# US 12,760,258 B2 · Cell culture process by intensified perfusion with continuous harvest and without cell bleeding

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- Number: US 12,760,258 B2
- Document ID: US12760258B2
- Status: Granted
- Kind: B2
- Application number: 17/289,332
- Filed: 2019-09-29
- Published: 2022-11-17
- Granted: 2026-10-13
- Earliest claimed: 2018-11-02
- Assignees: Wuxi Biologics Ireland Limited
- Applicants: Wuxi Biologics Ireland Limited
- Inventors: Weichang Zhou; Hang Zhou; Mingyue Fang; Siyuan Tang
- Web page: https://patentgrep.com/patent/US12760258B2

## CPC

- C12M 29/10: {Perfusion}
- C12M 29/06: {Nozzles; Sprayers; Spargers; Diffusers (per se B01F23/231, B01J19/26)}
- C12M 33/14: {with filters, sieves or membranes}
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- 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)
- 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)}

## Abstract

Provided are a method and a system for culturing cells and harvesting biologics. More particularly process for cell culture by intensified perfusion with continuous harvest and without cell bleeding is provided.

## Claims

### Claim 1

A method for producing a biological substance comprising:
(a) culturing a cell culture comprising Chinese Hamster Ovary (CHO) cells and a cell culture medium for culturing the CHO cells, wherein the CHO cells are cultured at a range of about 35° C. to about 37° C., and the cell culture is established by inoculating at about 0.6-0.8×106 viable cells/mL,
(b) perfusing the cell culture in a bioreactor with a basal medium and a feed medium, and
(c) harvesting the biological substance,
wherein the basal medium and the feed medium are fed to the cell culture at different rates, the cell culture is continuously passed through a separation system to harvest the biological substance from the bioreactor, and the cells are retained in the bioreactor without bleeding,
wherein the basal medium is perfused at a perfusion rate of about 0.3 to about 0.6 working volume per day (VVD) from the first day and the perfusion rate is increased to about 1.5 to about 2.0 VVD between days 4-5 of the culturing,
wherein the perfusion of the feed medium is started one day after the perfusion rate of the basal medium is increased and is started at a rate ranging from about 1 to 5% of the perfusion rate of the basal medium, then increased to about 7 to 9% between days 12-13 of the culturing and maintained at about 6 to 7% from days 18-19 of the culturing, and
wherein the method further comprises subjecting the cell culture to a temperature shift to a temperature in the range of about 32° C. to about 33° C. in response to a predetermined peak viable cell density (VCD), wherein the feed medium is a CHO cell culture medium or a combination of CHO cell culture media.

### Claim 2 (depends on 1)

The method of claim 1, wherein the separation system is an alternating tangential flow (ATF) device or tangential flow filtration (TFF) device.

### Claim 3 (depends on 1)

The method of claim 1, wherein the separation system comprises a hollow fiber filter, wherein the pore size of the hollow fiber filter is about 0.08 mm to about 0.5 mm.

### Claim 4 (depends on 3)

The method of claim 3, wherein the pore size of the hollow fiber filter is about 0.1 μm to about 0.5 μm.

### Claim 5 (depends on 3)

The method of claim 3, wherein the pore size is about 0.2 μm or about 0.45 μm.

### Claim 6 (depends on 1)

The method of claim 1, wherein an antifoam is added to the bioreactor.

### Claim 7 (depends on 1)

The method of claim 1, wherein a microsparger is used.

### Claim 8 (depends on 7)

The method of claim 7, wherein the microsparger delivers oxygen at a flow rate in a range of about 0.2 to about 0.5 volume of gas per volume of liquid per minute (VVM).

### Claim 9 (depends on 1)

The method of claim 1, wherein the biological substance is chosen from receptors, enzymes, fusion proteins, blood proteins, multifunctional proteins, viral or bacterial proteins, and immunoglobulins.

### Claim 10 (depends on 1)

The method of claim 1, wherein said method achieves an accumulative volumetric productivity (Pv) of about 10 g/L or more.

### Claim 11 (depends on 1)

The method of claim 1, wherein said method achieves an accumulative volumetric productivity (Pv) of about 15 g/L or more.

### Claim 12 (depends on 1)

The method of claim 1, wherein said method achieves an accumulative volumetric productivity (Pv) of about 20 g/L or more.

### Claim 13 (depends on 1)

The method of claim 1, further comprising subjecting the harvested biological substance to a continuous product capture process by at least one chromatography step.

### Claim 14 (depends on 1)

The method of claim 1, wherein the basal medium is a CHO cell culture medium.