Source: Elgundi, Z. et al., Laboratory Scale Production and Purification of a Therapeutic Antibody. J. Vis. Exp. (2017)
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.
The authors have nothing to disclose.
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. |