Mammalian Cell Culture Process Principles

At the core of mammalian cell culture process is reproducing the life-support systems that a mammal (like you) provides to their cells.

You - and by proxy, your cells - need oxygen, water, and nutrients. As well, you need warmth, the right pH balance and 'saltiness'. In addition to providing the nutrients and the environment, your body removes metabolic waste.

Large-scale cell culture is simply enabling a large stainless steel tank (called bioreactors) to support living mammalian cells. In reality, large-scale cell culture is filling a tank with sugar-water, spiking it with cells and letting it "stew."

Media

Cell culture is sometimes referred to as suspension cell culture because the cells are suspended in a nutrient-rich, pH-buffered liquid called media. Media is mostly water... extremely highly-purified water called "water for injection (WFI)." In it, you'll find nutrients like glucose or glutamine, sodium bicarbonate, trace amounts of metalic elements, folic acid and supplemental amino acids. Surfactant gets put in to help with the shearing (from the bubbles). The actual concentrations of these items are trade secrets; even if a firm outsources the media, the exact recipe is kept secret.

Sometimes, management-tampering happens right around now when a fully-defined media gets tested against the same media, except with peptones added in. Peptone is basically what you get when you take unused animal parts, grind it up, digest it with enzymes and make into powder. The idea here is, "Well, if it used to be a part of something that was once alive, then it ought to help support this here cell culture." Usually what happens is the undefined media outperforms the defined media and the peptone gets added in.

But fully-defined media is certainly the way to go if you're looking to reduce process variability: all your variables are known and controlled. Too many times, people blame variability in their undefined media as the source of their process variability. Some say that unknown nutrients is the secret sauce of peptones; others say it acts as a surfactant. Whatever it is - don't choose higher long-term process variability over short-term yields.

Environment

If you ever dump cells into water, you'll watch them sink to the bottom. In order to scatter the cells and make sure they are evenly distributed, you have to stir it with an agitator (impeller). The agitation also helps to evenly distribute:

• heat
• acid/base
• dissolved oxygen

The heat is added/removed by flowing hot/cold water around the outside of the bioreactor (called a jacket).

The acid is added by sparging carbon dioxide from the bottom of the reactor. As the CO2 bubbles its way to the top, it forms carbonic acid decreasing the pH. The base is added with carbonate as a fluid.

Oxygen is provided to the cells by sparging air/oxygen from the bottom of the reactor. As the air/O2 bubbles its way to the top, it is smeared into the media by the agitator, providing it to the cells.

The agitator speed is typically set by the power-to-volume ratio from dusty ChemE textbooks. In addition, the vessel is pressurized to ensure the direction of flow is away from the bioreactor.

Control systems are in place to maintain pH, Temperature, dissolved oxygen, pressure and agitation. Engineers have figured out how to control reactors since the beginning of time. The challenges of controlling bioreators is maintaining sterility since a cell-growth-promoting environment also promotes the growth of bacterial contaminants.

Cells

The final piece of cell culture is, of course, the cells. From a manufacturing standpoint, the cells are another ingredient... not much different than glucose solution in that it is added to the bioreactor to be stirred.

The genetically engineered cells are typically cryogenically frozen until they are needed. A vial of cells is thawed (think Wesley Snipes and Sylvester Stallone in Demolition Man) into successively larger bioreactors. Typically at the 20L scale, the cells are maintained by "solera." Solera is when the cell density has increased near the maximum the media can support, a large fraction of the cell culture is siphoned off, leaving a smaller fraction. The larger fraction is used to inoculate another bioreactor while the smaller fraction gets fresh media to continue growing.

Recap

There's a lot more to cell culture than can be described in a blog post. There's seed cultures/selective media, inoculum cultures/growth media, production cultures/production media. There's fed-batch operations, there's perfusion. But in the end, cell culture processes are a lot like cooking. You put the ingredients: water, nutrients, cells into a bioreactor and then you wait until the cells have consumed the media and then you're done.

What's surprising is how maintaining large-scale biological systems actually is.

How To Make Biologics

Drugs used in modern medicine are manufactured with mammalian cell culture.

Why Mammalian Cells?

Humans have been making therapeutic biological molecules utilizing fermentation since Alexander Fleming sneezed into a Petri dish and discovered penicillin. But complex biological molecules, like antibodies, cannot be produced by microbes because microbes cannot do post-translational processing. Post-translational what?

Basically when our bodies construct a human protein from the things we eat, our cells will "sign" the newly created protein with a carbohydrate. This "signature" not only helps the protein fold, but also helps our bodies recognize the protein as self-made. Microbes do not have the machinery to do this post-translational glycosolation such that proteins made by them simply unfold and lose their therapeutic value.

What is Cell Culture?

Cell Culture refers to the cultivation of (typically mammalian) cells. The reason companies use mammalian cells is because the active pharmaceutical ingredient (API) in drugs are complex molecules that cannot be made with chemical synthesis and also needs post-translational processing. To make the API, firms genetically engineer cells to secrete the complex molecule. Cell culture is the art/science/process of growing mammalian cells in at a large-scale so the purified API can be sold.

Inoculum/Seed Cultures

The first of two parts in cell culture is growing a sufficient amount of cells.  You'll hear people say, "scale biomass" when referring to "seed" or "inoculum" cultures. Note: we've assumed the cells have been engineered and are stable. The goal is to get as many cells in as short a time as possible - i.e. get fast growing cells. To do this, you get some cells and put it in a large volume of nutrients at growth-promoting pH (somewhere near 7) and temperature (between 30 to 40 degC). The cells will grow, consuming nutrients and secrete metabolic waste (CO2 and lactic acid etc). To keep the cells growing, you must replenish their nutrients or remove the waste.  Companies do this by transferring the cells to a larger bioreactor;  the classic "solution to pollution is dilution."

Production Cultures

The second and final part in a cell culture cultivation is having the cells secrete the API. After all, your sales comes from the API, not the cells themselves. The cells get their final transfer into a bioreactor with nutrients that encourage the secretion of API. The bioreactor is typically called the "production bioreactor" and the nutritious fluid is typically called "production media." After the cells produce the API, the cells are typically discarded while the API is "harvested" for purification - a.k.a. "downstream processing".

One Unit Operation

Many in the downstream world (Rob Caren) like to take pot-shots at cell culture scientists and engineers and ask, "How hard can this be? There's just one unit operation, right?" Sure. But on a large-scale, very few things in cell culture are well understood, which is why armies of chemical engineers are hired to do multivariate data analysis on cell culture.

So in addition to providing this brief primer on mammalian cell culture, the reason I'm putting this out there is that the ultimate goal of the data acquisition is to enable multivariate analysis that confers process understanding. This is something that Zymergi engineers, given our first-hand experience supporting large-scale cell culture processes, are able to do.

What Lean says about the Stanford Kicker

Mark Graban has an excellent post on the Lean Blog on the blaming of the Stanford kicker for their 41-38 loss to Oklahoma State in the Fiesta Bowl. Holding with the dogma of lean, failure of a system (football team to win the game) is rarely a single root cause (the kicker).

Mark's post is entertaining as it is informative. Here is yet another great application of lean thinking:

http://www.leanblog.org/2012/01/blame-the-stanford-kicker-blame-the-kicker/