How to find a suitable pressure endpoint criterion for freeze drying

Ending the primary and secondary drying step at the right moment is a key aspect to improving the efficiency of your freeze-drying process. Using pressure as an endpoint criterion is a great approach for determining the endpoint of these two lyophilization steps. Read on as I describe the tools you need for comparative pressure measurement. I also show you some experimental data that could come in handy when you try to establish the most suitable endpoint criterion for your applications.

Last weekend I went on a hiking trip in the mountains with a group of friends. We walked for a few hours before reaching a lovely hut right around lunch time. By this time, we were completely starved from all the physical activity and fresh mountain air. We sat down and ordered lots of yummy food before proceeding to stuff ourselves silly. I only stopped myself when I noticed pressure and aches building up in my belly. My stomach had uncomfortably expanded the waistline of my hiking shorts and was serving as a clear endpoint criterion to this lunchtime gluttony.

As I sat there, trying to digest and prepare myself for more walking, I became lost in thought. To be honest, whenever I am lost in thought, I am usually thinking about the lab. As I was feeling the pressure in my stomach subside, I was reminded of how pressure can be very helpful in determining the endpoint or more than just hefty meals. Pressure is also incredibly useful in evaluating the end of the primary or secondary drying phase in freeze drying.

I already discussed endpoint determination of the primary drying step using temperature in the previous lyophilization post. Here I’d like to offer you an alternative approach based on pressure.

In fact, comparative pressure measurement is a great non-invasive endpoint detection method for determining the end of the primary or secondary drying phase.

The technique uses two different pressure gauges, a Pirani sensor and a capacitance manometer. The Pirani sensor works on the principle that the thermal conductivity of a gas varies with pressure. The gauge measures the pressure using a thin wire suspended in a gas and heated with an electric current. At high pressure, the wire loses heat energy to the gas due to the high collision rate of the surrounding gas molecules with the wire. This principle is illustrated in the figure below.

pirani gauge, freeze drying, endpoint determination, pressure gauge

When the gas pressure is reduced by vacuum, the amount of gas molecules decreases together with the conductivity of the surrounding media. The media then starts to lose heat more slowly. Since this process relies on the thermal conductivity of the gas molecules and gas composition, a Pirani sensor will only display the correct pressure under its calibrated conditions, which are usually set at pure nitrogen or air environments.

In addition to the Pirani sensor, a capacitance manometer is used to measure the pressure independently of the gas composition. With this type of gauge, the difference in capacitance signal is produced by physical changes within the manometer and not by changes in gas properties.

Pressure measurement by capacitance manometer is thus independent of the gas composition.

If you are like me, you might be wondering exactly how these two tools work together in this type of endpoint determination.

Well, during the freeze-drying process, the gas in the drying chamber is almost completely composed of water vapor, due to the sublimation of ice. The more the sample dries, the more the gas composition changes. The water vapor is replaced by nitrogen or air until the chamber gas contains only pure nitrogen or air at the end of the drying process.

Since the thermal conductivity of water vapor is higher than that of nitrogen by a factor of approximately 1.6, the Pirani gauge has a measurement offset of approximately 60% in a pure water environment. The Pirani gauge and the capacitance manometer can only measure a similar pressure once the sample is dry and the composition of the chamber gas mainly consists of pure nitrogen or air. Hence, when the endpoint is reached, the two tools show the same pressure. The process is described graphically in the figure below.

comparative pressure measurement, endpoint determination, freeze drying, lyophilization, Priani sensor, capacitance mamometer

Importantly, pressure fluctuations prevent the difference between the two measurement tools from ever reaching zero.

A suitable endpoint criterion should be higher than the difference caused by pressure fluctuations. The value should also be low enough to ensure that the difference between the two displayed pressures is minimal over a given period before switching to the next freeze-drying step.

That sounds easy enough. But how do you really go about establishing a suitable endpoint criterion?

In order to find a suitable pressure difference, I recommend performing multiple test runs with a test formulation. I provide you with an example below.

In one study, I used a glycin solution (5% w/V in deionized water) as a test formulation. The solution was frozen over 24 hours at – 40°C and dried at a pressure of 0.300 mbar on a freeze dryer.

Importantly, before each freeze-drying cycle, a vacuum test to calibrate the Pirani gauge should be carried out. This step is mandatory to ensure that the Pirani gauge displays the correct pressure before and after the drying phases.

To find a suitable endpoint criterion, I carried out multiple test runs with different pressure differences as an endpoint criterion.

A successful endpoint detection was achieved when the temperature of the shelf met that of the sample and the pressure of the two manometers merged.

The results are displayed in the figure below. The top graph shows results with pressure differences of 0.05 mbar, the middle graph with 0.03 mbar and the bottom graph with 0.25 mbar as an endpoint criterion. The pressure differences were maintained for at least 60 minutes before the endpoint criterion was attained. The shelf temperature is represented by a yellow line, the sample temperature with a red line, the chamber pressure by a green line, the Pirani gauge with a blue line over primary drying (white shade) and secondary drying (grey shade). The time at which the pressure (black line) and temperature endpoint criterion (black, dashed line) are reached and the corresponding temperature and pressure differences at this point are also displayed.

comparative pressure measurement, endpoint criterion, freeze drying, lyophilization, endpoint determination, pressure

The results indicate that only in the cycle with a pressure difference of 0.025 mbar as an endpoint criterion, the pressure curves and temperature curves were merging simultaneously before switching over to secondary drying.

A pressure difference of 0.025 mbar or less that is maintained for more than 60 minutes can be considered a suitable endpoint criterion at the set pressure of 0.30 mbar.

That’s fine for the easy breezy glycin solution, but what about more challenging samples that need secondary drying?

Well, I decided to carry out a freeze-drying run using yummy strawberries on a freeze dryer. The strawberries were frozen for more than 24 hours at – 40°C and freeze dried at a pressure of 0.300 mbar, a shelf temperature of 25°C during primary drying and 40 °C during secondary drying. A pressure difference of 0.025 mbar was chosen as an endpoint criterion. The largest strawberry was carrying a thermocouple so that the sample temperature could be compared to the shelf temperature.

In the figure below, the top graph shows the full freeze-drying cycle, the figure at the bottom shows an extract of the secondary drying step. The shelf temperature is shown with a yellow line, the sample temperature with a red line, the chamber pressure with a green line, the Pirani gauge with a blue line. The white shade on the graphs represents primary drying, whereas the grey shade represents secondary drying. The time at which the endpoint criterion (black line) is reached and the corresponding temperature difference at this point are also shown.

The top graph from the figure below shows that the endpoint was reached after 37 hours. At this point, the temperature curves, as well as the pressure curve were merging simultaneously before the cycle changed to secondary drying.

During secondary drying of the strawberries, the Pirani gauge showed a clear peak once the shelf temperature was increased (bottom figure) and evaporation began. This peak is usually missed when using temperature measurements for endpoint determination, which demonstrates another clear advantage of the comparative pressure measurement.

pirani gauge, comparative pressure measurement, secondary drying step, endpoint determination, pressure measurement, endpoint criterion, freeze drying, lyophilization

This demonstrates that comparative pressure measurement also works in evaluating the endpoint criterion for challenging samples, such as strawberries.

For my parting words, I’d like to point out that a suitable endpoint criterion is highly dependent on the chamber pressure during the freeze-drying cycle. This is because the pressure difference during the drying phase is not absolute, but will always be at 60% of the chamber pressure.

The process to find an appropriate endpoint criterion hence needs to be repeated if the chamber pressure of a freeze-drying method is changes.

The endpoint criterion of the secondary drying stage should be adapted as well. Here, the maximal pressure difference usually does not reach 60% of the chamber pressure, since only a small portion of water is left in the sample. When compared to primary drying, it might be beneficial to consider a smaller pressure difference, which needs to last for a longer period of time as an endpoint criterion of secondary drying. This approach should also be validated with test runs first.

Now excuse me as I go grab some lunch. This time I will try to keep the pressure difference between my belly and the waistline of my pants to a minimum as well. Hope you also stay hungry for more freeze-drying and chromatography knowledge and keep dropping by the blog to satisfy your appetite.

Till next time,

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