Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

Overview

The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further use.

Protocol

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).

Materials

Name	Company	Catalog Number	Comments
pFUSE vector series	N/A	InvivoGen	Heavy and light antibody genes expressed in separate vectors that require co-transfection.
mAbXpress vector series	N/A	ACYTE Biotech Pty Ltd.	Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).
pVITRO1 vector	N/A	N/A	Heavy and light antibody genes are each driven by a separate promoter in a single vector.  Refer to: Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).
GS vector series	N/A	Lonza	Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.
Multi-cistronic vector series 1	N/A	N/A	Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. et al. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).
Multi-cistronic vector series 2	N/A	N/A	Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).
pVITRO1-Trastuzumab-IgG1/κ	61883	Addgene	Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/κ was a gift from Andrew Beavil.
Fast-Media Hygro Agar	fas-hg-s	Jomar Life Research	Used to prepare low salt LB agar containing 75 µg/ml hygromycin.
Fast-Media Hygro TB	fas-hg-l	Jomar Life Research	Used to prepare low salt TB broth containing 75 µg/ml hygromycin.
Glycerol, BioXtra, ≥99%	G6279	Sigma-Aldrich	Prepare to 80% with water and autoclave. Store at room temperature.
Jestar 2.0/LFU Plasmid Maxi Kit	G221020	Astral Scientific	Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).
FreeStyle 293-F Cells	R790-07	Life Technologies	HEK-293 cell line adapted to suspension culture in serum-free media.
FreeStyle 293 Expression Medium	12338-018	Life Technologies	Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.
Kolliphor P188	K4894	Sigma-Aldrich	Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 μm filter. Store at 4oC.
DMEM, high glucose	11995-065	Life Technologies
Heat-Inactivated Foetal Bovine Serum	10082-147	Life Technologies
Polyethylenimine, Linear, MW 25,000	23966	Polysciences, Inc.	Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 μm filter. Store at -80oC until use.
OptiPro SFM	12309-050	Life Technologies	Transfection formulated serum-free media
Hygromycin B Solution	ant-hg-1	Jomar Life Research
Dimethylsulphoxide (DMSO)	AJA2225	Thermo Fisher Scientific
Tryptone (casein peptone)	LP0042B	Thermo Fisher Scientific	Prepare to 20% in PBS and filter-sterilize using 0.22 μm filter. Store at 4oC.
BLItz System	45-5000	fortéBIO	Instrument used for bio-layer interferometry (BLI) measurements.