# US 2020/0399585 A1 · Product production method

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- Number: US 2020/0399585 A1
- Document ID: US20200399585A1
- Status: Published application
- Kind: A1
- Application number: 17/014,751
- Filed: 2020-09-08
- Published: 2020-12-24
- Earliest claimed: 2018-03-19
- Assignees: FUJIFILM Corporation
- Applicants: FUJIFILM Corporation
- Inventors: Naoto TAKAHASHI; Shinichi NAKAI
- Web page: https://patentgrep.com/patent/US20200399585A1

## CPC

- C12M 47/10: {Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/065: {Purification, fragmentation}
- C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis}
- C12M 29/10: {Perfusion}
- C12M 29/18: {External loop; Means for reintroduction of fermented biomass or liquid percolate (loop type reactors for chemical or physical processes B01J19/2435)}
- C12M 29/26: {Conditioning fluids entering or exiting the reaction vessel}
- C12M 33/14: {with filters, sieves or membranes}
- C12M 39/00: {Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms (apparatus for cleaning laboratory receptacles or instruments B01L13/02; cleaning in general B08B)}
- C12M 47/02: {Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02)}
- C12N 1/02: Separating microorganisms from their culture media
- C12N 5/06: {Animal cells or tissues; Human cells or tissues (preservation of excised living parts A01N1/10)}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C12N 15/09: Recombinant DNA-technology
- 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}
- 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)}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided is a product production method which includes: a step of culturing a cell which produces a product and is contained in a cell suspension accommodated in a culture vessel; a separation treatment step of extracting the cell suspension from the culture vessel and separating the cell suspension by a tangential filtration method using a separation membrane; a step of returning a return liquid to the culture vessel; a step of supplying a fresh medium into the culture vessel; and a step of collecting the product, and in which with respect to a live cell concentration Nc, a pore diameter Dp of the separation membrane, a filtration area S of the separation membrane, and a volume Vf of a primary side flow path of the separation membrane, a number density Nd of fine particles having a particle size of 8 Dp to 30 Dp other than live cells in the cell suspension satisfies Nc≤Nd≤S/(32×π×Vf×Dp2).

## Claims

### Claim 1

A product production method, comprising:
a step of culturing a cell which produces a product and is contained in a cell suspension accommodated in a culture vessel;
a separation treatment step of extracting the cell suspension from the culture vessel, and separating the cell suspension, using a separation membrane, into a return liquid having a cell concentration higher than that of the cell suspension and a permeated liquid having a cell concentration lower than that of the cell suspension and containing the product by a tangential filtration method;
a step of returning the return liquid to the culture vessel;
a step of supplying a fresh medium into the culture vessel; and
a step of collecting the product,
wherein in a case where a live cell concentration, which is expressed in a unit of cells/mL, in the cell suspension is denoted by Nc, a pore diameter, which is expressed in a unit of m, of the separation membrane is denoted by Dp, a filtration area, which is expressed in a unit of m2, of the separation membrane is denoted by S, and a volume, which is expressed in a unit of cm3, of a primary side flow path of the separation membrane is denoted by Vf, a number density Nd, which is expressed in a unit of particles/mL, of fine particles having a particle size of 8 Dp to 30 Dp other than live cells in the cell suspension satisfies Expression (1),
Nc≤Nd≤S/(32×π×Vf×Dp2) Expression (1).

### Claim 2 (depends on 1)

The production method according to claim 1,
wherein a duration of culture satisfying Expression (1) is 10 days or longer.

### Claim 3 (depends on 1)

The production method according to claim 1,
wherein a duration of culture satisfying Expression (1) is 10 days to 40 days.

### Claim 4 (depends on 1)

The production method according to claim 1,
wherein the live cell concentration Nc satisfies Expression (2),
2×107 cells/mL≤Nc≤20×107 cells/mL Expression (2).

### Claim 5 (depends on 1)

The production method according to claim 1, further comprising:
a bleeding step of extracting the cell suspension from the culture vessel and adding the same amount of the fresh medium as an extracted amount of the cell suspension into the culture vessel,
wherein the bleeding step includes measuring the number density Nd of the fine particles having a particle size of 8 Dp to 30 Dp other than the live cells in the cell suspension, and adjusting a bleeding amount such that Nd satisfies Expression (1).

### Claim 6 (depends on 5)

The production method according to claim 5,
wherein in a case where the number of times Nb of bleeding of the cell suspension per day is one or more, a volume, which is expressed in a unit of L, of the cell suspension in the culture vessel is denoted by Vc, a target value, which is expressed in a unit of particles/mL, of Nd is denoted by Nd′, and a number density, which is expressed in a unit of particles/mL, of fine particles having a particle size of 8 Dp to 30 Dp other than live cells in the cell suspension before n-th bleeding (n is any natural number which is Nb or less) is denoted by Ndn, an amount Vbn, which is expressed in a unit of L, of the n-th bleeding satisfies Expression (3) and Expression (4),
Vbn=Vc×(Ndn−Nd′)/Ndn Expression (3)
Nc≤Nd′≤S/(32×π×Vf×Dp2) Expression (4).

### Claim 7 (depends on 5)

The production method according to claim 5,
wherein in a case where the number of times Nb of bleeding of the cell suspension per day is one or more, a volume, which is expressed in a unit of L, of the cell suspension in the culture vessel is denoted by Vc, a target value, which is expressed in a unit of particles/mL, of Nd is denoted by Nd′, and a number density, which is expressed in a unit of particles/mL, of the fine particles having a particle size of 8 Dp to 30 Dp other than live cells in the cell suspension before n-th bleeding (n is any natural number which is Nb or less) is denoted by Ndn, the amount Vbn, which is expressed in a unit of L, of the n-th bleeding satisfies Expression (3) and Expression (5),
Vbn=Vc×(Ndn−Nd′)/Ndn Expression (3)
2×107 particles/mL≤Nd′≤2×109 particles/mL Expression (5).

### Claim 8 (depends on 1)

The production method according to claim 1,
wherein the step of culturing a cell which produces a product includes stirring the cell suspension in the culture vessel with a stirring blade, and
in a case where a power coefficient of the stirring blade is denoted by Np, a blade diameter, which is expressed in a unit of m, of the stirring blade is denoted by Di, a rotation speed, which is expressed in a unit of s−1, of the stirring blade is denoted by Ro, a density, which is expressed in a unit of kg/m3, of the cell suspension is denoted by ρ, and a volume, which is expressed in a unit of L, of the cell suspension in the culture vessel is denoted by Vc, stirring is performed with a stirring power Pv, which is defined by Expression (6) and expressed in a unit of W/m3, of the stirring blade in a range of 10 W/m3 to 150 W/m3, and in a case where a liquid level height, which is expressed in a unit of m, of the cell suspension based on a bottom of the culture vessel is denoted by Hc, a height Hb, which is expressed in a unit of m, of a bleeding extraction position is ½×Hc or lower,
Pv=Np×ρ×Ro3×Di5/(Vc×10−3) Expression (6).

### Claim 9 (depends on 1)

The production method according to claim 1,
wherein the step of culturing a cell which produces a product includes passing a gas through the cell suspension with a sparger, and
in a case where an interfacial tension, which is expressed in a unit of N/m, of the cell suspension is denoted by σ, a pore diameter, which is expressed in a unit of μm, of the sparger is denoted by Ds, a volume, which is expressed in a unit of mL, of a single gas generated from the sparger is denoted by Vg, a gas passage flow rate, which is expressed in a unit of mL/min, of the sparger is denoted by Q, a volume, which is expressed in a unit of L, of the cell suspension in the culture vessel is denoted by Vc, a density, which is expressed in a unit of kg/m3, of the cell suspension is denoted by ρ, and a gravitational acceleration expressed in a unit of m/s2 is denoted by g, the number Ng, which is defined by Expression (7) and Expression (8) and expressed in a unit of s−1·L−1, of passing gases per unit capacity and per unit time satisfies Expression (9),
Ng=Q/(Vg×Vc×60) Expression (7)
Vg=π×Ds×σ/(ρ×g) Expression (8)
100 s−1·L−1≤Ng≤5,000 s−1·L−1 Expression (9).

### Claim 10 (depends on 9)

The production method according to claim 9,
wherein the interfacial tension σ of the cell suspension and the pore diameter Ds of the sparger respectively satisfy Expression (10) and Expression (11),
10 mN/m≤σ≤100 mN/m Expression (10)
0.1 μm≤Ds≤200 μm Expression (11).

### Claim 11 (depends on 1)

The production method according to claim 1,
wherein the cell is a CHO cell.

### Claim 12 (depends on 1)

The production method according to claim 1,
wherein the pore diameter Dp of the separation membrane is 0.1 μm to 1 μm, and
during a culture period after the live cell concentration reaches a maximum live cell concentration or a set live cell concentration, Nd satisfies Expression (12) at all times,
2×107≤Nd≤2×109 Expression (12).

### Claim 13 (depends on 1)

The production method according to claim 1,
wherein a permeation rate of the product through the separation membrane, which is calculated as a proportion of a concentration of the product contained in the permeated liquid to a concentration of the product contained in the cell suspension, is 55% or greater.