In the world of GMP validation, a question repeatedly arises: should we draw a carbon dioxide concentration distribution map in a constant temperature chamber like we draw a temperature distribution map?

Why do temperature distribution maps differ

Temperature distribution diagram is a common requirement in GMP. We indirectly reflect product temperature by measuring temperature. Due to the fact that the product does not immediately reach equilibrium with changes in the surrounding air, drawing a distribution map helps to demonstrate its uniformity and stability. Relative humidity also varies with temperature, so it is important to draw a distribution map. But the behavior of carbon dioxide gas is different.

Physics of Carbon Dioxide Distribution:

Unlike temperature and relative humidity, gases equilibrate very quickly. Unless the probe is directly located in front of the gas inlet, carbon dioxide in the chamber will almost immediately diffuse. Modern incubators typically use fans to maintain temperature uniformity, which also accelerates the mixing of carbon dioxide.

I have been engaged in GMP validation and environmental monitoring work for over 20 years. During this period, I often used this metaphor to explain the balance of carbon dioxide: it is similar to water in a tank. You don't need to verify if the water surface is level; Due to gravity, the water surface is essentially horizontal. Similarly, due to the inherent behavior of gases, the concentration of carbon dioxide will stabilize throughout the entire constant temperature chamber within a few seconds.

Some clients I have worked with have raised an important point: although thermostat manufacturers claim to have "rapid diffusion" and "uniform carbon dioxide distribution," in a GMP environment, relying solely on supplier statements is not enough, even if these statements are true. The verification team must provide written evidence to prove that the thermostat is suitable for its intended use.

Buffer effect:

Even if the door is opened, the concentration of carbon dioxide drops from the typical 5% target to the ambient concentration (~0.04%), and the liquid culture medium in the culture dish and flask can still act as a buffer. Dissolved carbon dioxide will not immediately escape. This buffering effect helps to protect cell cultures when the door is briefly opened, just as frozen products do not immediately thaw in the refrigerator when the door is opened. In validation, this means that the recovery rate after opening the door may be a more meaningful parameter than drawing a carbon dioxide distribution map at multiple points inside the chamber.

What results do auditors expect?

The challenge lies here: there is no formal regulatory guidance to draw a carbon dioxide distribution map in a constant temperature chamber. USP, WHO, and ISPE provide guidance on temperature distribution maps, but do not provide guidance on carbon dioxide distribution maps.

But this does not mean that auditors will not raise questions. We have learned from experience that regulatory agencies may require written evidence to prove that the thermostat is "in a controlled state" and "meets its intended use". However, it is usually not required to provide a carbon dioxide distribution map. Instead, our focus is on proving that the thermostat can function as expected, rather than relying solely on the supplier's design statement.

My viewpoint is quite pragmatic:

Monitoring and controlling carbon dioxide is crucial.

2. Distribution maps are unlikely to reveal meaningful trends of change.

The evidence for carbon dioxide recovery rate and stability may be more convincing than distribution map studies.

Key points for validating the team:

Rapid equilibrium of carbon dioxide: Unlike temperature, distribution maps usually do not show trends of change.

Buffer effect protects the culture: Dissolved carbon dioxide in liquid culture medium can increase stability when the door is briefly opened.

Attention to recovery rate: Verification should emphasize the speed at which carbon dioxide concentration levels recover to the set point after interference.

Document control: Even without a distribution chart, validation data should demonstrate controlled status and applicability.

Get ready to answer the reviewer's question: The statement must undergo internal verification, not just rely on the manufacturer's manual.

Conclusion:

Although drawing a carbon dioxide distribution map in a constant temperature chamber may not be necessary or scientifically meaningful, validation of carbon dioxide control is necessary. By focusing on recovery rate, stability, and monitoring records, the verification team can meet regulatory expectations while respecting the unique physical characteristics of carbon dioxide behavior.