Single b-cell cultivation method

A method for producing an antibody binding to a target antigen comprising the following steps

• a) co-cultivating each B-cell of a population of B-cells, which has been deposited as single cell in an individual container, in the presence of feeder cells and a feeder mix,
• b) selecting a B-cell clone producing an antibody specifically binding to the target antigen,
• c) cultivating a cell, which contains a nucleic acid that encodes the antibody produced by the B-cell clone selected in step b) or a humanized variant thereof, and recovering the antibody from the cell or the cultivation supernatant and thereby producing the antibody.

The method according to any one of the preceding claims further comprising the step of incubating the population of B-cells in the co-cultivation medium prior to single cell depositing.

The method according to any one of the preceding claims further comprising the step of centrifuging the single cell deposited B-cells prior to the co-cultivation.

The method according to any one of the preceding claims, characterized in that the population of B-cells is isolated by density gradient centrifugation.

The method according to any one of the preceding claims, characterized in that the B-cells are mature B-cells.

The method according to any one of the preceding claims, characterized in that the labeling is with two or three fluorescence dyes.

The method according to any one of the preceding claims, characterized in that 0.1% to 2.5% of the cells of the total B-cell population are labeled.

The method according to any one of the preceding claims, characterized in that the B-cells are mouse B-cells, or hamster B-cells, or rabbit B-cells.

The method according to any one of the preceding claims, characterized in that the feeder cells are murine EL-4 B5 cells.

The method according to any one of the preceding claims, characterized in that the co-cultivating is in an RPMI 1640 medium supplemented with 10% (v/v) FCS, 1% (w/v) of a 200 mM glutamine solution that comprises penicillin and streptomycin, 2% (v/v) of a 100 mM sodium pyruvate solution, and 1% (v/v) of a 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES) buffer.

The method according to any one of the preceding claims, characterized in that the co-cultivating is in the presence of a synthetic feeder mix that comprises interleukin-1 beta and tumor necrosis factor alpha, and interleukin-2 and interleukin-10 and Staphylococcus aureus strain Cowans cells.