreactor system of claim 11, wherein the plurality of woven substrates is arranged in a stack such that one of the first and second side of a substrate is facing the first or second side of an adjacent substrate.

### Claim 13 (depends on 11)

The packed-bed bioreactor system of claim 11, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier, or are in physical contact with each other.

### Claim 14 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the inlet and the outlet are configured for at least one of supplying material to or removing material from the at least one reservoir.

### Claim 15 (depends on 14)

The packed-bed bioreactor system of claim 14, wherein the material comprises at least one of cell culture media, cells, or cell by-products.

### Claim 16 (depends on 9)

The packed-bed bioreactor system of claim 9, wherein each of the plurality of interwoven fibers comprises a first fiber with a first fiber diameter from about 50 μm to about 1000 μm, from about 50 μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

### Claim 17 (depends on 7)

The packed-bed bioreactor system of claim 7, wherein each of the plurality of openings comprises an opening diameter of from about 100 μm to about 1000 μm, from about 200 μm to about 900 μm, or from about 225 μm to about 800 μm.

### Claim 18 (depends on 9)

The packed-bed bioreactor system of claim 9, wherein a ratio of the opening diameter to the fiber diameter is from about 1.0 to about 3.5, from about 1.25 to about 3.25, from about 1.4 to about 3.0, from about 1.5 to about 2.9, from about 1.5 to about 2.4, or from about 2.4 to about 2.9.

### Claim 19 (depends on 11)

The packed-bed bioreactor system of claim 11, wherein the cell culture matrix is disposed in the bioreactor vessel such that a bulk flow direction of media through the bioreactor vessel is perpendicular to the first and second sides.

### Claim 20 (depends on 8)

The packed-bed bioreactor system of claim 8, wherein the structurally defined multi-layered substrate comprises a plurality of layers of the woven substrate, the plurality of layers of woven substrate comprising woven meshes of differing geometries, wherein the differing geometries are different in at least one of fiber diameter, opening diameter, or opening geometry.

### Claim 21 (depends on 20)

The packed-bed bioreactor system of claim 20, wherein the woven meshes of differing geometries are disposed in the bioreactor vessel in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

### Claim 22 (depends on 21)

The packed-bed bioreactor system of claim 21, wherein the desired flow characteristics comprise at least one of uniform perfusion of liquid media across the cell culture matrix, and distribution of cell growth across the cell culture matrix.

### Claim 23 (depends on 1)

The packed-bed bioreactor system of claim 1, further comprising means for harvesting the adherent cells or cell by-products from the reservoir.

### Claim 24 (depends on 23)

The packed-bed bioreactor system of claim 23, wherein the cell byproducts comprise at least one of proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvessicles, exosomes, and polysaccharides.

### Claim 25 (depends on 23)

The packed-bed bioreactor system of claim 23, wherein the bioreactor system exhibits a cell harvest rate of over 90% viable cells.

### Claim 26 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the cell culture matrix exhibits a cell culture density of at least 3×109 cells/cm2.

### Claim 27 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein each of the plurality of substrate layers has a diameter of about 30 mm to about 60 mm.

### Claim 28 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the cell culture matrix comprises a cell culture surface area of about 678 cm2 to 2.5×106 cm2.

### Claim 29 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the cell culture matrix is configured to culture transfected cells at a density capable of producing a viral genome (VG) concentration of at least about 1.74×1010 VG/cm2.

### Claim 30 (depends on 1)

The packed-bed bioreactor system of claim 1, wherein the cell culture matrix exhibits uniform fluid flow across at least 90% of a surface area of the cell culture matrix.