# US 2014/0154745 A1 · Process for the culturing of cells

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- Number: US 2014/0154745 A1
- Document ID: US20140154745A1
- Status: Published application
- Kind: A1
- Application number: 14/175,172
- Filed: 2014-02-07
- Published: 2014-06-05
- Earliest claimed: 2006-07-14
- Assignees: Dsm IP Assets B.V.
- Applicants: Dsm IP Assets B.V.
- Inventors: Gerben Meile Zijlstra; Robert Patrick Hoff; Jacob Schilder
- Web page: https://patentgrep.com/patent/US20140154745A1

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- B01D 15/26: characterised by the separation mechanism
- B01D 69/08: Hollow fibre membranes (manufacture of hollow fibres D01D5/24, D01F1/08)
- C07K 1/18: Ion-exchange chromatography
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 1/34: by filtration, ultrafiltration or reverse osmosis
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12M 23/28: {disposable or single use}
- C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis}
- 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 47/10: {Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02)}
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- C12N 5/0621: {Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062)}
- C12P 21/005: {Glycopeptides, glycoproteins}
- C12N 2500/32: Amino acids
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/92: Medium free of human- or animal-derived components
- C12N 2500/99: Serum-free medium
- C12N 2510/04: Immortalised cells
- 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)}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- 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)
- 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

The invention relates to a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cells culture comprising the cells, the biological substance and the cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.

## Claims

### Claim 1

Process for the culturing of cells in a reactor in suspension in a cell culture medium,
wherein the cells produce a biological substance, wherein at least one cell culture medium component is fed to the cell culture and
wherein the cell culture comprising the cells, the biological substance and cell culture medium is circulated over a separation system and
wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and
wherein the biological substance is retained in or fed back into the reactor.

### Claim 2 (depends on 1)

Process according to claim 1, wherein part of the substances of lower molecular weight is continuously removed from the cell culture.

### Claim 3 (depends on 1)

Process according to claim 1, wherein the cells are mammalian cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells.

### Claim 4 (depends on 1)

Process according to claim 1, wherein the biological substance is recombinant protein, preferably an antibody.

### Claim 5 (depends on 1)

Process according to claim 1, wherein the separation system is a filter, preferably a membrane filter, more preferably a hollow fiber filter.

### Claim 6 (depends on 5)

Process according to claim 5, wherein the cell culture is circulated over the filter in cross-flow, preferably an alternating tangential flow.

### Claim 7 (depends on 1)

Process according to claim 1, wherein cell culture is removed at least once from the reactor and liquid, preferably cell culture medium is added to the reactor to compensate for the cell culture removal.

### Claim 8 (depends on 1)

Process according to claim 1, wherein cell culture conditions are chosen such that cell growth rate and/or specific productivity of the cells is not limited.

### Claim 9 (depends on 8)

Process according to claim 8, wherein the cell culture conditions are chosen such that the concentration of at least one of the components of the cell culture medium remains constant.

### Claim 10 (depends on 1)

Process according to claim 1, wherein at least the depleted nutrients are partly or preferably in whole supplemented by feeding these nutrients to the reactor.

### Claim 11 (depends on 1)

Process according to claim 1, wherein optionally the biological substance is harvested from the cells and/or from the cell culture.

### Claim 12

Cell culture comprising mammalian cells having a viable cell density of at least 50×106 cells/mL and a concentration of biological substance of at least 5 g/L.

### Claim 13 (depends on 12)

Cell culture according to claim 12, wherein the concentration of biological substance is at least 10 g/L, preferably at least 11 g/L.

### Claim 14 (depends on 12)

Cell culture according to claim 12, wherein the mammalian cells are E1-immortalized HER cells, preferably PER.C6 cells.