# US 10,767,186 B2 · Organic compounds

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- Number: US 10,767,186 B2
- Document ID: US10767186B2
- Status: Granted
- Kind: B2
- Application number: 15/233,726
- Filed: 2016-08-10
- Published: 2016-12-08
- Granted: 2020-09-08
- Earliest claimed: 2007-12-21
- Assignees: Novartis AG
- Applicants: Novartis AG
- Inventors: Yehuda G. Assaraf; Thomas Jostock; Hans-Peter Knopf
- Web page: https://patentgrep.com/patent/US10767186B2

## CPC

- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)}
- C12N 15/65: using markers (enzymes used as markers C12N15/52)
- C12N 15/85: for animal cells
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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

The present invention relates to a novel selection system for use in a eukaryotic cell culture process and for expression of a recombinant product of interest. The selection system is based on the introduction of an exogenous functional membrane-bound folate receptor gene together with the polynucleotide or gene encoding the product of interest into a eukaryotic cell and can be widely utilized with eukaryotic cells for which cellular viability is dependent upon folic acid uptake.

## Claims

### Claim 1

A method for selection of a CHO cell capable of stably expressing a product of interest encoded by an expression vector which has been introduced into the cell, comprising
(i) providing a plurality of CHO cells for which cellular viability is dependent upon folate uptake, and into which cells a first polynucleotide located on an expression vector and encoding a functional membrane-bound folate receptor alpha as selection marker and a second polynucleotide located on an expression vector and encoding the product of interest have been introduced, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector or on separate expression vectors,
(ii) culturing said plurality of CHO cells in a cell culture medium having a limiting concentration of a folic acid, wherein the concentration in the cell culture medium is from 0.001 nM to 100 nM, thereby obtaining a CHO cell wherein stable expression of the product of interest is achieved, and
(iii) identifying and isolating a CHO cell wherein stable expression of the product of interest is achieved.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the plurality of CHO cells are CHO cells for which cellular viability is dependent on folate uptake and into which a first polynucleotide located on an expression vector and encoding a functional membrane-bound folate receptor alpha as a selection marker and a second polynucleotide located on an expression vector and encoding a product of interest have been stably introduced, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector.

### Claim 3 (depends on 1)

A process for production of a product of interest, comprising
(i) performing a method of selection according to claim 1,
(ii) and isolating the product of interest from said cell culture medium or from said cell.

### Claim 4 (depends on 3)

The process of claim 3, wherein the product of interest is expressed from a eukaryotic expression vector that comprises a first polynucleotide encoding a functional membrane-bound folate receptor alpha and a second polynucleotide encoding the product of interest.

### Claim 5 (depends on 3)

The process of claim 3, wherein the product of interest is a polypeptide.

### Claim 6 (depends on 5)

The process of claim 5, wherein the product of interest is a pharmaceutically or therapeutically active polypeptide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first polynucleotide and the second polynucleotide are each operably linked to its distinct transcription promotors.

### Claim 8 (depends on 7)

The method of claim 7, wherein the transcription promoters are the same.

### Claim 9 (depends on 7)

The method of claim 7, wherein the transcription promoters are different.

### Claim 10 (depends on 7)

The method of claim 7, wherein the promoter controlling the transcription of the first polynucleotide is an SV40 promoter.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first polynucleotide and the second polynucleotide are under the control of a common transcription promoter.

### Claim 12 (depends on 11)

The method of claim 11, wherein the common transcription promoter is an SV40 promoter.

### Claim 13 (depends on 11)

The method of claim 11, wherein said vector comprises an IRES element functionally located between the first polynucleotide and the second polynucleotide.

### Claim 14 (depends on 1)

The method of claim 1, wherein the functional membrane-bound folate receptor alpha encoded by the first polynucleotide is a human folate receptor alpha (FRα).

### Claim 15 (depends on 2)

The method of claim 2, wherein the CHO cells lack full activity of at least one endogenous functional membrane-bound folate receptor.

### Claim 16 (depends on 1)

The method of claim 1, wherein the concentration of folic acid in the cell culture medium is selected from 0.01 nM to 100 nM, 0.1 nM to 100 nM, 1 nM to 100 nM, 1 nM to 30 nM and 3 nM to 10 nM.

### Claim 17 (depends on 1)

The process of claim 1, wherein the endogenous folate receptor alpha gene of the cell is not deleted or attenuated prior to transfection.