In biopharma, cells are grown in bioreactors in order to generate protein products that are used as therapeutic agents. Biopharma companies mostly use mammalian cells such as CHO cells to develop the complex therapeutic proteins that are structurally similar to those produced by human cells. This means that if a protein is used as a drug for humans, it will be safer and much more effective.
1. Bioreactor Design
- Condenser: The media is held at 37°C in the vessel. Hence, it evaporates continuously. If this evaporated media escapes out of the vessel, it will alter the concentrations of various nutrients in the medium. Therefore, a condenser is used to maintain the concentration of culture. The evaporated material passes through the condenser in the gas exhaust line, cools down, and falls back into the vessel. It is basically a hollow metal coil.
- Agitator: It is basically a part of the stirring mechanism of the bioreactor. An impeller is attached to a motor-driven shaft which acts as the stirrer. The motor may be attached at the top or bottom of the bioreactor based on the design.
- Gas Lines: Gases like oxygen, carbon dioxide and nitrogen are passed into the bioreactor during cell culture through the gas lines. The gases pass through a sterilizing grade hydrophobic filter before entering into the culture vessel.
- Liquid Addition Lines: There are liquid addition lines, inoculum (cells), antifoam lines and alkali addition lines so that these liquids can be pushed into the vessel. These liquids are added to maintain the culture conditions.
- Temperature Control Jacket: Not only these jackets maintain the culture temperature at 37°C, but also serve an important role during the sterilization process. During steaming of the stainless steel culture vessel, this jacket helps in heating up and then cools down to bring the temperature back to the operational temperature.
- Probes: Probes are used to monitor the process parameters like the dissolved oxygen, pH and temperature. Novel Process Analytical Technology (PAT) like Raman Spectroscopy can be used to analyze the protein concentration and cell viability in real-time. Culture conditions can be adjusted if these values deviate from the standards.
2. Scaling Up
While scaling up the culture conditions, following 3 operating modes can be used:
- Batch Mode: Used when the goal is to increase the cell number. This is mostly used for the cell growth stage. Then one can shift to the following two modes for protein production.
- Fed-Batch Mode: To extend the culture and allow for longer growth by increasing the media volume with time
- Continuous Mode: To extend the culture and allow for longer growth with a static volume. In continuous mode, you constantly add fresh media and remove harvest. Thus, the nutrient level remains high and waste levels remain low. The harvested proteins can be stored at a lower temperature to increase their shelf life and prevent denaturation. A cell separation device is used to remove the cells and waste products. This allows the cells to be maintained at very high density. However, the continuous mode of culture is expensive with high media demand. There are more chances of equipment failure and increased risk of contamination.
3. Controlling Parameters
There are typically 4 controlling parameters, namely, agitation, dissolved oxygen, temperature and pH.
Why do we need Agitation?
- That’s because the contents remain homogenized in terms pH, temperature and nutrient concentration.
- Agitation leads to higher oxygen dissolution into the media
- Insufficient agitation can lead to cell settling and oxygen deprivation
3.2. Types of Impellers
Agitation is provided by impellers attached to the motors. There are two types of impellers:
- Axial Flow impellers: In these impellers, the blades are at an angle less than 90° to the plane of rotation. This is particularly useful for mammalian cell culture as they generate low levels of shear and are less likely to damage the fragile cells.
- Radial flow impellers: They provide good mixing but generate more shear. They are used to provide good aeration in the culture, distributing and breaking up bubbles.
Baffles are fixed near the sides of the vessel to generate turbulent motion. For large capacity bioreactors, baffles are needed to properly mix the nutrients.
3.2. Dissolved Oxygen
Less dissolved oxygen will cease cell growth and eventually lead to cell death. There are several factors that affect the oxygen transfer rate. The oxygen transfer rate increases due to longer bubble residence time, larger bubble surface area, thinner bubble film and increased supplied oxygen concentration.
We can use amperometric electrochemical probe (Clark electrodes) or an optical probe to measure the dissolved oxygen in the media. The amperometric probes measure higher current with higher dissolved oxygen concentration. Optical probes require less maintenance and operate by measuring the intensity of the light signal. Lower the intensity of the returned signal, higher the dissolved oxygen concentration.
Most mammalian cells are neutrophils – they will grow best at
and around a neutral pH. Optimum pH level for mammalian cells is 6.8-7.4.
Glucose in media is metabolized within the cell in order to generate energy. As part of this process, lactate and carbon dioxide may be generated. These can cause the pH of the media to become more acidic, especially at high cell densities and high growth rates. Ammonia is also released by the breakdown of amino acids like glutamine and this may cause a shift towards a more basic pH within the cell. It can be seen that the
cells are constantly changing the pH of their own environment due to their metabolic activity.
Enzymes are central to many cellular processes. For example, cytochromes catalyze the reactions that are necessary to generate energy within a cell but they will not function if the pH becomes too extreme, leaving the cell without a source of energy.
Also, post-translational modifications, such as glycosylation, that are relevant to the function of many proteins, including biopharmaceuticals, are enzymatically mediated and if these enzymes stop functioning there will be a direct impact on product quality. Therefore you have to control these changes in pH to ensure the cells remain healthy.
The pH of the culture is measured with a pH probe. The pH probe measures the voltage created between the internal electrolyte solution and the hydrogen ions (H+) that are in the media. If the pH is acidic, this voltage will
be positive. If it is basic it will be negative. And if it is neutral there should be no voltage measured.
3.3.1. Airflow and pH
Airflow can affect pH in the following way:
- High Airflow: High airflow can lead to high pH as the carbon dioxide may be pushed out in this process. Carbon dioxide is acidic in nature. Thus, reduced carbon dioxide leads to higher pH, that is, the culture is driven towards alkalinity.
- Low airflow: Oxygen limitation leads to higher lactic acid production because of the anaerobic breakdown of glucose.