# US 10,865,416 B2 · Recombinant host cell engineered to overexpress helper proteins

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- Number: US 10,865,416 B2
- Document ID: US10865416B2
- Status: Granted
- Kind: B2
- Application number: 15/304,050
- Filed: 2015-04-16
- Published: 2017-02-02
- Granted: 2020-12-15
- Earliest claimed: 2014-04-17
- Assignees: Boehringer Ingelheim Rcv GmbH & Co KG; Vaitdogen GmbH; Lonza Ltd.
- Applicants: Boehringer Ingelheim RCV GmbH & Co KG; Validogen GmBH; Lonza Ltd.
- Inventors: Brigitte Gasser; Diethard Mattanovich; Markus Buchetics
- Web page: https://patentgrep.com/patent/US10865416B2

## CPC

- C12N 15/67: General methods for enhancing the expression
- C07K 14/39: from yeasts
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- 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
- C07K 2317/55: Fab or Fab'
- 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 is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell's capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.

## Claims

### Claim 1

A method of manufacturing a heterologous protein of interest in an isolated host cell comprising:
a) providing an isolated host cell engineered to overexpress a polynucleotide encoding a helper protein having the amino acid sequence as shown in SEQ ID NO: 1 or a functional homologue thereof, wherein the amino acid sequence of the functional homologue has at least 95% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1, wherein the isolated host cell comprises a heterologous polynucleotide encoding the heterologous protein of interest, wherein the heterologous protein of interest and the helper protein are different proteins, and wherein the yield of the heterologous protein of interest produced by the isolated host cell is increased as compared to a corresponding isolated host cell that does not overexpress the polynucleotide encoding the helper protein or the functional homologue;
b) culturing the isolated host cell under suitable conditions to overexpress the helper protein or the functional homologue and express the heterologous protein of interest; and
c) isolating the heterologous protein of interest from the cell culture.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amino acid sequence of the functional homologue has at least 97% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amino acid sequence of the functional homologue has at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1.

### Claim 4 (depends on 1)

The method of claim 1, wherein the heterologous protein of interest comprises a mammalian polypeptide.

### Claim 5 (depends on 1)

The method of claim 1, wherein the heterologous protein of interest comprises a human polypeptide.

### Claim 6 (depends on 1)

The method of claim 1, wherein the heterologous protein of interest is an enzyme, a therapeutic protein, a food additive or a feed additive, or an antibody or an antibody fragment.