Monday, May 28, 2012

Mammalian Cell Culture Environment - pH Control Strategy

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):

cell culture process parameters
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).

ph control strategy
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.

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