Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used (i.e., heavy and light chain expression is driven by separate promoters). Trastuzumab heavy and light chains were previously cloned into the vector. This vector was a gift from Andrew Beavil, obtained through a not-for-profit plasmid repository.

1. Recovery and Scale-up of Vector DNA

NOTE: Vector DNA was received as a soft agar stab culture in Escherichia coli XL-1 Blue strain; vector carries hygromycin resistance.

1. Insert a sterile inoculation stab or loop into the soft agar of the stab culture, then streak a Luria Bertani (LB) agar plate prepared with 75 µg/ml hygromycin for isolated colonies. Incubate the plate at 37 °C for 18-24 hr.
2. Inoculate a single colony into 5 ml Terrific broth (TB) containing 75 µg/ml hygromycin. Incubate the culture at 37 °C for 18-24 hours with shaking at 225 rpm.
3. Use the overnight culture to prepare a glycerol stock of vector DNA by gently mixing 800 µl of culture with 200 µl 80% glycerol in a cryovial and freeze at -80 °C.
   1. Add 100 µl of the overnight culture to 100 ml TB containing 75 µg/ml hygromycin in a baffled shaker flask (i.e., 1/1,000 dilution). Incubate the culture at 37 °C for 18-24 hr with 225 rpm shaking.
4. After overnight culture, extract and purify the deoxyribose nucleic acid (DNA) according to the manufacturer's instructions for the midi/maxi preparation kit with the following exception; during the final step, elute or resuspend DNA with water (pH 7.0-8.5).
5. Check the concentration and purity of DNA by absorbance readings at 260 and 280 nm, then store DNA at -20 °C.     
   NOTE: Optional (highly recommended): Sequence DNA using vector-specific primers to confirm identity.

2. Stable Transfection of HEK-293 Cells

1. Grow and maintain HEK-293 cells (suspension cells) according to standard protocols in serum-free media supplemented with 0.1% non-ionic surfactant in Erlenmeyer flasks. Maintain at 2 x 10⁵ cells/ml and subculture every fourth day. Culture cells at 37 °C with 5% carbon dioxide (CO₂) and 120 rpm rotation.      
   NOTE: Cells should have been in culture for no less than 4 days and no more than 4 weeks prior to transfection. HEK-293 cells grown as monolayers in serum-containing media can also be used for this procedure.
2. On the day prior to transfection, seed HEK-293 cells at 3 x 10⁵ cells/ml in wells of a 12-well plate in 2 ml of Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). On the day of transfection, check that cells have reached 80-90% confluency
3. Dilute DNA and polyethylenimine (PEI) separately in the transfection medium and then mix them together.
   1. Dilute 1.25 µg vector DNA per well (15 µg for 12 wells) in 300 µl transfection media. Incubate at room temperature for 5 min.
   2. Dilute 2.5 µl of 1 mg/ml PEI solution (30 µl for 12 wells, i.e. 1:2 ratio of DNA to PEI) in 300 µl transfection media. Incubate at room temperature for 5 min.
   3. Add diluted DNA to diluted PEI, gently mix and incubate at room temperature for 15 min.
4. Add 50 µl of DNA/PEI mixture dropwise to each well of the plate. Rock the plate gently to distribute the transfection mixture, then place the plate at 37 °C, 5% CO₂ for 24 hr.
5. Add 50 µg/ml hygromycin B per well and return the plate to 37 °C, 5% CO₂ for 10 days.
   NOTE: By Day 10, un-transfected cells will have died and detached from the surface of the plate, whereas stably-transfected cells will be viable and attached to the plate.
6. Replace the media in wells with 1/4 volume serum-free media and 3/4 volume DMEM supplemented with 10% FBS (i.e. 0.5 ml serum-free media + 1.5 ml DMEM = 7.5% final serum concentration) and incubate for 4 days.
7. Replace the media in wells with 1/2 volume serum-free media and 1/2 volume DMEM supplemented with 10% FBS (i.e. 1 ml serum-free media + 1 ml DMEM = 5% final serum concentration) and incubate for 4 days.
8. Replace the media in wells with 3/4 volume serum-free media and 1/4 volume DMEM supplemented with 10% FBS (i.e. 1.5 ml serum-free media + 0.5 ml DMEM = 2.5% final serum concentration) and incubate for 4 days.
9. Replace the media in wells with 2 ml of serum-free media supplemented with 0.1% non-ionic surfactant (i.e. 0% final serum concentration) and incubate for 4 days.
   NOTE: Maintain 50 µg/ml hygromycin B selective pressure on cells during serum-free adaptation. Cells adapted to serum-free media detach from the surface of wells and may cluster in suspension. Refer to step 2.1 for culture conditions with the additional supplement of 50 µg/ml hygromycin B.
10. Using a pipette, gently mix the suspension cultures in the 12-well plate and pool cells into a separate tube.
    1. Using a hemocytometer, enumerate pooled cells and seed in 30 ml serum-free media in an Erlenmeyer flask at 2 x 10⁵ cells/ml. Culture cells at 37 °C with 5% CO₂ and 120 rpm rotation. Subculture and expand every fourth day.
11. Continue to expand culture size to the required cell density to obtain 10 vials at 1 x 10⁷ cells/vial for cryopreservation in liquid nitrogen. Freeze the cells in serum-free media containing 10% dimethylsulfoxide (DMSO).         
    NOTE: Conditioned media containing antibodies can be harvested at each subculture to confirm protein production or used to optimize purification conditions. To harvest conditioned media and maintain cells for subculture, centrifuge the culture at 300 x g for 5 min, then filter-sterilize supernatant through a 0.22 µm filter. Filtered supernatant may be kept at 4 °C for 1-2 weeks or -20 °C for longer-term storage.

3. Large-Scale Antibody Production from Batch Overgrow Culture

NOTE: Antibody production can be followed on from step 2.11, where existing cells are expanded to the required cell density based on the batch overgrow culture volume to be set up. Otherwise, cells that have been thawed from cryopreservation begin at this stage once expanded to an appropriate cell density and volume. Various culture supplements may be used to optimize antibody production; the use of tryptone to increase antibody yield is demonstrated in this protocol.

1. Seed cells at 2 x 10⁵ cells/ml in 100 ml serum-free media in two Erlenmeyer flasks (to compare antibody yields from un-supplemented and nutrient-supplemented cultures). Culture at 37 °C, 5% CO₂ and 120 rpm.
2. After 24 hr, add tryptone to a final concentration of 0.5% to the nutrient-supplemented culture. Equalize the volume of un-supplemented culture with serum-free media.
3. From day 8, perform cell counts of the cultures daily using a hemocytometer to monitor cell viability. Harvest the culture supernatants once cell viability is less than 80%.
   1. Harvest supernatant by centrifugation of the cultures at 3,000 x g for 15 min, then filter-sterilize the supernatant (containing antibody) through a 0.22 µm filter. Store the supernatants at 4 °C (short-term) or freeze at -20 °C (long-term).