Fundamentals
Biomanufacturing: Making Life-Saving Medications and High-Value Products with Cells
The curious cell lines, the indispensably elusive product, and the sewage-like liquid in between
Biomanufacturing is a general term that uses biological elements (most often cells) to manufacture a product
The first, and most common, use of biomanufacturing is for the manufacturing of medications, such as insulin or Herceptin. While we’ll be focused on medications in this article, there are many other exciting applications of biomanufacturing in the works, such as animal-free meat (Source).
I first set foot in a biomanufacturing plant in 2018 as an intern at Genentech’s biomanufacturing plant in Vacaville, and have been truly fascinated ever since. The fact that humans have figured out how to take cells that evolved as disease-causing bacteria or were literally part of the ovary of a Chinese hamster, and train them to produce massive quantities of life-saving medications sounds like science fiction but is actually a scientific fact. There are hundreds of biomanufactured medications on the market today, which generate hundreds of billions in revenue each year.
In order to manufacture a life-saving medication, we need to select the cellular machinery best suited to the task. Three commonly used cell types used to make medication are: E. coli, CHO cells, and sf9 insect cells. Each has its own unique benefits and drawbacks.
E.coli
E. coli is a type of bacterium that is used to manufacture simpler biomolecules, like hormones (Source). It is the first “workhorse” of the biomanufacturing industry and are used to make ~30% of all approved biopharmaceutical products (Source).
One of the first biomanufactured products to enter the market was Humulin, which was slow-acting insulin synthesized by E. coli. This life-saving insulin is still used by people with diabetes today to keep their blood sugar in check and prevent their disease from getting worse (Source, Source, and Source).
In order for E. coli to begin producing a biomolecule, we must provide the bacteria with specific instructions on how to make insulin. These instructions are in the form of genetic code (DNA), which is commonly referred to as a plasmid. The E. coli bacterium “reads” the plasmid instructions, and begins to synthesize the biomolecule. Once the E. coli makes biomolecules inside of its cell, it secretes them so that it is floating around outside the cell (Source).
E. coli was also the first cellular machinery I encountered. In my high school AP biology class, we provided E. coli with instructions called pGLO– genetic information that allow the bacterium to produce a fluorescent protein that made the petri dish the E coli was living in growing in the dark. Our lab-grade hinged on whether our petri dishes glowed after we closed the curtains and turned off all of the lights (thankfully, mine did).
CHO Cells (Chinese Hamster Ovary Cells)
Chinese hamster ovary cells, CHO cells, are used to manufacture more complex biomolecules. These cells were harvested from Chinese hamster ovaries in the 1950s, and are now growing in 2,000 L vats of media in biomanufacturing facilities all around the world. A majority of biomanufactured medications approved today are made with mammalian cell lines, including CHO cells. CHO cell products produce >$100 B in revenue annually (Source).
CHO cells are used to make a complex biomolecule called a monoclonal antibody (Source). The human body makes monoclonal antibodies to fight all kinds of diseases, and we have learned to tailor monoclonal antibodies to fight specific types of cancer (Source and Source). Important CHO-cell-made cancer medications include Herceptin and Rituxan.
Because CHO cells are closer to human cells than E. coli (CHO cells and human cells are both mammalian cells with the same fundamental machinery, whereas E. coli is a bacterial cell with some major differences in cellular machinery) it is easier to “teach” a CHO cell to synthesize complex biomolecules (including monoclonal antibodies) than it is to teach E. coli to synthesize a complex biomolecule.
When I first learned about how Genentech and other biotechnology companies refer to vats of hamster cells as the “workhorse” of monoclonal antibody production, I thought they were making some time of strange pun. It was only after I saw the 2,000 L vats of CHO cell culture that I realized that they weren’t kidding — CHO cells produce thousands of doses of life-saving medication each day.
Making very expensive fancy sewage
Once you have selected your cellular factory, you need to create and maintain the conditions required for your cells to multiply and make your product. Our goal during this part of the process is to make a very fancy and expensive sewage-like mixture called cell culture.
This first step in the process is called the “seed train”, where we take the “seed” cells from a frozen vial, thaw them out, and place them in a small flask to multiply in number (Source). These flasks contain media, a special nutrient-dense broth that the cells float around in. We also supply the cells with oxygen to make sure they don’t die, which is dissolved in the media.
The combination of media, oxygen, cells, and random other stuff floating around is commonly referred to as the “cell culture”. By definition, a cell culture is an artificial environment containing all of the stuff a group of cells needs to grow and multiply (Source).
The speed at which the cells multiply will vary based on the type of cellular factory you are using. Generally speaking, the more complicated your cellular factory, the longer it takes for it to multiply in number. Bacteria cellular factories, for example, tend to multiply faster than mammalian cell factories (Source).
As the cells multiply in number, they inevitably outgrow the vessel they are in. Right before the cells in the cell culture would die due to lack of room, they are transferred to a larger vessel. This process of transferring cell cultures between fancy containers is called “scale up”, as the scale of the vessel goes up over time (Source).
As the vessels increase in size, it becomes increasingly hard for the cells to survive in the culture. This is because as the vessel gets bigger, it’s harder to ensure that nutrients and oxygen are properly distributed. Larger scale cell cultures, in like 10 or 100 L vessels, are a bit like large scale, high stakes, cookie dough mixing operation. You need all of the ingredients in the dough (or cell culture) to be distributed equally, but if you do too much mixing all of your cells will die and you have to go back to the cell bank to start over (Source).
In an attempt to reduce the chances that our cells die during this process, we monitor the conditions of the cell culture very carefully. While exact parameters will depend on the type of cellular factory we are using, the environmental conditions we monitor are pH, temperature, dissolved oxygen, dissolved carbon dioxide, cellular density, electrolytes, glucose, and lactate (Source). In addition to monitoring these conditions, we will often try to control them by adding stuff to the media, like oxygen, powered glucose, or electrolytes.
Throughout the seed train and scale-up processes, cellular factories are producing your product (like Herceptin, for example, a breast cancer medication), and your cells are busy with two things: making more cells and making your product. The next step of the biomanufacturing process is to separate a single type of molecule, your product, from the rest of the cell culture (Source). Essentially, we need to isolate your product from all of the other stuff in our very expensive sewage-like mixture.
Getting the needle (your product) out of the haystack
Taking the needle out of the haystack, or isolating your product from the rest of your cell culture, is commonly referred to as “downstream manufacturing”. To isolate your product, a combination of physical and chemical isolation techniques is used. Typically, we start with more “general” and less complex isolation techniques and move to more “specific” and complex isolation techniques. We do this to save on cost and increase process efficiency (Source).
The specific methods used to extract your product from other products will heavily depend on the chemical properties of your product (Source). If your product is very polar, for example, you could use specific isolation methods to isolate polar molecules from non-polar molecules. This would not work at all if your product was somewhere in between polar and non-polar.
Generally speaking, downstream processing can be broken down into four different phases: pre-treatment, separation, concentration, and purification (Source). How each of these phases is done varies from product to product, but the goal of each phase is the same across product types.
The pre-treatment phase goals are:
- To get any product out of cells
- Add some chemicals to the cell culture to make future isolation steps easier.
Sometimes, particularly for bacterial cell factories, product can end up “stuck” inside individual cells.
To maximize the amount of product we are able to produce, we need to make sure we extract any product that is “stuck” inside the cell. We accomplish this by basically blowing up the cells so that there aren’t any cellular membranes trapping our product. There are a variety of ways to blow up cells, but my favorite methods are using a special enzyme (fancy molecule) to chew up the cell’s membranes and blenderizing them (literally putting them in a fancy blender that chops them into small pieces).
To maximize the effectiveness of our isolation processes, we will also add fancy chemicals to the cell culture to make it easier to work with later (Source). Commonly used fancy chemicals include coagulants, such as iron-based salts, which force molecules with specific chemical properties to stick together (Source).
Now that we have a bunch of our product sticking together, thanks to our coagulants, we can move to the next phase of the isolation process: separation. During this phase, we use a special machine called a centrifuge to physically separate molecules from each other. Centrifugation is a process where we spin our pre-treated cell culture around in a circle and use the centrifugal force to separate molecules based on size, shape, or other properties (Source). Because we used a coagulation chemical during the pre-treatment phase, it’s much easier to separate our product from other stuff using this centrifugation (Source).
After the separating process, we still need to concentrate on our product and remove other similar molecules that survived the centrifugation process. This is the concentration phase. One common way to concentrate product in this phase is called extraction, which is basically a fancy term for extracting one type of molecule from a larger group. One way to extract, and by extension, concentrate, a molecule is through solvent extraction. Solvent extraction uses a highly specific solvent, that dissolves our product, but does not dissolve other molecules in the mixture (Source).
The output of the concentration phase is commonly referred to as “crude” product because it still contains some impurities (such as other molecules with similar chemical properties or defective product molecules). To turn “crude” product into “pure” products we move to the next phase of the downstream process: purification.
My favorite — and one of the most expensive — ways to purify products is chromatography. Chromatography involves creating small fancy beads that “grab onto” our product under specific conditions (for example, at pH 7), and “let go” of our product under different conditions (for example, at a pH of 10) (Source). This is a highly specific isolation process, and chromatography beads can be specially engineered so that they grab onto only one type of molecule (such as our product).
By dumping the crude product through a chromatography column under specific conditions, the chromatography beads will “grab onto” our product, and allow everything else to pass through. Once the chromatography beads have “grabbed onto” our product, we can change the conditions of the chromatography column so that the product is released, and collect the product at the bottom of the column (Source).
Congratulations! The stuff at the bottom of the chromatography column is exclusively our product. We have successfully isolated our needle from the haystack.
Even after having worked at multiple biomanufacturing facilities, I am still surprised that a process this complex is used to make billions of dollars worth of medications each day. This technology continues to excite me because it has great untapped potential. As it matures, I’m looking forward to contributing to other novel applications of these processes across sectors, such as cellular agriculture (Source).
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