While monoclonal and polyclonal antibodies are readily available from several sources, fewer sources of recombinant antibodies (rAbs) exist (though Addgene has a great collection of ready-to-use rAbs and rAb plasmids!). Since recombinant antibodies conveniently allow for unlimited production, reliable expression, and easy distribution as DNA (Trimmer, 2020), you may be interested in using them in your own experiments. It is possible to produce your own recombinant antibodies with some molecular biology and cell culture experience. Let’s go over the basics of making rAbs.
Production
Recombinant antibody production can take place in bacterial, yeast, plant, or mammalian cells. Each of these systems has their advantages and disadvantages. Briefly, bacterial and yeast cells are cost-effective and faster-growing than mammalian cells, but mammalian cells can perform human-like post-translational modifications that bacterial and most yeast cells cannot. Your choice of production system may depend on what downstream applications you are planning, cost, and the amount of protein you need to produce.
The most common yeast strain used for recombinant antibody production is Komagataella phaffii (a.k.a Pichia pastoris) because it secretes fewer of its own proteins, making the purification process simpler. Due to its high yields, E. coli is often used for the production of Fab fragments where variable chains are secreted into the periplasmic space to be oxidized and form disulfide bonds (Frenzel et. al., 2013). If you’re using a mammalian production system, cell lines typically used include Human Embryonic Kidney (HEK) cells and Chinese Hamster Ovary (CHO) cells. (L'Abbé et al., 2018).
Recombinant antibodies can be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain genes are contained to one plasmid or if they’ve been separated onto two. You’ll first transfect your cells with your plasmid(s), and then wait 7-14 days (in mammalian cells) to collect the cell culture supernatant for further processing. It may be helpful to incorporate regular feeds during this incubation time to maintain cell viability and increase protein production (Schwarz et. al., 2020).
Harvest and purification
At its core, harvesting recombinant antibodies consists of separating antibodies from media, cells, and debris. Affinity chromatography is a popular choice for this. Chromatography columns containing appropriate immobilized ligands are incubated with cell culture supernatant containing antibodies. The antibodies bind the immobilized ligands and can be eluted once the other materials have been washed away (Figure 1).
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| Figure 1: The steps of rAb production. Created with biorender.com |
Another method of harvesting uses magnetic beads. In this method, beads are incubated with the cell suspension. Antibodies bind ligands on the beads and a magnet is used to separate the beads from the media (Brechmann et al. al., 2021) while the media is aspirated. The beads are then washed, followed by antibody elution.
Whatever method you choose, you may not be able to elute the antibodies directly into your preferred buffer. If that’s the case, a buffer exchange can be performed using a desalting column or an ultrafiltration concentrator. As the name suggests, you may also concentrate your prep to the desired concentration using these columns. If you are conjugating your antibody in a downstream step, you’ll want to ensure that your buffer components are compatible with your conjugate and conjugation chemistry.
Quantification
Once you’ve collected your antibody in the appropriate buffer, it’s time to quantify it. There are several options to choose from depending on the time, reagents, and equipment available to you. Whatever you choose, you’ll want an accurate concentration to give your downstream applications the best chance at being successful.
Using a spectrophotometer is among the faster methods of quantification. Many spectrophotometers have an IgG protein setting. If yours does not, be sure to set the absorbance to 280 nm (Pace et al., 1995). You’ll also need to know the extinction coefficient to calculate the concentration. At 280 nm, the extinction coefficient of IgG is around 1.35, though this can vary depending on the specific amino acid sequence (Maity et al., 2015). If your antibody is not IgG, make sure to look up the setting for your isotype.
Antibody concentration may also be measured through a protein stain, such as Coomassie blue. Though more time-consuming, this assay will also measure the purity of your prep. In this method, antibodies are separated by size on an SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and a protein standard. The density of the antibody bands can be measured against the protein standard and the concentration may be calculated using imaging software. Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands lower the purity of your antibody prep. If you have multiple contaminating bands or a few strong ones, consider increasing the number of washes performed during your harvest.
Validation
Recombinant antibodies should be validated to ensure they bind their antigens specifically. Possible applications for validation include western blot, immunohistochemistry, and immunocytochemistry. It’s important to note that some antibodies are better suited for certain applications. An antibody that performs well in immunohistochemistry may not perform well in a western blot. This can be due to conformational changes in the antigen or lower detection sensitivity of the assay. Where possible, it can be useful to compare recombinant antibodies to their parental hybridomas.
Aggregation and storage
Aggregation of antibodies may impact their quality and efficacy (Wang et. al., 2018). Aggregation can arise from a shift in temperature, pH, or salt concentration (Amin et. al., 2014). Antibodies may even aggregate just from dropping the tube they are contained in (Randolph et. al., 2018). At Addgene, we ensure the efficacy of our antibodies through stress testing. An aliquot of our recombinant antibodies is stored at 37 °C for a period of two weeks. After this time, quality control assays showed that the majority of antibodies are still equally effective compared to those aliquots frozen immediately post-harvest. Stress-tested antibodies that are not equally effective are shipped to customers on ice, preventing any temperature shifts. Of course, we are also cautious during the aliquoting and storage process to prevent dropped tubes.
Now that your antibody is produced, harvested, purified, quantified, and validated, it’s ready for use! You can learn more about common antibody applications in our Antibodies 101 blog series.
Resources and references
More resources on the Addgene blog
Plasmid-based Recombinant Antibodies
Resources on addgene.org
Addgene's Antibody Purification Protocol
Addgene's Video Antibody Purification Protocol
References
Trimmer JS. Recombinant Antibodies in Basic Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID: PMC7665837.
L'Abbé D, Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676.
Frenzel A, Hust M, Schirrmann T. Expression of recombinant antibodies. Front Immunol. 2013 Jul 29;4:217. doi: 10.3389/fimmu.2013.00217. PMID: 23908655; PMCID: PMC3725456.
Schwarz H, Zhang Y, Zhan C, Malm M, Field R, Turner R, Sellick C, Varley P, Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin. J Biotechnol. 2020 Feb 10;309:44-52. doi: 10.1016/j.jbiotec.2019.12.017. Epub 2019 Dec 28. PMID: 31891733.
Brechmann NA, Schwarz H, Eriksson PO, Eriksson K, Shokri A, Chotteau V. Antibody capture process based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021 Sep;118(9):3499-3510. doi: 10.1002/bit.27776. Epub 2021 May 4. PMID: 33811659.
Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411-23. doi: 10.1002/pro.5560041120. PMID: 8563639; PMCID: PMC2143013.
Maity H, Wei A, Chen E, Haidar JN, Srivastava A, Goldstein J. Comparison of predicted extinction coefficients of monoclonal antibodies with experimental values as measured by the Edelhoch method. Int J Biol Macromol. 2015;77:260-5. doi: 10.1016/j.ijbiomac.2015.03.027. Epub 2015 Mar 25. PMID: 25819219.
Wang W, Roberts CJ. Protein aggregation - Mechanisms, detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub 2018 Aug 23. PMID: 30145245.
Samiul Amin, Gregory V. Barnett, Jai A. Pathak, Christopher J. Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization & rheology, Current Opinion in Colloid & Interface Science, Volume 19, Issue 5, 2014, Pages 438-449, ISSN 1359-0294, https://doi.org/10.1016/j.cocis.2014.10.002.
Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci O, Schöneich C, Freund E, Ricci MS, Carpenter JF, Lengsfeld CS. Do not drop: mechanical shock in vials causes cavitation, protein aggregation, and particle formation. J Pharm Sci. 2015 Feb;104(2):602-11. doi: 10.1002/jps.24259. Epub 2014 Nov 21. PMID: 25418950; PMCID: PMC4312221.
Topics: Antibodies, antibodies 101
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