# US 2014/0154726 A1 · Perfusion Culturing Methods and Uses Thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0154726 A1
- Document ID: US20140154726A1
- Status: Published application
- Kind: A1
- Application number: 14/061,657
- Filed: 2013-10-23
- Published: 2014-06-05
- Earliest claimed: 2012-10-23
- Applicants: Genzyme Corporation
- Inventors: Jianguo Yang; Agata Villiger-Oberbek; Yang Yang
- Web page: https://patentgrep.com/patent/US20140154726A1

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12M 23/08: {Flask, bottle or test tube}
- C12M 23/50: {Means for positioning or orientating the apparatus (C12M41/08 takes precedence)}
- C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)}
- C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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

Provided herein are improved methods of culturing a mammalian cell in a conical container, and methods that utilize these culturing methods.

## Claims

### Claim 1

A method of culturing a mammalian cell, the method comprising:
providing a conical container containing a mammalian cell suspended in a first liquid culture medium that occupies about 4% to about 80% of the volume of the container;
incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with a rotary agitation of about 20 revolutions per minute (RPM) to about 1000 RPM; and
continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first volume of the first liquid culture medium is substantially free of mammalian cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first liquid culture medium occupies about 4% to about 30% of the volume of the container.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the CHO cell contains a nucleic acid encoding a recombinant protein.

### Claim 6 (depends on 5)

The method of claim 5, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

### Claim 7 (depends on 1)

The method of claim 1, wherein the container is incubated at about 40 degrees to about 55 degrees from horizontal.

### Claim 8 (depends on 1)

The method of claim 1, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed simultanesouly.

### Claim 9 (depends on 1)

The method of claim 1, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed continuously.

### Claim 10 (depends on 1)

The method of claim 1, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed periodically.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added are increased over time.

### Claim 12 (depends on 11)

The method of claim 11, wherein:
the container is incubated for a period of time greater than 7 days, and on days 1 through 3 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 50% of the volume of the first liquid culturing medium;
on days 4 through 6 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 70% of the volume of the first liquid culture medium; and
on day 7 and onwards of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 100% of the volume of the first liquid culture medium.

### Claim 13 (depends on 1)

The method of claim 1, wherein the conical container is a gas-permeable 50-mL to 600-mL conical container.

### Claim 14 (depends on 1)

The method of claim 1, wherein the mammalian cell is suspended in about 2 mL to about 15 mL of the first liquid culture medium.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first liquid culture medium and/or second liquid culture medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.

### Claim 16

A method of culturing a mammalian cell, the method comprising:
culturing in a gradient perfusion process a mammalian cell suspended in a liquid culture medium under conditions that generate in the medium a fluid sheer force and dissolved oxygen (O2) concentration that is essentially the same as that achieved in a medium occupying 4% to 40% of the volume of a gas-permeable conical container when the container is positioned at a reactor angle of about 5 degrees to about 85 degrees from horizontal, incubated at a temperature of about 31° C. to about 40° C., and agitated at a frequency of about 20 revolutions per minute (RPM) to about 1000 RPM.

### Claim 17 (depends on 16)

The method of claim 16, wherein the conical container is a gas-permeable 50-mL to 600-mL conical container.

### Claim 18 (depends on 16)

The method of claim 16, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

### Claim 19 (depends on 18)

The method of claim 18, wherein the CHO cell contains a nucleic acid encoding a recombinant protein.

### Claim 20 (depends on 19)

The method of claim 19, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

### Claim 21 (depends on 16)

The method of claim 16, wherein the liquid culture medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, or a protein-free medium.

### Claim 22

A method of producing a recombinant protein, the method comprising:
providing a conical container containing a mammalian cell containing a nucleic acid that encodes a recombinant protein, wherein the cell is suspended in a first liquid culture medium that occupys about 4% to about 80% of the volume of the container;
incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with a rotary agitation of about 20 revolutions per minute (RPM) to about 1000 RPM;
continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal; and
recovering the recombinant protein from the mammalian cell or from the first or second culture medium.

### Claim 23 (depends on 22)

The method of claim 22, wherein the first volume of the first liquid culture medium is substantially free of mammalian cells.

### Claim 24 (depends on 22)

The method of claim 22, wherein the first liquid culture medium occupies about 4% to about 30% of the volume of the container.

### Claim 25 (depends on 22)

The method of claim 22, wherein the conical container is a gas-permeable 50-mL to 600-mL conical container.

### Claim 26 (depends on 22)

The method of claim 22, wherein the mammalian cell is suspended in about 2 mL to about 15 mL of the first liquid culture medium.

### Claim 27 (depends on 22)

The method of claim 22, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

### Claim 28 (depends on 22)

The method of claim 22, wherein the recombinant protein is a secreted immunoglobulin, a secreted enzyme, a secreted growth factor, a secreted protein fragment, or a secreted engineered protein and wherein the recombinant protein is recovered from the first or second culture medium.

### Claim 29 (depends on 22)

The method of claim 22, wherein the recombinant protein is recovered from the mammalian cell.

### Claim 30 (depends on 29)

The method of claim 29, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

### Claim 31 (depends on 22)

The method of claim 22, wherein the container is incubated at about 40 degrees to about 55 degrees from horizontal.

### Claim 32 (depends on 22)

The method of claim 22, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed simultanesouly.

### Claim 33 (depends on 22)

The method of claim 22, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed continuously.

### Claim 34 (depends on 22)

The method of claim 22, wherein the removing of the first volume of the first liquid culture medium and the adding of the second volume of the second liquid culture medium is performed periodically.

### Claim 35 (depends on 22)

The method of claim 22, wherein the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added are increased over time.

### Claim 36 (depends on 35)

The method of claim 35, wherein:
the container is incubated for a period of time greater than 7 days, and on days 1 through 3 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 50% of the volume of the first liquid culturing medium;
on days 4 through 6 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 70% of the volume of the first liquid culture medium; and
on day 7 and onwards of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 100% of the volume of the first liquid culture medium.

### Claim 36 (depends on 22)

The method of claim 22, wherein the first liquid culture medium and/or second liquid culture medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.

### Claim 37

A method of producing a recombinant protein, the method comprising:
culturing in a gradient perfusion process a mammalian cell containing a nucleic acid that encodes a recombinant protein, wherein the cell is suspended in a liquid culture medium under conditions that generate in the medium a fluid sheer force and dissolved oxygen (O2) concentration that is essentially the same as that achieved in a volume of liquid culture medium occupying 4% to 40% of the volume of a gas-permeable conical container when the container is positioned at a reactor angle of about 5 degrees to about 85 degrees from horizontal, incubated at a temperature of about 31° C. to about 40° C., and agitated at a frequency of about 20 revolutions per minute (RPM) to about 1000 RPM, and
recovering the recombinant protein from the mammalian cell or the liquid culture medium.

### Claim 38 (depends on 37)

The method of claim 37, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

### Claim 39 (depends on 37)

The method of claim 37, wherein the recombinant protein is a secreted immunoglobulin, a secreted enzyme, a secreted growth factor, a secreted protein fragment, or a secreted engineered protein and wherein the recombinant protein is recovered from the liquid culture medium.

### Claim 40 (depends on 37)

The method of claim 37, wherein the recombinant protein is recovered from the mammalian cell.

### Claim 41 (depends on 40)

The method of claim 40, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

### Claim 42 (depends on 37)

The method of claim 37, wherein the liquid culture medium is selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.

### Claim 43

A method for testing a manufacturing process for making a recombinant protein, the method comprising:
providing a conical container containing a mammalian cell suspended in a first liquid culture medium occupying about 4% to about 80% of the volume of the container;
incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with an agitation of about 20 revolutions per minute (RPM) to about 1000 RPM;
continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal;
detecting the recombinant protein in the cell or in the first or second culture medium; and
comparing the amount of recombinant protein present in the cell or in the first or second culture medium to a reference level of recombinant protein.

### Claim 44 (depends on 43)

The method of claim 43, wherein the first volume of the first liquid culture medium is substantially free of mammalian cells.

### Claim 45 (depends on 43)

The method of claim 43, wherein the reference level of recombinant protein is a level of recombinant protein produced using a different culturing method.

### Claim 46 (depends on 45)

The method of claim 45, wherein the different culturing method utilizes a different first or second liquid culture medium, a different mammalian cell, a different temperature, a different level of agitation, or a different reactor angle of the conical container.

### Claim 47 (depends on 45)

The method of claim 45, wherein the different culturing method utilizes different raw materials, anti-clumping agents, or chemically-defined liquid culture media.

### Claim 48 (depends on 43)

The method of claim 43, wherein the method is used to perform high throughput cell culture experiments to perform a design-of-experiment (DOE) or a quality-by-design (QBD) study.

### Claim 49 (depends on 43)

The method of claim 43, wherein the first liquid culture medium occupies about 4% to about 30% of the volume of the container.

### Claim 50 (depends on 43)

The method of claim 43, wherein the conical container is a gas-permeable 50-mL to 600-mL conical container.

### Claim 51 (depends on 43)

The method of claim 43, wherein the mammalian cell is suspended in about 2 mL to about 15 mL of the first liquid culture medium.

### Claim 52 (depends on 43)

The method of claim 43, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

### Claim 53 (depends on 43)

The method of claim 43, wherein the recombinant protein is a secreted immunoglobulin, a secreted enzyme, a secreted growth factor, a secreted protein fragment, or an engineered protein and wherein the recombinant protein is recovered from the first or second culture medium.

### Claim 54 (depends on 43)

The method of claim 43, wherein the recombinant protein is recovered from the mammalian cell.

### Claim 55 (depends on 54)

The method of claim 54, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein.

### Claim 56 (depends on 43)

The method of claim 43, wherein the removing of the first volume of the first liquid culture medium and the adding of the second liquid culture medium is performed simultaneously.

### Claim 57 (depends on 43)

The method of claim 43, wherein the removing of the first volume of the first liquid culture medium and the adding of the second liquid culture medium is performed continuously.

### Claim 58 (depends on 43)

The method of claim 43, wherein the removing of the first volume of the first liquid culture medium and the adding of the second liquid culture medium is performed periodically.

### Claim 59 (depends on 43)

The method of claim 43, wherein the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added are increased over time.

### Claim 60 (depends on 59)

The method of claim 59, wherein:
the container is incubated for a period of time greater than 7 days, and on days 1 through 3 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 50% of the volume of the first liquid culturing medium;
on days 4 through 6 of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 70% of the volume of the first liquid culture medium; and
on day 7 and onwards of incubation, in each 24-hour period, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 100% of the volume of the first liquid culture medium.

### Claim 61 (depends on 43)

The method of claim 43, wherein the first liquid culture medium and/or the second liquid culture medium are selected from the group consisting of: a chemically-defined liquid culture medium, a serum-free liquid culture medium, a serum-containing liquid culture medium, an animal-derived component free liquid culture medium, and a protein-free medium.