Monoclonal antibodies are used in medical tests and treatments. They help with many different health problems.
Most recently, they have been used to help treat people with COVID-19. You can now also find information and access related to monoclonal antibody online – making it easier for researchers to stay informed.
The way these antibodies are made is well known, can be done on a large scale, and follows a standard process.
Due of this, making monoclonal antibodies is a good example of how modern biotechnology works. This overview will focus on how they are made.
| The method for making hybridoma cells (which produce monoclonal antibodies) was developed in the 1970s. It has improved very well today. The process of making antibodies from these cells follows a typical path used for making proteins from specially modified mammal cells. Here are the main steps in making monoclonal antibodies: Create and choose the best hybridoma cell (this becomes the starting point for production) Grow the cells in small containers like T-flasks, shake flasks, or roller bottles Transfer the growing cells to larger containers (like bench-scale bioreactors) to produce more Scale up the production to very large tanks, up to 20,000 liters Purify the antibodies (called downstream processing) and store them for use |
How To Choose the Best Hybridoma Clone for Production?
Scientists first create and pick a hybridoma cell (a special cell that produces one kind of antibody) to make monoclonal antibodies,. The best hybridoma clones are those that are stable and make a lot of antibodies.
| For example, your goal is to produce an anti-F2 antibody. Only those hybridomas that specifically generate antibodies targeting the F2 antigen would be selected. |
Here’s how it works:
- An animal, usually a mouse, is injected with a specific substance (called an antigen) to trigger an immune response.
- Then, immune cells (B-cells) are taken from the mouse’s spleen.
- These B-cells don’t live very long on their own, so they are combined (fused) with special cancer cells called myeloma cells that can grow forever.
- This fusion creates hybridoma cells, which can keep growing and making antibodies.
The myeloma cells can come from mice, humans, or other animals. Scientists can also create slightly different types of antibodies—like –
- bi-clonal (from two cells),
- polyclonal (from many), or
- chimeric (a mix of mouse and human cells)—to make them work better in human treatments.
Growing the Hybridoma Cells
Once the best clone is chosen, it’s grown in small lab containers, like T-flasks. It is done by using special liquid without animal serum. These cultures become the “master” cells and are frozen in liquid nitrogen so they can be used later to start large-scale production.
Alternatively, scientists might grow the cells in a small bioreactor to get a larger number of cells right away. These can then be used directly in bigger production tanks.
To ensure the cells remain reliable, the master cultures undergo regular testing to verify they continue to produce the correct antibodies efficiently.
Seed Train Process For Growing Cells
The seed train is the step-by-step process of growing more and more cells. It starts from a small frozen vial and moves up to larger containers. In each step, the number of cells increases, and the culture is transferred to a bigger flask.
However, each step comes with risks, like –
- contamination,
- poor cell growth, or
- reduced antibody production.
That’s why it’s better to keep the number of steps as low as possible. Newer methods are known as process intensification. It help reduce or skip some of these steps.
In the early stages, scientists often use a special incubator that shakes and controls CO₂ levels to grow the cells. At first, only about 20 mL of culture might be used, then it’s scaled up over two or three steps to over a liter.
To be safe, multiple flasks are used at each step. It helps in case one has problems and also ensures there’s enough healthy culture for the next stage.
Some labs use multi-level systems to run several steps at the same time.
| Here’s what a typical setup for growing hybridoma cells might look like: Temperature: 37°C (like body temperature) CO₂ Level: 8% Shaking Movement: 50 mm (how far the flask moves back and forth) Shaker Speed: 120 times per minute Humidity: About 80% (even though it’s not always listed, this is commonly used) |
Special protein-free liquids (called media) are available to help the cells grow. When using shake flasks, you usually only fill them about 20–25% full. Some flasks can handle a bit more for better growth.
There are typically two steps involved for larger flasks when you go from a small frozen sample to a 1-litre culture in a shake flask.
Expand to Bench-Top and Pilot Bioreactors
With the increase in production, hybridoma cells are grown in larger containers. These are called bioreactors and they usually hold 10 to 50 liters.
Further, these are often used in a perfusion mode, which keeps cell numbers high and may reduce the need for many small-scale steps.
Common Features of Cell Culture Bioreactors:
- A rounded or dish-shaped bottom
- A short height compared to width (a 2:1 ratio)
- A gentle mixing blade (called a marine impeller) to avoid damaging the cells
- Low gas flow, using small bubbles to mix in air
- A gas system that can handle 3 or 4 gases, including CO₂ to control pH
- A slow-spinning motor for gentle stirring
- If running continuously, a filter (like a spin filter) separates the liquid product from the cells
| Important Settings for Growing Hybridoma Cells in Bioreactors: Temperature: 37°C pH: 7 Dissolved Oxygen (DO): 50% Stirring Speed: 60–120 times per minute (faster stirring helps reduce cell loss when using filters) Gas Mix: Usually includes air and CO₂ |
You can run bioreactors in different ways:
- Fed-batch: Feed new nutrients each day
- Perfusion: Constant flow of fresh media through the system (0.5–1 times the culture volume per day)
- Batch: Everything is added at the beginning, and nothing is added or removed until the end
These cultures usually last 7–14 days that start with an initial batch phase of 3–4 days.
The exact timing and setup depend on the specific cell line and process used.
Finally
Making monoclonal antibodies with hybridoma cells started just before companies began using DNA technology to make human insulin. The method is a great example of a modern bioprocess that combines different areas of science.
Once scientists create a hybridoma cell that produces the right antibody, the rest of the process usually follows the same basic steps.
After they choose and prepare the target (called an antigen) and use it to make the hybridoma, most of the following steps stay the same. If they want to switch to a different antigen, they only need to make a few small changes to the process.
Disclaimer: For research use only. Not for diagnostic or therapeutic applications in humans.
