Mammalian Cell Culture Environment - pH Control Strategy

by Oliver Yu

A key requirement of a cell culture is to recreate in a bioreactor the cellular environment that the cells experience were they still in their mammalian hosts, so that the cells grow and secrete the active pharmaceutical ingredient.

Cellular Environment

Key parameters of the cellular environment include:

• pH
• osmolality
• shear forces
• temperature
• mixing

To control this cellular environment, pH, dO2, Temperature and Pressure Control strategies are developed in the process definition (a.k.a. Manufacturing Formula):


Bioprocess engineering textbooks and modern cell culture scientists all think that these parameters determine cell growth, cell viability, cell metabolism and ultimately product formation and product quality.

These parameters are what the cells "see" and "feel" during the course of their stay in the bioreactor and large-scale cell culture support actually means being hospitable to our guests by controlling these parameters.

The Mac Daddy of all cell culture parameters is arguably pH. The amount of protons (H+) hanging around can change the way proteins fold thereby changing the function of cellular machinery. These changes can speed and slow the rate of reactions tilting the cells to favor one metabolic pathway over another.

Studies claim that pH increments as small as 0.1 units can change glucose consumption and lactate production dynamics... though I know of no pH probe that has an error smaller than 0.1 units of pH.

pH Control Strategy

The pH control strategy is most simply achieved by using carbon dioxide to lower pH (make more acidic) and sodium carbonate to increase pH (more basic).


Like any carbonated beverage, water with excess carbonation (CO2) is acidic or sour. Incidentally, this is the same mechanism by which global warming alarmists believe that that greenhouse gases kill off our marine life by making our oceans more acidic.

To increase pH, one simply needs to add a base and if carbon dioxide is the acid, the complementary base is carbonate (a.k.a sodium carbonate).

Because pH between 6.8 and 7.4 is the proven acceptable range for cell culture, a buffer is added to the media to make sure the media stays within that range. The buffer is sodium bi-carbonate and acts to ensure that the pH titration does not overshoot in either direction.

One key feature of this pH control strategy is that the acid is gaseous which means it is sparged in ("blown in via pipes") from the bottom of the bioreactor and bubbles its way to the top. It also means that this acid can be removed from the bioreactor by competing gases like air or oxygen.

On the other hand, the base is liquid, which means that it is dripped in from the top of the bioreactor. This also means that once added, the sodium cannot be removed.

How pH works in reality.

During the early culture, CO2 is often demanded to maintain pH because it is constantly being stripped by the air/oxygen sparge. Once there are enough cells evolving their own carbon dioxide, CO2 demand dwindles. During the mid-culture as the cells are growing gangbusters, the culture becomes acidic demanding sodium carbonate. And if cells start to die off towards the end, the culture may demand more carbon dioxide and less sodium carbonate to maintain a fixed pH.

To summarize:

• Acid (CO2) is consumed during early and late culture.
• Carbonate is used during mid-culture.
• CO2 is a gas and is sparged in from the bottom.
• Sodium carbonate is a liquid and is dripped from the top
• Alkali, once added, cannot be removed and contributes to increases in osmolality.

Biologics Manufacturers in the News 5/23

Biologics News for this past week.

• Bristol Myers Squibb gets regulatory approval for it's biologics manufacturing facility in Devens (Boston.com).
• CMC Biologics named finalist for Washington Manufacturing Awards (MarketWatch).
• Takeda shuts down South San Francisco operations and heads to San Diego (Genetic Engineering News).
• Avaxia Biologics legal monopoly for radiation-caused damage to digestive tract (Sacramento Bee)

Is @SAFEbiologics Really About Safe Biologics?

by Oliver Yu

In a prepared statements made to the FDA on biosimilars, the Alliance for Safe Biologic Medicines (@SAFEbiologics) outlined 5 areas for deeming biosimilars to be interchangeable with the original drug [PDF]:

1. Clinical testing
2. Global supply chain and manufacturing monitoring
3. Track, trace and naming
4. Clear labeling and packaging
5. "Close deliberate scrutiny"

There aren't many involved in the current system of drug approval that will disagree with these five points. These steps are known to add cost to biosimilar manufacturers thereby making the biosimilar less competitive.

cGMP regulations of finished pharmaceuticals already cover manufacturing monitoring. As well, 21 CFR Part 211 Subpart G issues six sections of regulations that cover labeling and packaging. You can be certain that the other 3 items are also required of existing cGMP manufacturers.

What the Alliance for Safe Biologic Medicines appears to be urging the FDA is simply this:

Regulate biosimilars the same way that you regulate original biologics.

Which isn't a surprise; nor is this request unfair.

What is a surprise is how ASBM's chairman, practicing endocrinologist Dr. Dolinar, doesn't understand the Central Dogma of Molecular Biology:

Biologics are complex, large molecule drugs that are grown inside living cells using unique and proprietary processes. For this reason, no two biologics made from different cell lines or using different processes can be identical based on today's science.

This is simply not true in most cases. We know that the DNA is DNA and that cellular machinery for any biological organism can transcribe and translate that DNA into protein.

Two insulin molecules made from different cell lines (one E.Coli, the other human cells) or using different processes are, in fact, identical based on today's science.

In fact, the opposite of Dr. Dolinar's statement is exactly how we have a thriving biotech industry in the first place: that biologics made by microbes in big stainless steel bioreactors is equivalent to the large complex biological molecules produced by human cells in vivo.

Here's another doozy:

Biologics are also highly sensitive to the manufacturing process. In fact, altering a single manufacturing parameter can change a compound's identity and/or the precise effect it has on the human body.

This statement is pretty bizarre as well. Here's why:

Original biologics manufacturers are constantly changing their manufacturing processes... or worse, their manufacturing processes changes on them. At this very moment, FDA-approved cell culture processes are being upgraded... with changes to media and operating conditions. Some manufacturers are even changing the cell line and hoping that they don't have to go back to the clinic.

Secondly, manufacturing processes where altering a single manufacturing parameter can change product quality is not a viable process at all. I don't dispute that these processes existed; I dispute that they are passing regulatory muster and being approved by the FDA today.

The robustness of a process can be defined as how many changes the manufacturing process can endure before the product quality or productivity suffers. It's the year 2012 and back in 2007, process scientists were working on process capability and trying to understand parameter interactions such that this proverbial single change in a manufacturing parameter cannot alter the the identity of the product.

Lastly, the FDA has been pushing programs like Process Analytical Technologies (PAT) and Quality by Design (QbD) to explicitly avoid situations where changing a single parameter renders the process incapable of meeting product quality specs. Most of my customers have been applying these principles for years.

ASBM's chairman has talking points from the late 90's: his information was true at one point in time but has since become hyperbole.

Why is there an ASBM in the first place and why is their chairman publicly issuing misleading hyperbole? Let's have a look at ASBM's website (safebiologics.org)...and there you have it:


Genentech... Amgen... Biotechnology Industry Organization (Big Biotech's lobby).

I'm pretty certain that these guys truly have the good intentions of protecting patient safety.

I'm also pretty certain that these guys truly have the intentions of protecting their status-quo legal monopoly by throwing regulatory hurdles in front of their future competitors.

Further Reading

FDA Releases Draft Guidance on Biosimilars

How to Prepare Large-Scale Cell Culture Media

Cell culture media is to mammalian cells what a workout smoothie is to your human cells.


Media's purpose is to make a stainless steel bioreactor hospitable to CHO cells by providing volume where temperature, dissolved oxygen (dO2), and pH can be controlled.

As well, it must provide nutrients intended for cellular uptake as well as a place to absorb metabolic waste. At the start of cell culture, the nutrient supply is defined; by the end of cell culture, the nutrients are depleted. Despite the added nutrients, media is still largely water and can thus be modeled at 1g/mL.

You make cell culture media the same way you'd make a smoothie, except at large-scale, you're making 100 or 500 or even 15,000 liters and so there are differences.

1. Initial QS.
   This is where you add water-for-injection (WFI) into a clean media preparation tank.
2. Add media powder.
   Once the powder touches the water, the media can promote growth. Since the bioreactor is only clean (and not sterile), if you don't proceed quickly, you may have a contamination on your hands.
3. Add bicarbonate powder
   Bicarbonate is the buffer. This whole time, you are agitating, and pH control is OFF.
4. Add peptones (optional).
   Over the past decade, we've seen movement away from bovine (cow) to porcine (pig) peptone. I've read that we now use veggie peptone, but have never seen it.
5. Adjust pH
   Some will dispute the necessity of adjusting the pH in the media prep tank because once the media is transferred to the bioreactor the pH will get adjusted there.
6. Final QS
   This is where you add the rest of the water. If you have an osmolality specification, you'd measure it here as an in-process test before transferring the media to the bioreactor
7. Transfer/sterile filter the media
   While pumping the media over to the bioreactor, there will be sterile filters that remove 0.1 micron particles so that the media that ends up in the bioreactor is free of microbes. In some cases, the media is virally inactivated by passing through a "pasteurizer" that raises the temperature to 121 degC, holds it for 1 minute and cools it down.

by Oliver Yu

It's a bit more than making a smoothie since mixing in a blender is forgiving. But in the preparation of cell culture media where you are making thousands of liters of this stuff at 7 bucks per gallon ($2/liter), the large-scale media preparation procedure has to be written to be highly reproducible.

Credits: Image above is from the greatest movie of all time - The Matrix (1999).

How to Make IR Control Charts

by Oliver Yu

Suppose you support a batch process. The way you likely measure performance is to sample each batch and measure different parameters. These measurements are ideal for plotting on an IR control chart - one control chart for each parameter and each batch would be represented by one point on the control chart.

If you have statistical software like JMP, then you can just click around on the menu



...and...



control charts appear like magic:



But suppose Wall Street bankers crashed the economy by securitizing AAA-rated subprime mortgages and you are the collateral damage; forking over $1,250 for a single-user annual license or $1,895 for a single-user perpetual license of JMP isn't in the cards. What do you do?

Good news. William Shewhart developed control charting principles long before computers so if worse comes to worse, you could probably create a control chart from graph paper and a grease pencil.

Here's what you do:

1. Get the data into a column
2. Compute moving range
3. Multiply MR by 3 and divide by 1.128

We're not going to do it with a grease pencil and graph paper. We're going to do it with a spreadsheet.

Step 1: Get the data into a column

We haven't talked about this yet, but data for analysis needs to be structured. If you look at the numbers in a column and they represent what the column headers describe, then you got it right.

Step 2: Compute the Moving Range

This is where you take the absolute value of the difference between measurements. =B3-B2 would be the formula that you'd drag in column C. The average of the moving range is used to determine the width of the control limits.

Step 3: Compute distance to control limits

To get the distance to each control limit, compute 3 * Average( MovingRange ) / 1.128.



In this case, the average of the moving range is 3.90. Take 3.90 * 3 / 1.128 = 10.37.

The Upper Control Limit (UCL) is the 296 + 10.37 = 306

The Lower Control Limit (LCL) is the 296 - 10.37 = 286

What you do is calculate limits for every parameter you measure; apply it to a steady process and lock the limits and monitor the process against the locked-down limits to detect drift.

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