Method for selecting high producing cell lines

• US 9,534,246 B2
• Filed: 06/29/2011
• Issued: 01/03/2017
• Est. Priority Date: 07/01/2010
• Status: Active Grant

• Current Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Original Assignee: Glaxo Group Limited (Glaxosmithkline Holdings Ltd.)
• Inventors: Priest, Richard, Kotsopoulou, Ekaterini, Uden, Mark
• Primary Examiner(s): Gabel, Gail R
• Application Number:
  US13/704,711
• Publication Number:
  US 20130090259A1
• Time in Patent Office: 2,015 Days
• US Class Current: 1/1
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention provides methods for the rapid identification and selection of cell lines suitable for biopharmaceuticals production, which do no utilize animal derived components.

Claims

1. A method for selecting one or more mammalian cells expressing secreted antibody for use as a biopharmaceutical, the method comprising the steps of:

(a) providing a population of mammalian cells in a medium free from animal-derived components, wherein the cell population comprises at least one cell expressing said secreted antibody;

(b) contacting the cell population with a label which directly binds to said antibody on the surface of the cell;

(c) detecting said label bound to the antibody on the surface of said cell; and

(d) selecting said cell based on the presence of said label bound to the antibody on the surface of said cell;

wherein the label is selected from the group consisting of Protein A, Protein G or Protein L; and

whereby one or more mammalian cells expressing a secreted antibody for use as a biopharmaceutical is selected.

2. The method according to claim 1, wherein said Protein A, Protein G or Protein L is fluorescent.

3. The method according to claim 1, wherein the detecting step comprises detecting a relative amount of said label bound to the surface of said cell.

4. The method according to claim 1, wherein the selecting step comprises selecting the cell or cells from the population which show the highest amount of label bound thereto.

5. The method according to claim 1, further comprising the step of:

(e) isolating said selected cell or cells from the population of cells.

6. The method according to claim 1, wherein the secreted antibody is a domain antibody.

7. The method according to claim 1, wherein the mammalian cell is selected from the group consisting of CHO, CHO K1, CHO DG44, NSO, COS-1, COS-7, H EK293, H K21, PerC6, H EK293, 293T, Vero, AGE1.CR, HT1080, TE671, Namalwa, and SP2/0.

8. The method according to claim 1, wherein the steps of detecting, selecting and/or isolating said cell is carried out by fluorescence activated cell sorting.

9. The method according to claim 8, wherein said label is fluorescent Protein A, Protein G, or Protein L;

wherein the step of detection comprises determining a relative fluorescence of said label bound to the antibody on the surface of the cell, wherein a relatively higher fluorescence is indicative of a higher relative expression of said antibody; and

wherein selection step comprises selecting the cell or cells from the population that have a relatively higher level of fluorescence.