Method for cell line development

Patent metadata

Field	Value
Publication number	US-11802297-B2
Application number	US-201816490623-A
Country	US
Kind code	B2
Filing date	Feb 23, 2018
Priority date	Mar 3, 2017
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

The present invention relates to an improved method for cell line development (CLD) which is generally applicable to production of any therapeutic protein that can be produced using mammalian Cell lines and in particular Chinese Hamster Ovary (CHO) cells. The method combines site-directed integration (SDI), expression construct components improving the post-transcriptional processing of the gene of interest (GOI) and the introduction of a onetime pre-CLD host cell line selection workflow to generate a production competent cell line that can then be used in multiple CLD efforts using SDI from that point on.

First claim

The invention claimed is:

1. A method to obtain a cell suitable for expressing a protein of interest comprising the following steps:
a) integration of a nucleic acid molecule comprising a polynucleotide encoding a first protein of interest into a pre-defined site in the genome of a recipient cell, wherein the nucleic acid molecule comprises in 5′ to 3′ sequence order:
a first recombinase recognition sequence;
a first copy of a second recombinase recognition sequence;
a promoter-less selection marker gene and the polynucleotide encoding the first protein of interest, wherein the promoter-less selection marker and the polynucleotide encoding the first protein of interest are in any order and orientation; and
an additional copy of said second recombinase recognition sequence, wherein the first and the second recombinase recognition sequences are recognition sequences for different recombinases;

b) identification of cells expressing the first protein of interest;
c) excision of the nucleic acid molecule comprising a polynucleotide encoding the first protein of interest from the genome of the identified cells to provide a resulting cell containing the first recombinase recognition sequence and a copy of the second recombinase recognition sequence enabling targeted introduction of a nucleic acid molecule encoding a second protein of interest into said pre-defined site in the genome; and
d) subsequent to step c), integration of the nucleic acid molecule encoding the second protein of interest into the pre-defined site in the genome of the resulting cell.

2. The method according to claim 1 , further comprising creation of a cell line suitable for production of said second protein of interest as a recombinant protein.

3. The method according to claim 2 , wherein the recombinant protein is an active pharmacological ingredient.

4. The method according to claim 2 , further comprising providing the recombinant protein as an ingredient in a diagnostic reagent.

5. The method according to claim 2 , further comprising providing the recombinant protein as a reagent in an industrial process.

6. The method according to claim 1 , wherein the first protein of interest is a fluorescent protein.

7. The method according to claim 1 , wherein the first protein of interest is an immunoglobulin or immunoglobulin-like protein.

8. The method according to claim 1 , further comprising selecting a cell with increased capacity to express the first protein of interest relative to other cells identified as expressing the first protein of interest prior to excision of the nucleic acid molecule comprising a polynucleotide encoding the first protein of interest.

9. The method according to claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises isolating clones from a culture of said identified cells expressing the first protein of interest.

10. The method according to claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises isolating pools or clones from said identified cells expressing the first protein of interest after (1) different culture time in a continuous culture format such as a perfusion culture or (2) different number of individual cultures starting with inoculation of a first culture using said identified cells expressing the first protein of interest and using a volume from a finished culture to inoculate a next culture or (3) a combination of (1) and (2).

11. The method according to claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises performing targeted engineering by applying gene editing methods to introduce, remove or modify genetic material in the genome of said identified cells expressing the first protein of interest.

Assignees

• Ge Healthcare Bio Sciences Ab
• Cytiva Sweden Ab

Inventors

• Ivansson Daniel
• Castan Andreas

Classifications

• C12N15/907 Primary

  in mammalian cells · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N2510/02

  Cells for production · CPC title

• C12N15/1082 Primary

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title