Making a monoclonal antibody means producing millions of identical copies of a single antibody that recognizes one specific target. The classic method, called hybridoma technology, fuses an antibody-producing immune cell with a cancer cell to create an immortal cell line that churns out the same antibody indefinitely. Newer methods skip the animal entirely and select antibodies in a test tube. Here’s how each approach works, from the original technique to large-scale manufacturing.
The Classic Method: Hybridoma Technology
Developed by Köhler and Milstein in 1975, hybridoma technology remains the foundational approach. It involves five core steps: immunizing an animal, harvesting its immune cells, fusing those cells with a partner that can grow forever in culture, selecting the fused cells, and then growing the winners to produce antibody at scale.
Immunization
The process starts by injecting a mouse or rabbit with the target molecule (the antigen) you want an antibody against. Over several weeks, the animal receives a series of injections that train its immune system to recognize the target. This triggers a specific type of white blood cell, the B cell, to mature into antibody-secreting cells. Each B cell produces a slightly different antibody, and the goal is to eventually isolate the one that binds best.
Harvesting B Cells
Once the animal has mounted a strong immune response, its spleen is removed under sterile conditions. The spleen is packed with activated B cells, making it the richest source of antibody-producing cells. These cells are separated out and prepared for the next step.
Cell Fusion
Here’s the central problem: B cells taken from the spleen die within days in a lab dish. To keep them alive and dividing indefinitely, they’re fused with myeloma cells, a type of cancer cell that grows without limit in culture. A chemical agent or electrical pulse pushes the two cell types together, merging their contents into a single hybrid cell called a hybridoma. This hybridoma inherits the B cell’s ability to make a specific antibody and the myeloma cell’s ability to replicate endlessly.
Selecting the Fused Cells
After fusion, the dish contains three types of cells: unfused B cells, unfused myeloma cells, and the desired hybridomas. A special growth medium called HAT medium sorts them out over 10 to 14 days. It works by blocking the main pathway cells use to build new DNA. Cells can switch to a backup pathway, but only if they have a specific enzyme. The myeloma cells used in this process are deliberately chosen because they lack that enzyme, so they can’t survive when the main pathway is shut down. Unfused B cells die on their own because they simply don’t last in culture. Only the hybridomas survive: they inherit the missing enzyme from the B cell side, letting them use the backup pathway and keep dividing.
Screening and Scaling Up
Surviving hybridomas are tested to find which ones produce the antibody that binds the target most strongly. Each hybridoma colony is screened individually, and the best performers are selected, cloned (grown from a single cell to ensure uniformity), and then expanded into larger culture vessels. At this point, you have a stable cell line producing one specific monoclonal antibody that can be frozen, stored, and grown whenever needed.
Phage Display: Skipping the Animal Entirely
Hybridoma technology has a major limitation for making drugs: the antibodies it produces come from mice, and the human immune system often recognizes them as foreign. This triggers what’s known as a human anti-mouse antibody response, which can neutralize the drug or cause side effects. Scientists can modify mouse antibodies to look more human through a process called humanization, but this sometimes weakens how well the antibody binds its target.
Phage display solves this by starting with human antibody genes from the beginning. Instead of immunizing an animal, researchers build a massive library of human antibody fragments and attach each one to the surface of a bacteriophage, a virus that infects bacteria. This library can contain billions of different antibody variants.
The selection process is elegant. The phage library is washed over a surface coated with the target antigen. Phages carrying antibody fragments that stick to the target are kept, while everything else is washed away. The bound phages are then recovered, amplified by infecting bacteria, and put through the process again. After three to five rounds of this selection, the remaining phages are highly enriched for antibody fragments that bind the target with strong affinity. The whole process happens in vitro, making it faster and more cost-effective than hybridoma methods, with the added advantage of producing fully human antibodies ready for clinical use.
Transgenic Animals: The Best of Both Worlds
A third approach combines the strengths of the immune system with the need for human antibodies. Transgenic mice have been engineered so that their own antibody genes are replaced with human versions. When you immunize these animals, their immune systems respond normally, generating diversity through the same natural processes of gene recombination and mutation that happen during any immune response. The difference is that every antibody they produce has a fully human structure.
This strategy lets researchers harness the body’s natural ability to refine and improve antibodies over the course of an immune response, something that’s difficult to replicate in a test tube. While mice are the most common transgenic host, the same engineering has been applied to rats, rabbits, chickens, and cows. Increasingly, therapeutic antibodies reaching the clinic are discovered through these transgenic platforms.
Manufacturing at Scale
Once you’ve identified your antibody, whether through hybridoma, phage display, or transgenic animals, you need to produce it in large quantities. For research purposes, small-scale hybridoma cultures may be sufficient. For therapeutic use, the antibody gene is typically inserted into a mammalian cell line optimized for industrial production.
Chinese Hamster Ovary (CHO) cells are the industry workhorse. They’ve held that position since 1986, when they were used to produce the first approved recombinant biopharmaceutical. CHO cells reliably produce high yields, typically around 1 gram per liter in simple batch cultures and up to 10 grams per liter in optimized fed-batch processes. Other mammalian cell lines used for production include mouse-derived NS0 and Sp2/0 cells, as well as human cell lines like HEK293 and PER.C6, which can add sugar molecules to the antibody in patterns identical to those found on natural human proteins.
Purifying the Final Product
Antibodies secreted into the cell culture broth need to be separated from everything else: host cell proteins, DNA, growth media components, and potential viruses. The near-universal first step is Protein A chromatography. Protein A is a bacterial protein that naturally binds to a specific region on antibody molecules with high selectivity, grabbing the antibody while letting contaminants flow through. The captured antibodies are then released by lowering the pH of the solution, effectively washing them off the column in a concentrated, highly pure form.
This single capture step achieves substantial purification and concentration at once. Additional polishing steps follow to remove any remaining impurities, and a low-pH hold serves as a virus inactivation measure. The final product is formulated into a stable solution or powder suitable for storage and administration.
How the Landscape Looks Today
Between January 2015 and September 2024 alone, 86 monoclonal antibodies received FDA marketing authorization. They’re used across oncology, autoimmune disease, infectious disease, and more. About 21% of those approved antibodies had their dosing information updated after reaching the market, with changes happening at a median of roughly 37 months after approval. This reflects how much fine-tuning continues even after a monoclonal antibody is in clinical use, as real-world data refines how the drug is best administered.