# US 8,541,204 B2 · Polypeptide producing cells

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- Number: US 8,541,204 B2
- Document ID: US8541204B2
- Status: Granted
- Kind: B2
- Application number: 13/705,339
- Filed: 2012-12-05
- Published: 2013-05-02
- Granted: 2013-09-24
- Earliest claimed: 2006-05-17
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Josef Endl; Erhard Kopetzki; Oliver Ploettner; Ursula Schwarz; Georg Tiefenthaler
- Web page: https://patentgrep.com/patent/US8541204B2

## CPC

- C12N 15/85: for animal cells
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/065: {Purification, fragmentation}
- C07K 2319/03: containing a transmembrane segment
- 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)

## Abstract

The current invention describes a nucleic acid comprising in a 5′ to 3′ direction a) a first nucleic acid encoding a heterologous polypeptide without an in frame stop codon, b) a second nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site comprising an in frame translational stop codon and a polyadenylation signal, and c) a nucleic acid encoding i) at least a fragment of a transmembrane domain, or ii) a signal peptide for a GPI-anchor.

## Claims

### Claim 1

A method for the production of a heterologous polypeptide or protein encoded by a nucleic acid, said method comprising;
a) providing a eukaryotic cell,
b) transfecting said eukaryotic cell with a nucleic acid, wherein the nucleic aid comprises in a 5′ to 3′ direction
i) a first nucleic acid encoding an immunoglobulin heavy chain without an in frame stop codon at its 3′ terminus,
ii) a second nucleic acid beginning with a 5′ splice donor site and terminated by a 3′ splice acceptor site comprising an in frame translational stop codon and a polyadenylation signal of a human or mouse immunoglobulin α, ε, γ and μ heavy chain, and
iii) a third nucleic acid encoding at least 60% of a complete human immunoglobulin transmembrane stretch,
wherein said immunoglobulin heavy chain and a marker are expressed from the same nucleic acid, wherein the marker is a plasma-membrane bound form of the expressed immunoglobulin heavy chain,
c) culturing said transfected cell under conditions suitable for the expression of said nucleic acid, and
d) recovering said polypeptide or protein from the culture medium or the cytoplasm of the cells.