Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells
Published: April 30, 2024
Abstract
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.
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 <p class="jove_co…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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. |
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Citar este artículo
Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells. J. Vis. Exp. (Pending Publication), e22106, doi: (2024).